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Sample records for cell fusion

  1. Cell fusions in mammals

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  2. Cell fusion and nuclear fusion in plants.

    Science.gov (United States)

    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.

  3. What Makes Fusion Cells Effective?

    Science.gov (United States)

    2009-12-01

    disbanded to address specific operations (e.g., a fleeting hostage rescue operation). Creation of these issue-based fusion cells would be based off...HQ USSOCOM Library MacDill AFB, FL 6. JSOC Fort Bragg, NC 7. ASD/SOLIC Washington, D.C.

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

    Directory of Open Access Journals (Sweden)

    Vincitorio Massimo

    2011-09-01

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

  5. Flavivirus cell entry and membrane fusion

    NARCIS (Netherlands)

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

    2011-01-01

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

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

    Science.gov (United States)

    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.

  7. Optically-Induced Cell Fusion on Cell Pairing Microstructures

    Science.gov (United States)

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

    2016-02-01

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

  8. Changes in Parthenogenetic Imprinting Patterns during Reprogramming by Cell Fusion.

    Directory of Open Access Journals (Sweden)

    Hyun Sik Jang

    Full Text Available Differentiated somatic cells can be reprogrammed into the pluripotent state by cell-cell fusion. In the pluripotent state, reprogrammed cells may then self-renew and differentiate into all three germ layers. Fusion-induced reprogramming also epigenetically modifies the somatic cell genome through DNA demethylation, X chromosome reactivation, and histone modification. In this study, we investigated whether fusion with embryonic stem cells (ESCs also reprograms genomic imprinting patterns in somatic cells. In particular, we examined imprinting changes in parthenogenetic neural stem cells fused with biparental ESCs, as well as in biparental neural stem cells fused with parthenogenetic ESCs. The resulting hybrid cells expressed the pluripotency markers Oct4 and Nanog. In addition, methylation of several imprinted genes except Peg3 was comparable between hybrid cells and ESCs. This finding indicates that reprogramming by cell fusion does not necessarily reverse the status of all imprinted genes to the state of pluripotent fusion partner.

  9. Inflammation and proliferation act together to mediate intestinal cell fusion.

    Directory of Open Access Journals (Sweden)

    Paige S Davies

    Full Text Available 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-10-15

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

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

    Science.gov (United States)

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

    2014-10-15

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

  12. Genetic variability available through cell fusion

    Energy Technology Data Exchange (ETDEWEB)

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

    1977-01-01

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

  13. Cell Fusion as a Cause of Prostate Cancer Metastases

    Science.gov (United States)

    2011-04-01

    treated identically in the same dish, but became either binuclear ( tetraploid ) or mononuclear (diploid). We used time-lapse microscopy to score mitosis...expressing Bcl-2 did not increase clonogenic survival of tetraploid cells produced by either cytokinesis failure or cell fusion, while expressing a...clonogenic hybrids (Fig. 12), suggesting that cell fusion and cytokinesis failure have different potential to produce clonogenic tetraploid cells. The

  14. Dendritic-tumor fusion cells in cancer immunotherapy.

    Science.gov (United States)

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

    2015-03-01

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

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

    Science.gov (United States)

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

    2016-08-01

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

  16. Lineage development of cell fusion hybrids upon somatic reprogramming

    OpenAIRE

    2011-01-01

    Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2011 Somatic cell reprogramming has been extensively studied over the last years and opened new perspectives in the use of pluripotent cells for regenerative biomedical purposes. Spontaneous cell fusion has been suggested to be involved in regenerative processes in vivo. Strong evidences support the hypothesis that the reprogrammed hybrids resulting from the fusion between a pluripote...

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

    Science.gov (United States)

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

  18. Cancer Vaccine by Fusions of Dendritic and Cancer Cells

    Directory of Open Access Journals (Sweden)

    Shigeo Koido

    2009-01-01

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

  19. Fusion

    CERN Document Server

    Mahaffey, James A

    2012-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Bzik, D.J.; Person, S.

    1981-04-15

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

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

    Science.gov (United States)

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

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

  4. Homotypic fusion of endoplasmic reticulum membranes in plant cells

    Directory of Open Access Journals (Sweden)

    Junjie eHu

    2013-12-01

    Full Text Available The endoplasmic reticulum (ER is a membrane-bounded organelle whose membrane comprises a network of tubules and sheets. The formation of these characteristic shapes and maintenance of their continuity through homotypic membrane fusion appears to be critical for the proper functioning of the ER. The atlastins (ATLs, a family of ER-localized dynamin-like GTPases, have been identified as fusogens of the ER membranes in metazoans. Mutations of the ATL proteins in mammalian cells cause morphological defects in the ER, and purified Drosophila ATL mediates membrane fusion in vitro. Plant cells do not possess ATL, but a family of similar GTPases, named root hair defective 3 (RHD3, are likely the functional orthologs of ATLs. In this review, we summarize recent advances in our understanding of how RHD3 proteins play a role in homotypic ER fusion. We also discuss the possible physiological significance of forming a tubular ER network in plant cells.

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

    Directory of Open Access Journals (Sweden)

    Junko Maeda

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

  6. Cancer Vaccine by Fusions of Dendritic and Cancer Cells

    OpenAIRE

    Shigeo Koido; Eiichi Hara; Sadamu Homma; Yoshihisa Namiki; Toshifumi Ohkusa; Jianlin Gong; Hisao Tajiri

    2010-01-01

    Dendritic cells (DCs) are potent antigen-presenting cells and play a central role in the initiation and regulation of primary immune responses. Therefore, their use for the active immunotherapy against cancers has been studied with considerable interest. The fusion of DCs with whole tumor cells represents in many ways an ideal approach to deliver, process, and subsequently present a broad array of tumor-associated antigens, including those yet to be unidentified, in the context of DCs-derived...

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

    Science.gov (United States)

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

    2011-03-01

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

  8. Cell Therapy to Obtain Spinal Fusion

    Science.gov (United States)

    2006-02-01

    Cell viability in these experiments was determined to be greater than 90%. 6 *** marketed for DNA rather than virus transfer, we have determined...dead cells and debris were removed by washing with PBS and cells were passaged before confluence. Several vials of these cells were frozen in Origen

  9. Information Fusion for Anomaly Detection with the Dendritic Cell Algorithm

    CERN Document Server

    Greensmith, Julie; Tedesco, Gianni

    2010-01-01

    Dendritic cells are antigen presenting cells that provide a vital link between the innate and adaptive immune system, providing the initial detection of pathogenic invaders. Research into this family of cells has revealed that they perform information fusion which directs immune responses. We have derived a Dendritic Cell Algorithm based on the functionality of these cells, by modelling the biological signals and differentiation pathways to build a control mechanism for an artificial immune system. We present algorithmic details in addition to experimental results, when the algorithm was applied to anomaly detection for the detection of port scans. The results show the Dendritic Cell Algorithm is sucessful at detecting port scans.

  10. Studies on virus-induced cell fusion. Progress report, July 1, 1978-July 31, 1979

    Energy Technology Data Exchange (ETDEWEB)

    Person, S.

    1979-08-01

    We have determined the extent of fusion in mixed Herpes Simplex Virus type 1 (HSV-1) cell populations. Labeled, sparse, infected cells were surrounded by unlabeled, excess, uninfected (or infected) cells to determine if fusion inhibition is active when present in the same cell as fusion factor, when present in cells that do not contain fusion factor, or in both situations. We found that wild type infected cells fuse to the same extent with each other as with uninfected cells. Therefore fusion inhibitor is active when present in the same cell as fusion factor. Other experiments indicate that the inhibition activity can also cause a small decrease of fusion when fusion factor and fusion inhibitor are present in different, but neighboring, cells. Marked variations are found in the capacity of different cell types, such as human embryonic lung (HEDL) and HEp-2 to support fusion by particular syn mutants. The difference in fusion capacity of the two cell types may be due to a difference in a cellular factor affecting the activity of viral, fusion-associated molecules, and not to cell surface differences of the uninfected cells. (PCS)

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

    Science.gov (United States)

    Maeshima, Kazuhiro; Funakoshi, Tomoko; Imamoto, Naoko

    2014-01-01

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

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

    Science.gov (United States)

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

    2008-06-01

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

  13. Premature Activation of the Paramyxovirus Fusion Protein before Target Cell Attachment with Corruption of the Viral Fusion Machinery*

    Science.gov (United States)

    Farzan, Shohreh F.; Palermo, Laura M.; Yokoyama, Christine C.; Orefice, Gianmarco; Fornabaio, Micaela; Sarkar, Aurijit; Kellogg, Glen E.; Greengard, Olga; Porotto, Matteo; Moscona, Anne

    2011-01-01

    Paramyxoviruses, including the childhood pathogen human parainfluenza virus type 3, enter host cells by fusion of the viral and target cell membranes. This fusion results from the concerted action of its two envelope glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). The receptor-bound HN triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We proposed that, if the fusion process could be activated prematurely before the virion reaches the target host cell, infection could be prevented. We identified a small molecule that inhibits paramyxovirus entry into target cells and prevents infection. We show here that this compound works by an interaction with HN that results in F-activation prior to receptor binding. The fusion process is thereby prematurely activated, preventing fusion of the viral membrane with target cells and precluding viral entry. This first evidence that activation of a paramyxovirus F can be specifically induced before the virus contacts its target cell suggests a new strategy with broad implications for the design of antiviral agents. PMID:21799008

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

    Science.gov (United States)

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

    2013-07-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-07-15

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

  16. Plant protoplast fusion and growth of intergeneric hybrid cells.

    Science.gov (United States)

    Kao, K N; Constabel, F; Michayluk, M R; Gamborg, O L

    1974-01-01

    Interspecific and intergeneric fusions of plant protoplasts were induced by polyethylene glycol (PEG) 1540 or 4000. The frequency of heterokaryocyte formation (or rate of fusion) was much higher when PEG was eluted with a high pH-high Ca(2+) solution or a salt solution than when it was eluted with a protoplast culture medium. The frequency of heterokaryocyte formation was also affected by the types of enzymes used for wall degradation, duration of enzyme incubation and molality of the PEG solutions.The maximum frequency of heterokaryocyte formation was 23% for V. hajastana Grossh.-soybean (Glycine max L.) and barley (Hordeum vulgare L.)-soybean, 35% for pea (Pisum sativum L.)-soybean, 20% for pea-V. hajastana, 14% for corn (Zea mays L.)-soybean and 10% for V. villosa Roth-V. hajastana.40% of the barley-soybean, corn-soybean and pea-soybean heterokaryocytes divided at least once. Some divided many times and formed clusters of up to 100 cells in 2 weeks. The heterokaryocytes of soybean-V. hajastana, V. villosa-V. hajastana also divided. Of the PEG-treated protoplasts of N. langsdorffii and N. glauca 13.5% developed into tumor-like calli. The morphology of these calli was very much like that of the tumors produced on amphidiploid plants of N. langsdorffii x glauca.Nuclear staining indicated that heterokaryocytes of V. hajastana-soybean, pea-soybean, corn-soybean and barley-soybean could undergo mitosis. Nuclear divisions in a heterokaryocyte were usually synchronized or almost synchronized. Nuclear fusion and true hybrid formation usually occurred during the first mitotic division after protoplast fusion. A hybrid of barley-soybean in third cell division was observed. The frequency of heterokaryocytes which underwent nuclear fusion has not been determined. Multipole formation and chimeral cell colonies were also observed.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Jeffrey L. Platt

    2016-07-01

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

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

    Science.gov (United States)

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

    2014-07-01

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

  20. Cell Therapy To Obtain Spinal Fusion

    Science.gov (United States)

    2010-07-01

    peritoneal perforation, sacroiliac joint instability, and herniation of abdominal contents through defects in the ilium [8]. Furthermore, the volume of...M, Zhang Y, Senitzer D, Forman SJ, Emerson SG 2007 Hematopoietic stem-cell contribution to ectopic skeletogenesis. J Bone Joint Surg Am 89(2):347...becomes weight bearing. It readily fuses to the skeletal bone, and often leads to ankylosis of the joints . Harnessing this capacity in a targeted

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

    Science.gov (United States)

    Gambhir; Sanjiv , Pritha; Ray

    2009-04-28

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

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

    Science.gov (United States)

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

    1997-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Klier Ulrike

    2011-09-01

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

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1982-01-01

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

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

    Science.gov (United States)

    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.

  7. Intra-hematopoietic cell fusion as a source of somatic variation in the hematopoietic system.

    Science.gov (United States)

    Skinner, Amy M; Grompe, Markus; Kurre, Peter

    2012-06-15

    Cell fusion plays a well-recognized, physiological role during development. Bone-marrow-derived hematopoietic cells have been shown to fuse with non-hematopoietic cells in a wide variety of tissues. Some organs appear to resolve the changes in ploidy status, generating functional and mitotically-competent events. However, cell fusion exclusively involving hematopoietic cells has not been reported. Indeed, genomic copy number variation in highly replicative hematopoietic cells is widely considered a hallmark of malignant transformation. Here we show that cell fusion occurs between cells of the hematopoietic system under injury as well as non-injury conditions. Experiments reveal the acquisition of genetic markers in fusion products, their tractable maintenance during hematopoietic differentiation and long-term persistence after serial transplantation. Fusion events were identified in clonogenic progenitors as well as differentiated myeloid and lymphoid cells. These observations provide a new experimental model for the study of non-pathogenic somatic diversity in the hematopoietic system.

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

    Science.gov (United States)

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

    2016-10-01

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

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

    Science.gov (United States)

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

    2015-11-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-04-16

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-01-14

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

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

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

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

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

    Science.gov (United States)

    Oleinick, Alexander; Svir, Irina; Amatore, Christian

    2017-01-01

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

  14. Fine structure of fusion products from soybean cell culture and pea leaf protoplasts.

    Science.gov (United States)

    Fowke, L C; Constabel, F; Gamborg, O L

    1977-01-01

    Protoplasts from pea (Pisum sativum L.) leaves and cultured soybean (Glycine max L.) cells were fused by means of polyethylene glycol and subsequently cultured for one week. Both agglutinated protoplasts and cultured fusion products were examined by electron microscopy. Agglutination occurred over large areas of the plasma membranes. The membrane contanct was discontinuous and irregularly spaced. Many cultured fusion products regenerated cell walls and divided to form cell clusters. Fusion of pea and soybean interphase nuclei occurred in some cells. The detection of heterochromatin typical of pea in the synkaryon, even after division, suggests the cells were hybrids. The cytoplasm of the cells from the fusion products contained both soybean leucoplasts and pea chloroplasts. The chloroplasts had apparently ceased dividing and some showed signs of degenerating. Large multinucleate fusion products developed cell walls but failed to divide.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    and Purkinje neurons. However, through lineage fate-mapping we demonstrate that such in vivo fusion of lymphoid and myeloid blood cells does not occur to an appreciable extent in steady-state adult tissues or during normal development. Rather, fusion of blood cells with different non-haematopoietic cell types...... is induced by organ-specific injuries or whole-body irradiation, which has been used in previous studies to condition recipients of bone marrow transplants. Our findings demonstrate that blood cells of the lymphoid and myeloid lineages contribute to various non-haematopoietic tissues by forming rare fusion......Recent studies have suggested that regeneration of non-haematopoietic cell lineages can occur through heterotypic cell fusion with haematopoietic cells of the myeloid lineage. Here we show that lymphocytes also form heterotypic-fusion hybrids with cardiomyocytes, skeletal muscle, hepatocytes...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-10-15

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

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

    Science.gov (United States)

    Jiang, Shibo; Radigan, Lin; Zhang, Li

    2000-03-01

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

  18. Cell-based analysis of Chikungunya virus E1 protein in membrane fusion

    Directory of Open Access Journals (Sweden)

    Kuo Szu-Cheng

    2012-04-01

    Full Text Available Abstract Background Chikungunya fever is a pandemic disease caused by the mosquito-borne Chikungunya virus (CHIKV. E1 glycoprotein mediation of viral membrane fusion during CHIKV infection is a crucial step in the release of viral genome into the host cytoplasm for replication. How the E1 structure determines membrane fusion and whether other CHIKV structural proteins participate in E1 fusion activity remain largely unexplored. Methods A bicistronic baculovirus expression system to produce recombinant baculoviruses for cell-based assay was used. Sf21 insect cells infected by recombinant baculoviruses bearing wild type or single-amino-acid substitution of CHIKV E1 and EGFP (enhanced green fluorescence protein were employed to investigate the roles of four E1 amino acid residues (G91, V178, A226, and H230 in membrane fusion activity. Results Western blot analysis revealed that the E1 expression level and surface features in wild type and mutant substituted cells were similar. However, cell fusion assay found that those cells infected by CHIKV E1-H230A mutant baculovirus showed little fusion activity, and those bearing CHIKV E1-G91D mutant completely lost the ability to induce cell-cell fusion. Cells infected by recombinant baculoviruses of CHIKV E1-A226V and E1-V178A mutants exhibited the same membrane fusion capability as wild type. Although the E1 expression level of cells bearing monomeric-E1-based constructs (expressing E1 only was greater than that of cells bearing 26S-based constructs (expressing all structural proteins, the sizes of syncytial cells induced by infection of baculoviruses containing 26S-based constructs were larger than those from infections having monomeric-E1 constructs, suggesting that other viral structure proteins participate or regulate E1 fusion activity. Furthermore, membrane fusion in cells infected by baculovirus bearing the A226V mutation constructs exhibited increased cholesterol-dependences and lower pH thresholds

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

    Energy Technology Data Exchange (ETDEWEB)

    Person, S.

    1978-07-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-12-18

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

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

    Institute of Scientific and Technical Information of China (English)

    Feng Xu; Ying-Jiang Ye; Shan Wang

    2004-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Alexander Krivokharchenko

    Full Text Available 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    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.

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

    Science.gov (United States)

    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.

  7. Spontaneous telomere to telomere fusions occur in unperturbed fission yeast cells

    OpenAIRE

    Almeida, H.; Godinho Ferreira, M.

    2013-01-01

    Telomeres protect eukaryotic chromosomes from illegitimate end-to-end fusions. When this function fails, dicentric chromosomes are formed, triggering breakage-fusion-bridge cycles and genome instability. How efficient is this protection mechanism in normal cells is not fully understood. We created a positive selection assay aimed at capturing chromosome-end fusions in Schizosaccharomyces pombe. We placed telomere sequences with a head to head arrangement in an intron of a selectable marker co...

  8. Human Metapneumovirus Is Capable of Entering Cells by Fusion with Endosomal Membranes.

    Directory of Open Access Journals (Sweden)

    Reagan G Cox

    2015-12-01

    Full Text Available Human metapneumovirus (HMPV, a member of the Paramyxoviridae family, is a leading cause of lower respiratory illness. Although receptor binding is thought to initiate fusion at the plasma membrane for paramyxoviruses, the entry mechanism for HMPV is largely uncharacterized. Here we sought to determine whether HMPV initiates fusion at the plasma membrane or following internalization. To study the HMPV entry process in human bronchial epithelial (BEAS-2B cells, we used fluorescence microscopy, an R18-dequenching fusion assay, and developed a quantitative, fluorescence microscopy assay to follow virus binding, internalization, membrane fusion, and visualize the cellular site of HMPV fusion. We found that HMPV particles are internalized into human bronchial epithelial cells before fusing with endosomes. Using chemical inhibitors and RNA interference, we determined that HMPV particles are internalized via clathrin-mediated endocytosis in a dynamin-dependent manner. HMPV fusion and productive infection are promoted by RGD-binding integrin engagement, internalization, actin polymerization, and dynamin. Further, HMPV fusion is pH-independent, although infection with rare strains is modestly inhibited by RNA interference or chemical inhibition of endosomal acidification. Thus, HMPV can enter via endocytosis, but the viral fusion machinery is not triggered by low pH. Together, our results indicate that HMPV is capable of entering host cells by multiple pathways, including membrane fusion from endosomal compartments.

  9. Fusion of proinsulin-producing bone marrow-derived cells with hepatocytes in diabetes.

    Science.gov (United States)

    Fujimiya, Mineko; Kojima, Hideto; Ichinose, Masumi; Arai, Ryohachi; Kimura, Hiroshi; Kashiwagi, Atsunori; Chan, Lawrence

    2007-03-06

    We previously reported that diabetes in mice is associated with the appearance of proinsulin-producing (Proins-P) cells in the liver. It was unclear, however, whether these Proins-P bone marrow-derived cells (BMDC) merely transit through the liver or undergo fusion with hepatocytes, normally an extremely rare event. In this study, we found that, in diabetes, BMDC in the liver produce not only Proins but also TNF-alpha, suggesting that diabetes reprograms gene expression in BMDC, turning on "inappropriate" genes. Bone marrow transplantation using genetically marked donor and recipient mice showed that fusion occurs between Proins-P BMDC and hepatocytes. Cell fusion is further supported by the presence of the Y chromosome in Proins-P cells in female mice that received male bone marrow transplantation cells. Morphologically, Proins-P fusion cells are albumin-producing hepatocytes that constitute approximately 2.5% of the liver section area 5 months after diabetes induction. An extensive search failed to reveal any fusion cells in nondiabetic mice. Thus, diabetes causes fusion between Proins-P BMDC and hepatocytes in vivo, an observation that has implications for the pathophysiology of diabetes as well as the fundamental biology of heterotypic cell fusion.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-11-01

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

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

    Science.gov (United States)

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

    2012-08-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    1999-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Aiko Amagai

    2012-01-01

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

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

    Science.gov (United States)

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

    2011-02-15

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

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

    Science.gov (United States)

    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.

  16. Viral glycoprotein-mediated cell fusion assays using vaccinia virus vectors.

    Science.gov (United States)

    Bossart, Katharine N; Broder, Christopher C

    2004-01-01

    The vaccinia virus-based expression of viral envelope glycoprotein genes-derived from enveloped viruses that infect their respective host cells through a pH-independent mechanism of membrane fusion-has been a powerful tool in helping to characterize these important attachment and fusion proteins. The cellular expression of these viral envelope glycoproteins has allowed for the measurement of membrane fusion events using cell-cell fusion or syncytia formation. This method has been enhanced by the addition of a reporter-gene system to the vaccinia virus-based cell-cell fusion assay. This improvement has provided a high-throughput and quantitative aspect to this assay, which can serve as a surrogate for virus entry and is therefore ideally suited in the characterization of numerous enveloped viruses, including biological safety level-4 (BSL-4) agents. This chapter will detail the methods of the vaccinia virus-based reporter-gene fusion assay and how it may be used to characterize the fusion mediated by the BSL-4-classified Hendra and Nipah viruses.

  17. The choriocarcinoma cell line BeWo: syncytial fusion and expression of syncytium-specific proteins.

    Science.gov (United States)

    Orendi, Kristina; Gauster, Martin; Moser, Gerit; Meiri, Hamutal; Huppertz, Berthold

    2010-11-01

    Fusion of the trophoblast-derived choriocarcinoma cell line BeWo can be triggered by forskolin. BeWo cells are regularly used as a cell culture model to mimic in vivo syncytialisation of placental villous trophoblast. The β subunit of human chorionic gonadotropin (CGB), placental alkaline phosphatase as well as placental protein 13 (PP13, LGALS13) are exclusively expressed in the syncytiotrophoblast of the human placenta, and CGB is commonly used as a marker of syncytial differentiation. Here we tested the hypothesis that syncytial fusion precedes CGB and LGALS13 expression in trophoblast-derived BeWo cells. BeWo cells were cultured for 48 h in the presence or absence of forskolin and varying concentrations of H-89, a protein kinase A inhibitor that interferes with the forskolin-mediated pathway of syncytial fusion. LGALS13 and CGB expression were quantified by DELFIA and real-time PCR. Cell fusion was determined by morphological analysis and cell counting after immunofluorescence staining. In forskolin-stimulated BeWo cells that were hindered to fuse by treatment with H-89, levels of CGB protein expression were not altered, while LGALS13 protein and mRNA expression decreased significantly to control levels without forskolin. The LGALS13 protein expression data coincided with a significant decrease in syncytial fusion, while CGB protein expression was unaffected by rates of cell fusion and proliferation. We postulate that CGB protein expression is not necessarily linked to syncytial fusion, and thus CGB should be used with great caution as a marker of BeWo cell fusion.

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

    Energy Technology Data Exchange (ETDEWEB)

    Song, Kai; Zhu, Fei; Zhang, Han-zhong [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST), Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Shang, Zheng-jun, E-mail: shangzhengjun@hotmail.com [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST), Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); First Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, Wuhan (China)

    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

  19. Osteoclast Fusion

    DEFF Research Database (Denmark)

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

    2017-01-01

    suggesting that fusion partners may specifically select each other and that heterogeneity between the partners seems to play a role. Therefore, we set out to directly test the hypothesis that fusion factors have a heterogenic involvement at different stages of nuclearity. Therefore, we have analyzed...... on the nuclearity of fusion partners. While CD47 promotes cell fusions involving mono-nucleated pre-osteoclasts, syncytin-1 promotes fusion of two multi-nucleated osteoclasts, but also reduces the number of fusions between mono-nucleated pre-osteoclasts. Furthermore, CD47 seems to mediate fusion mostly through......Investigations addressing the molecular keys of osteoclast fusion are primarily based on end-point analyses. No matter if investigations are performed in vivo or in vitro the impact of a given factor is predominantly analyzed by counting the number of multi-nucleated cells, the number of nuclei per...

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

    Science.gov (United States)

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

    2008-04-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2015-10-12

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

  5. Ultrastructure of fusion products from soybean cell culture and sweet clover leaf protoplasts.

    Science.gov (United States)

    Fowke, L C; Rennie, P J; Kirkpatrick, J W; Constabel, F

    1976-01-01

    Protoplasts from cultured cells of soybean (Glycine max L.) and from sweet clover (Melilotus officinalis L.) mesophyll cells were fused with polyethylene glycol and subsequently cultured for six days. The resulting fusion products as well as unfused protoplasts of each parental species regenerated cell walls and divided. The fusion products were characterized by the presence of soybean leucoplasts and sweet clover chloroplasts. The chloroplasts appeared to be degenerating but other cytoplasmic organelles were typical of actively growing plant cells. The fate of individual nuclei could not be determined.

  6. Fusions of Dendritic Cells and C6 Cells Transfected with TGF-β1 Antisense in Treatment of Intracranial Gliomas

    Institute of Scientific and Technical Information of China (English)

    Jin Gui-shan; Liu Fu-sheng; Chai Qi; Wang Jian-jao; Li Jun-hua

    2007-01-01

    Objective: To investigate the immunotherapy efficacy of fusion cells (dendritic-C6anti-TGF-β1 cells) in the treatment of intracranial gliomas. Methods: Dendritic cells were isolated from rat bone-marrow precursors stimulated in vitro with granulocyte-macrophage colony-stimulating factor (GM-CSF) and Interleukin-4 (IL-4). C6anti-TGF-β1 cells originally from C6 cell line of a rat glioblastoma were transfected with plasmid of TGF-β1 anti-sense gene. Fusions of dendritic cells and C6anti-TGF-β1 cells were prepared by polyethylene glycol (PEG). The DC/C6anti-TGF-β1 fusion cells were observed and confirmed by light microscopy and scanning electron microscopy. Experimental rats were divided into three groups at random: C6 cells (Ⅰ), dendritic-C6anti-TGF-β1 fusion cells and C6 cells (Ⅱ) and IMDM medium only (Ⅲ). The cells were injected into right parietal lobe region of the rat with stereotaxic technique. Histology, tumor necrosis and survival time were evaluated. Results: Compared with the rats that received C6 cells (survival median time was less than 20 days, tumor region was seen in all fields of observed), the rats injected with dendritic-C6anti-TGF-β1 fusion cells and C6 cells got a more prolonged life span (more than 59 days), as well as less tumor region (5.01%-6.2%). There was no tumor necrosis, but some glias were seen in surroundings. All rats were survived and no necrosis was observed in negative control group. Statistical analysis showed that group Ⅱ had significant difference compared with group Ⅰ. Conclusions: Dendritic-C6anti-TGF-β1 fusion cells could prolong the life span of rats, providing a strategy to achieve an antitumor response against tumors in the central nervous system.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Sreenivasa R Sankavaram

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

  10. Spontaneous cancer-stromal cell fusion as a mechanism of prostate cancer androgen-independent progression.

    Directory of Open Access Journals (Sweden)

    Ruoxiang Wang

    Full Text Available We have previously shown that human prostate cancer cells are capable of acquiring malignant attributes through interaction with stromal cells in the tumor microenvironment, while the interacting stromal cells can also become affected with both phenotypic and genotypic alterations. This study used a co-culture model to investigate the mechanism underlying the co-evolution of cancer and stromal cells. Red fluorescent androgen-dependent LNCaP prostate cancer cells were cultured with a matched pair of normal and cancer-associated prostate myofibroblast cells to simulate cancer-stromal interaction, and cellular changes in the co-culture were documented by tracking the red fluorescence. We found frequent spontaneous fusions between cancer and stromal cells throughout the co-culture. In colony formation assays assessing the fate of the hybrid cells, most of the cancer-stromal fusion hybrids remained growth-arrested and eventually perished. However, some of the hybrids survived to form colonies from the co-culture with cancer-associated stromal cells. These derivative clones showed genomic alterations together with androgen-independent phenotype. The results from this study reveal that prostate cancer cells are fusogenic, and cancer-stromal interaction can lead to spontaneous fusion between the two cell types. While a cancer-stromal fusion strategy may allow the stromal compartment to annihilate invading cancer cells, certain cancer-stromal hybrids with increased survival capability may escape annihilation to form a derivative cancer cell population with an altered genotype and increased malignancy. Cancer-stromal fusion thus lays a foundation for an incessant co-evolution between cancer and the cancer-associated stromal cells in the tumor microenvironment.

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

    Directory of Open Access Journals (Sweden)

    Waka Omata

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

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

    Science.gov (United States)

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

    2016-06-02

    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.

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

    Science.gov (United States)

    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.

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

    DEFF Research Database (Denmark)

    Bahadori, Azra

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

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

    Institute of Scientific and Technical Information of China (English)

    LI Ying; TIEN Po

    2007-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-12-12

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

  17. Characterization of Mason--Pfizer monkey virus-induced cell fusion. [uv radiation

    Energy Technology Data Exchange (ETDEWEB)

    Chatterjee, S.; Hunter, E.

    1979-06-01

    The characteristics and requirements of multinucleate cell (syncytium) induction by Mason--Pfizer monkey virus (M-PMV) on human and non-human primate cells have been investigated. Multinucleate cell induction by this D-type retrovirus shows single-hit kinetics on human foreskin and rhesus monkey fetal lung cells. The peak of syncytium-forming activity in an isopycnic sucrose gradient coincides with the peak of M-PMV virions as assessed by electron microscopy and analysis of viral polypeptides. Unlike the paramyxoviruses, M-PMV does not induce early cell fusion when added in high concentrations to the target cells. Furthermore, multinucleate cell formation is maximal 48 hr postinfection and the size of the syncytia remains constant after this time. Ultraviolet irradiation of M-PMV reduces its ability to form syncytia and to replicate with single-hit kinetics, suggesting that a functional viral genome is required for syncytium formation. Proviral DNA synthesis and assembly of virions are not necessary for cell fusion since the addition of cytosine arabinoside at concentrations which block virus replication has little effect on multinucleate cell formation. Moreover both multinucleate cells lacking detectable intracellular virus polypeptides, and groups of individual, nonfused but brightly staining cells can be observed in immunofluorescence assays at times when multinucleate cell formation is maximal. Cell fusion is inhibited by the addition of cycloheximide during the first 12 hr of infection, suggesting that de novo protein synthesis is required for multinucleate cell formation. The possibility that the translation of genomic RNA yields a fusion-inducing product is discussed.

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

    KAUST Repository

    Maruyama, Daisuke

    2015-05-01

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

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

    Science.gov (United States)

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

    2015-04-21

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

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

    Directory of Open Access Journals (Sweden)

    Luisa Cironi

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

  1. Cell Fusion as a Cofactor in Prostate Cancer Metastasis

    Science.gov (United States)

    2010-01-01

    mechanisms that control the fate of fused cells, and the fate of tetraploid cells in general, are poorly understood. To learn these mechanisms better...of the resulting binuclear cells, and delineated basic mechanisms through which apoptosis was induced. Remarkably, we found that tetraploid cells...survival of tetraploid cells, while expressing a dominant negative mutant of the tumor suppressor p53 did (Figure 2). These findings implied that

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

    Directory of Open Access Journals (Sweden)

    María D. Cuenca-López

    2014-12-01

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

  3. Evaluation of posterolateral lumbar fusion in sheep using mineral scaffolds seeded with cultured bone marrow cells.

    Science.gov (United States)

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

    2014-12-16

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-02-03

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

  8. Sodium butyrate induces DRP1-mediated mitochondrial fusion and apoptosis in human colorectal cancer cells.

    Science.gov (United States)

    Tailor, Dhanir; Hahm, Eun-Ryeong; Kale, Raosaheb K; Singh, Shivendra V; Singh, Rana P

    2014-05-01

    Sodium butyrate (NaBt) is the byproduct of anaerobic microbial fermentation inside the gastro-intestinal tract that could reach up to 20mM, and has been shown to inhibit the growth of various cancers. Herein, we evaluated its effect on mitochondrial fusion and associated induction of apoptosis in colorectal cancer cells (CRC). NaBt treatment at physiological (1-5mM) concentrations for 12 and 24h decreased the cell viability and induced G2-M phase cell cycle arrest in HCT116 (12h) and SW480 human CRC cells. This cell cycle arrest was associated with mitochondria-mediated apoptosis accompanied by a decrease in survivin and Bcl-2 expression, and generation of reactive oxygen species (ROS). Furthermore, NaBt treatment resulted in a significant decrease in the mitochondrial mass which is an indicator of mitochondrial fusion. Level of dynamin-related protein 1 (DRP1), a key regulator of mitochondrial fission and fusion where its up-regulation correlates with fission, was found to be decreased in CRC cells. Further, at early treatment time, DRP1 down-regulation was noticed in mitochondria which later became drastically reduced in both mitochondria as well as cytosol. DRP1 is activated by cyclin B1-CDK1 complex by its ser616 phosphorylation in which both cyclin B1-CDK1 complex and phospho-DRP1 (ser616) were strongly reduced by NaBt treatment. DRP1 was observed to be regulated by apoptosis as pan-caspase inhibitor showing rescue from NaBt-induced apoptosis also caused the reversal of DRP1 to the normal level as in control proliferating cells. Together, these findings suggest that NaBt can modulate mitochondrial fission and fusion by regulating the level of DRP1 and induce cell cycle arrest and apoptosis in human CRC cells.

  9. A novel fusion protein diphtheria toxin-stem cell factor (DT-SCF)-purification and characterization.

    Science.gov (United States)

    Potala, Sirisha; Verma, Rama Shanker

    2010-11-01

    Fusion toxins are an emerging class of targeted therapeutics for the treatment of cancer. Diphtheria toxin-stem cell factor (DT-SCF) is one such novel fusion toxin designed to target malignancies expressing c-kit. Since, c-kit overexpression has been reported on many types of cancers, it appeared to be a reasonably good molecule to target. In the present study, we report construction, expression, purification, and characterization of DT-SCF. DT-SCF gene coding for 1-387 amino acids of diphtheria toxin, His-Ala linker, 2-141 amino acids of SCF was cloned into expression vector with C terminal His tag. The induced DT-SCF protein was exclusively expressed in insoluble fraction. Purification of DT-SCF was achieved by inclusion body isolation and metal affinity chromatography under denaturing and reducing conditions. Purified DT-SCF was renatured partially on-column by gradually reducing denaturant concentration followed by complete refolding through rapid dilution technique. Cell viability assay provided the evidence that DT-SCF is a potent cytotoxic agent selective to cells expressing c-kit. The novelty of this study lies in employing SCF as a ligand in construction of fusion toxin to target wide range of malignancies expressing c-kit. Efficacy of DT-SCF fusion toxin was demonstrated over a range of malignancies such as chronic myeloid leukemia (K562), acute lymphoblastic leukemia (MOLT4), pancreatic carcinoma (PANC-1), and cervical carcinoma (HeLa 229). This is the first study reporting specificity and efficacy of DT-SCF against tumor cells expressing c-kit. There was significant correlation (P = 0.007) between c-kit expression on cells and their sensitivity to DT-SCF fusion toxin.

  10. Fusions of Breast Carcinoma and Dendritic Cells as a Vaccine for the Treatment of Metatastic Breast Cancer

    Science.gov (United States)

    2012-07-01

    Dendritic Cells as a Vaccine for the Treatment of Metatastic Breast Cancer PRINCIPAL INVESTIGATOR: Donald Kufe, M.D...COVERED 1 July 2011 – 30 June 2012 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Fusions of Breast Carcinoma and Dendritic Cells as a Vaccine for...have been enrolled thus far. We reported in detail the characterization of the tumor cells, the generated dendritic cells and the DC/tumor fusions

  11. Recovery of infectious human immunodeficiency virus type 1 after fusion of defectively infected clones of U-937 cells.

    OpenAIRE

    1991-01-01

    Polyethylene glycol was used to induce polykaryon formation among U-937 cell subclones carrying defective human immunodeficiency virus (HIV) type 1 proviral DNA. Fusion of cells which produced gp120-defective virions (UHC15.7) with cells unable to generate reverse transcriptase (RT) activity (UHC8 and UHC18) yielded polykaryons which made infectious viral progeny that showed normal protein profiles. Southern blot analysis of proviral DNA of cells infected with such fusion-derived virus reveal...

  12. Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture.

    Science.gov (United States)

    Imsoonthornruksa, Sumeth; Pruksananonda, Kamthorn; Parnpai, Rangsun; Rungsiwiwut, Ruttachuk; Ketudat-Cairns, Mariena

    2015-01-01

    To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins.

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

    Science.gov (United States)

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

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Mcgraw, T.F.; Davis, R.R.

    1998-07-01

    The fuel cell industry possesses much of the required manufacturing equipment and knowledge-base (e.g., proton conduction and hydrogen safety) necessary to develop cold fusion systems. Key factors in making a transition to cold fusion technology are discussed. Loading of reaction material can be provided by electrolytic charging and high gas over-pressure. Effective pressures over 10,000 atmospheres are required in cold fusion systems, giving a loading of H/M = 1; and a combination of loading methods is highly desirable. Systems must be designed to provide continuous flow of hydrogen ions ({much{underscore}gt}10{sup 17}/sec for ten kilowatts), with an input power source of 50 watts (est.). Cold fusion experiments have shown that helium is formed during the reaction, and physical changes occur in the reaction material. These revelations impact design and operation of cold fusion systems, as the reaction material must be replaced periodically, while the systems must maintain integrity during operation. Safety and cost are also highly important considerations.

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

    Directory of Open Access Journals (Sweden)

    Chen Chaoping

    2010-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Qian Liu

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

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

    Directory of Open Access Journals (Sweden)

    Yi-Shin Pan

    2010-01-01

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2008-11-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  1. SET-NUP214 fusion in acute myeloid leukemia- and T-cell acute lymphoblastic leukemia-derived cell lines

    Directory of Open Access Journals (Sweden)

    Zaborski Margarete

    2009-01-01

    Full Text Available Abstract Background SET-NUP214 fusion resulting from a recurrent cryptic deletion, del(9(q34.11q34.13 has recently been described in T-cell acute lymphoblastic leukemia (T-ALL and in one case of acute myeloid leukemia (AML. The fusion protein appears to promote elevated expression of HOXA cluster genes in T-ALL and may contribute to the pathogenesis of the disease. We screened a panel of ALL and AML cell lines for SET-NUP214 expression to find model systems that might help to elucidate the cellular function of this fusion gene. Results Of 141 human leukemia/lymphoma cell lines tested, only the T-ALL cell line LOUCY and the AML cell line MEGAL expressed the SET(TAF-Iβ-NUP214 fusion gene transcript. RT-PCR analysis specifically recognizing the alternative first exons of the two TAF-I isoforms revealed that the cell lines also expressed TAF-Iα-NUP214 mRNA. Results of fluorescence in situ hybridization (FISH and array-based copy number analysis were both consistent with del(9(q34.11q34.13 as described. Quantitative genomic PCR also confirmed loss of genomic material between SET and NUP214 in both cell lines. Genomic sequencing localized the breakpoints of the SET gene to regions downstream of the stop codon and to NUP214 intron 17/18 in both LOUCY and MEGAL cells. Both cell lines expressed the 140 kDa SET-NUP214 fusion protein. Conclusion Cell lines LOUCY and MEGAL express the recently described SET-NUP214 fusion gene. Of special note is that the formation of the SET exon 7/NUP214 exon 18 gene transcript requires alternative splicing as the SET breakpoint is located downstream of the stop codon in exon 8. The cell lines are promising model systems for SET-NUP214 studies and should facilitate investigating cellular functions of the the SET-NUP214 protein.

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

    Science.gov (United States)

    2009-07-01

    Dendritic Cells as a Vaccine for the Treatment of Metatastic Breast Cancer PRINCIPAL INVESTIGATOR: Donald W. Kufe, M.D...COVERED 1 Jul 2008 – 30 Jun 2009 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Fusions of Breast Carcinoma and Dendritic Cells as a Vaccine for the...David Avigan, MD, Beth Israel Deaconess Medical Center, Boston, MA, in Support of Proposal, "Fusions of Breast Carcinoma and Dendritic Cells as a

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

    Science.gov (United States)

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

    2016-06-21

    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.

  4. Association of six YFP-myosin XI-tail fusions with mobile plant cell organelles

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    Hanson Maureen R

    2007-02-01

    Full Text Available Abstract Background Myosins are molecular motors that carry cargo on actin filaments in eukaryotic cells. Seventeen myosin genes have been identified in the nuclear genome of Arabidopsis. The myosin genes can be divided into two plant-specific subfamilies, class VIII with four members and class XI with 13 members. Class XI myosins are related to animal and fungal myosin class V that are responsible for movement of particular vesicles and organelles. Organelle localization of only one of the 13 Arabidopsis myosin XI (myosin XI-6; At MYA2, which is found on peroxisomes, has so far been reported. Little information is available concerning the remaining 12 class XI myosins. Results We investigated 6 of the 13 class XI Arabidopsis myosins. cDNAs corresponding to the tail region of 6 myosin genes were generated and incorporated into a vector to encode YFP-myosin tail fusion proteins lacking the motor domain. Chimeric genes incorporating tail regions of myosin XI-5 (At MYA1, myosin XI-6 (At MYA2, myosin XI-8 (At XI-B, myosin XI-15 (At XI-I, myosin XI-16 (At XI-J and myosin XI-17 (At XI-K were expressed transiently. All YFP-myosin-tail fusion proteins were targeted to small organelles ranging in size from 0.5 to 3.0 μm. Despite the absence of a motor domain, the fluorescently-labeled organelles were motile in most cells. Tail cropping experiments demonstrated that the coiled-coil region was required for specific localization and shorter tail regions were inadequate for targeting. Myosin XI-6 (At MYA2, previously reported to localize to peroxisomes by immunofluorescence, labeled both peroxisomes and vesicles when expressed as a YFP-tail fusion. None of the 6 YFP-myosin tail fusions interacted with chloroplasts, and only one YFP-tail fusion appeared to sometimes co-localize with fluorescent proteins targeted to Golgi and mitochondria. Conclusion 6 myosin XI tails, extending from the coiled-coil region to the C-terminus, label specific vesicles and

  5. 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.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 (Pfusion cells could efficiently stimulate T lymphocytes to generate specific CTL targeting HepG2 cells.It might be a promising strategy of immunotherapy for HCC.

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

    Science.gov (United States)

    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.

  8. Mannose-exposing myeloid leukemia cells detected by the sCAR-PPA fusion protein.

    Science.gov (United States)

    Li, Gong Chu; Li, Na; Zhang, Yan Hong; Li, Xin; Wang, Yi Gang; Liu, Xin Yuan; Qian, Wen Bin; Liu, Xiao Chuan

    2009-06-01

    Altered glycosylation may be a hallmark of malignant transformation and cancer progression. In the work described, a specific mannose-binding lectin, Pinellia pedatisecta agglutinin (PPA), was genetically fused with the extracellular domain of coxsackie-adenovirus receptor (CAR) to generate the soluble CAR (sCAR)-PPA fusion protein. The adenoviral transduction of acute myeloid leukemia (AML) cell lines Kasumi-1 and HL-60 was increased by sCAR-PPA, indicating that a fraction of AML cells exposing mannose residues was detected by PPA. However, sCAR-PPA did not increase the adenoviral infection of KG-1 cells, suggesting the mannose exposure of AML cells may be cell type specific. Furthermore, the infectious efficiency of Ad-EGFP in chronic myeloid leukemia cell line K562 was significantly increased by sCAR-PPA as well. We, herein, report that PPA recognized a fraction of myeloid leukemia cells showing mannose-exposing phenotype. The sCAR-PPA fusion protein combined with the adenoviral vector system may provide a useful tool for investigating myeloid leukemia cells exposing mannose residues and further elucidating the role of these cells in the leukemia development.

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

    Directory of Open Access Journals (Sweden)

    Zsuzsanna Kurgyis

    2016-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Katie Bentley

    2009-10-01

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

  11. Rapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity.

    Directory of Open Access Journals (Sweden)

    Manuel A F V Gonçalves

    Full Text Available Cell-to-cell fusion is involved in multiple fundamental biological processes. Prominent examples include osteoclast and giant cell formation, fertilization and skeletal myogenesis which involve macrophage, sperm-egg and myoblast fusion, respectively. Indeed, the importance of cell fusion is underscored by the wide range of homeostatic as well as pathologic processes in which it plays a key role. Therefore, rapid and sensitive systems to trace and measure cell fusion events in various experimental systems are in demand. Here, we introduce a bipartite cell fusion monitoring system based on a genetic switch responsive to the site-specific recombinase FLP. To allow flexible deployment in both dividing as well as non-dividing cell populations, inducer and reporter modules were incorporated in lentivirus vector particles. Moreover, the recombinase-inducible transcription units were designed in such a way as to minimize basal activity and chromosomal position effects in the "off" and "on" states, respectively. The lentivirus vector-based conditional gene expression assay was validated in primary human mesenchymal stem cells and in a differentiation model based on muscle progenitor cells from a Duchenne muscular dystrophy patient using reporter genes compatible with live- and single-cell imaging and with whole population measurements. Using the skeletal muscle cell differentiation model, we showed that the new assay displays low background activity, a 2-log dynamic range, high sensitivity and is amenable to the investigation of cell fusion kinetics. The utility of the bipartite cell fusion monitoring system was underscored by a study on the impact of drug- and RNAi-mediated p38 MAPK inhibition on human myocyte differentiation. Finally, building on the capacity of lentivirus vectors to readily generate transgenic animals the present FLP-inducible system should be adaptable, alone or together with Cre/loxP-based assays, to cell lineage tracing and

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

    Science.gov (United States)

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

    2013-10-21

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

  13. Sodium butyrate induces DRP1-mediated mitochondrial fusion and apoptosis in human colorectal cancer cells

    OpenAIRE

    Tailor, Dhanir; Hahm, Eun-Ryeong; Kale, Raosaheb K.; Singh, Shivendra V.; Singh, Rana P.

    2013-01-01

    Sodium butyrate (NaBt) is the byproduct of anaerobic microbial fermentation inside the gastro-intestinal tract that could reach upto 20 mM, and has been shown to inhibit the growth of various cancers. Herein, we evaluated its effect on mitochondrial fusion and associated induction of apoptosis in colorectal cancer cells (CRC). NaBt treatment at physiological (1-5 mM) concentrations for 12 and 24 h decreased the cell viability and induced G2-M phase cell cycle arrest in HCT116 (12h) and SW480 ...

  14. Cell Fusion as a Cause of Prostate Cancer Metastasis

    Science.gov (United States)

    2008-01-01

    cultured cells. 1 Bornavirida e Bornavirus Human Bornavirus 9 Borna Disease Virus, 10, 11 Brona Disease Virus- Obesity?12 Autism /other behavioral...disease virus. J Neurovirol 9, 259-73 (2003). 15. Boyd, A., Fazakerley, J. K. & Bridgen, A. Pathogenesis of Dugbe virus infection in wild-type and...Jeor, S. C. S. C. Hantaviruses: molecular biology, evolution and pathogenesis . Current molecular medicine 5, 773-790 (2005). 19. Soldan, S. S. S. S

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Özel Cem

    2012-04-01

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

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

    Institute of Scientific and Technical Information of China (English)

    钟山

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Papaioannou Virginia E

    2004-12-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-02-15

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2008-06-20

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

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

    Science.gov (United States)

    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

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    were assessed with 2D sections and 3D reconstruction images and fusion was defined as intertransverse bridging. Results: Spinal fusion was found in 72 % of levels receiving silk fibre enhanced ABM/P15 graft material. In levels with silk fibre enhanced AMB fusion rate was 41 %. These findings...... on the surface of bone forming cells. The binding initiates natural intra- and extracellular signalling pathways, inducing production of growth factors, bone morphogenic proteins and cytokines. P15 peptide has previously shown to improve osteoinductive properties when coated on graft materials. Purpose...

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

    Directory of Open Access Journals (Sweden)

    Ying Su

    2015-06-01

    Full Text Available Basal-like and luminal breast tumors have distinct clinical behavior and molecular profiles, yet the underlying mechanisms are poorly defined. To interrogate processes that determine these distinct phenotypes and their inheritance pattern, we generated somatic cell fusions and performed integrated genetic and epigenetic (DNA methylation and chromatin profiling. We found that the basal-like trait is generally dominant and is largely defined by epigenetic repression of luminal transcription factors. Definition of super-enhancers highlighted a core program common in luminal cells but a high degree of heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells are sufficient to induce a luminal-to-basal phenotypic switch, implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required for luminal-basal fusions, and we identified EN1, TBX18, and TCF4 as candidate transcriptional regulators of the luminal-to-basal switch. Our findings highlight the remarkable epigenetic plasticity of breast cancer cells.

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

    Science.gov (United States)

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

    2015-07-07

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

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

    Directory of Open Access Journals (Sweden)

    Mareike K. S. van Duijl-Richter

    2015-07-01

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

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

    Science.gov (United States)

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

    2015-11-15

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

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

    Science.gov (United States)

    Sinkovics, Joseph G

    2009-09-01

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

  9. Ewing sarcoma cells secrete EWS/Fli-1 fusion mRNA via microvesicles.

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    Masanori Tsugita

    Full Text Available 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.

  10. Expression, refolding, and characterization of recombinant thrombopoietin/stem cell factor fusion protein in Escherichia coli.

    Science.gov (United States)

    Zang, Yuhui; Zhang, Xu; Jiang, Xiaoling; Li, Haoran; Zhu, Jie; Zhang, Chi; Peng, Wei; Qin, Junchuan

    2007-03-01

    Thrombopoietin/stem cell factor (TPO/SCF) is a novel fusion protein that combines the complementary biological effects of TPO and SCF into a single molecule. In this study, TPO/SCF gene was cloned into pET32a and expressed as a thioredoxin (Trx) fusion protein with a C-terminal 6His-tag in Escherichia coli BL21(DE3) under the control of T7 promoter. Trx-TPO/SCF protein approximately accounted for 20% of the total bacterial proteins and was found to accumulate in inclusion bodies. Inclusion bodies were separated from cellular debris, washed with buffer containing 2 M urea, and solubilized with 8 M urea. The refolding of Trx-TPO/SCF was then carried out by an on-column method. Soluble Trx-TPO/SCF was characterized for its dose-dependent effects on promoting cells proliferation in both TF1 and Mo7e cell lines. rhTPO/SCF was released by thrombin digestion and further purified by Ni(2+) affinity chromatography. Western blot analysis confirmed the identities of Trx-TPO/SCF and rhTPO/SCF.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-07-31

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

  12. Glycoprotein processing in mutants of HSV-1 that induce cell fusion

    Energy Technology Data Exchange (ETDEWEB)

    Person, S.; Kousoulas, K.G.; Knowles, R.W.; Read, G.S.; Holland, T.C.; Keller, P.M.; Warner, S.C.

    1982-01-01

    The synthesis of viral-specified glycoproteins, and their appearance on cell surfaces, were compared for cells infected either with syncytial mutants of HSV-1 or with the parental strains from which the mutants were derived. The mutants MP and tsB5, representatives of two different viral genes that affect fusion, were employed in these studies. Cells infected with either mutant gave rise to reduce surface labeling of major viral-specified glycoproteins throughout infection relative to the parental strains. Putative glycoprotein precursors were synthesized in similar amounts throughout infection in mutant and wild-type infected cells. However, during a chase period after a pulse of labeling, fully glycosylated species of glycoproteins accumulated more slowly in mutant than in parent infected cells. The effect was clearly visible early in MP-infected cells and by 9 hr after infection for tsB5-infected cells, and increased thereafter. Apparently the decrease in appearance of glycoproteins on the cell surface of mutant-infected cells is due to a decrease in their rate of processing. 26 references, 7 figures.

  13. Molecular Process Producing Oncogene Fusion in Lung Cancer Cells by Illegitimate Repair of DNA Double-Strand Breaks

    Directory of Open Access Journals (Sweden)

    Yoshitaka Seki

    2015-09-01

    Full Text Available Constitutive activation of oncogenes by fusion to partner genes, caused by chromosome translocation and inversion, is a critical genetic event driving lung carcinogenesis. Fusions of the tyrosine kinase genes ALK (anaplastic lymphoma kinase, ROS1 (c-ros oncogene 1, or RET (rearranged during transfection occur in 1%–5% of lung adenocarcinomas (LADCs and their products constitute therapeutic targets for kinase inhibitory drugs. Interestingly, ALK, RET, and ROS1 fusions occur preferentially in LADCs of never- and light-smokers, suggesting that the molecular mechanisms that cause these rearrangements are smoking-independent. In this study, using previously reported next generation LADC genome sequencing data of the breakpoint junction structures of chromosome rearrangements that cause oncogenic fusions in human cancer cells, we employed the structures of breakpoint junctions of ALK, RET, and ROS1 fusions in 41 LADC cases as “traces” to deduce the molecular processes of chromosome rearrangements caused by DNA double-strand breaks (DSBs and illegitimate joining. We found that gene fusion was produced by illegitimate repair of DSBs at unspecified sites in genomic regions of a few kb through DNA synthesis-dependent or -independent end-joining pathways, according to DSB type. This information will assist in the understanding of how oncogene fusions are generated and which etiological factors trigger them.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    genetic and epigenetic (DNA methylation and chromatin) profiling. We found that the basal-like trait is generally dominant and is largely defined by epigenetic repression of luminal transcription factors. Definition of super-enhancers highlighted a core program common in luminal cells but a high degree......Basal-like and luminal breast tumors have distinct clinical behavior and molecular profiles, yet the underlying mechanisms are poorly defined. To interrogate processes that determine these distinct phenotypes and their inheritance pattern, we generated somatic cell fusions and performed integrated...... of heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells are sufficient to induce a luminal-to-basal phenotypic switch, implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required...

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

    2016-10-01

    Beta-site APP cleaving enzyme 1 (BACE1) is a type I transmembrane aspartyl protease. In the amyloidogenic pathway, BACE1 provides β-secretase activity that cleaves the amyloid precursor protein (APP) that leads to amyloid beta (Aβ) peptides. The aggregation of these Aβ will ultimately results in amyloid plaque formation, a hallmark of Alzheimer's disease (AD). Amyloid aggregation leads to progressive memory impairment and neural loss. Recent detergent protein extraction studies suggest that the untreated BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. Here, we examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using fluorescence correlation spectroscopy (FCS). Cells were transfected with a BACE1-EGFP fusion protein construct and imaged using confocal and DIC microscopy to monitor labeled BACE1 localization and distribution within the cell. Our one-photon fluorescence fluctuation autocorrelation of BACE1- EGFP on the plasma membrane of HEK cells is modeled using two diffusing species on the plasma membrane with estimated diffusion coefficients of 1.39 x 10-7 cm2/sec and 2.8 x 10-8 cm2/sec under resting conditions and STA-200 inhibition, respectively. Anomalous diffusion model also provided adequate description of the observed autocorrelation function of BACE1- EGFP on the plasma membrane with an estimate exponent (α) of 0.8 and 0.5 for resting and STA-200 treated cells, respectively. The corresponding hydrodynamic radius of this transmembrane fusion protein was estimated using the measured diffusion coefficients assuming both Stokes-Einstein and Saffman-Delbruck models. Our results suggest a complex diffusion pattern of BACE1-EGFP on the plasma membrane of HEK cells with the possibility for dimer formation, especially under STA-200 inhibition.

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

    Directory of Open Access Journals (Sweden)

    Lin Song-Shu

    2010-03-01

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

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

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    Benjamin Berndt

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

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

    Directory of Open Access Journals (Sweden)

    Fujun Qin

    2015-02-01

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

  20. Oligophrenin-1 Connects Exocytotic Fusion to Compensatory Endocytosis in Neuroendocrine Cells.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    Nedukha, Olena

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

  3. Systematic mapping of occluded genes by cell fusion reveals prevalence and stability of cis-mediated silencing in somatic cells

    Science.gov (United States)

    Looney, Timothy J.; Zhang, Li; Chen, Chih-Hsin; Lee, Jae Hyun; Chari, Sheila; Mao, Frank Fuxiang; Pelizzola, Mattia; Zhang, Lu; Lister, Ryan; Baker, Samuel W.; Fernandes, Croydon J.; Gaetz, Jedidiah; Foshay, Kara M.; Clift, Kayla L.; Zhang, Zhenyu; Li, Wei-Qiang; Vallender, Eric J.; Wagner, Ulrich; Qin, Jane Yuxia; Michelini, Katelyn J.; Bugarija, Branimir; Park, Donghyun; Aryee, Emmanuel; Stricker, Thomas; Zhou, Jie; White, Kevin P.; Ren, Bing; Schroth, Gary P.; Ecker, Joseph R.; Xiang, Andy Peng; Lahn, Bruce T.

    2014-01-01

    Both diffusible factors acting in trans and chromatin components acting in cis are implicated in gene regulation, but the extent to which either process causally determines a cell's transcriptional identity is unclear. We recently used cell fusion to define a class of silent genes termed “cis-silenced” (or “occluded”) genes, which remain silent even in the presence of trans-acting transcriptional activators. We further showed that occlusion of lineage-inappropriate genes plays a critical role in maintaining the transcriptional identities of somatic cells. Here, we present, for the first time, a comprehensive map of occluded genes in somatic cells. Specifically, we mapped occluded genes in mouse fibroblasts via fusion to a dozen different rat cell types followed by whole-transcriptome profiling. We found that occluded genes are highly prevalent and stable in somatic cells, representing a sizeable fraction of silent genes. Occluded genes are also highly enriched for important developmental regulators of alternative lineages, consistent with the role of occlusion in safeguarding cell identities. Alongside this map, we also present whole-genome maps of DNA methylation and eight other chromatin marks. These maps uncover a complex relationship between chromatin state and occlusion. Furthermore, we found that DNA methylation functions as the memory of occlusion in a subset of occluded genes, while histone deacetylation contributes to the implementation but not memory of occlusion. Our data suggest that the identities of individual cell types are defined largely by the occlusion status of their genomes. The comprehensive reference maps reported here provide the foundation for future studies aimed at understanding the role of occlusion in development and disease. PMID:24310002

  4. 5-Fluorocytosine combined with Fcy-hEGF fusion protein targets EGFR-expressing cancer cells

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer EGFR-expressing epithelial cancers account for significant portion of cancer deaths. Black-Right-Pointing-Pointer EGF-EGFR signaling pathway is validated as an important anticancer drug target. Black-Right-Pointing-Pointer EGF and Fcy fusion protein (Fcy-hEGF) can bind to EGFR and convert 5-FC to 5-FU. Black-Right-Pointing-Pointer Fcy-hEGF combined with 5-FC preferentially inhibits EGFR-expressing cells viability. -- Abstract: Human epithelial cancers account for approximately 50% of all cancer deaths. This type of cancer is characterized by excessive activation and expression of the epidermal growth factor receptor (EGFR). The EGFR pathway is critical for cancer cell proliferation, survival, metastasis and angiogenesis. The EGF-EGFR signaling pathway has been validated as an important anticancer drug target. Increasing numbers of targeted therapies against this pathway have been either approved or are currently under development. Here, we adopted a prodrug system that uses 5-fluorocytosine (5-FC) and human EGF (hEGF) fused with yeast cytosine deaminase (Fcy) to target EGFR-overexpressing cancer cells and to convert 5-FC to a significantly more toxic chemotherapeutic, 5-fluorouracil (5-FU). We cloned and purified the Fcy-hEGF fusion protein from Pichia pastoris yeast. This fusion protein specifically binds to EGFR with a similar affinity as hEGF, approximately 10 nM. Fcy-hEGF binds tightly to A431 and MDA-MB-468 cells, which overexpress EGFR, but it binds with a lower affinity to MDA-MB-231 and MCF-7, which express lower levels of EGFR. Similarly, the viability of EGFR-expressing cells was suppressed by Fcy-hEGF in the presence of increasing concentrations of 5-FC, and the IC{sub 50} values for A431 and MDA-MB-468 were approximately 10-fold lower than those of MDA-MB-231 and MCF-7. This novel prodrug system, Fcy-hEGF/5-FC, might represent a promising addition to the available class of inhibitors that specifically target EGFR

  5. A protein G fragment from the salmonid viral hemorrhagic septicemia rhabdovirus induces cell-to-cell fusion and membrane phosphatidylserine translocation at low pH.

    Science.gov (United States)

    Estepa, A M; Rocha, A I; Mas, V; Pérez, L; Encinar, J A; Nuñez, E; Fernandez, A; Gonzalez Ros, J M; Gavilanes, F; Coll, J M

    2001-12-07

    The fusion-related properties of segments p9, p3, p4, and p9 + p2 surrounding the p2 phospholipid-binding domain of the protein G (pG) of the salmonid rhabdovirus of viral hemorrhagic septicemia (VHS) (Nuñez, E., Fernandez, A. M., Estepa, A., Gonzalez-Ros, J. M., Gavilanes, F., and Coll, J. M. (1998) Virology 243, 322-330; Estepa, A., and Coll, J. M. (1996) Virology 216, 60-70), have been studied at neutral and fusion (low) pH values by using its derived peptides. Cell-to-cell fusion, translocation of phosphatidylserine, and inhibition of fusion of pG-transfected cells defined the p9 + p2 (fragment 11, sequence 56-110) as a fragment with higher specific activity for anionic phospholipid aggregation than the previously reported p2. While fragment 11, p2, and p3 showed interactions with anionic phospholipids, p9 and p4 showed no interactions with any phospholipids. When added to a cell monolayer model at low pH, fragment 11 induced pH-dependent cell-to-cell fusion and translocated phosphatidylserine from the inner to the outer leaflet of the membrane. At low pH and in the presence of anionic phospholipids, fragment 11 showed more than 80% beta-sheet conformation (IR and CD spectroscopies). Finally, anti-fragment 11 antibodies inhibited low pH-dependent pG-transfected cell-to-cell fusion. All of the data support the conclusion that fragment 11 is a primary determinant of some of the viral cell fusion events in VHSV.

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

    Directory of Open Access Journals (Sweden)

    Shigeo Koido

    2011-01-01

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

  7. Discovery and characterization of a novel CCND1/MRCK gene fusion in mantle cell lymphoma

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    Chioniso Patience Masamha

    2016-03-01

    Full Text Available Abstract 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.

  8. Fusion Machinery

    DEFF Research Database (Denmark)

    Sørensen, Jakob Balslev; Milosevic, Ira

    2015-01-01

    the vesicular SNARE VAMP2/synaptobrevin-2 and the target (plasma membrane) SNAREs SNAP25 and syntaxin-1 results in fusion and release of neurotransmitter, synchronized to the electrical activity of the cell by calcium influx and binding to synaptotagmin. Formation of the SNARE complex is tightly regulated...... and appears to start with syntaxin-1 bound to an SM (Sec1/Munc18-like) protein. Proteins of the Munc13-family are responsible for opening up syntaxin and allowing sequential binding of SNAP-25 and VAMP2/synaptobrevin-2. N- to C-terminal “zippering” of the SNARE domains leads to membrane fusion...

  9. Nuclei of non-muscle cells bind centrosome proteins upon fusion with differentiating myoblasts.

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    Xavier Fant

    Full Text Available BACKGROUND: In differentiating myoblasts, the microtubule network is reorganized from a centrosome-bound, radial array into parallel fibres, aligned along the long axis of the cell. Concomitantly, proteins of the centrosome relocalize from the pericentriolar material to the outer surface of the nucleus. The mechanisms that govern this relocalization are largely unknown. METHODOLOGY: In this study, we perform experiments in vitro and in cell culture indicating that microtubule nucleation at the centrosome is reduced during myoblast differentiation, while nucleation at the nuclear surface increases. We show in heterologous cell fusion experiments, between cultures of differentiating mouse myoblasts and human cells of non-muscular origin, that nuclei from non-muscle cells recruit centrosome proteins once fused with the differentiating myoblasts. This recruitment still occurs in the presence of cycloheximide and thus appears to be independent of new protein biosynthesis. CONCLUSIONS: Altogether, our data suggest that nuclei of undifferentiated cells have the dormant potential to bind centrosome proteins, and that this potential becomes activated during myoblast differentiation.

  10. Development of a rapid cell-fusion-based phenotypic HIV-1 tropism assay

    Directory of Open Access Journals (Sweden)

    Phairote Teeranaipong

    2013-09-01

    Full Text Available Introduction: A dual split reporter protein system (DSP, recombining Renilla luciferase (RL and green fluorescent protein (GFP split into two different constructs (DSP1–7 and DSP8–11, was adapted to create a novel rapid phenotypic tropism assay (PTA for HIV-1 infection (DSP-Pheno. Methods: DSP1–7 was stably expressed in the glioma-derived NP-2 cell lines, which expressed CD4/CXCR4 (N4X4 or CD4/CCR5 (N4R5, respectively. An expression vector with DSP8–11 (pRE11 was constructed. The HIV-1 envelope genes were subcloned in pRE11 (pRE11-env and transfected into 293FT cells. Transfected 293FT cells were incubated with the indicator cell lines independently. In developing the assay, we selected the DSP1–7-positive clones that showed the highest GFP activity after complementation with DSP8–11. These cell lines, designated N4R5-DSP1–7, N4X4-DSP1–7 were used for subsequent assays. Results: The env gene from the reference strains (BaL for R5 virus, NL4-3 for X4 virus, SF2 for dual tropic virus subcloned in pRE11 and tested, was concordant with the expected co-receptor usage. Assay results were available in two ways (RL or GFP. The assay sensitivity by RL activity was comparable with those of the published phenotypic assays using pseudovirus. The shortest turnaround time was 5 days after obtaining the patient's plasma. All clinical samples gave positive RL signals on R5 indicator cells in the fusion assay. Median RLU value of the low CD4 group was significantly higher on X4 indicator cells and suggested the presence of more dual or X4 tropic viruses in this group of patients. Comparison of representative samples with Geno2Pheno [co-receptor] assay was concordant. Conclusions: A new cell-fusion-based, high-throughput PTA for HIV-1, which would be suitable for in-house studies, was developed. Equipped with two-way reporter system, RL and GFP, DSP-Pheno is a sensitive test with short turnaround time. Although maintenance of cell lines and

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

    Science.gov (United States)

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

    2012-03-01

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

  12. Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells

    Directory of Open Access Journals (Sweden)

    Veenhuis Marten

    2005-10-01

    Full Text Available Abstract Background Native as well as recombinant bacterial cell surface layer (S-layer protein of Geobacillus (G. stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested the expression and assembly of a fusion protein, consisting of the mature part (aa 31–1099 of the S-layer protein and EGFP (enhanced green fluorescent protein, in eukaryotic host cells, the yeast Saccharomyces cerevisiae and human HeLa cells. Results Upon expression in E. coli the recombinant mSbsC-EGFP fusion protein was recovered from the insoluble fraction. After denaturation by Guanidine (Gua-HCl treatment and subsequent dialysis the fusion protein assembled in solution and yielded green fluorescent cylindric structures with regular symmetry comparable to that of the authentic SbsC. For expression in the eukaryotic host Saccharomyces (S. cerevisiae mSbsC-EGFP was cloned in a multi-copy expression vector bearing the strong constitutive GPD1 (glyceraldehyde-3-phosophate-dehydrogenase promoter. The respective yeast transfomants were only slightly impaired in growth and exhibited a needle-like green fluorescent pattern. Transmission electron microscopy (TEM studies revealed the presence of closely packed cylindrical structures in the cytosol with regular symmetry comparable to those obtained after in vitro recrystallization. Similar structures are observed in HeLa cells expressing mSbsC-EGFP from the Cytomegalovirus (CMV IE promoter. Conclusion The mSbsC-EGFP fusion protein is stably expressed both in the yeast, Saccharomyces cerevisiae, and in HeLa cells. Recombinant mSbsC-EGFP combines properties of both fusion partners: it assembles both in vitro and in vivo to cylindrical structures that show an intensive green fluorescence. Fusion of proteins to S-layer proteins may be a useful tool for high level expression in yeast and HeLa cells of

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

    Science.gov (United States)

    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.

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

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    Frederic Vigant

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

  15. Viral membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-05-15

    Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism.

  16. Enhancement of the influenza A hemagglutinin (HA-mediated cell-cell fusion and virus entry by the viral neuraminidase (NA.

    Directory of Open Access Journals (Sweden)

    Bin Su

    Full Text Available BACKGROUND: The major role of the neuraminidase (NA protein of influenza A virus is related to its sialidase activity, which disrupts the interaction between the envelope hemagglutinin (HA protein and the sialic acid receptors expressed at the surface of infected cells. This enzymatic activity is known to promote the release and spread of progeny viral particles following their production by infected cells, but a potential role of NA in earlier steps of the viral life cycle has never been clearly demonstrated. In this study we have examined the impact of NA expression on influenza HA-mediated viral membrane fusion and virion infectivity. METHODOLOGY/PRINCIPAL FINDINGS: The role of NA in the early stages of influenza virus replication was examined using a cell-cell fusion assay that mimics HA-mediated membrane fusion, and a virion infectivity assay using HIV-based pseudoparticles expressing influenza HA and/or NA proteins. In the cell-cell fusion assay, which bypasses the endocytocytosis step that is characteristic of influenza virus entry, we found that in proper HA maturation conditions, NA clearly enhanced fusion in a dose-dependent manner. Similarly, expression of NA at the surface of pseudoparticles significantly enhanced virion infectivity. Further experiments using exogenous soluble NA revealed that the most likely mechanism for enhancement of fusion and infectivity by NA was related to desialylation of virion-expressed HA. CONCLUSION/SIGNIFICANCE: The NA protein of influenza A virus is not only required for virion release and spread but also plays a critical role in virion infectivity and HA-mediated membrane fusion.

  17. Construction of Recombinant Bacmid Containing M2e-Ctxb and Producing the Fusion Protein in Insect Cell Lines

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Science.gov (United States)

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

    2015-04-13

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

  20. Oncogenic fusion proteins expressed in immature hematopoietic cells fail to recapitulate the transcriptional changes observed in human AML

    DEFF Research Database (Denmark)

    Rapin, N; Porse, B T

    2014-01-01

    in acute promyelocytic leukemia. Hematopoietic stem/progenitor (HSPCs) cells transduced with oncogenic fusion genes are regarded as promising in vitromodels of their corresponding AML subtypes. Here, we critically assessed the potential of such in vitro models using an integrative bioinformatics approach...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-03-22

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

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

    Science.gov (United States)

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

    2008-08-08

    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.

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

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  5. Analysis of EML4-ALK Gene Fusion Mutation in Patients 
with Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Xuzhou WANG

    2015-02-01

    Full Text Available Background and objective Non-small cell lung cancer (NSCLC is the main type of lung cancer, and the related locus mutation detection research has become a hot direction of molecular targeted therapy, studying on gene mutation status of echinodem microtubule associated protein like 4-Anaplastic lymphoma kinase (EML4-ALK and epidermal growth factor receptor (EGFR, detecting the sensitivity of EML4-ALK gene fusion and gene mutation of EGFR. Methods EML4-ALK gene fusion in 85 cases of paraffin embedded tumor tissue and adjacent lung tissue was detected with the application of immunohistochemistry (IHC, Scorpions amplification refractory mutation system (Scorpions ARMS fluorescence quantitative PCR and fluorescence in situ hybridization (FISH technology, and EGFR gene in 18, 19, 20 and 21 exon mutation status was detected with the application of ARMS method. Results In 115 cases of NSCLC, IHC showed 32 cases with ALK (D5F3 expression, the expression rate was 27.8%; ARMS showed 27 cases with EML4-ALK fusion gene mutation, the mutation detection rate was 23.5%; 53 cases were detected with EGFR mutation, the mutation rate was 46%. While FISH showed 23 cases with EML4-ALK fusion gene mutation, the detection rate was 20%, slightly lower than the ARMS detection results, suggesting that ARMS more sensitive. Conclusion The application of IHC, ARMS fluorescence quantitative PCR and FISH technology can make a rapid and accurate evaluation of EML4-ALK gene fusion.

  6. Expression and bioactivity of recombinant human serum albumin and dTMP fusion proteins in CHO cells.

    Science.gov (United States)

    Ru, Yi; Zhi, Dejuan; Guo, Dingding; Wang, Yong; Li, Yang; Wang, Meizhu; Wei, Suzhen; Wang, Haiqing; Wang, Na; Che, Jingmin; Li, Hongyu

    2016-09-01

    The 14-amino acid (IEGPTLRQWLAARA) thrombopoietin mimetic peptide (TMP) shares no sequence homology with native thrombopoietin (TPO). When dimerized, it displays a high-binding affinity for the TPO receptor and has equipotent bioactivity with recombinant human TPO (rhTPO) in stimulating proliferation and maturation of megakaryocytes in vitro. However, TMP is limited for clinical usage because of its short half-life in vivo. In this study, fusion proteins that composed of tandem dimer of TMP (dTMP) genetically fused at the C- or N-terminus of human serum albumin (HSA) were separately expressed in Chinese hamster ovary (CHO) cells. In vitro bioactivity assays showed that purified fusion proteins promoted the proliferation of megakaryocytes in a dose-dependent manner and activated signal transducer and activator of transcription (STAT) pathway in TPO receptor-dependent manner. Following subcutaneous administration, both HSA-dTMP and dTMP-HSA significantly elevated peripheral platelet counts in normal mice in a dose-dependent manner. In addition, fusion with HSA successfully prolonged dTMP half-life in mice. However, when HSA was fused at the C-terminus of dTMP, the bioactivity of dTMP-HSA was about half of that of HSA-dTMP. In conclusion, these results suggested that HSA/dTMP fusion proteins might be potential drugs for thrombocytopenia and, when HSA was fused at the N-terminus of dTMP, the fusion protein had a higher activity.

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

    Science.gov (United States)

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

    Institute of Scientific and Technical Information of China (English)

    李朝军; 吕品; 张东才

    1999-01-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  10. Vaccination with Dendritic Cell Myeloma Fusions in Conjunction With Stem Cell Transplantation and PD1 Blockade

    Science.gov (United States)

    2010-05-01

    activated versus inhibitory T cell populations by quantifying the percentage of cells that expressed interferon gamma (IFNγ) as compared to IL-10. IFNγ...of T cells was noted particularly with CT-011, resulting in a greater than 5 fold increase in T cell proliferation. Interferon gamma secretion by CD4...example (A) and mean values of 5 experiments with associated standard errors of the mean (B) are shown. Figure 6. Interferon gamma production by

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

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    Qi Zhou

    2016-05-01

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

  12. The membrane fusion step of vaccinia virus entry is cooperatively mediated by multiple viral proteins and host cell components.

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    Jason P Laliberte

    2011-12-01

    Full Text Available For many viruses, one or two proteins allow cell attachment and entry, which occurs through the plasma membrane or following endocytosis at low pH. In contrast, vaccinia virus (VACV enters cells by both neutral and low pH routes; four proteins mediate cell attachment and twelve that are associated in a membrane complex and conserved in all poxviruses are dedicated to entry. The aim of the present study was to determine the roles of cellular and viral proteins in initial stages of entry, specifically fusion of the membranes of the mature virion and cell. For analysis of the role of cellular components, we used well characterized inhibitors and measured binding of a recombinant VACV virion containing Gaussia luciferase fused to a core protein; viral and cellular membrane lipid mixing with a self-quenching fluorescent probe in the virion membrane; and core entry with a recombinant VACV expressing firefly luciferase and electron microscopy. We determined that inhibitors of tyrosine protein kinases, dynamin GTPase and actin dynamics had little effect on binding of virions to cells but impaired membrane fusion, whereas partial cholesterol depletion and inhibitors of endosomal acidification and membrane blebbing had a severe effect at the later stage of core entry. To determine the role of viral proteins, virions lacking individual membrane components were purified from cells infected with members of a panel of ten conditional-lethal inducible mutants. Each of the entry protein-deficient virions had severely reduced infectivity and except for A28, L1 and L5 greatly impaired membrane fusion. In addition, a potent neutralizing L1 monoclonal antibody blocked entry at a post-membrane lipid-mixing step. Taken together, these results suggested a 2-step entry model and implicated an unprecedented number of viral proteins and cellular components involved in signaling and actin rearrangement for initiation of virus-cell membrane fusion during poxvirus entry.

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

    Institute of Scientific and Technical Information of China (English)

    Zhang Yingang; Guo Xiong; Liu Zheng; Wang Shijie

    2007-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

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

  15. Expression and purification of chimeric peptide comprising EGFR B-cell epitope and measles virus fusion protein T-cell epitope in Escherichia coli.

    Science.gov (United States)

    Wu, Meizhi; Zhao, Lin; Zhu, Lei; Chen, Zhange; Li, Huangjin

    2013-03-01

    Chimeric peptide MVF-EGFR(237-267), comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His(6)-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His(6)-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0 mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×10(4) titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides.

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

    Science.gov (United States)

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

    2007-06-01

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

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

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    Yasushi Yoneda

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

  18. A cell-permeable fusion protein based on Clostridium botulinum C2 toxin for delivery of p53 tumorsuppressor into cancer cells.

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    Jörg Fahrer

    Full Text Available Genetically engineered bacterial protein toxins are attractive systems for delivery of exogenous proteins into the cytosol of mammalian cells. The binary C2 toxin from C. botulinum has emerged as powerful delivery vehicle, which rests on its binding/translocation component C2IIa and the genetically modified adaptor domain C2IN that act in concert to trigger cell uptake. The p53 tumor suppressor protein has a crucial function in suppressing carcinogenesis and is frequently inactivated by diverse mechanisms in human tumor cells. Therefore, we constructed a C2IN-p53 fusion protein, which is internalized into cancer cells by C2IIa. To this end, the C2IN-p53 fusion construct was overexpressed in E. coli with good solubility, purified by heparin affinity chromatography and protein identity was confirmed by immunoblotting. We demonstrated that the fusion protein is capable of binding to the p53 consensus-DNA with high affinity in a p53-specific manner in vitro. Next, the internalization of C2IN-p53 was monitored in HeLa cells by cell fractionation and immunoblot analysis, which revealed a C2IIa-mediated translocation of the fusion protein into the cytosol. The uptake was also shown in A549 and Saos-2 cells with similar efficiency. These findings were further corroborated by confocal immunofluorescence analyses of C2IN-p53/C2IIa-treated HeLa and A549 cells, displaying predominantly cytoplasmic localization of the fusion construct.

  19. Targeting the unfolded protein response in glioblastoma cells with the fusion protein EGF-SubA.

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    Antony Prabhu

    Full Text Available Rapidly growing tumors require efficient means to allow them to adapt to fluctuating microenvironments consisting of hypoxia, nutrient deprivation, and acidosis. The unfolded protein response (UPR represents a defense mechanism allowing cells to respond to these adverse conditions. The chaperone protein GRP78 serves as a master UPR regulator that is aberrantly expressed in a variety of cancers, including glioma. Therefore, cancer cells may be particularly reliant upon the adaptive mechanisms offered by the UPR and targeting GRP78 may represent a unique therapeutic strategy. Here we report that diffuse expression of GRP78 protein is present in Grade III-IV, but not Grade I-II glioma. To determine the role GRP78 plays in glioblastoma tumorigenesis, we explored the anti-tumor activity of the novel fusion protein EGF-SubA, which combines EGF with the cytotoxin SubA that has been recently shown to selectively cleave GRP78. EGF-SubA demonstrated potent tumor-specific proteolytic activity and cytotoxicity in glioblastoma lines and potentiated the anti-tumor activity of both temozolomide and ionizing radiation. To determine if the tumor microenvironment influences EGF-SubA activity, we maintained cells in acidic conditions that led to both UPR activation and increased EGF-SubA induced cytotoxicity. EGF-SubA was well tolerated in mice and led to a significant tumor growth delay in a glioma xenograft mouse model. The UPR is emerging as an important adaptive pathway contributing to glioma tumorigenesis. Targeting its primary mediator, the chaperone protein GRP78, through specific, proteolytic cleavage with the immunotoxin EGF-SubA represents a novel and promising multi-targeted approach to cancer therapy.

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

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    Adil Bakayan

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

  1. Production of cloned dogs by decreasing the interval between fusion and activation during somatic cell nuclear transfer.

    Science.gov (United States)

    Kim, Sue; Park, Sun Woo; Hossein, Mohammad Shamim; Jeong, Yeon Woo; Kim, Joung Joo; Lee, Eugine; Kim, Yeun Wook; Hyun, Sang Hwan; Shin, Taeyoung; Hwang, Woo Suk

    2009-05-01

    To improve the efficiency of somatic cell nuclear transfer (SCNT) in dogs, we evaluated whether or not the interval between fusion and activation affects the success rate of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells from a male or female Golden Retriever. In total, 151 and 225 reconstructed oocytes were transferred to 9 and 14 naturally synchronized surrogates for male and female donor cells, respectively. Chromosomal morphology was evaluated in 12 oocytes held for an interval of 2 hr between fusion and activation and 14 oocytes held for an interval of 4 hr. Three hundred seventy-six and 288 embryos were transferred to 23 and 16 surrogates for the 2 and 4 hr interval groups, respectively. Both the male (two pregnant surrogates gave birth to three puppies) and female (one pregnant surrogate gave birth to one puppy) donor cells gave birth to live puppies (P > 0.05). In the 2 hr group, significantly more reconstructed oocytes showed condensed, metaphase-like chromosomes compared to the 4 hr group (P dogs carried pregnancies to term and four puppies were born. These results demonstrate that decreasing the interval between fusion and activation increases the success rate of clone production and pregnancy. These results may increase the overall efficiency of SCNT in the canine family.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  3. Parallel mesh support for particle-in-cell methods in magnetic fusion simulations

    Science.gov (United States)

    Yoon, Eisung; Shephard, Mark S.; Seol, E. Seegyoung; Kalyanaraman, Kaushik; Ibanez, Daniel

    2016-10-01

    As supercomputing power continues to increase Particle-In-Cell (PIC) methods are being widely adopted for transport simulations of magnetic fusion devices. Current implementations place a copy of the entire continuum mesh and its fields used in the PIC calculations on every node. This is in general not a scalable solution as computational power continues to grow faster than node level memory. To address this scalability issue, while still maintaining sufficient mesh per node to control costly inter-node communication, a new unstructured mesh distribution methods and associated mesh based PIC calculation procedure is being developed building on the parallel unstructured mesh infrastructure (PUMI). Key components to be outlined in the presentation include (i) the mesh distribution strategy, (ii) how the particles are tracked during a push cycle taking advantage of the unstructured mesh adjacency structures and searches based on that structure, and (iii) how the field solve steps and particle migration are controlled. Performance comparisons to the current approach will also be presented.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-07-25

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

  5. An important role for syndecan-1 in herpes simplex virus type-1 induced cell-to-cell fusion and virus spread.

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    Ghadah A Karasneh

    Full Text Available Herpes simplex virus type-1 (HSV-1 is a common human pathogen that relies heavily on cell-to-cell spread for establishing a lifelong latent infection. Molecular aspects of HSV-1 entry into host cells have been well studied; however, the molecular details of the spread of the virus from cell-to-cell remain poorly understood. In the past, the role of heparan sulfate proteoglycans (HSPG during HSV-1 infection has focused solely on the role of HS chains as an attachment receptor for the virus, while the core protein has been assumed to perform a passive role of only carrying the HS chains. Likewise, very little is known about the involvement of any specific HSPGs in HSV-1 lifecycle. Here we demonstrate that a HSPG, syndecan-1, plays an important role in HSV-1 induced membrane fusion and cell-to-cell spread. Interestingly, the functions of syndecan-1 in fusion and spread are independent of the presence of HS on the core protein. Using a mutant CHO-K1 cell line that lacks all glycosaminoglycans (GAGs on its surface (CHO-745 we demonstrate that the core protein of syndecan-1 possesses the ability to modulate membrane fusion and viral spread. Altogether, we identify a new role for syndecan-1 in HSV-1 pathogenesis and demonstrate HS-independent functions of its core protein in viral spread.

  6. Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication? [version 1; referees: 2 approved

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    Simon Imhof

    2016-04-01

    Full Text Available Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature.

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

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    Joshua Mayourian

    2016-07-01

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

  8. ZNF384-related fusion genes define a subgroup of childhood B-cell precursor acute lymphoblastic leukemia with a characteristic immunotype

    Science.gov (United States)

    Hirabayashi, Shinsuke; Ohki, Kentaro; Nakabayashi, Kazuhiko; Ichikawa, Hitoshi; Momozawa, Yukihide; Okamura, Kohji; Yaguchi, Akinori; Terada, Kazuki; Saito, Yuya; Yoshimi, Ai; Ogata-Kawata, Hiroko; Sakamoto, Hiromi; Kato, Motohiro; Fujimura, Junya; Hino, Moeko; Kinoshita, Akitoshi; Kakuda, Harumi; Kurosawa, Hidemitsu; Kato, Keisuke; Kajiwara, Ryosuke; Moriwaki, Koichi; Morimoto, Tsuyoshi; Nakamura, Kozue; Noguchi, Yasushi; Osumi, Tomoo; Sakashita, Kazuo; Takita, Junko; Yuza, Yuki; Matsuda, Koich; Yoshida, Teruhiko; Matsumoto, Kenji; Hata, Kenichiro; Kubo, Michiaki; Matsubara, Yoichi; Fukushima, Takashi; Koh, Katsuyoshi; Manabe, Atsushi; Ohara, Akira; Kiyokawa, Nobutaka

    2017-01-01

    Fusion genes involving ZNF384 have recently been identified in B-cell precursor acute lymphoblastic leukemia, and 7 fusion partners have been reported. We further characterized this type of fusion gene by whole transcriptome sequencing and/or polymerase chain reaction. In addition to previously reported genes, we identified BMP2K as a novel fusion partner for ZNF384. Including the EP300-ZNF384 that we reported recently, the total frequency of ZNF384-related fusion genes was 4.1% in 291 B-cell precursor acute lymphoblastic leukemia patients enrolled in a single clinical trial, and TCF3-ZNF384 was the most recurrent, with a frequency of 2.4%. The characteristic immunophenotype of weak CD10 and aberrant CD13 and/or CD33 expression was revealed to be a common feature of the leukemic cells harboring ZNF384-related fusion genes. The signature gene expression profile in TCF3-ZNF384-positive patients was enriched in hematopoietic stem cell features and related to that of EP300-ZNF384-positive patients, but was significantly distinct from that of TCF3-PBX1-positive and ZNF384-fusion-negative patients. However, clinical features of TCF3-ZNF384-positive patients are markedly different from those of EP300-ZNF384-positive patients, exhibiting higher cell counts and a younger age at presentation. TCF3-ZNF384-positive patients revealed a significantly poorer steroid response and a higher frequency of relapse, and the additional activating mutations in RAS signaling pathway genes were detected by whole exome analysis in some of the cases. Our observations indicate that ZNF384-related fusion genes consist of a distinct subgroup of B-cell precursor acute lymphoblastic leukemia with a characteristic immunophenotype, while the clinical features depend on the functional properties of individual fusion partners. PMID:27634205

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

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    James W. Gillespie

    2015-06-01

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

  10. Anti-tumor effects of fusion vaccine prepared by renal cell carcinoma 786-O cell line and peripheral blood dendritic cells of healthy volunteers in vitro and in human immune reconstituted SCID mice.

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    Hu, Zhi; Liu, Shihui; Mai, Xuancheng; Hu, Zili; Liu, Chuan

    2010-01-01

    Dendritic cells (DC), as professional antigen presenting cells, play the central role in the process of body initiating the anti-tumor immunity, and the study on DC anti-tumor vaccine has become heated in recent years. In this study, we used polyethylene glycol (PEG) to induce renal cell carcinoma (RCC) 786-O cell line fused with peripheral blood DC of healthy volunteers, and discuss the biological characteristics of fusion vaccine and its anti-tumor effects in vitro and in human immune reconstituted SCID mice model of RCC. The study found that PEG could effectively induce cell fusion, and the expressions of CD86 and HLA-DR in fusion vaccine group were significantly up-regulated compared with the DC control group; the secretion of IL-12 was much higher and longer than that of the control; the functions of dendritic cell-tumor fusion vaccine to stimulate the proliferation of allogenic T lymphocytes and to kill RCC786-O cells in vitro were significantly higher than those of the control group, and after the killing, apoptosis body was observed in the target cells; after the injection of fusion vaccine into human immune reconstituted SCID mice model of RCC786-O via vena caudalis, the volume of mice tumor was reduced significantly, proliferation index of tumor cells decreased obviously compared with that of the control group, and more hemorrhage and putrescence focuses presented, accompanying large quantity of lymphocytes soakage. The results of this experimental study shows that fusion vaccine of RCC786-O cell line and DC can significantly stimulate the proliferation of allogenic T cells and specifically inhibit and kill RCC cells in vitro and in vivo, which makes the DC-RCC786-O fusion vaccine a possible new way of effective RCC immunotherapy.

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

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

    2015-09-15

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

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

    Science.gov (United States)

    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.

  13. TBL1XR1/TP63: a novel recurrent gene fusion in B-cell non-Hodgkin lymphoma | Office of Cancer Genomics

    Science.gov (United States)

    Recently, the landscape of single base mutations in diffuse large B-cell lymphoma (DLBCL) was described. Here we report the discovery of a gene fusion between TBL1XR1 and TP63, the only recurrent somatic novel gene fusion identified in our analysis of transcriptome data from 96 DLBCL cases. Based on this cohort and a further 157 DLBCL cases analyzed by FISH, the incidence in de novo germinal center B cell-like (GCB) DLBCL is 5% (6 of 115).

  14. Post-fusion treatment with MG132 increases transcription factor expression in somatic cell nuclear transfer embryos in pigs.

    Science.gov (United States)

    You, Jinyoung; Lee, Joohyeong; Kim, Jinyoung; Park, Junhong; Lee, Eunsong

    2010-02-01

    The objective of this study was to examine the effect of post-fusion treatment of somatic cell nuclear transfer (SCNT) oocytes with the proteasomal inhibitor MG132 on maturation promoting factor (MPF) activity, nuclear remodeling, embryonic development, and gene expression of cloned pig embryos. Immediately after electrofusion, SCNT oocytes were treated with MG132 and/or caffeine for 2 hr, vanadate for 0.5 hr, or vanadate for 0.5 hr followed by MG132 for 1.5 hr. Of the MG132 concentrations tested (0-5 microM), the 1 microM concentration showed a higher rate of blastocyst formation (25.9%) than 0 (14.2%), 0.5 (16.9%), and 5 microM (16.9%). Post-fusion treatment with MG132, caffeine, and both MG132 and caffeine improved blastocyst formation (22.1%, 21.4%, and 24.4%, respectively), whereas vanadate treatment inhibited blastocyst formation (6.5%) compared to the control (11.1%). When examined 2 hr after fusion and 1 hr after activation, MPF activity remained at a higher (P fusion with caffeine and/or MG132, but it was decreased by vanadate. The rate of oocytes showing premature chromosome condensation was not altered by MG132 but was decreased by vanadate treatment. In addition, formation of single pronuclei was increased by MG132 compared to control and vanadate treatment. MG132-treated embryos showed increased expression of POU5F1, DPPA2, DPPA3, DPPA5, and NDP52l1 genes compared to control embryos. Our results demonstrate that post-fusion treatment of SCNT oocytes with MG132 prevents MPF degradation and increases expression of transcription factors in SCNT embryos, which are necessary for normal development of SCNT embryos.

  15. In-vivo fusion of human cancer and hamster stromal cells permanently transduces and transcribes human DNA.

    Science.gov (United States)

    Goldenberg, David M; Rooney, Robert J; Loo, Meiyu; Liu, Donglin; Chang, Chien-Hsing

    2014-01-01

    After demonstrating, with karyotyping, polymerase chain reaction (PCR) and fluorescence in-situ hybridization, the retention of certain human chromosomes and genes following the spontaneous fusion of human tumor and hamster cells in-vivo, it was postulated that cell fusion causes the horizontal transmission of malignancy and donor genes. Here, we analyzed gene expression profiles of 3 different hybrid tumors first generated in the hamster cheek pouch after human tumor grafting, and then propagated in hamsters and in cell cultures for years: two Hodgkin lymphomas (GW-532, GW-584) and a glioblastoma multiforme (GB-749). Based on the criteria of MAS 5.0 detection P-values ≤0.065 and at least a 2-fold greater signal expression value than a hamster melanoma control, we identified 3,759 probe sets (ranging from 1,040 to 1,303 in each transplant) from formalin-fixed, paraffin-embedded sections of the 3 hybrid tumors, which unambiguously mapped to 3,107 unique Entrez Gene IDs, representative of all human chromosomes; however, by karyology, one of the hybrid tumors (GB-749) had a total of 15 human chromosomes in its cells. Among the genes mapped, 39 probe sets, representing 33 unique Entrez Gene IDs, complied with the detection criteria in all hybrid tumor samples. Five of these 33 genes encode transcription factors that are known to regulate cell growth and differentiation; five encode cell adhesion- and transmigration-associated proteins that participate in oncogenesis and/or metastasis and invasion; and additional genes encode proteins involved in signaling pathways, regulation of apoptosis, DNA repair, and multidrug resistance. These findings were corroborated by PCR and reverse transcription PCR, showing the presence of human alphoid (α)-satellite DNA and the F11R transcripts in additional tumor transplant generations. We posit that in-vivo fusion discloses genes implicated in tumor progression, and gene families coding for the organoid phenotype. Thus, cancer cells

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

    Directory of Open Access Journals (Sweden)

    Suh JS

    2014-03-01

    Full Text Available Jin Sook Suh,1,* Jue Yeon Lee,2,* Yoon Jung Choi,1 Hyung Keun You,3 Seong-Doo Hong,4 Chong Pyoung Chung,2 Yoon Jeong Park1,2 1Dental Regenerative Biotechnology, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, 2Central Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC, Seoul, 3Department of Periodontology, College of Dentistry, Wonkwang University, Iksan, 4Department of Oral Pathology, School of Dentistry, Seoul National University, Seoul, Republic of Korea *These authors contributed equally to this work Abstract: Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP, and a transcriptional coactivator with a PDZ-binding motif (TAZ protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sharad P Adekar

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

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

    Science.gov (United States)

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

    2015-12-01

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

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

    Science.gov (United States)

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

    2009-01-01

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

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

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

    2009-04-24

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

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

    Science.gov (United States)

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

    2009-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Craig D Blanchette

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

  4. Autographa californica multicapsid nucleopolyhedrovirus efficiently infects Sf9 cells and transduces mammalian cells via direct fusion with the plasma membrane at low pH.

    Science.gov (United States)

    Dong, Sicong; Wang, Manli; Qiu, Zhijuan; Deng, Fei; Vlak, Just M; Hu, Zhihong; Wang, Hualin

    2010-05-01

    The budded virus (BV) of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infects insect cells and transduces mammalian cells mainly through the endocytosis pathway. However, this study revealed that the treatment of the virus bound to Sf9 cells at low pH could efficiently rescue the infectivity of AcMNPV in the presence of endocytosis pathway inhibitors. A colocalization assay of the major capsid protein VP39 with the early endosome marker EEA1 showed that at low pH, AcMNPV entered Sf9 cells via an endosome-independent pathway. Using a fluorescent probe (R18), we showed that at low pH, the viral nucleocapsid entered Sf9 cells via direct fusion at the cell surface. By using the myosin-specific inhibitor 2,3-butanedione monoxime (BDM) and the microtubule inhibitor nocodazole, the low pH-triggered direct fusion was demonstrated to be dependent on myosin-like proteins and independent of microtubules. The reverse transcription-PCR of the IE1 gene as a marker for viral entry showed that the kinetics of AcMNPV in cells triggered by low pH was similar to that of the normal entry via endocytosis. The low pH-mediated infection assay and VP39 and EEA1 colocalization assay also demonstrated that AcMNPV could efficiently transduce mammalian cells via direct membrane fusion at the cell surface. More importantly, we found that a low-pH trigger could significantly improve the transduction efficiency of AcMNPV in mammalian cells, leading to the potential application of this method when using baculovirus as a vector for heterologous gene expression and for gene therapy.

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

    Science.gov (United States)

    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.

  6. Injection of duck recombinant follistatin fusion protein into duck muscle tissues stimulates satellite cell proliferation and muscle fiber hypertrophy.

    Science.gov (United States)

    Liu, He-he; Wang, Ji-wen; Yu, Hai-yue; Zhang, Rong-ping; Chen, Xi; Jin, Hai-bo; Dai, Fei; Li, Liang; Xu, Feng

    2012-06-01

    Follistatin (FST) can inhibit the expression of myostatin, which is a predominant inhibitor of muscle development. The potential application of myostatin-based technology has been prompted in different ways in agriculture. We previously constructed an expression vector of duck FST and isolated the FST fusion protein. After the protein was purified and refolded, it was added to the medium of duck myoblasts cultured in vitro. The results show that the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide value of the myoblasts in the duck FST treatment group is higher than that in the control group, which indicates that the duck FST fusion protein exhibits the biological activities that can accelerate myoblast proliferation. To further investigate the roles of duck FST on muscle development, we injected the protein into the duck muscle tissues in vivo. The results show that both the duck muscle fiber cross-sectional area and the satellite cell activation frequency are influenced more in the FST treatment group than they are in the control group. In addition to these phenomena, expression of MyoD and Myf5 were increased, and the expression of myostatin was decreased. Together, these results suggest the potential for using duck FST fusion protein to inhibit myostatin activity and subsequently to enhance muscle growth in vivo. The mechanism by which FST regulates muscle development in the duck is similar to that in mammals and fishes.

  7. An inducible cell-cell fusion system with integrated ability to measure the efficiency and specificity of HIV-1 entry inhibitors.

    Directory of Open Access Journals (Sweden)

    Alon Herschhorn

    Full Text Available HIV-1 envelope glycoproteins (Envs mediate virus entry by fusing the viral and target cell membranes, a multi-step process that represents an attractive target for inhibition. Entry inhibitors with broad-range activity against diverse isolates of HIV-1 may be extremely useful as lead compounds for the development of therapies or prophylactic microbicides. To facilitate the identification of such inhibitors, we have constructed a cell-cell fusion system capable of simultaneously monitoring inhibition efficiency and specificity. In this system, effector cells stably express a tetracycline-controlled transactivator (tTA that enables tightly inducible expression of both HIV-1 Env and the Renilla luciferase (R-Luc reporter protein. Target cells express the HIV-1 receptors, CD4 and CCR5, and carry the firefly luciferase (F-Luc reporter gene under the control of a tTA-responsive promoter. Thus, Env-mediated fusion of these two cell types allows the tTA to diffuse to the target cell and activate the expression of the F-Luc protein. The efficiency with which an inhibitor blocks cell-cell fusion is measured by a decrease in the F-Luc activity, while the specificity of the inhibitor is evaluated by its effect on the R-Luc activity. The system exhibited a high dynamic range and high Z'-factor values. The assay was validated with a reference panel of inhibitors that target different steps in HIV-1 entry, yielding inhibitory concentrations comparable to published virus inhibition data. Our system is suitable for large-scale screening of chemical libraries and can also be used for detailed characterization of inhibitory and cytotoxic properties of known entry inhibitors.

  8. CRACC-targeting Fc-fusion protein induces activation of NK cells and DCs and improves T cell immune responses to antigenic targets.

    Science.gov (United States)

    Aldhamen, Yasser A; Rastall, David P W; Chen, Weimin; Seregin, Sergey S; Pereira-Hicks, Cristiane; Godbehere, Sarah; Kaminski, Norbert E; Amalfitano, Andrea

    2016-06-08

    The CD2-like receptor activating cytotoxic cell (CRACC) receptor is a member of the SLAM family of receptors that are found on several types of immune cells. We previously demonstrated that increasing the abundance of the adaptor protein EAT-2 during vaccination enhanced innate and adaptive immune responses to vaccine antigens. Engagement of the CRACC receptor in the presence of the EAT-2 adaptor generally results in immune cell activation, while activating CRACC signaling in cells that lack EAT-2 adaptor inhibits their effector and regulatory functions. As EAT-2 is the only SAP adaptor that interacts with the CRACC receptor, we hypothesized that technologies that specifically modulate CRACC signaling during vaccination may also improve antigen specific adaptive immune responses. To test this hypothesis, we constructed a CRACC-targeting Fc fusion protein and included it in vaccination attempts. Indeed, mice co-vaccinated with the CRACC-Fc fusion protein and an adenovirus vaccine expressing the HIV-Gag protein had improved Gag-specific T cell responses, as compared to control mice. These responses are characterized by increased numbers of Gag-specific tetramer+ CD8+ T cells and increases in production of IFNγ, TNFα, and IL2, by Gag-specific CD8+ T cells. Moreover, our results revealed that use of the CRACC-Fc fusion protein enhances vaccine-elicited innate immune responses, as characterized by increased dendritic cells (DCs) maturation and IFNγ production from NK cells. This study highlights the importance of CRACC signaling during the induction of an immune response generally, and during vaccinations specifically, and also lends insight into the mechanisms underlying our prior results noting EAT-2-dependent improvements in vaccine efficacy.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    Exocytosis requires formation of SNARE complexes between vesicle- and target-membranes. Recent assessments in reduced model systems have produced divergent estimates of the number of SNARE complexes needed for fusion. Here, we used a titration approach to answer this question in intact, cultured ...

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

    NARCIS (Netherlands)

    Holm, C.K.; Jensen, S.B.; Jakobsen, M.R.; Cheshenko, N.; Horan, K.A.; Moeller, H.B.; Gonzalez-Dosal, R.; Rasmussen, S.B.; Christensen, M.H.; Yarovinsky, T.O.; Rixon, F.J.; Herold, B.C.; Fitzgerald, K.A.; Paludan, S.R.

    2012-01-01

    The innate immune system senses infection by detecting either evolutionarily conserved molecules essential for the survival of microbes or the abnormal location of molecules. Here we demonstrate the existence of a previously unknown innate detection mechanism induced by fusion between viral envelope

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

  12. Production, secretion, and cell surface display of recombinant Sporosarcina ureae S-layer fusion proteins in Bacillus megaterium.

    Science.gov (United States)

    Knobloch, Denise; Ostermann, Kai; Rödel, Gerhard

    2012-01-01

    Monomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report that Bacillus megaterium is an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA from Sporosarcina ureae ATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-terminally truncated, and N- and C-terminally truncated forms of the S-layer SslA protein tagged with the human influenza hemagglutinin epitope. For secretion of fusion proteins, the open reading frames were cloned into the Escherichia coli-Bacillus megaterium shuttle vector pHIS1525. After transformation of the respective plasmids into Bacillus megaterium protoplasts, the recombinant genes were successfully expressed and the proteins were secreted into the growth medium. The isolated S-layer proteins are able to assemble in vitro into highly ordered, crystalline, sheetlike structures with the fused HA tag accessible to antibody. We further show by fluorescent labeling that the secreted S-layer fusion proteins are also clustered on the cell envelope of Bacillus megaterium, indicating that the cell surface can serve in vivo as a nucleation point for crystallization. Thus, this system can be used as a display system that allows the dense and periodic presentation of S-layer proteins or the fused tags.

  13. The role of blood cell membrane lipids on the mode of action of HIV-1 fusion inhibitor sifuvirtide

    Energy Technology Data Exchange (ETDEWEB)

    Matos, Pedro M.; Freitas, Teresa; Castanho, Miguel A.R.B. [Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisbon, Av. Prof. Egas Moniz, 1649-028 Lisboa (Portugal); Santos, Nuno C., E-mail: nsantos@fm.ul.pt [Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisbon, Av. Prof. Egas Moniz, 1649-028 Lisboa (Portugal)

    2010-12-17

    Research highlights: {yields} Sifuvirtide interacts with erythrocyte and lymphocyte membrane in a concentration dependent manner by decreasing its dipole potential. {yields} Dipole potential variations in lipid vesicles show sifuvirtide's lipid selectivity towards saturated phosphatidylcholines. {yields} This peptide-membrane interaction may direct the drug towards raft-like membrane domains where the receptors used by HIV are located, facilitating its inhibitory action. -- Abstract: Sifuvirtide is a gp41 based peptide that inhibits HIV-1 fusion with the host cells and is currently under clinical trials. Previous studies showed that sifuvirtide partitions preferably to saturated phosphatidylcholine lipid membranes, instead of fluid-phase lipid vesicles. We extended the study to the interaction of the peptide with circulating blood cells, by using the dipole potential sensitive probe di-8-ANEPPS. Sifuvirtide decreased the dipole potential of erythrocyte and lymphocyte membranes in a concentration dependent manner, demonstrating its interaction. Also, the lipid selectivity of the peptide towards more rigid phosphatidylcholines was confirmed based on the dipole potential variations. Overall, the interaction of the peptide with the cell membranes is a contribution of different lipid preferences that presumably directs the peptide towards raft-like domains where the receptors are located, facilitating the reach of the peptide to its molecular target, the gp41 in its pre-fusion conformation.

  14. N-terminal fusion tags for effective production of g-protein-coupled receptors in bacterial cell-free systems.

    Science.gov (United States)

    Lyukmanova, E N; Shenkarev, Z O; Khabibullina, N F; Kulbatskiy, D S; Shulepko, M A; Petrovskaya, L E; Arseniev, A S; Dolgikh, D A; Kirpichnikov, M P

    2012-10-01

    G-protein-coupled receptors (GPCR) constitute one of the biggest families of membrane proteins. In spite of the fact that they are highly relevant to pharmacy, they have remained poorly explored. One of the main bottlenecks encountered in structural-functional studies of GPCRs is the difficulty to produce sufficient amounts of the proteins. Cell-free systems based on bacterial extracts fromE. colicells attract much attention as an effective tool for recombinant production of membrane proteins. GPCR production in bacterial cell-free expression systems is often inefficient because of the problems associated with the low efficiency of the translation initiation process. This problem could be resolved if GPCRs were expressed in the form of hybrid proteins with N-terminal polypeptide fusion tags. In the present work, three new N-terminal fusion tags are proposed for cell-free production of the human β2-adrenergic receptor, human M1 muscarinic acetylcholine receptor, and human somatostatin receptor type 5. It is demonstrated that the application of an N-terminal fragment (6 a.a.) of bacteriorhodopsin fromExiguobacterium sibiricum(ESR-tag), N-terminal fragment (16 а.о.) of RNAse A (S-tag), and Mistic protein fromB. subtilisallows to increase the CF synthesis of the target GPCRs by 5-38 times, resulting in yields of 0.6-3.8 mg from 1 ml of the reaction mixture, which is sufficient for structural-functional studies.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-08-02

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

  16. Membrane fusion

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    At Stanford University, Boxer lab, I worked on membrane fusion of small unilamellar lipid vesicles to flat membranes tethered to glass surfaces. This geometry closely resembles biological systems in which liposomes fuse to plasma membranes. The fusion mechanism was studied using DNA zippering...... between complementary strands linked to the two apposing membranes closely mimicking the zippering mechanism of SNARE fusion complexes....

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

    Science.gov (United States)

    2015-06-01

    whether viral fusion proteins enable or catalyze this event. If successful, this discovery would dramatically change our approach to breast cancer...would dramatically change our approach to breast cancer therapy in the following specific ways. First, the studies proposed here will help identify...participated in study design, offered helpful consultation in the course of the studies and participated in writing the manuscript. All authors read

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

    NARCIS (Netherlands)

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

    1988-01-01

    The fusion of liposomes with the plasma membrane of influenza virus-infected monolayers of an epithelial cell line, Madin-Darby canine kidney cells (van Meer et al., 1985. Biochemistry, 24: 3593-3602), has been analyzed by morphological techniques. The distribution of liposomal lipids over the apica

  19. Fusion rings and fusion ideals

    DEFF Research Database (Denmark)

    Andersen, Troels Bak

    by the so-called fusion ideals. The fusion rings of Wess-Zumino-Witten models have been widely studied and are well understood in terms of precise combinatorial descriptions and explicit generating sets of the fusion ideals. They also appear in another, more general, setting via tilting modules for quantum...

  20. Regulation of Varicella-Zoster Virus-Induced Cell-to-Cell Fusion by the Endocytosis-Competent Glycoproteins gH and gE

    OpenAIRE

    Pasieka, Tracy Jo; Maresova, Lucie; Shiraki, Kimiyasu; Grose, Charles

    2004-01-01

    The gH glycoprotein of varicella-zoster virus (VZV) is a major fusogen. The realigned short cytoplasmic tail of gH (18 amino acids) harbors a functional endocytosis motif (YNKI) that mediates internalization in both VZV-infected and transfected cells (T. J. Pasieka, L. Maresova, and C. Grose, J. Virol. 77: 4194-4202, 2003). During subsequent confocal microscopy studies of endocytosis-deficient gH mutants, we observed that cells transfected with the gH tail mutants exhibited marked fusion. The...

  1. Efficient production and purification of extracellular domain of human FGFR-Fc fusion proteins from Chinese hamster ovary cells.

    Science.gov (United States)

    Sokolowska-Wedzina, Aleksandra; Borek, Aleksandra; Chudzian, Julia; Jakimowicz, Piotr; Zakrzewska, Malgorzata; Otlewski, Jacek

    2014-07-01

    The family of fibroblast growth factor receptors (FGFRs) plays an important role in cell growth, survival, differentiation and angiogenesis. The three immunoglobulin-like extracellular domains of FGFR (D1, D2, and D3) are critical for ligand binding and specificity towards fibroblast growth factor and heparan sulfate. Fibroblast growth factor receptors are overexpressed in a wide variety of tumors, such as breast, bladder, and prostate cancer, and therefore they are attractive targets for different types of anticancer therapies. In this study, we have cloned, expressed in CHO cells and purified Fc-fused extracellular domains of different types of FGFRs (ECD_FGFR1a-Fc, ECD_FGFR1b-Fc, ECD_FGFR2a-Fc, ECD_FGFR2b-Fc, ECD_FGFR3a-Fc, ECD_FGFR3b-Fc, ECD_FGFR4a-Fc, ECD_FGFR4b-Fc), which could be used as molecular targets for the selection of specific antibodies. The fusion proteins were analyzed using gel electrophoresis, Western blotting and mass spectrometry. To facilitate their full characterization, the fusion proteins were deglycosylated using PNGase F enzyme. With an optimized transient transfection protocol and purification procedure we were able to express the proteins at a high level and purify them to homogeneity.

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

    Directory of Open Access Journals (Sweden)

    Alexandra J Spencer

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

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

    Directory of Open Access Journals (Sweden)

    Laura Arribillaga

    2013-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-01-06

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

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

    Directory of Open Access Journals (Sweden)

    Bárbara Hissa

    lysosomes are available in the cell and that cholesterol depletion may modulate the fusion of pre-docked lysosomes at the cell cortex.

  6. [Expression of SET-NUP214 fusion gene in patients with T-cell acute lymphoblastic leukemia and its clinical significance].

    Science.gov (United States)

    Dai, Hai-Ping; Wang, Qian; Wu, Li-Li; Ping, Na-Na; Wu, Chun-Xiao; Xie, Jun-Dan; Pan, Jin-Lan; Xue, Yong-Quan; Wu, De-Pei; Chen, Su-Ning

    2012-10-01

    This study was aimed to investigate the occurrence and clinical significance of the SET-NUP214 fusion gene in patients with T-cell acute lymphoblastic leukemia (T-ALL), analyse clinical and biological characteristics in this disease. RT-PCR was used to detect the expression of SET-NUP214 fusion gene in 58 T-ALL cases. Interphase FISH and Array-CGH were used to detect the deletion of 9q34. Direct sequencing was applied to detect mutations of PHF6 and NOTCH1. The results showed that 6 out of 58 T-ALL cases (10.3%) were detected to have the SET-NUP214 fusion gene by RT-PCR. Besides T-lineage antigens, expression of CD13 and(or) CD33 were detected in all the 6 cases. Deletions of 9q34 were detected in 4 out of the 6 patients by FISH. Array-CGH results of 3 SET-NUP214 positive T-ALL patients confirmed that this fusion gene was resulted from a cryptic deletion of 9q34.11q34.13. PHF6 and NOTCH1 gene mutations were found in 4 and 5 out of 6 SET-NUP214 positive T-ALL patients, respectively. It is concluded that SET-NUP214 fusion gene is often resulted from del(9)(q34). PHF6 and NOTCH1 mutations may be potential leukemogenic event in SET-NUP214 fusion gene.

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

    Science.gov (United States)

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

    2013-01-01

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

  8. Dielectrophoresis-assisted massively parallel cell pairing and fusion based on field constriction created by a micro-orifice array sheet.

    Science.gov (United States)

    Kimura, Yuji; Gel, Murat; Techaumnat, Boonchai; Oana, Hidehiro; Kotera, Hidetoshi; Washizu, Masao

    2011-09-01

    In this paper, we present a novel electrofusion device that enables massive parallelism, using an electrically insulating sheet having a two-dimensional micro-orifice array. The sheet is sandwiched by a pair of micro-chambers with immersed electrodes, and each chamber is filled with the suspensions of the two types of cells to be fused. Dielectrophoresis, assisted by sedimentation, is used to position the cells in the upper chamber down onto the orifices, then the device is flipped over to position the cells on the other side, so that cell pairs making contact in the orifice are formed. When a pulse voltage is applied to the electrodes, most voltage drop occurs around the orifice and impressed on the cell membrane in the orifice. This makes possible the application of size-independent voltage to fuse two cells in contact at all orifices exclusively in 1:1 manner. In the experiment, cytoplasm of one of the cells is stained with a fluorescence dye, and the transfer of the fluorescence to the other cell is used as the indication of fusion events. The two-dimensional orifice arrangement at the pitch of 50 μm realizes simultaneous fusion of 6 × 10³ cells on a 4 mm diameter chip, and the fusion yield of 78-90% is achieved for various sizes and types of cells.

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

    Science.gov (United States)

    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.

  10. Alphavirus Entry and Membrane Fusion

    Directory of Open Access Journals (Sweden)

    Margaret Kielian

    2010-03-01

    Full Text Available The study of enveloped animal viruses has greatly advanced our understanding of the general properties of membrane fusion and of the specific pathways that viruses use to infect the host cell. The membrane fusion proteins of the alphaviruses and flaviviruses have many similarities in structure and function. As reviewed here, alphaviruses use receptor-mediated endocytic uptake and low pH-triggered membrane fusion to deliver their RNA genomes into the cytoplasm. Recent advances in understanding the biochemistry and structure of the alphavirus membrane fusion protein provide a clearer picture of this fusion reaction, including the protein’s conformational changes during fusion and the identification of key domains. These insights into the alphavirus fusion mechanism suggest new areas for experimental investigation and potential inhibitor strategies for anti-viral therapy.

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

    Institute of Scientific and Technical Information of China (English)

    John M. Pawelek

    2014-01-01

    This perspective article highlights the leukocyte-cancer cellhybrid 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 cellis a secondary disease superimposed upon the early tumor, giving birth to a new, malignant cellwith a leukocyte-cancer cellhybrid epigenome.

  12. [Construction of eukaryotic recombinant vector and expression in COS7 cell of LipL32-HlyX fusion gene from Leptospira serovar Lai].

    Science.gov (United States)

    Huang, Bi; Bao, Lang; Zhong, Qi; Zhang, Huidong; Zhang, Ying

    2009-04-01

    This study was conducted to construct eukaryotic recombinant vector of LipL32-HlyX fusion gene from Leptospira serovar Lai and express it in mammalian cell. Both of LipL32 gene and HlyX gene were amplified from Leptospira strain O17 genomic DNA by PCR. Then with the two genes as template, LipL32-HlyX fusion gene was obtained by SOE PCR (gene splicing by overlap extension PCR). The fusion gene was then cloned into pcDNA3.1 by restriction nuclease digestion. Having been transformed into E. coli DH5alpha, the recombiant plasmid was identified by restriction nuclease digestion, PCR analysis and sequencing. The recombinant plasmid was then transfected into COS7 cell whose expression was detected by RT-PCR and Western blotting analysis. RT-PCR amplified a fragment about 2000 bp and Western blotting analysis found a specific band about 75 KD which was consistent with the expected fusion protein size. In conclusion, the successful construction of eukaryotic recombinant vector containing LipL32-HlyX fusion gene and the effective expression in mammalian have laid a foundation for the application of Leptospira DNA vaccine.

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

    Science.gov (United States)

    Cárdenas, Ana María; Marengo, Fernando D

    2016-06-01

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

  14. Laser-induced tobacco protoplast fusion

    Institute of Scientific and Technical Information of China (English)

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

    1999-01-01

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

  15. Development of a soluble PTD-HPV18E7 fusion protein and its functional characterization in eukaryotic cells

    Institute of Scientific and Technical Information of China (English)

    Xiaofei Yan; Shah Walayat; Qinfeng Shi; Jin Zheng; Yili Wang

    2009-01-01

    Though accumulated evidence has demonstrated the transformation capacity of human papillomavirus (HPV) type 18 protein E7, the underlying mechanism is still arguable. Developing a protein transduction domain (PTD)-iinked E7 molecule is a suitable strategy for assessing the biological functions of the protein. In the present study, HPVI8 E7 protein fused to an N-terminal PTD was expressed in the form of giutathione S-trans-ferase fusion protein in Escherichia coil with pGEX-4T-3 vector. After giutathione-Sepharose 4B bead affinity purification, immunobiot identification and thrombin cleavage, the PTD-18E7 protein showed structural and functional activity in that it potently transduced the cells and localized into their nuclei. The PTD-18E7 protein transduced the NIH3T3 cells in 30 min and remained stable for at least 24 h. In addition, the PTD-18E7 protein interacted with retinoblastoma protein (pRB) and caused pRB degradation in the transduced NIH3T3 cells. In contrast to the pRB level, p27 protein level was elevated in the transduced NIH3T3 cells. The PTD-18E7 protein gives us a new tool to study the biological functions of the HPV E7 protein.

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

    DEFF Research Database (Denmark)

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

    Abstract Mechanical Stimulation of C2C12 Cells Increases m-calpain Expression and Activity, Focal Adhesion Plaque Degradation and Cell Fusion A. Grossi, A. H. Karlsson, M. A. Lawson; Department of Dairy and Food Science, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark...... to the activity of ubiquitous proteolytic enzymes known as calpains has been reported. Whether there is a link between stretch- or load induced signaling and calpain expression and activation is not known. Using a magnetic bead stimulation assay and C2C12 mouse myoblasts cell population, we have demonstrated...... that mechanical stimulation via laminin receptors leads to an increase in m-calpain expression, but no increase in the expression of other calpain isoforms. Our study revealed that after a short period of stimulation, m-calpain relocates into focal adhesion complexes and is followed by a breakdown of specific...

  17. A plant cell-expressed recombinant anti-TNF fusion protein is biologically active in the gut and alleviates immune-mediated hepatitis and colitis.

    Science.gov (United States)

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

    2017-03-01

    The orally administered BY-2 plant cell-expressed recombinant anti-TNF fusion protein (PRX-106) (n=6) consists of the soluble form of the human TNF receptor (TNFR) fused to the Fc component of a human antibody IgG1 domain.

  18. Rapid detection of BCR-ABL fusion genes using a novel combined LUX primer, in-cell RT-PCR and flow cytometric method.

    Science.gov (United States)

    Shi, Yan; Li, Li-Zhen; Sun, Jian-Zhi; Zhang, Ti; Peng, Jun; Xu, Cong-Gao

    2008-01-01

    Currently, quantitative and semiquantitative assays for minimal residual disease detection include fluorescence in situ hybridisation, multiparameter flow cytometric immunophenotyping and real-time quantitative polymerase chain reaction (RQ-PCR). We have developed a new approach to detect hybrid breakpoint cluster region and Abelson proto-oncogene (BCR-ABL) transcripts inside suspension cells using in situ RT-PCR and light upon extension (LUX) primer, followed by rapid quantitative analysis with flow cytometry. After cellular permeabilization and fixation of single cell suspension, the neoplastic mRNA was reverse transcribed and amplified by PCR with LUX primer. The results demonstrated that a strong positive yellow-green signal was observed in 99-100% cells of K562 cell line, only the red nucleus was detected in NB4 cell line and normal controls. The technique has been utilised to study 12 patients with chronic myeloid leukemia, and the results were compared with those of BCR-ABL fusion mRNA by RT-PCR and BCR-ABL fusion gene of the interphase cells by fluorescence in situ hybridization (FISH). In the five diagnosed patients, 90-98% cells were strongly positive. Four patients, including three patients treated with interferon-alpha and hydroxyurea and one patient treated with imatinib mesylate, had 26-82.5% positive cells. Three patients treated with imatinib mesylate were negative. The in situ RT-PCR results demonstrated complete concordance with the results of I-FISH and RT-PCR. A fluorescence signal was detectable at 1/10(4) cells and became negative below this threshold with flow cytometry. The results of the present study suggest that (1) LUX primers can be used to efficiently detect BCR-ABL fusion mRNA by in-cell RT-PCR; (2) the novel technique is a specific and sensitive way of detecting fusion gene with potential clinical usefulness.

  19. The elementary fusion modalities of osteoclasts

    DEFF Research Database (Denmark)

    Søe, Kent; Hobolt-Pedersen, Anne-Sofie; Delaisse, Jean-Marie

    2015-01-01

    , are not known for the osteoclast. Here we show that osteoclast fusion partners are characterized by differences in mobility, nuclearity, and differentiation level. Our demonstration was based on time-laps videos of human osteoclast preparations from three donors where 656 fusion events were analyzed. Fusions......The last step of the osteoclast differentiation process is cell fusion. Most efforts to understand the fusion mechanism have focused on the identification of molecules involved in the fusion process. Surprisingly, the basic fusion modalities, which are well known for fusion of other cell types...... between a mobile and an immobile partner were most frequent (62%), while fusion between two mobile (26%) or two immobile partners (12%) was less frequent (pfusion partner contained more nuclei than the mobile one (p

  20. A novel thrombopoietin-stem-cell factor fusion protein possesses enhanced potential in stimulating megakaryocyte proliferation and differentiation.

    Science.gov (United States)

    Zang, Yuhui; Zhang, Yumin; Peng, Wei; Chen, Bin; Zhu, Jie; Zhang, Chi; Ouyang, Jian; Qin, Junchuan

    2007-11-01

    TPO (thrombopoietin) and SCF (stem-cell factor) are functionally related cytokines with overlapping but distinct haematopoietic effects. In the present study, a novel TPO-SCF fusion protein that combined the complementary biological effects of TPO and SCF into a single molecule was expressed in, and purified from, Sf9 [Spodoptera frugiperda (fall armyworm)] insect cells. The specific activity of rhTPO (recombinant human TPO)-SCF in megakaryoblastic Mo7e cell proliferation assays was 2.90+/-0.35 x 10(7) units/micromol, approx. 1.7 times as high as that of rhTPO. The specific activity of rhTPO-SCF in TF-1 cells proliferation assays was 7.10+/-0.95 x 10(6) units/micromol, approx. 1.2 times as high as that of rhSCF (recombinant human SCF). In a megakaryocyte-colony-forming assay using human peripheral-blood CD34(+) cells, the SCF moiety of rhTPO-SCF worked in a synergistic way to augment the colony number and exhibited a higher potential to stimulate megakaryocyte colony growth. According to the results of EMSA (electrophoretic mobility-shift assay) and semi-quantitative RT (reverse transcriptase)-PCR, the synergistic effects of the SCF moiety were also reflected in increased STAT5 (signal transducer and activator of transcription 5) DNA binding and enhanced up-regulation of p21 expression in Mo7e cells treated by rhTPO-SCF, suggesting that rhTPO-SCF could be more potent in promoting megakaryocyte proliferation and differentiation.

  1. Enhanced antitumor immunity of nanoliposome-encapsulated heat shock protein 70 peptide complex derived from dendritic tumor fusion cells.

    Science.gov (United States)

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

    2015-06-01

    Tumor-derived heat shock proteins peptide complex (HSP.PC-Tu) has been regarded as a promising antitumor agent. However, inadequate immunogenicity and low bioavailability limit the clinical uses of this agent. In a previous study, we first produced an improved HSP70.PC-based vaccine purified from dendritic cell (DC)-tumor fusion cells (HSP70.PC-Fc) which had increased immunogenicity due to enhanced antigenic tumor peptides compared to HSP70.PC-Tu. In order to increase the bioavailability of HSP70.PC-Fc, the peptide complex was encapsulated with nanoliposomes (NL-HSP70.PC-Fc) in this study. After encapsulation, the tumor immunogenicity was observed using various assays. It was demonstrated that the NL-HSP70.PC-Fc has acceptable stability. The in vivo antitumor immune response was increased with regard to T-cell activation, CTL response and tumor therapy efficiency compared to that of HSP70.PC-Fc. In addition, it was shown that DC maturation was improved by NL-HSP70.PC-Fc, which added to the antitumor immunity. The results obtained for NL-HSP70.PC-Fc, which improved immunogenicity and increases the bioavailability of HSP70.PC, may represent superior heat shock proteins (HSPs)-based tumor vaccines. Such vaccines deserve further investigation and may provide a preclinical rationale to translate findings into early phase trials for patients with breast tumors.

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

    Science.gov (United States)

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

    2016-02-26

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

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

    Directory of Open Access Journals (Sweden)

    Janine Kimpel

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

  4. A novel luciferase fusion protein for highly sensitive optical imaging: from single-cell analysis to in vivo whole-body bioluminescence imaging.

    Science.gov (United States)

    Mezzanotte, Laura; Blankevoort, Vicky; Löwik, Clemens W G M; Kaijzel, Eric L

    2014-09-01

    Fluorescence and bioluminescence imaging have different advantages and disadvantages depending on the application. Bioluminescence imaging is now the most sensitive optical technique for tracking cells, promoter activity studies, or for longitudinal in vivo preclinical studies. Far-red and near-infrared fluorescence imaging have the advantage of being suitable for both ex vivo and in vivo analysis and have translational potential, thanks to the availability of very sensitive imaging instrumentation. Here, we report the development and validation of a new luciferase fusion reporter generated by the fusion of the firefly luciferase Luc2 to the far-red fluorescent protein TurboFP635 by a 14-amino acid linker peptide. Expression of the fusion protein, named TurboLuc, was analyzed in human embryonic kidney cells, (HEK)-293 cells, via Western blot analysis, fluorescence microscopy, and in vivo optical imaging. The created fusion protein maintained the characteristics of the original bioluminescent and fluorescent protein and showed no toxicity when expressed in living cells. To assess the sensitivity of the reporter for in vivo imaging, transfected cells were subcutaneously injected in animals. Detection limits of cells were 5 × 10(3) and 5 × 10(4) cells for bioluminescent and fluorescent imaging, respectively. In addition, hydrodynamics-based in vivo gene delivery using a minicircle vector expressing TurboLuc allowed for the analysis of luminescent signals over time in deep tissue. Bioluminescence could be monitored for over 30 days in the liver of animals. In conclusion, TurboLuc combines the advantages of both bioluminescence and fluorescence and allows for highly sensitive optical imaging ranging from single-cell analysis to in vivo whole-body bioluminescence imaging.

  5. An IL-12/Shh-C domain fusion protein-based IL-12 autocrine loop for sustained natural killer cell activation.

    Science.gov (United States)

    Zhu, Lining; Zhao, Zhihui; Wei, Yanzhang; Marcotte, William; Wagner, Thomas E; Yu, Xianzhong

    2012-08-01

    The dependency of activated natural killer (NK) cells on the continuous support of exogenous interleukin (IL)-2 for their in vivo survival, tumor localization and consequently, their antitumor effect, is a major obstacle for NK cell-mediated tumor therapy. In the present study, a fusion gene between IL-12 and mouse sonic hedgehog C-terminal domain (Shh-C) was constructed. The fusion protein was autocatalytically processed to form cholesterol-modified IL-12 molecules and an autocrine loop of IL-12 was established for the sustained activation of NK cells. The transduced NK cells matured more rapidly in vitro with the enhanced expression of granule-related proteins. NKIL-12/Shh-C cells reached the same proliferation rate as NK cells transduced with enhanced green fluorescent protein (EGFP)/Shh-C (NKEGFP/Shh-C) with Shh-C cells 5 and 7 days after transduction was significantly higher than that in the supernatants of NKIL-12 cells. Immunofluorescent staining of lung tissues from B16-bearing mice which had received an intravenous injection of lentivirus-transduced NK cells without exogenous IL-2 confirmed that donor NK cells successfully infiltrated into the lung tissues. The survival time of the mice which had received NKIL-12/Shh-C cells was significantly prolonged compared to the mice which had received NKEGFP/Shh-C cells.

  6. A cholesterol recognition amino acid consensus domain in GP64 fusion protein facilitates anchoring of baculovirus to mammalian cells.

    Science.gov (United States)

    Luz-Madrigal, Agustin; Asanov, Alexander; Camacho-Zarco, Aldo R; Sampieri, Alicia; Vaca, Luis

    2013-11-01

    Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV.

  7. A Cholesterol Recognition Amino Acid Consensus Domain in GP64 Fusion Protein Facilitates Anchoring of Baculovirus to Mammalian Cells

    Science.gov (United States)

    Luz-Madrigal, Agustin; Asanov, Alexander; Camacho-Zarco, Aldo R.; Sampieri, Alicia

    2013-01-01

    Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV. PMID:23986592

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  10. Cold fusion

    Energy Technology Data Exchange (ETDEWEB)

    Suh, Suk Yong; Sung, Ki Woong; Kang, Joo Sang; Lee, Jong Jik [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of)

    1995-02-01

    So called `cold fusion phenomena` are not confirmed yet. Excess heat generation is very delicate one. Neutron generation is most reliable results, however, the records are erratic and the same results could not be repeated. So there is no reason to exclude the malfunction of testing instruments. The same arguments arise in recording {sup 4}He, {sup 3}He, {sup 3}H, which are not rich in quantity basically. An experiment where plenty of {sup 4}He were recorded is attached in appendix. The problem is that we are trying to search cold fusion which is permitted by nature or not. The famous tunneling effect in quantum mechanics will answer it, however, the most fusion rate is known to be negligible. The focus of this project is on the theme that how to increase that negligible fusion rate. 6 figs, 4 tabs, 1512 refs. (Author).

  11. Spinal Fusion

    Science.gov (United States)

    ... results in predictable healing. Autograft is currently the “gold standard” source of bone for a fusion. The ... pump. With this technique, the patient presses a button that delivers a predetermined amount of narcotic pain ...

  12. Application of Quality by Design to the characterization of the cell culture process of an Fc-Fusion protein.

    Science.gov (United States)

    Rouiller, Yolande; Solacroup, Thomas; Deparis, Véronique; Barbafieri, Marco; Gleixner, Ralf; Broly, Hervé; Eon-Duval, Alex

    2012-06-01

    The production bioreactor step of an Fc-Fusion protein manufacturing cell culture process was characterized following Quality by Design principles. Using scientific knowledge derived from the literature and process knowledge gathered during development studies and manufacturing to support clinical trials, potential critical and key process parameters with a possible impact on product quality and process performance, respectively, were determined during a risk assessment exercise. The identified process parameters were evaluated using a design of experiment approach. The regression models generated from the data allowed characterizing the impact of the identified process parameters on quality attributes. The main parameters having an impact on product titer were pH and dissolved oxygen, while those having the highest impact on process- and product-related impurities and variants were pH and culture duration. The models derived from characterization studies were used to define the cell culture process design space. The design space limits were set in such a way as to ensure that the drug substance material would consistently have the desired quality.

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

    Directory of Open Access Journals (Sweden)

    Gale Smith

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

  14. Phosphomimetic mutation of cysteine string protein-α increases the rate of regulated exocytosis by modulating fusion pore dynamics in PC12 cells.

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    Ning Chiang

    Full Text Available BACKGROUND: Cysteine string protein-α (CSPα is a chaperone to ensure protein folding. Loss of CSPα function associates with many neurological diseases. However, its function in modulating regulated exocytosis remains elusive. Although cspα-knockouts exhibit impaired synaptic transmission, overexpression of CSPα in neuroendocrine cells inhibits secretion. These seemingly conflicting results lead to a hypothesis that CSPα may undergo a modification that switches its function in regulating neurotransmitter and hormone secretion. Previous studies implied that CSPα undergoes phosphorylation at Ser10 that may influence exocytosis by altering fusion pore dynamics. However, direct evidence is missing up to date. METHODOLOGY/PRINCIPAL FINDINGS: Using amperometry, we investigated how phosphorylation at Ser10 of CSPα (CSPα-Ser10 modulates regulated exocytosis and if this modulation involves regulating a specific kinetic step of fusion pore dynamics. The real-time exocytosis of single vesicles was detected in PC12 cells overexpressing control vector, wild-type CSPα (WT, the CSPα phosphodeficient mutant (S10A, or the CSPα phosphomimetic mutants (S10D and S10E. The shapes of amperometric signals were used to distinguish the full-fusion events (i.e., prespike feet followed by spikes and the kiss-and-run events (i.e., square-shaped flickers. We found that the secretion rate was significantly increased in cells overexpressing S10D or S10E compared to WT or S10A. Further analysis showed that overexpression of S10D or S10E prolonged fusion pore lifetime compared to WT or S10A. The fraction of kiss-and-run events was significantly lower but the frequency of full-fusion events was higher in cells overexpressing S10D or S10E compared to WT or S10A. Advanced kinetic analysis suggests that overexpression of S10D or S10E may stabilize open fusion pores mainly by inhibiting them from closing. CONCLUSIONS/SIGNIFICANCE: CSPα may modulate fusion pore dynamics

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

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    Shigeo Koido

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

  16. Perspective for special Gurdon issue for differentiation: can cell fusion inform nuclear reprogramming?

    Science.gov (United States)

    Burns, David; Blau, Helen M

    2014-07-01

    Nuclear reprogramming was first shown to be possible by Sir John Gurdon over a half century ago. The process has been revolutionized by the production of induced pluripotent cells by overexpression of the four transcription factors discovered by Shinya Yamanaka, which now enables mammalian applications. Yet, reprogramming by a few transcription factors remains incomplete and inefficient, whether to pluripotent or differentiated cells. We propose that a better understanding of mechanistic insights based on developmental principles gained from heterokaryon studies may inform the process of directing cell fate, fundamentally and clinically.

  17. Graft vascularization is a critical rate-limiting step in skeletal stem cell-mediated posterolateral spinal fusion.

    Science.gov (United States)

    Giannicola, Giuseppe; Ferrari, Emiliano; Citro, Gennaro; Sacchetti, Benedetto; Corsi, Alessandro; Riminucci, Mara; Cinotti, Gianluca; Bianco, Paolo

    2010-06-01

    The ability of skeletal stem cells (SSCs) to direct spinal fusion (SF) upon transplantation in conjunction with osteoconductive biomaterials was investigated in a rabbit model. When tested in a mouse heterotopic transplantation assay, rabbit SSCs and Pro-Osteon 500R was osteoconductive and supported osteogenesis. When used in a SF model, the same constructs induced bone formation in periapophyseal regions (PARs). In this respect, they proved to be superior to grafts of cell-free carrier or total uncultured bone marrow-carrier constructs, used as controls. However, interapophyseal regions (IARs) remained devoid of new bone, such that true bony bridging of adjacent transverse apophyses (true SF) could not be achieved. Interestingly, this could not be predicted from high-resolution radiography. A systematic histological survey of the entire graft harvested at 6 months was essential for proper assessment of the transplantation procedure outcome. Immunohistochemical analysis of microvessel density revealed that IARs remained undervascularized, as compared to PARs, suggesting that differential vascularization could account for the absence or presence of new bone formation in the same regions. SF is an extreme model of stem cell-directed bone regeneration, requiring a combination of orthotopic (PAR) and heterotopic (IAR) bone formation. Our data show that, in this setting, graft size can be critical with respect to the necessary neovascularization, a crucial variable independent of proper osteogenic and osteoconductive competence of the cells and materials employed. Furthermore, stringent histological studies are mandatory for proper assessment of outcomes in SF studies, in which the use of mineralized materials can make radiographic assessment misleading.

  18. Myoblast fusion in Drosophila

    Energy Technology Data Exchange (ETDEWEB)

    Haralalka, Shruti [Stowers Institute for Medical Research, Kansas City, MO 64110 (United States); Abmayr, Susan M., E-mail: sma@stowers.org [Stowers Institute for Medical Research, Kansas City, MO 64110 (United States); Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, MO 66160 (United States)

    2010-11-01

    The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process . With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.

  19. Development of a 3D co-culture model using human stem cells for studying embryonic palatal fusion.

    Science.gov (United States)

    Morphogenetic tissue fusion is a critical and complex event in embryonic development and failure of this event leads to birth defects, such as cleft palate. Palatal fusion requires adhesion and subsequent dissolution of the medial epithelial layer of the mesenchymal palatal shelv...

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

    Directory of Open Access Journals (Sweden)

    Lei DONG

    2014-10-01

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

  1. Molecular Characterization of TMPRSS2-ERG Gene Fusion in the NCI-H660 Prostate Cancer Cell Line: A New Perspective for an Old Model

    Directory of Open Access Journals (Sweden)

    Kirsten D. Mertz

    2007-03-01

    Full Text Available Recent studies have established that a significant fraction of prostate cancers harbor a signature gene fusion between the 5' region of androgen-regulated TMPRSS2 and an ETS family transcription factor, most commonly ERG. Studies on the molecular mechanisms and functional consequences of this important chromosomal rearrangement are currently limited to the VCaP cell line derived from a vertebral bone metastasis of a hormone-refractory prostate tumor. Here we report on the NCI-H660 cell line, derived from a metastasic site of an extrapulmonary small cell carcinoma arising from the prostate. NCI-H660 harbors TMPRSS2-ERG fusion with a homozygous intronic deletion between TMPRSS2 and ERG. We demonstrate this by real-time quantitative polymerase chain reaction, a two-stage dual-color interphase fluorescence in situ hybridization (FISH assay testing for TMPRSS2 and ERG break-aparts, and single-nucleotide polymorphism oligonucleotide arrays. The deletion is consistent with the common intronic deletion found on chromosome 21q22.2-3 in human prostate cancer samples. We demonstrate the physical juxtaposition of TMPRSS2 and ERG on the DNA level by fiber FISH. The androgen receptor-negative NCI-H660 cell line expresses ERG in an androgen-independent fashion. This in vitro model system has the potential to provide important pathobiologic insights into TMPRSS2-ERG fusion prostate cancer.

  2. Activation of Notch in lgd mutant cells requires the fusion of late endosomes with the lysosome.

    Science.gov (United States)

    Schneider, Markus; Troost, Tobias; Grawe, Ferdi; Martinez-Arias, Alfonso; Klein, Thomas

    2013-01-15

    The tumour suppressor Lethal (2) giant discs (Lgd) is a regulator of endosomal trafficking of the Notch signalling receptor as well as other transmembrane proteins in Drosophila. The loss of its function results in an uncontrolled ligand-independent activation of the Notch signalling receptor. Here, we investigated the consequences of loss of lgd function and the requirements for the activation of Notch. We show that the activation of Notch in lgd cells is independent of Kuz and dependent on γ-secretase. We found that the lgd cells have a defect that delays degradation of transmembrane proteins, which are residents of the plasma membrane. Furthermore, our results show that the activation of Notch in lgd cells occurs in the lysosome. By contrast, the pathway is activated at an earlier phase in mutants of the gene that encodes the ESCRT-III component Shrub, which is an interaction partner of Lgd. We further show that activation of Notch appears to be a general consequence of loss of lgd function. In addition, electron microscopy of lgd cells revealed that they contain enlarged multi-vesicular bodies. The presented results further elucidate the mechanism of uncontrolled Notch activation upon derailed endocytosis.

  3. Proteolytic Activation of the Porcine Epidemic Diarrhea Coronavirus Spike Fusion Protein by Trypsin in Cell Culture.

    NARCIS (Netherlands)

    Wicht, Oliver; Li, Wentao; Willems, Lione; Meuleman, Tom J; Wubbolts, Richard W; van Kuppeveld, Frank J M; Rottier, Peter J M; Bosch, Berend Jan

    2014-01-01

    Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infec

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

    Directory of Open Access Journals (Sweden)

    Birgit Hotz

    2010-10-01

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

  5. Exo-endo cellulase fusion protein

    Science.gov (United States)

    Bower, Benjamin S [Palo Alto, CA; Larenas, Edmund A [Palo Alto, CA; Mitchinson, Colin [Palo Alto, CA

    2012-01-17

    The present invention relates to a heterologous exo-endo cellulase fusion construct, which encodes a fusion protein having cellulolytic activity comprising a catalytic domain derived from a fungal exo-cellobiohydrolase and a catalytic domain derived from an endoglucanase. The invention also relates to vectors and fungal host cells comprising the heterologous exo-endo cellulase fusion construct as well as methods for producing a cellulase fusion protein and enzymatic cellulase compositions.

  6. Trophoblast fusion.

    Science.gov (United States)

    Huppertz, Berthold; Gauster, Martin

    2011-01-01

    The villous trophoblast of the human placenta is the epithelial cover of the fetal chorionic villi floating in maternal blood. This epithelial cover is organized in two distinct layers, the multinucleated syncytiotrophoblast directly facing maternal blood and a second layer of mononucleated cytotrophoblasts. During pregnancy single cytotrophoblasts continuously fuse with the overlying syncytiotrophoblast to preserve this end-differentiated layer until delivery. Syncytial fusion continuously supplies the syncytiotrophoblast with compounds of fusing cytotrophoblasts such as proteins, nucleic acids and lipids as well as organelles. At the same time the input of cytotrophoblastic components is counterbalanced by a continuous release of apoptotic material from the syncytiotrophoblast into maternal blood. Fusion is an essential step in maintaining the syncytiotrophoblast. Trophoblast fusion was shown to be dependant on and regulated by multiple factors such as fusion proteins, proteases and cytoskeletal proteins as well as cytokines, hormones and transcription factors. In this chapter we focus on factors that may be involved in the fusion process of trophoblast directly or that may prepare the cytotrophoblast to fuse.

  7. Sulphated Polysaccharides from Ulva clathrata and Cladosiphon okamuranus Seaweeds both Inhibit Viral Attachment/Entry and Cell-Cell Fusion, in NDV Infection

    Directory of Open Access Journals (Sweden)

    José Alberto Aguilar-Briseño

    2015-01-01

    Full Text Available Sulphated polysaccharides (SP extracted from seaweeds have antiviral properties and are much less cytotoxic than conventional drugs, but little is known about their mode of action. Combination antiviral chemotherapy may offer advantages over single agent therapy, increasing efficiency, potency and delaying the emergence of resistant virus. The paramyxoviridae family includes pathogens causing morbidity and mortality worldwide in humans and animals, such as the Newcastle Disease Virus (NDV in poultry. This study aims at determining the antiviral activity and mechanism of action in vitro of an ulvan (SP from the green seaweed Ulva clathrata, and of its mixture with a fucoidan (SP from Cladosiphon okamuranus, against La Sota NDV strain. The ulvan antiviral activity was tested using syncytia formation, exhibiting an IC50 of 0.1 μg/mL; ulvan had a better anti cell-cell spread effect than that previously shown for fucoidan, and inhibited cell-cell fusion via a direct effect on the F0 protein, but did not show any virucidal effect. The mixture of ulvan and fucoidan showed a greater anti-spread effect than SPs alone, but ulvan antagonizes the effect of fucoidan on the viral attachment/entry. Both SPs may be promising antivirals against paramyxovirus infection but their mixture has no clear synergistic advantage.

  8. Stromal Cell-Derived Growth Factor-1 Alpha-Elastin Like Peptide Fusion Protein Promotes Cell Migration and Revascularization of Experimental Wounds in Diabetic Mice

    Science.gov (United States)

    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

  9. Cell-specific targeting of lentiviral vectors mediated by fusion proteins derived from Sindbis virus, vesicular stomatitis virus, or avian sarcoma/leukosis virus

    Directory of Open Access Journals (Sweden)

    Marino Michael P

    2010-01-01

    Full Text Available Abstract Background The ability to efficiently and selectively target gene delivery vectors to specific cell types in vitro and in vivo remains one of the formidable challenges in gene therapy. We pursued two different strategies to target lentiviral vector delivery to specific cell types. In one of the strategies, vector particles bearing a membrane-bound stem cell factor sequence plus a separate fusion protein based either on Sindbis virus strain TR339 glycoproteins or the vesicular stomatitis virus G glycoprotein were used to selectively transduce cells expressing the corresponding stem cell factor receptor (c-kit. An alternative approach involved soluble avian sarcoma/leukosis virus receptors fused to cell-specific ligands including stem cell factor and erythropoietin for targeting lentiviral vectors pseudotyped with avian sarcoma/leukosis virus envelope proteins to cells that express the corresponding receptors. Results The titers of unconcentrated vector particles bearing Sindbis virus strain TR339 or vesicular stomatitis virus G fusion proteins plus stem cell factor in the context of c-kit expressing cells were up to 3.2 × 105 transducing units per ml while vector particles lacking the stem cell factor ligand displayed titers that were approximately 80 fold lower. On cells that lacked the c-kit receptor, the titers of stem cell factor-containing vectors were approximately 40 times lower compared to c-kit-expressing cells. Lentiviral vectors pseudotyped with avian sarcoma/leukosis virus subgroup A or B envelope proteins and bearing bi-functional bridge proteins encoding erythropoietin or stem cell factor fused to the soluble extracellular domains of the avian sarcoma/leukosis virus subgroup A or B receptors resulted in efficient transduction of erythropoietin receptor or c-kit-expressing cells. Transduction of erythropoietin receptor-expressing cells mediated by bi-functional bridge proteins was found to be dependent on the dose, the

  10. Fusion expression of DDR2 extracellular domain in insect cells and its purification and function characterization.

    Science.gov (United States)

    Zhang, Wei; Ding, Tianbing; Zhang, Jian; Su, Jin; Yu, Jiangtian; Li, Jipeng; Li, Fuyang; Wang, Chunmei; Liu, Nannan; Liu, Xinping; Ma, Wenyu; Yao, Libo

    2007-09-01

    Discoidin domain receptor 2 (DDR2) is a kind of protein tyrosine kinases associated with cell proliferation and tumor metastasis, and collagen, a ligand for DDR2, up-regulates matrix metalloproteinase 1 (MMP-1) and MMP-2 expression in extracellular matrix (ECM). To investigate the role of DDR2 in cartilage destruction in rheumatoid arthritis (RA), we expressed the extracellular domain (ECD) of DDR2 (without signal peptide and transmembrane domain, designated DR) in insect cells, purified and characterized DR, hoping to use it as a specific antagonist of DDR2. By using Bac-To-Bac Expression System with a His tag, we successfully obtained the recombinant bacularvirus containing DDR2 ECD, purified it and characterized its function. The soluble fraction of DR was about 12% of the total fused protein. After chromatographic purification, DR with 92% purity was obtained. Competitive inhibition assay demonstrated that DR blocked the binding between DDR2 and natural DDR2 receptors on NIH3T3 and synovial cells. Results of RT-PCR, Western blotting, and gelatinase zymography showed that DR was capable of inhibiting MMP-1 and MMP-2 secretion from NIH3T3 and RA synoviocytes stimulated by collagen II. For MMP-1, inhibition was displayed at the levels of mRNA and protein, whereas for MMP-2 it was at the level of protein. These findings suggested that the expressed DR inhibited the activity of natural DDR2 and relevant MMP-1 and MMP-2 expression in RA synoviocytes and NIH3T3 cells provoked by collagen II.

  11. Particle-in-cell simulations of an alpha channeling scenario: electron current drive arising from lower hybrid drift instability of fusion-born ions

    Science.gov (United States)

    Cook, James; Chapman, Sandra; Dendy, Richard

    2010-11-01

    Particle-in-cell (PIC) simulations of fusion-born protons in deuterium plasmas demonstrate a key alpha channeling phenomenon for tokamak fusion plasmas. We focus on obliquely propagating modes at the plasma edge, excited by centrally born fusion products on banana orbits, known to be responsible for observations of ion cyclotron emission in JET and TFTR. A fully self-consistent electromagnetic 1D3V PIC code evolves a ring-beam distribution of 3MeV protons in a 10keV thermal deuterium-electron plasma with realistic mass ratio. A collective instability occurs, giving rise to electromagnetic field activity in the lower hybrid range of frequencies. Waves spontaneously excited by this lower hybrid drift instability undergo Landau damping on resonant electrons, drawing out an asymmetric tail in the distribution of electron parallel velocities, which constitutes a net current. These simulations demonstrate a key building block of some alpha channeling scenarios: the direct collisionless coupling of fusion product energy into a form which can help sustain the equilibrium of the tokamak.

  12. The efficacy and cost-effectiveness of cell saver use in instrumented posterior correction and fusion surgery for scoliosis in school-aged children and adolescents.

    Science.gov (United States)

    Miao, Yu-Liang; Ma, Hua-Song; Guo, Wen-Zhi; Wu, Ji-Gong; Liu, Yan; Shi, Wen-Zhu; Wang, Xiao-Ping; Mi, Wei-Dong; Fang, Wei-Wu

    2014-01-01

    Posterior spinal instrumentation and fusion surgery in school-aged children and adolescents is associated with the potential for massive intraoperative blood loss, which requires significant allogeneic blood transfusion. Until now, the intraoperative use of the cell saver has been extensively adopted; however, its efficacy and cost-effectiveness have not been well established. Therefore, the aim of this study is to determine the efficacy and cost-effectiveness of intraoperative cell saver use. This study was a single-center, retrospective study of 247 school-aged and adolescent patients who underwent posterior spinal instrumentation and fusion surgery between August 2007 and June 2013. A cell saver was used intraoperatively in 67 patients and was not used in 180 patients. Matched case-control pairs were selected using a propensity score to balance potential confounders in baseline characteristics. Allogeneic red blood cell (RBC) and plasma transfusions as well as blood transfusion costs were analyzed. The propensity score matching produced 60 matched pairs. Compared to the control group, the cell saver group had significantly fewer intraoperative allogeneic RBC transfusions (P = 0.012). However, when the combined postoperative and total perioperative periods were evaluated for the use of allogeneic RBC transfusion, no significant differences were observed between the two groups (P = 0.813 and P = 0.101, respectively). With regard to the total cost of perioperative transfusion of all blood products (RBC and plasma), costs for the control group were slightly lower than those of the cell saver group, but this variance did not reach statistical significance (P = 0.095). The use of the cell saver in posterior spinal instrumentation and fusion surgery in school-aged children and adolescents was able to decrease the amount of intraoperative allogeneic RBC transfusion but failed to decrease total perioperative allogeneic RBC transfusion. Moreover, the use of the cell saver

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  14. Analysis of cathepsin and furin proteolytic enzymes involved in viral fusion protein activation in cells of the bat reservoir host.

    Directory of Open Access Journals (Sweden)

    Farah El Najjar

    Full Text Available Bats of different species play a major role in the emergence and transmission of highly pathogenic viruses including Ebola virus, SARS-like coronavirus and the henipaviruses. These viruses require proteolytic activation of surface envelope glycoproteins needed for entry, and cellular cathepsins have been shown to be involved in proteolysis of glycoproteins from these distinct virus families. Very little is currently known about the available proteases in bats. To determine whether the utilization of cathepsins by bat-borne viruses is related to the nature of proteases in their natural hosts, we examined proteolytic processing of several viral fusion proteins in cells derived from two fruit bat species, Pteropus alecto and Rousettus aegyptiacus. Our work shows that fruit bat cells have homologs of cathepsin and furin proteases capable of cleaving and activating both the cathepsin-dependent Hendra virus F and the furin-dependent parainfluenza virus 5 F proteins. Sequence analysis comparing Pteropus alecto furin and cathepsin L to proteases from other mammalian species showed a high degree of conservation; however significant amino acid variation occurs at the C-terminus of Pteropus alecto furin. Further analysis of furin-like proteases from fruit bats revealed that these proteases are catalytically active and resemble other mammalian furins in their response to a potent furin inhibitor. However, kinetic analysis suggests that differences may exist in the cellular localization of furin between different species. Collectively, these results indicate that the unusual role of cathepsin proteases in the life cycle of bat-borne viruses is not due to the lack of active furin-like proteases in these natural reservoir species; however, differences may exist between furin proteases present in fruit bats compared to furins in other mammalian species, and these differences may impact protease usage for viral glycoprotein processing.

  15. Fusion of a Short HA2-Derived Peptide Sequence to Cell-Penetrating Peptides Improves Cytosolic Uptake, but Enhances Cytotoxic Activity

    Directory of Open Access Journals (Sweden)

    Igor Kitanovic

    2009-09-01

    Full Text Available Cell-penetrating peptides (CPP have become a widely used tool for efficient cargo delivery into cells. However, one limiting fact is their uptake by endocytosis causing the enclosure of the CPP-cargo construct within endosomes. One often used method to enhance the outflow into the cytosol is the fusion of endosome-disruptive peptide or protein sequences to CPP. But, until now, no studies exist investigating the effects of the fusion peptide to the cellular distribution, structural arrangements and cytotoxic behaviour of the CPP. In this study, we attached a short modified sequence of hemagglutinin subunit HA2 to different CPP and analysed the biologic activity of the new designed peptides. Interestingly, we observed an increased cytosolic distribution but also highly toxic activities in the micromolar range against several cell lines. Structural analysis revealed that attachment of the fusion peptide had profound implications on the whole conformation of the peptide, which might be responsible for membrane interaction and endosome disruption.

  16. A Short-Term Advantage for Syngamy in the Origin of Eukaryotic Sex: Effects of Cell Fusion on Cell Cycle Duration and Other Effects Related to the Duration of the Cell Cycle-Relationship between Cell Growth Curve and the Optimal Size of the Species, and Circadian Cell Cycle in Photosynthetic Unicellular Organisms.

    Science.gov (United States)

    Mancebo Quintana, J M; Mancebo Quintana, S

    2012-01-01

    The origin of sex is becoming a vexatious issue for Evolutionary Biology. Numerous hypotheses have been proposed, based on the genetic effects of sex, on trophic effects or on the formation of cysts and syncytia. Our approach addresses the change in cell cycle duration which would cause cell fusion. Several results are obtained through graphical and mathematical analysis and computer simulations. (1) In poor environments, cell fusion would be an advantageous strategy, as fusion between cells of different size shortens the cycle of the smaller cell (relative to the asexual cycle), and the majority of mergers would occur between cells of different sizes. (2) The easiest-to-evolve regulation of cell proliferation (sexual/asexual) would be by modifying the checkpoints of the cell cycle. (3) A regulation of this kind would have required the existence of the G2 phase, and sex could thus be the cause of the appearance of this phase. Regarding cell cycle, (4) the exponential curve is the only cell growth curve that has no effect on the optimal cell size in unicellular species; (5) the existence of a plateau with no growth at the end of the cell cycle explains the circadian cell cycle observed in unicellular algae.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-11-05

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

  18. Modern Gyrokinetic Particle-In-Cell Simulation of Fusion Plasmas on Top Supercomputers

    CERN Document Server

    Wang, Bei; Tang, William; Ibrahim, Khaled; Madduri, Kamesh; Williams, Samuel; Oliker, Leonid

    2015-01-01

    The Gyrokinetic Toroidal Code at Princeton (GTC-P) is a highly scalable and portable particle-in-cell (PIC) code. It solves the 5D Vlasov-Poisson equation featuring efficient utilization of modern parallel computer architectures at the petascale and beyond. Motivated by the goal of developing a modern code capable of dealing with the physics challenge of increasing problem size with sufficient resolution, new thread-level optimizations have been introduced as well as a key additional domain decomposition. GTC-P's multiple levels of parallelism, including inter-node 2D domain decomposition and particle decomposition, as well as intra-node shared memory partition and vectorization have enabled pushing the scalability of the PIC method to extreme computational scales. In this paper, we describe the methods developed to build a highly parallelized PIC code across a broad range of supercomputer designs. This particularly includes implementations on heterogeneous systems using NVIDIA GPU accelerators and Intel Xeon...

  19. Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

    Energy Technology Data Exchange (ETDEWEB)

    Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina; Daum, Gabor; Kuckwa, Jessica; Kastl, Lena; Buttgereit, Detlev [Developmental Biology, Department of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35037 Marburg (Germany); Renkawitz-Pohl, Renate, E-mail: renkawit@biologie.uni-marburg.de [Developmental Biology, Department of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35037 Marburg (Germany)

    2013-02-15

    Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity.

  20. Reactivity of murine cytokine fusion toxin, diphtheria toxin390-murine interleukin-3 (DT390-mIL-3), with bone marrow progenitor cells.

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-05-10

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

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

    Science.gov (United States)

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

    2010-09-01

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

  3. Fusion of the BCL9 HD2 domain to E1A increases the cytopathic effect of an oncolytic adenovirus that targets colon cancer cells

    Directory of Open Access Journals (Sweden)

    Pittet Anne-Laure

    2006-10-01

    Full Text Available Abstract Background The Wnt signaling pathway is activated by mutations in the APC and β-catenin genes in many types of human cancer. β-catenin is stabilized by these mutations and activates transcription in part by acting as a bridge between Tcf/LEF proteins and the HD2 domain of the BCL9 coactivator. We have previously described oncolytic adenoviruses with binding sites for Tcf/LEF transcription factors inserted into the early viral promoters. These viruses replicate selectively in cells with activation of the Wnt pathway. To increase the activity of these viruses we have fused the viral transactivator E1A to the BCL9 HD2 domain. Methods Luciferase assays, co-immunoprecipitation and Western blotting, immunofluorescent cell staining and cytopathic effect assays were used to characterize the E1A-HD2 fusion protein and virus in vitro. Growth curves of subcutaneous SW620 colon cancer xenografts were used to characterize the virus in vivo. Results The E1A-HD2 fusion protein binds to β-catenin in vivo and activates a Tcf-regulated luciferase reporter better than wild-type E1A in cells with activated Wnt signaling. Expression of the E1A-HD2 protein promotes nuclear import of β-catenin, mediated by the strong nuclear localization signal in E1A. Tcf-regulated viruses expressing the fusion protein show increased expression of viral proteins and a five-fold increase in cytopathic effect (CPE in colorectal cancer cell lines. There was no change in viral protein expression or CPE in HeLa cells, indicating that E1A-HD2 viruses retain selectivity for cells with activation of the Wnt signaling pathway. Despite increasing the cytopathic effect of the virus in vitro, fusion of the HD2 domain to E1A did not increase the burst size of the virus in vitro or the anti-tumor effect of the virus in an SW620 xenograft model in vivo. Conclusion Despite an increase in the nuclear pool of β-catenin, the effects on viral activity in colon cancer cells were small

  4. Membrane fusion during poxvirus entry.

    Science.gov (United States)

    Moss, Bernard

    2016-12-01

    Poxviruses comprise a large family of enveloped DNA viruses that infect vertebrates and invertebrates. Poxviruses, unlike most DNA viruses, replicate in the cytoplasm and encode enzymes and other proteins that enable entry, gene expression, genome replication, virion assembly and resistance to host defenses. Entry of vaccinia virus, the prototype member of the family, can occur at the plasma membrane or following endocytosis. Whereas many viruses encode one or two proteins for attachment and membrane fusion, vaccinia virus encodes four proteins for attachment and eleven more for membrane fusion and core entry. The entry-fusion proteins are conserved in all poxviruses and form a complex, known as the Entry Fusion Complex (EFC), which is embedded in the membrane of the mature virion. An additional membrane that encloses the mature virion and is discarded prior to entry is present on an extracellular form of the virus. The EFC is held together by multiple interactions that depend on nine of the eleven proteins. The entry process can be divided into attachment, hemifusion and core entry. All eleven EFC proteins are required for core entry and at least eight for hemifusion. To mediate fusion the virus particle is activated by low pH, which removes one or more fusion repressors that interact with EFC components. Additional EFC-interacting fusion repressors insert into cell membranes and prevent secondary infection. The absence of detailed structural information, except for two attachment proteins and one EFC protein, is delaying efforts to determine the fusion mechanism.

  5. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Roehrig, John T., E-mail: jtr1@cdc.gov [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Butrapet, Siritorn; Liss, Nathan M. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Bennett, Susan L. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Luy, Betty E.; Childers, Thomas; Boroughs, Karen L.; Stovall, Janae L.; Calvert, Amanda E. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Blair, Carol D. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Huang, Claire Y.-H. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States)

    2013-07-05

    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants.

  6. Magnetic fusion; La fusion magnetique

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2002-07-01

    This document is a detailed lecture on thermonuclear fusion. The basic physics principles are recalled and the technological choices that have led to tokamaks or stellarators are exposed. Different aspects concerning thermonuclear reactors such as safety, economy and feasibility are discussed. Tore-supra is described in details as well as the ITER project.

  7. Antibodies to several conformation-dependent epitopes of gp120/gp41 inhibit CCR-5-dependent cell-to-cell fusion mediated by the native envelope glycoprotein of a primary macrophage-tropic HIV-1 isolate

    OpenAIRE

    Verrier, Florence C.; Charneau, Pierre; Altmeyer, Ralf; Laurent, Stephanie; Borman, Andrew M.; Girard, Marc

    1997-01-01

    The β-chemokine receptor CCR-5 is essential for the efficient entry of primary macrophage-tropic HIV-1 isolates into CD4+ target cells. To study CCR-5-dependent cell-to-cell fusion, we have developed an assay system based on the infection of CD4+ CCR-5+ HeLa cells with a Semliki Forest virus recombinant expressing the gp120/gp41 envelope (Env) from a primary clade B HIV-1 isolate (BX08), or from a laboratory T cell line-adapted strain (LAI). In this system, gp120/gp41 of the “nonsyncytium-ind...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-01-05

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

  9. Tame Fusion

    Institute of Scientific and Technical Information of China (English)

    S.D. Scott

    2003-01-01

    The first section of this paper covers preliminaries. Essentially, the next four cover units. It is shown that a compatible nearring with DCCR is Nnilpotent if and only if every maximal right N-subgroup is a right ideal. The last five sections relate to fusion (I.e., N-groups minimal for being generated by Nsubgroups, where each is N-isomorphic to a given N-group). Right N-subgroups of a tame nearring N with DCCR, minimal for not annihilating a minimal ideal from the left, are self monogenic and N-isomorphic. That this holds for any collection of minimal ideals is significant. Here, the right N-subgroup involved is a 'fusion product' of the 'components'.

  10. Carpal Fusion

    OpenAIRE

    2012-01-01

    Carpal fusion may be seen in hereditary and nonhereditary conditions such as acrocallosal syndrome,acromegaly, Apert syndrome, arthrogryposis, Carpenter syndrome, chromosomal abnormalities, ectrodactyly-ectodermal dysplasia-cleft (EEC) syndrome, the F form of acropectorovertebral dysgenesis or the F syndrome, fetal alcohol syndrome, Holt-Oram syndrome, Leopard syndrome, multiple synostosis syndrome, oligosyndactyly syndrome, Pfeiffer-like syndrome, scleroderma, split hand and foot malformatio...

  11. Fusion rules of equivariantizations of fusion categories

    OpenAIRE

    2012-01-01

    We determine the fusion rules of the equivariantization of a fusion category $\\mathcal{C}$ under the action of a finite group $G$ in terms of the fusion rules of $\\mathcal{C}$ and group-theoretical data associated to the group action. As an application we obtain a formula for the fusion rules in an equivariantization of a pointed fusion category in terms of group-theoretical data. This entails a description of the fusion rules in any braided group-theoretical fusion category.

  12. Detection of a rare BCR-ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS).

    Science.gov (United States)

    Breitkopf, Susanne B; Yuan, Min; Pihan, German A; Asara, John M

    2012-10-02

    Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.

  13. BCR and its mutants, the reciprocal t(9;22-associated ABL/BCR fusion proteins, differentially regulate the cytoskeleton and cell motility

    Directory of Open Access Journals (Sweden)

    Puccetti Elena

    2006-11-01

    Full Text Available Abstract Background The reciprocal (9;22 translocation fuses the bcr (breakpoint cluster region gene on chromosome 22 to the abl (Abelson-leukemia-virus gene on chromosome 9. Depending on the breakpoint on chromosome 22 (the Philadelphia chromosome – Ph+ the derivative 9+ encodes either the p40(ABL/BCR fusion transcript, detectable in about 65% patients suffering from chronic myeloid leukemia, or the p96(ABL/BCR fusion transcript, detectable in 100% of Ph+ acute lymphatic leukemia patients. The ABL/BCRs are N-terminally truncated BCR mutants. The fact that BCR contains Rho-GEF and Rac-GAP functions strongly suggest an important role in cytoskeleton modeling by regulating the activity of Rho-like GTPases, such as Rho, Rac and cdc42. We, therefore, compared the function of the ABL/BCR proteins with that of wild-type BCR. Methods We investigated the effects of BCR and ABL/BCRs i. on the activation status of Rho, Rac and cdc42 in GTPase-activation assays; ii. on the actin cytoskeleton by direct immunofluorescence; and iii on cell motility by studying migration into a three-dimensional stroma spheroid model, adhesion on an endothelial cell layer under shear stress in a flow chamber model, and chemotaxis and endothelial transmigration in a transwell model with an SDF-1α gradient. Results Here we show that both ABL/BCRs lost fundamental functional features of BCR regarding the regulation of small Rho-like GTPases with negative consequences on cell motility, in particular on the capacity to adhere to endothelial cells. Conclusion Our data presented here describe for the first time an analysis of the biological function of the reciprocal t(9;22 ABL/BCR fusion proteins in comparison to their physiological counterpart BCR.

  14. The Neurospora Transcription Factor ADV-1 Transduces Light Signals and Temporal Information to Control Rhythmic Expression of Genes Involved in Cell Fusion

    Directory of Open Access Journals (Sweden)

    Rigzin Dekhang

    2017-01-01

    Full Text Available Light and the circadian clock have a profound effect on the biology of organisms through the regulation of large sets of genes. Toward understanding how light and the circadian clock regulate gene expression, we used genome-wide approaches to identify the direct and indirect targets of the light-responsive and clock-controlled transcription factor ADV-1 in Neurospora crassa. A large proportion of ADV-1 targets were found to be light- and/or clock-controlled, and enriched for genes involved in development, metabolism, cell growth, and cell fusion. We show that ADV-1 is necessary for transducing light and/or temporal information to its immediate downstream targets, including controlling rhythms in genes critical to somatic cell fusion. However, while ADV-1 targets are altered in predictable ways in Δadv-1 cells in response to light, this is not always the case for rhythmic target gene expression. These data suggest that a complex regulatory network downstream of ADV-1 functions to generate distinct temporal dynamics of target gene expression relative to the central clock mechanism.

  15. Effect of the time interval between fusion and activation on epigenetic reprogramming and development of bovine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Liu, Jun; Wang, Yongsheng; Su, Jianmin; Wang, Lijun; Li, Ruizhe; Li, Qian; Wu, Yongyan; Hua, Song; Quan, Fusheng; Guo, Zekun; Zhang, Yong

    2013-04-01

    Previous studies have shown that the time interval between fusion and activation (FA interval) play an important role in nuclear remodeling and in vitro development of somatic cell nuclear transfer (SCNT) embryos. However, the effects of FA interval on the epigenetic reprogramming and in vivo developmental competence of SCNT embryos remain unknown. In the present study, the effects of different FA intervals (0 h, 2 h, and 4 h) on the epigenetic reprogramming and developmental competence of bovine SCNT embryos were assessed. The results demonstrated that H3 lysine 9 (H3K9ac) levels decreased rapidly after fusion in all three groups. H3K9ac was practically undetectable 2 h after fusion in the 2-h and 4-h FA interval groups. However, H3K9ac was still evidently detectable in the 0-h FA interval group. The H3K9ac levels increased 10 h after fusion in all three groups, but were higher in the 2-h and 4-h FA interval groups than that in the 0-h FA interval group. The methylation levels of the satellite I region in day-7 blastocysts derived from the 2-h or 4-h FA interval groups was similar to that of in vitro fertilization blastocysts and is significantly lower than that of the 0-h FA interval group. SCNT embryos derived from 2-h FA interval group showed higher developmental competence than those from the 0-h and 4-h FA interval groups in terms of cleavage rate, blastocyst formation rate, apoptosis index, and pregnancy and calving rates. Hence, the FA interval is an important factor influencing the epigenetic reprogramming and developmental competence of bovine SCNT embryos.

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

    Science.gov (United States)

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

    2013-01-01

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

  17. FUSION WORLD

    Institute of Scientific and Technical Information of China (English)

    Caroline; 黄颖(翻译)

    2009-01-01

    Fusion World”科技展示体验中心是英国设计公司MET Studio为新加坡科技研究局(A*Star)的科学工程委员会(SERC)所设计的,位于启汇城的办公地点,用于展示该委员会的精选技术作品,以吸引潜在的客户和启汇城内的学生购买群体。

  18. Temsirolimus in the treatment of renal cell carcinoma associated with Xp11.2 translocation/TFE gene fusion proteins: a case report and review of literature

    Directory of Open Access Journals (Sweden)

    James Brown

    2009-12-01

    Full Text Available Xp11.2 translocation renal cell carcinomas (TRCCs are a rare family of tumors newly recognized by the World Health Organization (WHO in 2004. These tumors result in the fusion of partner genes to the TFE3 gene located on Xp11.2. They are most common in the pediatric population, but have been recently implicated in adult renal cell carcinoma (RCC presenting at an early age. TFE3-mediated direct transcriptional upregulation of the Met tyrosine kinase receptor triggers dramatic activation of downstream signaling pathways including the protein kinase B (Akt/phosphatidylinositol-3 kinase (PI3K and mammalian target of rapamycin (mTOR pathways. Temsirolimus is an inhibitor of mammalian target of rapamycin (mTOR kinase, a component of intracellular signaling pathways involved in the growth and proliferation of malignant cells. Here we present a case of a 22-year old female who has been treated with temsirolimus for her Xp11.2/TFE3 gene fusion RCC.

  19. Fusion-Expressed CTB Improves Both Systemic and Mucosal T-Cell Responses Elicited by an Intranasal DNA Priming/Intramuscular Recombinant Vaccinia Boosting Regimen

    Directory of Open Access Journals (Sweden)

    Sugan Qiu

    2014-01-01

    Full Text Available Previous study showed that CTB (Cholera toxin subunit B can be used as a genetic adjuvant to enhance the systemic immune responses. To further investigate whether it can also be used as a genetic adjuvant to improve mucosal immune responses, we constructed DNA and recombinant Tiantan vaccinia (rTTV vaccines expressing OVA-CTB fusion antigen. Female C57BL/6 mice were immunized with an intranasal DNA priming/intramuscular rTTV boosting regimen. OVA specific T-cell responses were measured by IFN-γ ELISPOT and specific antibody responses were determined by ELISA. Compared to the nonadjuvant group (pSV-OVA intranasal priming/rTTV-OVA intramuscular boosting, pSV-OVA-CTB intranasal priming/rTTV-OVA-CTB intramuscular boosting group significantly improved the magnitudes of T-cell responses at spleen (1562±567 SFCs/106 splenocytes versus 330±182 SFCs/106 splenocytes, P<0.01, mesenteric LN (96±83 SFCs/106 lymphocytes versus 1±2 SFCs/106 lymphocytes, P<0.05, draining LNs of respiratory tract (109±60 SFCs/106 lymphocytes versus 2±2 SFCs/106 lymphocytes, P<0.01 and female genital tract (89±48 SFCs/106 lymphocytes versus 23±21 SFCs/106 lymphocytes, P<0.01. These results collectively demonstrated that fusion-expressed CTB could act as a potent adjuvant to improve both systemic and mucosal T-cell responses.

  20. The uptake machinery of clostridial actin ADP-ribosylating toxins--a cell delivery system for fusion proteins and polypeptide drugs.

    Science.gov (United States)

    Barth, Holger; Blöcker, Dagmar; Aktories, Klaus

    2002-12-01

    Several bacterial protein toxins, including Clostridium botulinum C2 toxin, Clostridum perfringens iota toxin, Clostridium difficile ADP-ribosyltransferase, and the Bacillus-produced vegetative insecticidal proteins, target the cytoskeleton by ADP-ribosylation of actin. All these toxins are binary in structure and consist of an enzyme component, possessing ADP-ribosyltransferase activity and a separated binding and translocation component, which is involved in the delivery of the enzyme component into the cell. The toxins are not only important virulence factors but also cell biological tools to study the function of the actin cytoskeleton. Moreover, the binary toxins turned out to be effective transporter systems for the delivery of specific fusion toxins (e.g., Rho-ADP-ribosylating C3 exoenzyme) into cells. The present review describes the biological functions of the toxins, focuses on recent studies on the uptake and delivery mechanism and discusses the usage as a drug delivery system.

  1. Carpal Fusion

    Directory of Open Access Journals (Sweden)

    Jalal Jalalshokouhi*

    2012-05-01

    Full Text Available Carpal fusion may be seen in hereditary and nonhereditary conditions such as acrocallosal syndrome,acromegaly, Apert syndrome, arthrogryposis, Carpenter syndrome, chromosomal abnormalities, ectrodactyly-ectodermal dysplasia-cleft (EEC syndrome, the F form of acropectorovertebral dysgenesis or the F syndrome, fetal alcohol syndrome, Holt-Oram syndrome, Leopard syndrome, multiple synostosis syndrome, oligosyndactyly syndrome, Pfeiffer-like syndrome, scleroderma, split hand and foot malformation, Stickler syndrome, thalidomide embryopathy, Turner syndrome and many other conditions as mentioned in Rubinstein-Taybi's book. Sometimes there is no known causative disease.Diagnosis is usually made by plain X-ray during studying a syndrome or congenital disease or could be an incidental finding like our patients. Hand bone anomalies are more common in syndromes or other congenital or non-hereditary conditions, but polydactyly, syndactyly or oligodactyly and carpal fusions are interesting. X-ray is the modality of choice, but MRI and X-ray CT with multiplanar reconstructions may be used for diagnosis.

  2. Results from the CDE phase activity on neutron dosimetry for the international fusion materials irradiation facility test cell

    CERN Document Server

    Esposito, B; Maruccia, G; Petrizzi, L; Bignon, G; Blandin, C; Chauffriat, S; Lebrun, A; Recroix, H; Trapp, J P; Kaschuck, Y

    2000-01-01

    The international fusion materials irradiation facility (IFMIF) project deals with the study of an accelerator-based, deuterium-lithium source, producing high energy neutrons at sufficient intensity and irradiation volume to test samples of candidate materials for fusion energy reactors. IFMIF would also provide calibration and validation of data from fission reactor and other accelerator based irradiation tests. This paper describes the activity on neutron/gamma dosimetry (necessary for the characterization of the specimens' irradiation) performed in the frame of the IFMIF conceptual design evaluation (CDE) neutronics tasks. During the previous phase (conceptual design activity (CDA)) the multifoil activation method was proposed for the measurement of the neutron fluence and spectrum and a set of suitable foils was defined. The cross section variances and covariances of this set of foils have now been used for tests on the sensitivity of the IFMIF neutron spectrum determination to cross section uncertainties...

  3. Fusions of Breast Carcinoma and Dendritic Cells as a Vaccine for the Treatment of Metatastic Breast Cancer

    Science.gov (United States)

    2010-07-31

    stimulate anti-tumor immunity . We demonstrated that DC/breast carcinoma fusions strongly express costimulatory, adhesion, and maturation markers as...can initiate the study. Our correspondence is summarized below: 1. Request for further information from DOD issued 3.4.09 2. Reponses to...days 3 and 5. Measures of tumor specific cellular and humoral immunity will be obtained at serial time points following vaccination. Time to disease

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

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

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

  5. Intermittent hypoxia stimulates formation of binuclear neurons in brain cortex- a role of cell fusion in neuroprotection?

    Science.gov (United States)

    Paltsyn, Alexander A; Manukhina, Eugenia B; Goryacheva, Anna V; Downey, H Fred; Dubrovin, Ivan P; Komissarova, Svetlana V; Kubatiev, Aslan A

    2014-05-01

    Oligodendrocyte fusion with neurons in the brain cortex is a part of normal ontogenesis and is a possible means of neuroregeneration. Following such fusion, the oligodendrocyte nucleus undergoes neuron-specific reprogramming, resulting in the formation of binuclear neurons, which doubles the functional capability of the neuron. In this study, we tested the hypothesis that the formation of binuclear neurons is involved in long-term adaptation of the brain to intermittent hypobaric hypoxia, which is known to be neuroprotective. Rats were adapted to hypoxia in an altitude chamber at a simulated altitude of 4000 m above sea level for 14 days (30 min increasing to 4 h, daily). One micrometer sections of the left motor cortex were analyzed by light microscopy. Phases of the fusion and reprogramming process were recorded, and the number of binuclear neurons was counted for all section areas containing pyramidal neurons of layers III-V. For the control group subjected to sham hypoxia, the density of binuclear neurons was 4.49 ± 0.32 mm(2). In the hypoxia-adapted group, this density increased to 5.71 ± 0.39 mm(2) (P neurons did not differ from the number observed in the control group. We suggest that the increased content of binuclear neurons may serve as a structural basis for the neuroprotective effects of the adaptation to hypoxia.

  6. Recombinant ESAT-6-CFP10 Fusion Protein Induction of Th1/Th2 Cytokines and FoxP3 Expressing Treg Cells in Pulmonary TB.

    Directory of Open Access Journals (Sweden)

    Dolly Jackson-Sillah

    Full Text Available Early secretory antigenic target 6 (ESAT-6 and culture filtrate protein 10 (CFP-10 are Mycobacterium tuberculosis (Mtb-specific antigens that are secreted by actively metabolising bacteria and contribute to the virulence of the bacteria. Their ability to induce Treg and Th2 responses, particularly during the first two weeks of treatment, has not been comprehensively examined to date. The purpose of this work was to characterise Th1, Th2 and Treg responses to rESAT-6-CFP10 fusion protein in TB patients before and during the intensive phase of treatment and in healthy M.bovis BCG vaccinated donors.Forty-six newly diagnosed, HIV-negative, smear-positive pulmonary TB patients and 20 healthy donors were recruited in the UK and Ghana. Their peripheral blood mononuclear cells (PBMC were used in ex vivo ELISPOT and in vitro cultures to identify immunological parameters of interest.The study confirmed that protective immune responses to rESAT-6-CFP10 are impaired in active TB but improved during treatment: circulating antigen-specific IL-4-producing T-cells were increased in untreated TB but declined by two weeks of treatment while the circulating antigen-specific IFN-γ producing T cells which showed a transient rise at one week of treatment, persisted at baseline levels at two months of treatment. In vitro T cell proliferation and IFN-γ production were reduced, while IL-4 and CD4(+FoxP3(+CD25(hi cell expression were increased in response to rESAT-6-CFP10 fusion protein in untreated TB. These responses were reversed during early treatment of TB.These observations support further investigations into the possible utility of these parameters as markers of active disease and favourable treatment outcomes.

  7. Catalysed fusion

    CERN Document Server

    Farley, Francis

    2012-01-01

    A sizzling romance and a romp with subatomic particles at CERN. Love, discovery and adventure in the city where nations meet and beams collide. Life in a large laboratory. As always, the challenges are the same. Who leads? Who follows? Who succeeds? Who gets the credit? Who gets the women or the men? Young Jeremy arrives in CERN and joins the quest for green energy. Coping with baffling jargon and manifold dangers, he is distracted by radioactive rats, lovely ladies and an unscrupulous rival. Full of doubts and hesitations, he falls for a dazzling Danish girl, who leads him astray. His brilliant idea leads to a discovery and a new route to cold fusion. But his personal life is scrambled. Does it bring fame or failure? Tragedy or triumph?

  8. The Cytoplasmic Tail Domain of Epstein-Barr Virus gH Regulates Membrane Fusion Activity through Altering gH Binding to gp42 and Epithelial Cell Attachment.

    Science.gov (United States)

    Chen, Jia; Jardetzky, Theodore S; Longnecker, Richard

    2016-11-15

    Epstein-Barr virus (EBV) is associated with infectious mononucleosis and a variety of cancers as well as lymphoproliferative disorders in immunocompromised patients. EBV mediates viral entry into epithelial and B cells using fusion machinery composed of four glycoproteins: gB, the gH/gL complex, and gp42. gB and gH/gL are required for both epithelial and B cell fusion. The specific role of gH/gL in fusion has been the most elusive among the required herpesvirus entry glycoproteins. Previous mutational studies have focused on the ectodomain of EBV gH and not on the gH cytoplasmic tail domain (CTD). In this study, we chose to examine the function of the gH CTD by making serial gH truncation mutants as well as amino acid substitution mutants to determine the importance of the gH CTD in epithelial and B cell fusion. Truncation of 8 amino acids (aa 698 to 706) of the gH CTD resulted in diminished fusion activity using a virus-free syncytium formation assay and fusion assay. The importance of the amino acid composition of the gH CTD was also investigated by amino acid substitutions that altered the hydrophobicity or hydrophilicity of the CTD. These mutations also resulted in diminished fusion activity. Interestingly, some of the gH CTD truncation mutants and hydrophilic tail substitution mutants lost the ability to bind to gp42 and epithelial cells. In summary, our studies indicate that the gH CTD is an important functional domain.

  9. The Cytoplasmic Tail Domain of Epstein-Barr Virus gH Regulates Membrane Fusion Activity through Altering gH Binding to gp42 and Epithelial Cell Attachment

    Directory of Open Access Journals (Sweden)

    Jia Chen

    2016-11-01

    Full Text Available Epstein-Barr virus (EBV is associated with infectious mononucleosis and a variety of cancers as well as lymphoproliferative disorders in immunocompromised patients. EBV mediates viral entry into epithelial and B cells using fusion machinery composed of four glycoproteins: gB, the gH/gL complex, and gp42. gB and gH/gL are required for both epithelial and B cell fusion. The specific role of gH/gL in fusion has been the most elusive among the required herpesvirus entry glycoproteins. Previous mutational studies have focused on the ectodomain of EBV gH and not on the gH cytoplasmic tail domain (CTD. In this study, we chose to examine the function of the gH CTD by making serial gH truncation mutants as well as amino acid substitution mutants to determine the importance of the gH CTD in epithelial and B cell fusion. Truncation of 8 amino acids (aa 698 to 706 of the gH CTD resulted in diminished fusion activity using a virus-free syncytium formation assay and fusion assay. The importance of the amino acid composition of the gH CTD was also investigated by amino acid substitutions that altered the hydrophobicity or hydrophilicity of the CTD. These mutations also resulted in diminished fusion activity. Interestingly, some of the gH CTD truncation mutants and hydrophilic tail substitution mutants lost the ability to bind to gp42 and epithelial cells. In summary, our studies indicate that the gH CTD is an important functional domain.

  10. A Novel Human TGF-β1 Fusion Protein in Combination with rhBMP-2 Increases Chondro-Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells

    Science.gov (United States)

    Claros, Silvia; Rico-Llanos, Gustavo A.; Becerra, José; Andrades, José A.

    2014-01-01

    Transforming growth factor-beta (TGF-β) is involved in processes related to the differentiation and maturation of osteoprogenitor cells into osteoblasts. Rat bone marrow (BM) cells were cultured in a collagen-gel containing 0.5% fetal bovine serum (FBS) for 10 days in the presence of rhTGF (recombinant human TGF)-β1-F2, a fusion protein engineered to include a high-affinity collagen-binding decapeptide derived from von Willebrand factor. Subsequently, cells were moderately expanded in medium with 10% FBS for 4 days and treated with a short pulse of rhBMP (recombinant human bone morphogenetic protein)-2 for 4 h. During the last 2 days, dexamethasone and β-glycerophosphate were added to potentiate osteoinduction. Concomitant with an up-regulation of cell proliferation, DNA synthesis levels were determined. Polymerase chain reaction was performed to reveal the possible stemness of these cells. Osteogenic differentiation was evaluated in terms of alkaline phosphatase activity and mineralized matrix formation as well as by mRNA expression of osteogenic marker genes. Moreover, cells were placed inside diffusion chambers and implanted subcutaneously into the backs of adult rats for 4 weeks. Histological study provided evidence of cartilage and bone-like tissue formation. This experimental procedure is capable of selecting cell populations from BM that, in the presence of rhTGF-β1-F2 and rhBMP-2, achieve skeletogenic potential in vitro and in vivo. PMID:24968268

  11. A Novel Human TGF-β1 Fusion Protein in Combination with rhBMP-2 Increases Chondro-Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Silvia Claros

    2014-06-01

    Full Text Available Transforming growth factor-beta (TGF-β is involved in processes related to the differentiation and maturation of osteoprogenitor cells into osteoblasts. Rat bone marrow (BM cells were cultured in a collagen-gel containing 0.5% fetal bovine serum (FBS for 10 days in the presence of rhTGF (recombinant human TGF-β1-F2, a fusion protein engineered to include a high-affinity collagen-binding decapeptide derived from von Willebrand factor. Subsequently, cells were moderately expanded in medium with 10% FBS for 4 days and treated with a short pulse of rhBMP (recombinant human bone morphogenetic protein-2 for 4 h. During the last 2 days, dexamethasone and β-glycerophosphate were added to potentiate osteoinduction. Concomitant with an up-regulation of cell proliferation, DNA synthesis levels were determined. Polymerase chain reaction was performed to reveal the possible stemness of these cells. Osteogenic differentiation was evaluated in terms of alkaline phosphatase activity and mineralized matrix formation as well as by mRNA expression of osteogenic marker genes. Moreover, cells were placed inside diffusion chambers and implanted subcutaneously into the backs of adult rats for 4 weeks. Histological study provided evidence of cartilage and bone-like tissue formation. This experimental procedure is capable of selecting cell populations from BM that, in the presence of rhTGF-β1-F2 and rhBMP-2, achieve skeletogenic potential in vitro and in vivo.

  12. Prokaryotic expression and purification of fusion protein HSP70-EGFP and its application in the study of dendritic cells internalization antigen

    Institute of Scientific and Technical Information of China (English)

    Ping QU; Yanfang SUI; Libing LIU; Jiahai MA; Guangsheng CHEN; Jiankang CHEN; Fang'e LIU

    2008-01-01

    To study the endocytic activity of dendritic cells (DCs) by obtaining fusion protein HSP70-EGFP as exogenous antigen and loading it with DCs derived from human peripheral blood. Fusion protein HSP70-EGFP was prokaryotically expressed, isolated and puri-fied. DCs were isolated and cultured from human peri-pheral blood. The DCs were divided into 3 groups in the endocytic experiment. There were 106 DCs in each group. Group 1 and 2 were respectively incubated for 30 min. with HSP70-EGFP and EGFP. Group 3 was incubated with HSP70 for 30 min, and then incubated for 30 min. with HSP70-EGFP. Subsequently, 3 groups were placed in an incubator at 37℃ for 0.5, 1,2 and 24 h. Flow cytometry (FCM) was adopted to detect the amount of DCs with EGFP inside. IL-12 Eli-spot was adopted to detect the amount of DCs which secreted IL-12. There were 5 types in the experiment: LPS, inactive LPS, HSP70-EGFP, EGFP and no antigen. Fusion pro-tein HSP70-EGFP was successfully obtained and its molecular weight was 97 000. It accounted for 35.32% of the total protein. Under irradiation of an ultraviolet lamp, the protein solution sent out viridescent fluor-escence. The result detected by FCM indicated that after incubation for 0.5 h at 37℃, the positive rate in group 1 was 63%, while the other 2 groups were negative. After incubation for 1, 2 and 24 h at 37℃, the positive rates in the 3 groups were above 80%. The IL-12 Eli-spot exam-ination shows that with HSP70-EGFP being loaded, the amount of DCs secreting IL-12 was 134.09±31.78/105 cells, a little lower than that of DCs with LPS loaded (with the average point of 156.36±15.73). There was no significant difference between the 2 groups (P<0.01). By contrast, both of them were significantly higher than inactive LPS-(33.78±1.40)/105 cells and EGFP-loaded (23.13±4,57)/105 cells DC groups in the amount of DCs secreting IL-12 (P<0.01). The results suggest that receptor-mediated phagocytosis plays a main role in the preliminary stage of DCs

  13. A Model for Membrane Fusion

    Science.gov (United States)

    Ngatchou, Annita

    2010-01-01

    Pheochromocytoma is a tumor of the adrenal gland which originates from chromaffin cells and is characterized by the secretion of excessive amounts of neurotransmitter which lead to high blood pressure and palpitations. Pheochromocytoma contain membrane bound granules that store neurotransmitter. The release of these stored molecules into the extracellular space occurs by fusion of the granule membrane with the cell plasma membrane, a process called exocytosis. The molecular mechanism of this membrane fusion is not well understood. It is proposed that the so called SNARE proteins [1] are the pillar of vesicle fusion as their cleavage by clostridial toxin notably, Botulinum neurotoxin and Tetanus toxin abrogate the secretion of neurotransmitter [2]. Here, I describe how physical principles are applied to a biological cell to explore the role of the vesicle SNARE protein synaptobrevin-2 in easing granule fusion. The data presented here suggest a paradigm according to which the movement of the C-terminal of synaptobrevin-2 disrupts the lipid bilayer to form a fusion pore through which molecules can exit.

  14. A CheR/CheB fusion protein is involved in cyst cell development and chemotaxis in Azospirillum brasilense Sp7.

    Science.gov (United States)

    Wu, Lixian; Cui, Yanhua; Hong, Yuanyuan; Chen, Sanfeng

    2011-12-20

    We here report the sequence and functional analysis of cstB of Azospirillum brasilense Sp7. The predicted cstB contains C-terminal two PAS domains and N-terminal part which has similarity with CheB-CheR fusion protein. cstB mutants had reduced swarming ability compared to that of A. brasilense wild-type strain, implying that cstB was involved in chemotaxis in A. brasilense. A microscopic analysis revealed that cstB mutants developed mature cyst cells more quickly than wild type, indicating that cstB is involved in cyst formation. cstB mutants were affected in colony morphology and the production of exopolysaccharides (EPS) which are essential for A. brasilense cells to differentiate into cyst-like forms. These observations suggested that cstB was a multi-effector involved in cyst development and chemotaxis in A. brasilense.

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

    Directory of Open Access Journals (Sweden)

    Poussard S

    2015-02-01

    Full Text Available Sylvie Poussard,1,2 Marion Decossas,1,2 Olivier Le Bihan,1,2 Stéphane Mornet,3 Grégoire Naudin,1,2 Olivier Lambert1,2 1Institute of Chemistry and Biology of Membranes and Nanoobjects, University of Bordeaux, UMR5248, Pessac, France; 2Institute of Chemistry and Biology of Membranes and Nanoobjects, Centre National de la Recherche Scientifique, Institute of Chemistry and Biology of Membranes and Nanoobjects, UMR5248, Pessac, France; 3ICMCB, Institut de Chimie de la Matière Condensée de Bordeaux, CNRS UPR9048, Université de Bordeaux, Pessac, France Abstract: The use of silica nanoparticles for their cellular uptake capability opens up new fields in biomedical research. Among the toxicological effects associated with their internalization, silica nanoparticles induce apoptosis that has been recently reported as a biochemical cue required for muscle regeneration. To assess whether silica nanoparticles could affect muscle regeneration, we used the C2C12 muscle cell line to study the uptake of fluorescently labeled NPs and their cellular trafficking over a long period. Using inhibitors of endocytosis, we determined that the NP uptake was an energy-dependent process mainly involving macropinocytosis and clathrin-mediated pathway. NPs were eventually clustered in lysosomal structures. Myoblasts containing NPs were capable of differentiation into myotubes, and after 7 days, electron microscopy revealed that the NPs remained primarily within lysosomes. The presence of NPs stimulated the formation of myotubes in a dose-dependent manner. NP internalization induced an increase of apoptotic myoblasts required for myoblast fusion. At noncytotoxic doses, the NP uptake by skeletal muscle cells did not prevent their differentiation into myotubes but, instead, enhanced the cell fusion. Keywords: silica, nanoparticle, muscle, cell encapsulation, transmission electron microscopy, apoptosis

  16. The rGel/BLyS Fusion Toxin Inhibits Diffuse Large B-cell Lymphoma Growth In Vitro and In Vivo

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    Mi-Ae Lyu

    2010-05-01

    Full Text Available Diffuse large B-cell lymphoma (DLBCL is an aggressive subtype of B-cell non-Hodgkin lymphoma (NHL and accounts for 30%to 40%of NHL. Molecules targeting nuclear factor-κB (NF-κB are expected to be of therapeutic value in those tumors where NF-κB seems to play a unique survival role such as activated B-cell (ABC-subtype DLBCL. We previously generated a rGel/BLyS fusion toxin for receptor-mediated delivery of the rGel toxin specifically to malignant B cells. In this study, we examined this fusion toxin for its ability to suppress DLBCL growth in vitro and in vivo. rGel/BLyS was specifically cytotoxic to DLBCL lines expressing all three BLyS receptors and constitutively active NF-κB. Treatment with rGel/BLyS induced down-regulation of the phosphorylation of inhibitory subunit of NF-κB (IκB-α, inhibition of NF-κB DNA-binding activity, and accumulation of IκB-α. In agreement with these results, we additionally found that rGel/BLyS downregulated levels of several NF-κB targets including Bcl-xL, Mcl-1, survivin, and x-chromosome linked inhibitor-of-apoptosis. Treatment also induced up-regulation of Bax and apoptosis through caspase-3 activation and poly ADP-ribose polymerase cleavage. Importantly, rGel/BLyS significantly inhibited tumor growth (P < .05 in a DLBCL xenograft model. Thus, our results indicate that rGel/BLyS is an excellent candidate for the treatment of aggressive NHLs that are both dependent on NF-κB and are resistant to conventional chemotherapeutic regimens.

  17. Depressed excitability and ion currents linked to slow exocytotic fusion pore in chromaffin cells of the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Calvo-Gallardo, Enrique; de Pascual, Ricardo; Fernández-Morales, José-Carlos; Arranz-Tagarro, Juan-Alberto; Maroto, Marcos; Nanclares, Carmen; Gandía, Luis; de Diego, Antonio M G; Padín, Juan-Fernando; García, Antonio G

    2015-01-01

    Altered synaptic transmission with excess glutamate release has been implicated in the loss of motoneurons occurring in amyotrophic lateral sclerosis (ALS). Hyperexcitability or hypoexcitability of motoneurons from mice carrying the ALS mutation SOD1(G93A) (mSOD1) has also been reported. Here we have investigated the excitability, the ion currents, and the kinetics of the exocytotic fusion pore in chromaffin cells from postnatal day 90 to postnatal day 130 mSOD1 mice, when motor deficits are already established. With respect to wild-type (WT), mSOD1 chromaffin cells had a decrease in the following parameters: 95% in spontaneous action potentials, 70% in nicotinic current for acetylcholine (ACh), 35% in Na(+) current, 40% in Ca(2+)-dependent K(+) current, and 53% in voltage-dependent K(+) current. Ca(2+) current was increased by 37%, but the ACh-evoked elevation of cytosolic Ca(2+) was unchanged. Single exocytotic spike events triggered by ACh had the following differences (mSOD1 vs. WT): 36% lower rise rate, 60% higher decay time, 51% higher half-width, 13% lower amplitude, and 61% higher quantal size. The expression of the α3-subtype of nicotinic receptors and proteins of the exocytotic machinery was unchanged in the brain and adrenal medulla of mSOD1, with respect to WT mice. A slower fusion pore opening, expansion, and closure are likely linked to the pronounced reduction in cell excitability and in the ion currents driving action potentials in mSOD1, compared with WT chromaffin cells.

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

    Science.gov (United States)

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

    2013-09-18

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

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

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    Nordheim Alfred

    2004-08-01

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

  20. Renal Cell Carcinoma Associated with Xp11.2 Translocation/TFE3 Gene Fusion: A Rare Case Report with Review of the Literature

    Directory of Open Access Journals (Sweden)

    Puneet Ahluwalia

    2013-01-01

    Full Text Available Introduction. The recently recognized renal cell carcinomas associated with Xp11.2 translocations are rare tumors predominantly reported in children. Chromosome Xp11.2 translocation results in gene fusion related to transcription factor E3 (TFE3 that plays an important role in proliferation and survival. Case Report. Herein, we present two cases of a TFE3 translocation-associated RCC in young female adults, one detected incidentally and the other one presenting with gross hematuria. Tumor is characterized by immunohistochemistry and a literature review with optimal treatment regimen is presented. Discussion. Xp11.2 translocation RCCs in adult patients are associated with advanced stages, large tumors, and extracapsular disease and usually have an aggressive clinical course. Conclusion. In TFE3 RCC, the genetic background may not only contribute to tumorigenesis, but also determine the response to chemotherapy and targeted therapy. Therefore it is necessary to diagnose this tumor entity accurately. Because of the small number of TFE3 gene fusion-related renal tumors described in the literature, the exact biologic behavior and impact of current treatment modalities remain to be uncertain.

  1. The efficient elimination of solid tumor cells by EGFR-specific and HER2-specific scFv-SNAP fusion proteins conjugated to benzylguanine-modified auristatin F.

    Science.gov (United States)

    Woitok, Mira; Klose, Diana; Niesen, Judith; Richter, Wolfgang; Abbas, Muhammad; Stein, Christoph; Fendel, Rolf; Bialon, Magdalena; Püttmann, Christiane; Fischer, Rainer; Barth, Stefan; Kolberg, Katharina

    2016-10-28

    Antibody-drug conjugates (ADCs) combine the potency of cytotoxic drugs with the specificity of monoclonal antibodies (mAbs). Most ADCs are currently generated by the nonspecific conjugation of drug-linker reagents to certain amino acid residues in mAbs, resulting in a heterogeneous product. To overcome this limitation and prepare ADCs with a defined stoichiometry, we use SNAP-tag technology as an alternative conjugation strategy. This allows the site-specific conjugation of O(6)-benzylguanine (BG)-modified small molecules to SNAP-tag fusion proteins. To demonstrate the suitability of this system for the preparation of novel recombinant ADCs, here we conjugated SNAP-tagged single chain antibody fragments (scFvs) to a BG-modified version of auristatin F (AURIF). We used two scFv-SNAP fusion proteins targeting members of the epidermal growth factor receptor (EGFR) family that are frequently overexpressed in breast cancer. The conjugation of BG-AURIF to EGFR-specific 425(scFv)-SNAP and HER2-specific αHER2(scFv)-SNAP resulted in two potent recombinant ADCs that specifically killed breast cancer cell lines by inducing apoptosis when applied at nanomolar concentrations. These data confirm that SNAP-tag technology is a promising tool for the generation of novel recombinant ADCs.

  2. Generation of a Stable Transgenic Swine Model Expressing a Porcine Histone 2B-eGFP Fusion Protein for Cell Tracking and Chromosome Dynamics Studies

    Science.gov (United States)

    Simpson, Sean; Collins, Bruce; Sommer, Jeff; Petters, Robert M.; Caballero, Ignacio; Platt, Jeff L.

    2017-01-01

    Transgenic pigs have become an attractive research model in the field of translational research, regenerative medicine, and stem cell therapy due to their anatomic, genetic and physiological similarities with humans. The development of fluorescent proteins as molecular tags has allowed investigators to track cell migration and engraftment levels after transplantation. Here we describe the development of two transgenic pig models via SCNT expressing a fusion protein composed of eGFP and porcine Histone 2B (pH2B). This fusion protein is targeted to the nucleosomes resulting a nuclear/chromatin eGFP signal. The first model (I) was generated via random insertion of pH2B-eGFP driven by the CAG promoter (chicken beta actin promoter and rabbit Globin poly A; pCAG-pH2B-eGFP) and protected by human interferon-β matrix attachment regions (MARs). Despite the consistent, high, and ubiquitous expression of the fusion protein pH2B-eGFP in all tissues analyzed, two independently generated Model I transgenic lines developed neurodegenerative symptoms including Wallerian degeneration between 3–5 months of age, requiring euthanasia. A second transgenic model (II) was developed via CRISPR-Cas9 mediated homology-directed repair (HDR) of IRES-pH2B-eGFP into the endogenous β-actin (ACTB) locus. Model II transgenic animals showed ubiquitous expression of pH2B-eGFP on all tissues analyzed. Unlike the pCAG-pH2B-eGFP/MAR line, all Model II animals were healthy and multiple pregnancies have been established with progeny showing the expected Mendelian ratio for the transmission of the pH2B-eGFP. Expression of pH2B-eGFP was used to examine the timing of the maternal to zygotic transition after IVF, and to examine chromosome segregation of SCNT embryos. To our knowledge this is the first viable transgenic pig model with chromatin-associated eGFP allowing both cell tracking and the study of chromatin dynamics in a large animal model. PMID:28081156

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

    Science.gov (United States)

    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.

  4. Single-particle kinetics of influenza virus membrane fusion

    NARCIS (Netherlands)

    Floyd, Daniel L.; Ragains, Justin R.; Skehel, John J.; Harrison, Stephen C.; Oijen, Antoine M. van; Harrison, Stephen C.

    2008-01-01

    Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays are generally limited to observation of ensembles of multiple fusion events, confounding more detailed analysis of the sequence of the molecular steps involved. We have developed an in vitro

  5. Myoblast fusion: Experimental systems and cellular mechanisms.

    Science.gov (United States)

    Schejter, Eyal D

    2016-12-01

    Fusion of myoblasts gives rise to the large, multi-nucleated muscle fibers that power and support organism motion and form. The mechanisms underlying this prominent form of cell-cell fusion have been investigated by a variety of experimental approaches, in several model systems. The purpose of this review is to describe and discuss recent progress in the field, as well as point out issues currently unresolved and worthy of further investigation. Following a description of several new experimental settings employed in the study of myoblast fusion, a series of topics relevant to the current understanding of the process are presented. These pertain to elements of three major cellular machineries- cell-adhesion, the actin-based cytoskeleton and membrane-associated elements- all of which play key roles in mediating myoblast fusion. Among the issues raised are the diversity of functions ascribed to different adhesion proteins (e.g. external cell apposition and internal recruitment of cytoskeleton regulators); functional significance of fusion-associated actin structures; and discussion of alternative mechanisms employing single or multiple fusion pore formation as the basis for muscle cell fusion.

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

    Science.gov (United States)

    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 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 nuclear aneuploidy.

  7. Analysis of cytoskeleton dynamics and cell migration in drosophila ovaries using GFP-actin and E-cadherin-GFP fusion molecules

    Science.gov (United States)

    Verkhusha, Vladyslav V.; Tsukita, Shoichiro; Oda, Hiroki

    1999-06-01

    Coordination of cell migration and adhesion is essential for movement of tissues during morphogenesis. During Drosophila oogenesis so called border cells (BCs) break from an anterior epithelium of egg chamber, acquire a mesenchymal-like morphology, and migrate posteriorly between nurse cells to oocyte. The confocal microscopic observation of BCs has revealed well-developed forepart lamellipodium stained with Drosophila E-cadherin (DE-cadherin), PS2 integrin, cytoplasmic myosin and F-actin. To investigate mechanism of BC migration in vivo we have constructed a DE-cadherin-GFP and a GFP-actin fusion proteins and induced their expression BCs utilizing the UAS/GAL4 system. The DE-cadherin-GFP signal as well as immunostaining of PS2 integrin visualized a track of migrating BCs providing an evidence that adhesive molecules are pulled out and left behind on the surface of nurse cells. Our data suggest that two distinct adhesive systems, DE-cadherins and PS2 integrins simultaneously mediate the migration of BCs. Release of adhesive contacts in the tail region is a rate- limited event in BC migration. The spatial-temporal sequence of actin-based events visualized by the GFP-actin suggest a treadmilling model for actin behavior in BC lamellipodium. BC migration can be considered as simultaneous reiterating processes of lamellipodium extension and adhesive attachment, cytoskeletal contraction, and rear detachment.

  8. Non - small cell lung cancer novel target ROS1 fusion%非小细胞肺癌新靶点 ROS1融合基因

    Institute of Scientific and Technical Information of China (English)

    张东芳; 魏万里

    2016-01-01

    Non - small cell lung cancer(NSCLC)is one of the biggest killers all over the world,and its five - year survival rate low than 20% . Screening and confirmed the tumor drive genes has become a top priority of the future re-search and development of targeted drugs. Recently,more and more scholars focus shifted to ROS1 gene fusion,and have the relevant data and studies have shown that ROS1 fusion genes have been confirmed for NSCLC new potential therapeutic targets. In this review,we summarize the research progress of ROS1 fusion gene in NSCLC.%非小细胞肺癌(non - small cell lung cancer,NSCLC)是造成人类死亡最多的恶性肿瘤之一,其五年生存率一直徘徊在20%以下。自肺癌领域首个分子靶向药物吉非替尼上市以来,靶向药物因其低毒、高效、便于给药的临床特点,己逐渐成为治疗 NSCLC 的重要选择之一。因此,筛选和证实肿瘤驱动基因已经成为未来靶向药物研发的重中之重。近来,越来越多的学者把焦点转移到 ROS1融合基因上,并且已经有相关数据及研究表明 ROS1融合基因被证实为 NSCLC 新的有潜力的治疗靶点,因此我们现就 ROS1融合基因在NSCLC 中的相关研究进展做一综述。

  9. Correcting mitochondrial fusion by manipulating mitofusin conformations

    Science.gov (United States)

    Franco, Antonietta; Kitsis, Richard N.; Fleischer, Julie A.; Gavathiotis, Evripidis; Kornfeld, Opher S.; Gong, Guohua; Biris, Nikolaos; Benz, Ann; Qvit, Nir; Donnelly, Sara K; Chen, Yun; Mennerick, Steven; Hodgson, Louis; Mochly-Rosen, Daria; Dorn, Gerald W

    2017-01-01

    Summary Mitochondria are dynamic organelles, remodeling and exchanging contents during cyclic fusion and fission. Genetic mutations of mitofusin (Mfn) 2 interrupt mitochondrial fusion and cause the untreatable neurodegenerative condition, Charcot Marie Tooth disease type 2A (CMT2A). It has not been possible to directly modulate mitochondrial fusion, in part because the structural basis of mitofusin function is incompletely understood. Here we show that mitofusins adopt either a fusion-constrained or fusion-permissive molecular conformation directed by specific intramolecular binding interactions, and demonstrate that mitofusin-dependent mitochondrial fusion can be regulated by targeting these conformational transitions. Based on this model we engineered a cell-permeant minipeptide to destabilize fusion-constrained mitofusin and promote the fusion-permissive conformation, reversing mitochondrial abnormalities in cultured fibroblasts and neurons harboring CMT2A gene defects. The relationship between mitofusin conformational plasticity and mitochondrial dynamism uncovers a central mechanism regulating mitochondrial fusion whose manipulation can correct mitochondrial pathology triggered by defective or imbalanced mitochondrial dynamics. PMID:27775718

  10. Fusion of Breast Carcinoma and Dendritic Cells as a Vaccine for the Treatment of Metastatic Breast Cancer. Addendum

    Science.gov (United States)

    2011-08-01

    patients. This brief report details the characterization of tumor cells and dendritic cells generated from patient BV01 with metastatic breast cancer following isolation from pleural effusions and leukapheresis, respectively.

  11. Detection of Echinoderm Microtubule Associated Protein Like 4-Anaplastic Lymphoma Kinase Fusion Genes in Non-small Cell Lung Cancer Clinical Samples by a Real-time Quantitative Reverse Transcription Polymerase Chain Reaction Method.

    Science.gov (United States)

    Zhao, Jing; Zhao, Jin-Yin; Chen, Zhi-Xia; Zhong, Wei; Li, Long-Yun; Liu, Li-Cheng; Hu, Xiao-Xu; Chen, Wei-Jun; Wang, Meng-Zhao

    2016-12-20

    Objective To establish a real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) for the rapid, sensitive, and specific detection of echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion genes in non-small cell lung cancer. Methods The specific primers for the four variants of EML4-ALK fusion genes (V1, V2, V3a, and V3b) and Taqman fluorescence probes for the detection of the target sequences were carefully designed by the Primer Premier 5.0 software. Then, using pseudovirus containing EML4-ALK fusion genes variants (V1, V2, V3a, and V3b) as the study objects, we further analyzed the lower limit, sensitivity, and specificity of this method. Finally, 50 clinical samples, including 3 ALK-fluorescence in situ hybridization (FISH) positive specimens, were collected and used to detect EML4-ALK fusion genes using this method. Results The lower limit of this method for the detection of EML4-ALK fusion genes was 10 copies/μl if no interference of background RNA existed. Regarding the method's sensitivity, the detection resolution was as high as 1% and 0.5% in the background of 500 and 5000 copies/μl wild-type ALK gene, respectively. Regarding the method's specificity, no non-specific amplification was found when it was used to detect EML4-ALK fusion genes in leukocyte and plasma RNA samples from healthy volunteers. Among the 50 clinical samples, 47 ALK-FISH negative samples were also negative. Among 3 ALK-FISH positive samples, 2 cases were detected positive using this method, but another was not detected because of the failure of RNA extraction. Conclusion The proposed qRT-PCR assay for the detection of EML4-ALK fusion genes is rapid, simple, sensitive, and specific, which is deserved to be validated and widely used in clinical settings.

  12. Sulindac sulfide reverses aberrant self-renewal of progenitor cells induced by the AML-associated fusion proteins PML/RARα and PLZF/RARα.

    Science.gov (United States)

    Steinert, Gunnar; Oancea, Claudia; Roos, Jessica; Hagemeyer, Heike; Maier, Thorsten; Ruthardt, Martin; Puccetti, Elena

    2011-01-01

    Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARα, PLZF/RARα, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML). One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARα and PLZF/RARα or AML-1/ETO activate Wnt signaling by upregulating γ-catenin and β-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARα-positive progenitor cell models. Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both β-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs) expressing PML/RARα, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings.

  13. Sulindac sulfide reverses aberrant self-renewal of progenitor cells induced by the AML-associated fusion proteins PML/RARα and PLZF/RARα.

    Directory of Open Access Journals (Sweden)

    Gunnar Steinert

    Full Text Available Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARα, PLZF/RARα, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML. One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARα and PLZF/RARα or AML-1/ETO activate Wnt signaling by upregulating γ-catenin and β-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID, where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARα-positive progenitor cell models. Sulindac Sulfide (SSi is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both β-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs expressing PML/RARα, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings.

  14. A system for the measurement of gene targeting efficiency in human cell lines using an antibiotic resistance-GFP fusion gene.

    Science.gov (United States)

    Konishi, Yuko; Karnan, Sivasundaram; Takahashi, Miyuki; Ota, Akinobu; Damdindorj, Lkhagvasuren; Hosokawa, Yoshitaka; Konishi, Hiroyuki

    2012-09-01

    Gene targeting in a broad range of human somatic cell lines has been hampered by inefficient homologous recombination. To improve this technology and facilitate its widespread application, it is critical to first have a robust and efficient research system for measuring gene targeting efficiency. Here, using a fusion gene consisting of hygromycin B phosphotransferase and 3'-truncated enhanced GFP (HygR-5' EGFP) as a reporter gene, we created a molecular system monitoring the ratio of homologous to random integration (H/R ratio) of targeting vectors into the genome. Cell clones transduced with a reporter vector containing HygR-5' EGFP were efficiently established from two human somatic cell lines. Established HygR-5' EGFP reporter clones retained their capacity to monitor gene targeting efficiency for a longer duration than a conventional reporter system using an unfused 5' EGFP gene. With the HygR-5' EGFP reporter system, we reproduced previous findings of gene targeting frequency being up-regulated by the use of an adeno-associated viral (AAV) backbone, a promoter-trap system, or a longer homology arm in a targeting vector, suggesting that this system accurately monitors H/R ratio. Thus, our HygR-5' EGFP reporter system will assist in the development of an efficient AAV-based gene targeting technology.

  15. The recurrent SET-NUP214 fusion as a new HOXA activation mechanism in pediatric T-cell acute lymphoblastic leukemia.

    Science.gov (United States)

    Van Vlierberghe, Pieter; van Grotel, Martine; Tchinda, Joëlle; Lee, Charles; Beverloo, H Berna; van der Spek, Peter J; Stubbs, Andrew; Cools, Jan; Nagata, Kyosuke; Fornerod, Maarten; Buijs-Gladdines, Jessica; Horstmann, Martin; van Wering, Elisabeth R; Soulier, Jean; Pieters, Rob; Meijerink, Jules P P

    2008-05-01

    T-cell acute lymphoblastic leukemia (T-ALL) is mostly characterized by specific chromosomal abnormalities, some occurring in a mutually exclusive manner that possibly delineate specific T-ALL subgroups. One subgroup, including MLL-rearranged, CALM-AF10 or inv (7)(p15q34) patients, is characterized by elevated expression of HOXA genes. Using a gene expression-based clustering analysis of 67 T-ALL cases with recurrent molecular genetic abnormalities and 25 samples lacking apparent aberrations, we identified 5 new patients with elevated HOXA levels. Using microarray-based comparative genomic hybridization (array-CGH), a cryptic and recurrent deletion, del (9)(q34.11q34.13), was exclusively identified in 3 of these 5 patients. This deletion results in a conserved SET-NUP214 fusion product, which was also identified in the T-ALL cell line LOUCY. SET-NUP214 binds in the promoter regions of specific HOXA genes, where it interacts with CRM1 and DOT1L, which may transcriptionally activate specific members of the HOXA cluster. Targeted inhibition of SET-NUP214 by siRNA abolished expression of HOXA genes, inhibited proliferation, and induced differentiation in LOUCY but not in other T-ALL lines. We conclude that SET-NUP214 may contribute to the pathogenesis of T-ALL by enforcing T-cell differentiation arrest.

  16. A VIN1 GUS::GFP fusion reveals activated sucrose metabolism programming occurring in interspersed cells during tomato fruit ripening.

    Science.gov (United States)

    Estornell, Leandro Hueso; Pons, Clara; Martínez, Alicia; O'Connor, José Enrique; Orzaez, Diego; Granell, Antonio

    2013-08-15

    The tomato is a model for fleshy fruit development and ripening. Here we report on the identification of a novel unique cell autonomous/cellular pattern of expression that was detected in fruits of transgenic tomato lines carrying a GFP GUS driven by the fruit specific vacuolar invertase promoter VIN1. The VIN1 promoter sequence faithfully reproduced the global endogenous VIN expression by conferring a biphasic pattern of expression with a second phase clearly associated to fruit ripening. A closer view revealed a salt and pepper pattern of expression characterized by individual cells exhibiting a range of expression levels (from high to low) surrounded by cells with no expression. This type of pattern was detected across different fruit tissues and cell types with some preferences for vascular, sub-epidermal layer and the inner part of the fruit. Cell ability to show promoter activity was neither directly associated with overall ripening - as we find VIN+ and - VIN- cells at all stages of ripening, nor with cell size. Nevertheless the number of cells with active VIN-driven expression increased with ripening and the activity of the VIN promoter seems to be inversely correlated with cell size in VIN+ cells. Gene expression analysis of FACS-sorted VIN+ cells revealed a transcriptionally distinct subpopulation of cells defined by increased expression of genes related to sucrose metabolism, and decreased activity in protein synthesis and chromatin remodeling. This finding suggests that local micro heterogeneity may underlie some aspects (i.e. the futile cycles involving sucrose metabolism) of an otherwise more uniform looking ripening program.

  17. Repair of Staphylococcus aureus-infected wound with gene-modified C3H10T1/2 cells expressing BPI-BD3 fusion antibiotic peptide

    Directory of Open Access Journals (Sweden)

    Xin-ran ZHANG

    2015-10-01

    Full Text Available Objective To study the antibacterial and tissue reparative effect of BPI-BD3 gene-modified mesenchymal stem cells in a mouse model of wound infection. Methods C3H10T1/2 cells were transfected with recombinant adenovirus vector pAdxsi-BPI-BD3, the expression of BPI-BD3 fusion protein was verified by RT-PCR and Western blotting. Excision wound with a diameter of 1cm was inoculated with Staphylococcus aureuswas made on the back of 30 mice. The mice were randomly divided into 3 groups (10 each. Mice in group T were injected with BPI-BD3 gene-modified C3H10T1/2 cells through caudal vein, those in group C were injected with unmodified C3H10T1/2 cells, and in group N were injected with PBS as control. The wound repair result was evaluated by estimation of the percentage of remaining wound area and the amount of wound bacteria under the scar, followed by observation of pathological changes. Inflammatory reactions of the wounds were assessed accordingly. Results The amount of bacteria under the scar was less in group T than in the other two groups (P<0.05. It was also found that the wound healing process was faster in group T than in group C and group N. Pathological observation showed that the inflammatory reaction in group T was also significantly milder than in the other two groups. Conclusion BPI-BD3 gene-modified mesenchymal stem cells may enhance wound repair by controlling infection and promoting tissue regeneration, thus it may be promising in clinical application. DOI: 10.11855/j.issn.0577-7402.2015.09.07

  18. Switch-like reprogramming of gene expression after fusion of multinucleate plasmodial cells of two Physarum polycephalum sporulation mutants

    Energy Technology Data Exchange (ETDEWEB)

    Walter, Pauline; Hoffmann, Xenia-Katharina; Ebeling, Britta; Haas, Markus; Marwan, Wolfgang, E-mail: wolfgang.marwan@ovgu.de

    2013-05-24

    Highlights: •We investigate reprogramming of gene expression in multinucleate single cells. •Cells of two differentiation control mutants are fused. •Fused cells proceed to alternative gene expression patterns. •The population of nuclei damps stochastic fluctuations in gene expression. •Dynamic processes of cellular reprogramming can be observed by repeated sampling of a cell. -- Abstract: Nonlinear dynamic processes involving the differential regulation of transcription factors are considered to impact the reprogramming of stem cells, germ cells, and somatic cells. Here, we fused two multinucleate plasmodial cells of Physarum polycephalum mutants defective in different sporulation control genes while being in different physiological states. The resulting heterokaryons established one of two significantly different expression patterns of marker genes while the plasmodial halves that were fused to each other synchronized spontaneously. Spontaneous synchronization suggests that switch-like control mechanisms spread over and finally control the entire plasmodium as a result of cytoplasmic mixing. Regulatory molecules due to the large volume of the vigorously streaming cytoplasm will define concentrations in acting on the population of nuclei and in the global setting of switches. Mixing of a large cytoplasmic volume is expected to damp stochasticity when individual nuclei deliver certain RNAs at low copy number into the cytoplasm. We conclude that spontaneous synchronization, the damping of molecular noise in gene expression by the large cytoplasmic volume, and the option to take multiple macroscopic samples from the same plasmodium provide unique options for studying the dynamics of cellular reprogramming at the single cell level.

  19. Secretory Transactivating Transcription-apoptin fusion protein induces apoptosis in hepatocellular carcinoma HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Su-Xia Han; Jin-Lu Ma; Yi Lv; Chen Huang; Hai-Hua Liang; Kang-Min Duan

    2008-01-01

    AIM: To determine whether SP-TAT-apoptin induces apoptosis and also maintains its tumor cell specificity.METHODS: In this study, we designed a secretory protein by adding a secretory signal peptide (SP) to the N terminus of Transactivating Transcription (TAT)-apoptin (SP-TAT-apoptin), to test the hypothesis that it gains an additive bystander effect as an anti-cancer therapy. We used an artificial human secretory SP whose amino acid sequence and corresponding cDNA sequence were generated by the SP hidden Markov model.RESULTS: In human liver carcinoma HepG2 cells, SP-TAT-apoptin expression showed a diffuse pattern in the early phase after transfection. After 48h, however, it translocated into the nuclear compartment and caused massive apoptotic cell death, as determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and annexin-V binding assay. SP-TAT-apoptin did not, however, cause any cell death in non-malignant human umbilical vein endothelial cells (HUVECs). Most importantly, the conditioned medium from Chinese hamster ovary (CHO) cells transfected with SP-TAT-apoptin also induced significant cell death in HepG2 cells, but not in HUVECs.CONCLUSION: The data demonstrated that SP-TAT-apoptin induces apoptosis only in malignant cells, and its secretory property might greatly increase its potency once it is delivered in vivo for cancer therapy.

  20. Cold fusion research

    Energy Technology Data Exchange (ETDEWEB)

    None

    1989-11-01

    I am pleased to forward to you the Final Report of the Cold Fusion Panel. This report reviews the current status of cold fusion and includes major chapters on Calorimetry and Excess Heat, Fusion Products and Materials Characterization. In addition, the report makes a number of conclusions and recommendations, as requested by the Secretary of Energy.

  1. Fusions of Breast Carcinoma and Dendritic Cells as a Vaccine for the Treatment of Metatastic Breast Cancer.

    Science.gov (United States)

    2013-07-01

    specific CD4+ helper T cell responses in cancer patients. Blood, 106:1008- 1011 , 2005. (2) Bell D, Chomarat P, Broyles D, Netto G, Harb GM, Lebecque S...regulatory t cell proliferation. J Exp Med, 2005, 202: 919- 929 . (4) Rabinowich H, Suminami Y, Reichert TE, Crowley-Nowick P, Bell M, Edwards R

  2. EpCAM-selective elimination of carcinoma cells by a novel MAP-based cytolytic fusion protein

    NARCIS (Netherlands)

    Hristodorov, Dmitrij; Amoury, Manal; Mladenov, Radoslav; Niesen, Judith; Arens, Katharina; Berges, Nina; Hein, Lea; di Fiore, Stefano; Pham, Anh-Tuan; Huhn, Michael; Helfrich, Wijnand; Fischer, Rainer; Thepen, Theo; Barth, Stefan

    2014-01-01

    In normal epithelia, the epithelial cell adhesion molecule (EpCAM) expression is relatively low and only present at the basolateral cell surface. In contrast, EpCAM is aberrantly overexpressed in various human carcinomas. Therefore, EpCAM is considered to be a highly promising target for antibody-ba

  3. Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability.

    Science.gov (United States)

    Webb, Stacy; Nagy, Tamas; Moseley, Hunter; Fried, Michael; Dutch, Rebecca Ellis

    2017-02-17

    Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a meta-stable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements which control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith EC, et al. Trimeric transmembrane domain interactions in paramyxovirus fusion proteins. 2013. J Biol Chem. 288, 35726). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability.

  4. Fluorescent vital staining of plant sexual cell nuclei with DNA—specific fluorochromes and its application in gametoplast fusion

    Institute of Scientific and Technical Information of China (English)

    YANGHONGYUAN; XINLIWU; 等

    1993-01-01

    DNA-binding fluorochromes are often used for vital staining of plant cell nuclei.However,it is not always sure whether the cells after staining still remain in living state.We chose several criteria to estimate the validity of real vital staining for sexual cell nuclei.These were:the cytoplasmic streaming in pollen tubes whose nuclei were stined,the simultaneous visualization of fluorochromatic reaction and nucleus staining in isolated generative cells,and the capability of isolated.prestained generative or sperm cells to fuse with other protoplasts.The results confirmed that 4,6-diamidino-2-phenylindole(DAPI),Hoechst 33258 and mithramycin could be used as real vital stains,though their efficiency varied from case to case;among them DAPI showed best effect.The fluo rescent vital staining technique offered a useful means foridentification and selection of heterokaryons in gametoplast manipulation studies.

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

    Science.gov (United States)

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

    2015-12-01

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

  6. Production of tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid and dog zona pellucida glycoprotein-3 for contraceptive vaccine development.

    Science.gov (United States)

    Gupta, Neha; Shrestha, Abhinav; Panda, Amulya Kumar; Gupta, Satish Kumar

    2013-07-01

    Affinity tags can interfere in various physicochemical properties and immunogenicity of the recombinant proteins. In the present study, tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker and dog zona pellucida glycoprotein-3 (ZP3; aa residues 23-348) (TT-KK-ZP3) was expressed in Escherichia coli. The recombinant protein, expressed as inclusion bodies (IBs), was purified by isolation of IBs, processed to remove host cell proteins, followed by solubilization and refolding. A specific 39 kDa protein including ZP3 was identified by SDS-PAGE. CD spectra showed the presence of α-helices and β-sheets, and fluorescent spectroscopy revealed emission maxima of 265 A.U. at 339 nm for refolded protein and showed red shift in the presence of 6 M guanidine hydrochloride. Immunization of inbred FvB/J female mice with purified recombinant TT-KK-ZP3 (25 μg/animal) led to generation of high antibody titers against the recombinant protein. The antibodies reacted specifically with ZP matrix surrounding mouse oocytes. Immunized mice showed significant reduction in fertility as compared to the control group. The studies described herein provide a simple method to produce and purify tag-free recombinant protein for the development of a contraceptive vaccine.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-01-22

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

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

    Science.gov (United States)

    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.

  9. Growth hormone promotes skeletal muscle cell fusion independent of insulin-like growth factor 1 up-regulation

    OpenAIRE

    Sotiropoulos, Athanassia; Ohanna, Mickaël; Kedzia, Cécile; Menon, Ram K.; Kopchick, John J.; Kelly, Paul A; Pende, Mario

    2006-01-01

    Growth hormone (GH) participates in the postnatal regulation of skeletal muscle growth, although the mechanism of action is unclear. Here we show that the mass of skeletal muscles lacking GH receptors is reduced because of a decrease in myofiber size with normal myofiber number. GH signaling controls the size of the differentiated myotubes in a cell-autonomous manner while having no effect on size, proliferation, and differentiation of the myoblast precursor cells. The GH hypertrophic action ...

  10. DNA fusion gene vaccines

    DEFF Research Database (Denmark)

    Holst, Peter Johannes; Bassi, Maria Rosaria; Thomsen, Allan Randrup

    2010-01-01

    DNA vaccines are versatile and safe, but limited immunogenicity has prevented their use in the clinical setting. Experimentally, immunogenicity may be enhanced by the use of new delivery technologies, by coadministration of cytokines and pathogen-associated molecular patterns, or by fusion...... of antigens into molecular domains that enhance antigen presentation. More specifically, the immunogenicity of DNA vaccines may benefit from increased protein synthesis, increased T-cell help and MHC class I presentation, and the addition of a range of specific cytokines and pathogen-associated molecular...... with viral-vectored vaccines, various synergistic components may need to be incorporated into DNA vaccines. From the perspective of the future clinical use of DNA vaccines, it has been suggested that antigen presentation should be improved and cytokine coadministration attempted. However, even...

  11. Fusion dual-tracer SPECT-based hepatic dosimetry predicts outcome after radioembolization for a wide range of tumour cell types

    Energy Technology Data Exchange (ETDEWEB)

    Lam, Marnix G.E.H. [Stanford University, Division of Interventional Radiology, Stanford, CA (United States); University Medical Center Utrecht, Department of Radiology and Nuclear Medicine, Heidelberglaan 100, CX, Utrecht (Netherlands); Banerjee, Arjun; Louie, John D.; Sze, Daniel Y. [Stanford University, Division of Interventional Radiology, Stanford, CA (United States); Goris, Michael L.; Iagaru, Andrei H.; Mittra, Erik S. [Stanford University, Division of Nuclear Medicine and Molecular Imaging, Stanford, CA (United States)

    2015-07-15

    Fusion dual-tracer SPECT imaging enables physiological rather than morphological voxel-based partitioning and dosimetry for {sup 90}Y hepatic radioembolization (RE). We evaluated its prognostic value in a large heterogeneous cohort of patients with extensive hepatic malignancy. A total of 122 patients with primary or secondary liver malignancy (18 different cell types) underwent SPECT imaging after intraarterial injection of {sup 99m}Tc macroaggregated albumin (TcMAA) as a simulation of subsequent {sup 90}Y microsphere distribution, followed by administration of an excess of intravenous {sup 99m}Tc-labelled sulphur colloid (TcSC) as a biomarker for functional liver, and a second SPECT scan. TcMAA distribution was used to estimate {sup 90}Y radiation absorbed dose in tumour (D{sub T}) and in functional liver. Laboratory and clinical follow-up were recorded for 12 weeks after RE, and radiographic responses according to (m)RECIST were evaluated at 3 and 6 months. Dose-response relationships were determined for efficacy and toxicity. Patients were treated with a median of 1.73 GBq activity of resin microspheres (98 patients) or glass microspheres (24 patients), in a whole-liver approach (97 patients) or a lobar approach (25 patients). The objective response rate was 41 % at 3 months and 48 % at 6 months. Response was correlated with D{sub T} (P < 0.01). Median overall survival was 10.1 months (95 % confidence interval 7.4 - 12.8 months). Responders lived for 36.0 months compared to 8.7 months for nonresponders (P < 0.01). Stratified for tumour cell type, D{sub T} was independently associated with survival (P < 0.01). Absorbed dose in functional liver was correlated with toxicity grade change (P < 0.05) and RE-induced liver disease (P < 0.05). Fusion dual-tracer SPECT imaging offers a physiology-based functional imaging tool to predict efficacy and toxicity of RE. This technique can be refined to define dosing thresholds for specific tumour types and treatments, but

  12. Auto-fusion and the shaping of neurons and tubes.

    Science.gov (United States)

    Soulavie, Fabien; Sundaram, Meera V

    2016-12-01

    Cells adopt specific shapes that are necessary for specific functions. For example, some neurons extend elaborate arborized dendrites that can contact multiple targets. Epithelial and endothelial cells can form tiny seamless unicellular tubes with an intracellular lumen. Recent advances showed that cells can auto-fuse to acquire those specific shapes. During auto-fusion, a cell merges two parts of its own plasma membrane. In contrast to cell-cell fusion or macropinocytic fission, which result in the merging or formation of two separate membrane bound compartments, auto-fusion preserves one compartment, but changes its shape. The discovery of auto-fusion in C. elegans was enabled by identification of specific protein fusogens, EFF-1 and AFF-1, that mediate cell-cell fusion. Phenotypic characterization of eff-1 and aff-1 mutants revealed that fusogen-mediated fusion of two parts of the same cell can be used to sculpt dendritic arbors, reconnect two parts of an axon after injury, or form a hollow unicellular tube. Similar auto-fusion events recently were detected in vertebrate cells, suggesting that auto-fusion could be a widely used mechanism for shaping neurons and tubes.

  13. Cell-specific modulation of drug resistance in acute myeloid leukemic blasts by diphtheria fusion toxin, DT388-GMCSF.

    Science.gov (United States)

    Frankel, A E; Hall, P D; McLain, C; Safa, A R; Tagge, E P; Kreitman, R J

    1998-01-01

    Radiochemotherapy-resistant blasts commonly cause treatment failure in acute myeloid leukemia (AML), and their resistance is due, in part, to overexpression of multidrug resistance (mdr) proteins. We reasoned that targeted delivery of protein synthesis inactivating toxins to leukemic blasts would reduce the cellular concentrations of relatively short half-life resistance proteins and sensitize the cells to cytotoxic drugs. To test this hypothesis, we employed human granulocyte-macrophage colony-stimulating factor fused to truncated diphtheria toxin (DT388-GMCSF). The human AML cell line HL60 and its vincristine-resistant sublines, HL60Vinc and HL60VCR, were incubated in vitro for 24 h with varying concentrations of toxin. Doxorubicin was added for an additional 24 h, and cell cytotoxicity was assayed by thymidine incorporation and colony formation in semisolid medium. DT388-GMCSF sensitized HL60Vinc and HL60VCR but not HL60 to doxorubicin. Combination indices for three log cell kill varied from 0.2 to 0.3. In contrast, pretreatment with doxorubicin followed by toxins failed to show synergy. At least in the case of the vincristine-resistant cell lines, modulation of drug resistance correlated with reduction in membrane P-glycoprotein concentrations based on immunoblots with C219 antibody, flow cytometry with MRK16 antibody, and cell uptake of doxorubicin. These observations suggest clinical trials of combination therapy may be warranted in patients with refractory AML. Further, targeted toxins may represent a novel class of cell-specific modulators of drug resistance for a number of malignancies.

  14. Switch-like reprogramming of gene expression after fusion of multinucleate plasmodial cells of two Physarum polycephalum sporulation mutants.

    Science.gov (United States)

    Walter, Pauline; Hoffmann, Xenia-Katharina; Ebeling, Britta; Haas, Markus; Marwan, Wolfgang

    2013-05-24

    Nonlinear dynamic processes involving the differential regulation of transcription factors are considered to impact the reprogramming of stem cells, germ cells, and somatic cells. Here, we fused two multinucleate plasmodial cells of Physarum polycephalum mutants defective in different sporulation control genes while being in different physiological states. The resulting heterokaryons established one of two significantly different expression patterns of marker genes while the plasmodial halves that were fused to each other synchronized spontaneously. Spontaneous synchronization suggests that switch-like control mechanisms spread over and finally control the entire plasmodium as a result of cytoplasmic mixing. Regulatory molecules due to the large volume of the vigorously streaming cytoplasm will define concentrations in acting on the population of nuclei and in the global setting of switches. Mixing of a large cytoplasmic volume is expected to damp stochasticity when individual nuclei deliver certain RNAs at low copy number into the cytoplasm. We conclude that spontaneous synchronization, the damping of molecular noise in gene expression by the large cytoplasmic volume, and the option to take multiple macroscopic samples from the same plasmodium provide unique options for studying the dynamics of cellular reprogramming at the single cell level.

  15. Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity.

    Science.gov (United States)

    Lv, Qiang; Yang, Xu-Zhong; Fu, Long-Yun; Lu, Yv-Ting; Lu, Yan-Hua; Zhao, Jian; Wang, Fu-Jun

    2015-07-01

    MAP30 (Momordica Antiviral Protein 30 Kd), a single-stranded type-I ribosome inactivating protein, possesses versatile biological activities including anti-tumor abilities. However, the low efficiency penetrating into tumor cells hampers the tumoricidal effect of MAP30. This paper describes MAP30 fused with a human-derived cell penetrating peptide HBD which overcome the low uptake efficiency by tumor cells and exhibits higher anti-tumor bioactivity. MAP30 gene was cloned from the genomic DNA of Momordica charantia and the recombinant plasmid pET28b-MAP30-HBD was established and transferred into Escherichia coli BL21 (DE3). The recombinant MAP30-HBD protein (rMAP30-HBD) was expressed in a soluble form after being induced by 0.5mM IPTG for 14h at 15°C. The recombinant protein was purified to greater than 95% purity with Ni-NTA affinity chromatography. The rMAP30-HBD protein not only has topological inactivation and protein translation inhibition activity but also showed significant improvements in cytotoxic activity compared to that of the rMAP30 protein without HBD in the tested tumor cell lines, and induced higher apoptosis rates in HeLa cells analyzed by Annexin V-FITC with FACS. This paper demonstrated a new method for improving MAP30 protein anti-tumor activity and might have potential applications in cancer therapy area.

  16. In vitro translocation experiments with RxLR-reporter fusion proteins of Avr1b from Phytophthora sojae and AVR3a from Phytophthora infestans fail to demonstrate specific autonomous uptake in plant and animal cells.

    Science.gov (United States)

    Wawra, Stephan; Djamei, Armin; Albert, Isabell; Nürnberger, Thorsten; Kahmann, Regine; van West, Pieter

    2013-05-01

    Plant-pathogenic oomycetes have a large set of secreted effectors that can be translocated into their host cells during infection. One group of these effectors are the RxLR effectors for which it has been shown, in a few cases, that the RxLR motif is important for their translocation. It has been suggested that the RxLR-leader sequences alone are enough to translocate the respective effectors into eukaryotic cells through binding to surface-exposed phosphoinositol-3-phosphate. These conclusions were primary based on translocation experiments conducted with recombinant fusion proteins whereby the RxLR leader of RxLR effectors (i.e., Avr1b from Phytophthora sojae) were fused to the green fluorescent protein reporter-protein. However, we failed to observe specific cellular uptake for a comparable fusion protein where the RxLR leader of the P. infestans AVR3a was fused to monomeric red fluorescent protein. Therefore, we reexamined the ability of the reported P. sojae AVR1b RxLR leader to enter eukaryotic cells. Different relevant experiments were performed in three independent laboratories, using fluorescent reporter fusion constructs of AVR3a and Avr1b proteins in a side-by-side comparative study on plant tissue and human and animal cells. We report that we were unable to obtain conclusive evidence for specific RxLR-mediated translocation.

  17. Economics of fusion research

    Energy Technology Data Exchange (ETDEWEB)

    None, None

    1977-10-15

    This report provides the results of a study of methods of economic analysis applied to the evaluation of fusion research. The study recognizes that a hierarchy of economic analyses of research programs exists: standard benefit-cost analysis, expected value of R and D information, and expected utility analysis. It is shown that standard benefit-cost analysis, as commonly applied to research programs, is inadequate for the evaluation of a high technology research effort such as fusion research. A methodology for performing an expected value analysis is developed and demonstrated and an overview of an approach to perform an expected utility analysis of fusion research is presented. In addition, a potential benefit of fusion research, not previously identified, is discussed and rough estimates of its magnitude are presented. This benefit deals with the effect of a fusion research program on optimal fossil fuel consumption patterns. The results of this study indicate that it is both appropriate and possible to perform an expected value analysis of fusion research in order to assess the economics of a fusion research program. The results indicate further that the major area of benefits of fusion research is likely due to the impact of a fusion research program on optimal fossil fuel consumption patterns and it is recommended that this benefit be included in future assessments of fusion research economics.

  18. Materials research for fusion

    Science.gov (United States)

    Knaster, J.; Moeslang, A.; Muroga, T.

    2016-05-01

    Fusion materials research started in the early 1970s following the observation of the degradation of irradiated materials used in the first commercial fission reactors. The technological challenges of fusion energy are intimately linked with the availability of suitable materials capable of reliably withstanding the extremely severe operational conditions of fusion reactors. Although fission and fusion materials exhibit common features, fusion materials research is broader. The harder mono-energetic spectrum associated with the deuterium-tritium fusion neutrons (14.1 MeV compared to hydrogen and helium as transmutation products that might lead to a (at present undetermined) degradation of structural materials after a few years of operation. Overcoming the historical lack of a fusion-relevant neutron source for materials testing is an essential pending step in fusion roadmaps. Structural materials development, together with research on functional materials capable of sustaining unprecedented power densities during plasma operation in a fusion reactor, have been the subject of decades of worldwide research efforts underpinning the present maturity of the fusion materials research programme.

  19. Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells

    NARCIS (Netherlands)

    Blecha, Andreas; Zarschler, Kristof; Sjollema, Klaas A.; Veenhuis, Marten; Rödel, Gerhard; Rodel, G.

    2005-01-01

    Background: Native as well as recombinant bacterial cell surface layer (S-layer) protein of Geobacillus (G.) stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested

  20. Transient fusion ensures granule replenishment to enable repeated release after IgE-mediated mast cell degranulation.

    Science.gov (United States)

    Balseiro-Gomez, Santiago; Flores, Juan A; Acosta, Jorge; Ramirez-Ponce, M Pilar; Ales, Eva

    2016-11-01

    To ensure normal immune function, mast cells employ different pathways to release mediators. Here, we report a thus far unknown capacity of mast cells to recycle and reuse secretory granules after an antigen-evoked degranulation process under physiological conditions; this phenomenon involves the existence of a recycling secretory granule pool that is available for release in a short time scale. Rapid endocytic modes contributed to the recycling of ∼60% of the total secretory granule population, which involved kiss-and-run and cavicapture mechanisms, causing retention of the intragranular matrix. We found the presence of normal-size granules and giant actomyosin- and dynamin-dependent granules, which were characterized by large quantal content. These large structures allowed the recovered mast cells to release a large amount of 5-HT, compensating for the decrease in the number of exocytosed secretory granules. This work uncovers a new physiological role of the exo-endocytosis cycle in the immunological plasticity of mast cells and reveals a new property of their biological secretion.

  1. 三阴乳腺癌细胞-树突状细胞融合疫苗的抗肿瘤免疫效应%Antitumor effect of a triple negative breast cancer-dendritic cell fusion vaccine

    Institute of Scientific and Technical Information of China (English)

    张鹏; 刘瑞磊; 姜华; 刘宇; 张翘楚; 黄勇

    2012-01-01

    目的 利用细胞电融合技术制备外周血来源树突状细胞(DC)和三阴乳腺癌细胞(MDA-MB-231)全抗原肿瘤疫苗,观察其特异性抗肿瘤免疫效应.方法 从健康人外周血中分离培养DC,利用电融合技术,将DC和三阴乳腺癌细胞融合;荧光显微镜观察融合疫苗的形态;流式细胞仪进行融合疫苗的表型鉴定;ELlSA试剂盒检测IL-12、IFN-y的分泌情况;CCK-8试剂盒测定融合疫苗刺激同源异体T淋巴细胞增殖和细胞毒性效应.结果 成功分离培养DC,其表面高表达DC的分子标记CD83、CD11c、CD86、HLA-DR;融合疫苗形态不规则,其表面共同表达DC和三阴乳腺癌的标记分子;T淋巴细胞增殖实验证明融合疫苗有很强的免疫刺激活性;细胞毒性实验证明融合疫苗具有比对照组更强的杀伤肿瘤细胞作用.结论 电融合技术成功诱导DC和三阴乳腺癌细胞融合,全抗原融合疫苗体外实验可显著增强抗肿瘤免疫效应.%Objective To test the antitumor effect of a human triple-negative breast cancer cell-dendritic cell (DC) fusion vaccine.Methods DCs were isolated from fresh peripheral blood of healthy donors.The fusion vaccine was prepared by fusing the DCs and MDA-MB-231 cells via electrofusion.The morphology of the vaccine was indentified under inverted fluorescence microscope and the phenotypes were analyzed with flow cytometry.The production of interleukin-12 (JL-12) and interferon-y (IFN-y) by the fusion cells was assessed using ELISA.A CCK-8 kit was used to examine the effect of the vaccine in stimulating the proliferation and cytotoxiciry of autologous T lymphocytes.Results The DCs isolated from peripheral blood monomiclear cells highly expressed CD83,CD86,CDllc and HLA-DR on the cell surface.The fusion cells were irregular in shape and coexpressed the phenotypes of DCs and MDA-MB-231 cells.The fusion cells possessed a strong ability to stimulate the proliferation of T lymphocytes in vitro

  2. Distinct roles for key karyogamy proteins during yeast nuclear fusion.

    Science.gov (United States)

    Melloy, Patricia; Shen, Shu; White, Erin; Rose, Mark D

    2009-09-01

    During yeast mating, cell fusion is followed by the congression and fusion of the two nuclei. Proteins required for nuclear fusion are found at the surface (Prm3p) and within the lumen (Kar2p, Kar5p, and Kar8p) of the nuclear envelope (NE). Electron tomography (ET) of zygotes revealed that mutations in these proteins block nuclear fusion with different morphologies, suggesting that they act in different steps of fusion. Specifically, prm3 zygotes were blocked before formation of membrane bridges, whereas kar2, kar5, and kar8 zygotes frequently contained them. Membrane bridges were significantly larger and occurred more frequently in kar2 and kar8, than in kar5 mutant zygotes. The kinetics of NE fusion in prm3, kar5, and kar8 mutants, measured by live-cell fluorescence microscopy, were well correlated with the size and frequency of bridges observed by ET. However the kar2 mutant was defective for transfer of NE lumenal GFP, but not diffusion within the lumen, suggesting that transfer was blocked at the NE fusion junction. These observations suggest that Prm3p acts before initiation of outer NE fusion, Kar5p may help dilation of the initial fusion pore, and Kar2p and Kar8p act after outer NE fusion, during inner NE fusion.

  3. Repair of rabbit cartilage defect based on the fusion of rabbit bone marrow stromal cells and Nano-HA/PLLA composite material.

    Science.gov (United States)

    Zhu, Weimin; Guo, Daiqi; Peng, Liangquan; Chen, Yun Fang; Cui, Jiaming; Xiong, Jianyi; Lu, Wei; Duan, Li; Chen, Kang; Zeng, Yanjun; Wang, Daping

    2017-02-01

    Objective To assess the effect of the fusion of rabbit bone marrow stromal cells (rBMSCs) and Nano-hydroxyapatite/poly (l-lactic acid) (Nano-HA/PLLA) in repairing the rabbit knee joint with full-thickness cartilage defect. Method The rBMSCs were isolated and cultured in vitro, and the third generation of rBMSCs was co-cultured with the Nano-HA/PLLA to construct the tissue-engineered cartilage (TEC). Eighteen New Zealand white rabbits were selected and randomly divided into three groups, namely, TEC group, Nano-HA/PLLA group, and control group. A cartilage defect model with the diameter of 4.5 mm and depth of 5 mm was constructed on the articular surface of medial malleolus of rabbit femur. General observation, histological observation, and Wakitani's histological scoring were conducted in the 12th and 24th week postoperatively. Results The results of TEC group indicated that new cartilage tissue was formed on the defect site and subchondral bone achieved physiological integration basically. Histological and immunohistochemical analyses indicated the generation of massive extracellular matrix. In contrast, limited regeneration and reconstruction of cartilage was achieved in the Nano-HA/PLLA group and control group, with a significant difference from the TEC group (p Nano-HA/PLLA combined with BMSCs promoted the repair of weight-bearing bone of adult rabbit's knee joint with cartilage defect.

  4. Fully non-linear multi-species Fokker-Planck-Landau collisions for gyrokinetic particle-in-cell simulations of fusion plasma

    Science.gov (United States)

    Hager, Robert; Yoon, E. S.; Ku, S.; D'Azevedo, E. F.; Worley, P. H.; Chang, C. S.

    2015-11-01

    We describe the implementation, and application of a time-dependent, fully nonlinear multi-species Fokker-Planck-Landau collision operator based on the single-species work of Yoon and Chang [Phys. Plasmas 21, 032503 (2014)] in the full-function gyrokinetic particle-in-cell codes XGC1 [Ku et al., Nucl. Fusion 49, 115021 (2009)] and XGCa. XGC simulations include the pedestal and scrape-off layer, where significant deviations of the particle distribution function from a Maxwellian can occur. Thus, in order to describe collisional effects on neoclassical and turbulence physics accurately, the use of a non-linear collision operator is a necessity. Our collision operator is based on a finite volume method using the velocity-space distribution functions sampled from the marker particles. Since the same fine configuration space mesh is used for collisions and the Poisson solver, the workload due to collisions can be comparable to or larger than the workload due to particle motion. We demonstrate that computing time spent on collisions can be kept affordable by applying advanced parallelization strategies while conserving mass, momentum, and energy to reasonable accuracy. We also show results of production scale XGCa simulations in the H-mode pedestal and compare to conventional theory. Work supported by US DOE OFES and OASCR.

  5. Training Enhances Immune Cells Mitochondrial Biosynthesis, Fission, Fusion, and Their Antioxidant Capabilities Synergistically with Dietary Docosahexaenoic Supplementation

    OpenAIRE

    2016-01-01

    Exercise training induces adaptations in mitochondrial metabolism, dynamics, and oxidative protection. Omega-3 fatty acids change membrane lipid composition and modulate mitochondrial function. The aim was to investigate the effect of 8-week training and docosahexaenoic acid (DHA) supplementation (1.14 g/day) on the mitochondria dynamics and antioxidant status in peripheral blood mononuclear cells (PBMCs) from sportsmen. Subjects were assigned to an intervention (N = 9) or placebo groups (N =...

  6. Muon Catalyzed Fusion

    Science.gov (United States)

    Armour, Edward A.G.

    2007-01-01

    Muon catalyzed fusion is a process in which a negatively charged muon combines with two nuclei of isotopes of hydrogen, e.g, a proton and a deuteron or a deuteron and a triton, to form a muonic molecular ion in which the binding is so tight that nuclear fusion occurs. The muon is normally released after fusion has taken place and so can catalyze further fusions. As the muon has a mean lifetime of 2.2 microseconds, this is the maximum period over which a muon can participate in this process. This article gives an outline of the history of muon catalyzed fusion from 1947, when it was first realised that such a process might occur, to the present day. It includes a description of the contribution that Drachrnan has made to the theory of muon catalyzed fusion and the influence this has had on the author's research.

  7. 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⁺.

    Science.gov (United States)

    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.

  8. Magnetic fusion technology

    CERN Document Server

    Dolan, Thomas J

    2014-01-01

    Magnetic Fusion Technology describes the technologies that are required for successful development of nuclear fusion power plants using strong magnetic fields. These technologies include: ? magnet systems, ? plasma heating systems, ? control systems, ? energy conversion systems, ? advanced materials development, ? vacuum systems, ? cryogenic systems, ? plasma diagnostics, ? safety systems, and ? power plant design studies. Magnetic Fusion Technology will be useful to students and to specialists working in energy research.

  9. Fusion research principles

    CERN Document Server

    Dolan, Thomas James

    2013-01-01

    Fusion Research, Volume I: Principles provides a general description of the methods and problems of fusion research. The book contains three main parts: Principles, Experiments, and Technology. The Principles part describes the conditions necessary for a fusion reaction, as well as the fundamentals of plasma confinement, heating, and diagnostics. The Experiments part details about forty plasma confinement schemes and experiments. The last part explores various engineering problems associated with reactor design, vacuum and magnet systems, materials, plasma purity, fueling, blankets, neutronics

  10. Efficient production of sTNFRII-gAD fusion protein in large quantity by use of the modified CHO-S cell expression system.

    Directory of Open Access Journals (Sweden)

    Qinzhen Cai

    Full Text Available TNFα is one of the initial and important mediators to activate downstream signaling pathways by binding to trimerized TNFα receptors (TNFR, and thus is an ideal drug target for cancer therapy. Taking advantage of intrinsic homotimerization of the globular domain of adiponectin (gAD, we have developed a novel TNFα antagonist, the trimerized fusion protein named sTNFRII-gAD. However, our previously-used CHO expression system yielded less than 10 mg/L of sTNFRII-gAD. To produce large quantities of sTNFRII-gAD efficiently, we used a modified CHO-S cell expression system, which is based on a pMH3 vector with non-coding GC-rich DNA fragments for high-level gene expression. We obtained stable clones that produced 75 mg/L of sTNFRII-gAD in the 96-well plate, adapted the clones to 40 ml suspension serum-free batch culture, then optimized the culturing conditions to scale up the fed-batch culture in a 3 L shake-flask and finally in a 5 L AP30 bioreactor. We achieved a final yield of 52 mg/L of sTNFRII-gAD. The trimerized sTNFRII-gAD exhibited the higher affinity to TNFα with a dissociation constant (Kd of 5.63 nM than the dimerized sTNFRII-Fc with a Kd of 13.4 nM, and further displayed the higher TNFα-neutralizing activity than sTNFRII-Fc (p<0.05 in a L929 cytotoxicity assay. Therefore, the strategy employed in this study may provide an efficient avenue for large-scale production of other recombinant proteins by use of the modified CHO-S cell expression system.

  11. Autographa californica Multicapsid Nucleopolyhedrovirus efficiently infects Sf9 cells and transduces mammalian cells via direct fusion with the plasma membrane at low pH

    NARCIS (Netherlands)

    Dong, S.; Wang, M.; Qiu, Z.; Deng, F.; Vlak, J.M.; Hu, Z.H.; Wang, H.L.

    2010-01-01

    The budded virus (BV) of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infects insect cells and transduces mammalian cells mainly through the endocytosis pathway. However, this study revealed that the treatment of the virus bound to Sf9 cells at low pH could efficiently rescue

  12. Magnetic fusion reactor economics

    Energy Technology Data Exchange (ETDEWEB)

    Krakowski, R.A.

    1995-12-01

    An almost primordial trend in the conversion and use of energy is an increased complexity and cost of conversion systems designed to utilize cheaper and more-abundant fuels; this trend is exemplified by the progression fossil fission {yields} fusion. The present projections of the latter indicate that capital costs of the fusion ``burner`` far exceed any commensurate savings associated with the cheapest and most-abundant of fuels. These projections suggest competitive fusion power only if internal costs associate with the use of fossil or fission fuels emerge to make them either uneconomic, unacceptable, or both with respect to expensive fusion systems. This ``implementation-by-default`` plan for fusion is re-examined by identifying in general terms fusion power-plant embodiments that might compete favorably under conditions where internal costs (both economic and environmental) of fossil and/or fission are not as great as is needed to justify the contemporary vision for fusion power. Competitive fusion power in this context will require a significant broadening of an overly focused program to explore the physics and simbiotic technologies leading to more compact, simplified, and efficient plasma-confinement configurations that reside at the heart of an attractive fusion power plant.

  13. Magnetic-confinement fusion

    Science.gov (United States)

    Ongena, J.; Koch, R.; Wolf, R.; Zohm, H.

    2016-05-01

    Our modern society requires environmentally friendly solutions for energy production. Energy can be released not only from the fission of heavy nuclei but also from the fusion of light nuclei. Nuclear fusion is an important option for a clean and safe solution for our long-term energy needs. The extremely high temperatures required for the fusion reaction are routinely realized in several magnetic-fusion machines. Since the early 1990s, up to 16 MW of fusion power has been released in pulses of a few seconds, corresponding to a power multiplication close to break-even. Our understanding of the very complex behaviour of a magnetized plasma at temperatures between 150 and 200 million °C surrounded by cold walls has also advanced substantially. This steady progress has resulted in the construction of ITER, a fusion device with a planned fusion power output of 500 MW in pulses of 400 s. ITER should provide answers to remaining important questions on the integration of physics and technology, through a full-size demonstration of a tenfold power multiplication, and on nuclear safety aspects. Here we review the basic physics underlying magnetic fusion: past achievements, present efforts and the prospects for future production of electrical energy. We also discuss questions related to the safety, waste management and decommissioning of a future fusion power plant.

  14. Frontiers in fusion research

    CERN Document Server

    Kikuchi, Mitsuru

    2011-01-01

    Frontiers in Fusion Research provides a systematic overview of the latest physical principles of fusion and plasma confinement. It is primarily devoted to the principle of magnetic plasma confinement, that has been systematized through 50 years of fusion research. Frontiers in Fusion Research begins with an introduction to the study of plasma, discussing the astronomical birth of hydrogen energy and the beginnings of human attempts to harness the Sun's energy for use on Earth. It moves on to chapters that cover a variety of topics such as: * charged particle motion, * plasma kinetic theory, *

  15. A novel in vitro system for gamete fusion in maize

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Various systems by using electric pulse, calcium, or polyethylene glycol have been developed in the past decade for the in vitro fusion of plant gametes. These in vitro systems provide a new way to study the fertilization mechanisms of plants. In this study, we developed a bovine serum albumin (BSA)-mediated fusion system for the in vitro fusion of maize gametes. The in vitro fusion of the isolated single egg cell and sperm cell of maize was observed microscopically in the BSA solution and the fertilized egg cell showed normal cell wall regeneration and nuclear division. The effects of the BSA concentration, pH value and calcium level on the efficiency of the maize gamete fusion were also assessed. BSA concentration and pH value did significantly affect the efficiency of the gamete fusion. Calcium was not necessary for the gamete fusion when BSA was present. The optimal solution for the gamete fusion contained 0.1% BSA, pH 6.0. The fusion frequency was as high as 96.7% in that optimal solution. This new in vitro fertilization system offers an alternative tool for the in vitro study of fertilization mechanisms with much simpler manipulating procedure than PEG system,and it will be especially useful for the in vitro study of the calcium dynamics during plant fertilization.

  16. Nuclear fusion inside condense matters

    Institute of Scientific and Technical Information of China (English)

    HE Jing-tang

    2007-01-01

    This article describes in detail the nuclear fusion inside condense matters--the Fleischmann-Pons effect, the reproducibility of cold fusions, self-consistentcy of cold fusions and the possible applications.

  17. In vitro and in vivo study of pluripotency in intraspecific hybrid cells obtained by fusion of murine embryonic stem cells with splenocytes.

    Science.gov (United States)

    Matveeva, N M; Shilov, A G; Kaftanovskaya, E M; Maximovsky, L P; Zhelezova, A I; Golubitsa, A N; Bayborodin, S I; Fokina, M M; Serov, O L

    1998-06-01

    Hypoxanthine phosphoribosyltransferase-deficient (HPRT-) mouse embryonic stem (ES) cells, HM-1 cells (genotype XY), were fused with adult female DD/c mouse spleen cells. As a result, a set of HAT-resistant clones was isolated. Four hybrid clones most similar in morphology and growth characteristics to the HM-1 cells were studied in detail with respect to their pluripotency. Of these, three clones contained 41-43 chromosomes, and one clone was nearly tetraploid. All the clones had the XXY set of sex chromosomes and expressed the HPRT of the somatic partner only. The hybrid clones shared features with the HM-1 cells, indicating that they retained their pluripotent properties: (1) embryonic ECMA-7 antigen, not TROMA-1 antigen, was present in most cells; (2) the hybrid cells showed high activity of endogenous alkaline phosphatase (AP); (3) all the hybrid clones were able to form complex embryoid bodies containing derivatives of all the embryonic germinal layers; (4) the hybrid cells contained synchronously replicating X chromosomes, indicating that they were in an active state; and (5) a set of chimeric animals was generated by injecting hybrid cells into BALB/c and C57BL/6J mouse blastocysts. Evidence for chimerism was provided by the spotted coat derived from 129/Ola mice and identification of 129/Ola glucose phosphate isomerase (GPI) in many organs. Thus the results obtained demonstrated that the hybrid cells retain their high pluripotency level despite the close contact of the "pluripotent" HM-1 genome with the "somatic" spleen cell genome during hybrid cell formation and the presence of the "somatic" X chromosome during many cell generations. The presence of HPRT of the somatic partner in many organs and tissues, including the testes in chimeric animals, shows that the "somatic" X chromosome segregates weakly, if at all, during development of the chimeras. There were no individuals with the 129/Ola genotype among the more than 50 offspring from chimeric mice. The

  18. Training Enhances Immune Cells Mitochondrial Biosynthesis, Fission, Fusion, and Their Antioxidant Capabilities Synergistically with Dietary Docosahexaenoic Supplementation

    Science.gov (United States)

    Busquets-Cortés, Carla; Capó, Xavier; Tur, Josep A.; Sureda, Antoni

    2016-01-01

    Exercise training induces adaptations in mitochondrial metabolism, dynamics, and oxidative protection. Omega-3 fatty acids change membrane lipid composition and modulate mitochondrial function. The aim was to investigate the effect of 8-week training and docosahexaenoic acid (DHA) supplementation (1.14 g/day) on the mitochondria dynamics and antioxidant status in peripheral blood mononuclear cells (PBMCs) from sportsmen. Subjects were assigned to an intervention (N = 9) or placebo groups (N = 7) in a randomized double-blind trial. Nutritional intervention significantly increased the DHA content in erythrocyte membranes from the experimental group. No significant differences were reported in terms of circulating PBMCs, Mn-superoxide dismutase protein levels, and their capability to produce reactive oxygen species. The proteins related to mitochondrial dynamics were, in general, increased after an 8-week training and this increase was enhanced by DHA supplementation. The content in mitofusins Mtf-1 and Mtf-2, optic atrophy protein-1 (Opa-1), and mitochondrial transcription factor A (Tfam) were significantly higher in the DHA-supplemented group after intervention. Cytochrome c oxidase (COX-IV) activity and uncoupling proteins UCP-2 and UCP-3 protein levels were increased after training, with higher UCP-3 levels in the supplemented group. In conclusion, training induced mitochondrial adaptations which may contribute to improved mitochondrial function. This mitochondrial response was modulated by DHA supplementation. PMID:27698953

  19. Training Enhances Immune Cells Mitochondrial Biosynthesis, Fission, Fusion, and Their Antioxidant Capabilities Synergistically with Dietary Docosahexaenoic Supplementation

    Directory of Open Access Journals (Sweden)

    Carla Busquets-Cortés

    2016-01-01

    Full Text Available Exercise training induces adaptations in mitochondrial metabolism, dynamics, and oxidative protection. Omega-3 fatty acids change membrane lipid composition and modulate mitochondrial function. The aim was to investigate the effect of 8-week training and docosahexaenoic acid (DHA supplementation (1.14 g/day on the mitochondria dynamics and antioxidant status in peripheral blood mononuclear cells (PBMCs from sportsmen. Subjects were assigned to an intervention (N=9 or placebo groups (N=7 in a randomized double-blind trial. Nutritional intervention significantly increased the DHA content in erythrocyte membranes from the experimental group. No significant differences were reported in terms of circulating PBMCs, Mn-superoxide dismutase protein levels, and their capability to produce reactive oxygen species. The proteins related to mitochondrial dynamics were, in general, increased after an 8-week training and this increase was enhanced by DHA supplementation. The content in mitofusins Mtf-1 and Mtf-2, optic atrophy protein-1 (Opa-1, and mitochondrial transcription factor A (Tfam were significantly higher in the DHA-supplemented group after intervention. Cytochrome c oxidase (COX-IV activity and uncoupling proteins UCP-2 and UCP-3 protein levels were increased after training, with higher UCP-3 levels in the supplemented group. In conclusion, training induced mitochondrial adaptations which may contribute to improved mitochondrial function. This mitochondrial response was modulated by DHA supplementation.

  20. Complementary Advanced Fusion Exploration

    Science.gov (United States)

    2005-08-01

    homographic computer vision image fusion, out-of-sequence measurement and track data handling, Nash bargaining approaches to sensor management... homographic fusion notions are identified together with the Nash approach, the pursuit-evasion approach to threat situation outcome determination, and the

  1. Controlled Nuclear Fusion.

    Science.gov (United States)

    Glasstone, Samuel

    This publication is one of a series of information booklets for the general public published by The United States Atomic Energy Commission. Among the topics discussed are: Importance of Fusion Energy; Conditions for Nuclear Fusion; Thermonuclear Reactions in Plasmas; Plasma Confinement by Magnetic Fields; Experiments With Plasmas; High-Temperature…

  2. Fusion helps diversification

    NARCIS (Netherlands)

    S. Liang; Z. Ren; M. de Rijke

    2014-01-01

    A popular strategy for search result diversification is to first retrieve a set of documents utilizing a standard retrieval method and then rerank the results. We adopt a different perspective on the problem, based on data fusion. Starting from the hypothesis that data fusion can improve performance

  3. Fusion of biological membranes

    Indian Academy of Sciences (India)

    K Katsov; M Müller; M Schick

    2005-06-01

    The process of membrane fusion has been examined by Monte Carlo simulation, and is found to be very different than the conventional picture. The differences in mechanism lead to several predictions, in particular that fusion is accompanied by transient leakage. This prediction has recently been verified. Self-consistent field theory is applied to examine the free energy barriers in the different scenarios.

  4. Controlled thermonuclear fusion

    CERN Document Server

    Bobin, Jean Louis

    2014-01-01

    The book is a presentation of the basic principles and main achievements in the field of nuclear fusion. It encompasses both magnetic and inertial confinements plus a few exotic mechanisms for nuclear fusion. The state-of-the-art regarding thermonuclear reactions, hot plasmas, tokamaks, laser-driven compression and future reactors is given.

  5. Effective Delivery of Antigen-Encapsulin Nanoparticle Fusions to Dendritic Cells Leads to Antigen-Specific Cytotoxic T Cell Activation and Tumor Rejection.

    Science.gov (United States)

    Choi, Bongseo; Moon, Hyojin; Hong, Sung Joon; Shin, Changsik; Do, Yoonkyung; Ryu, Seongho; Kang, Sebyung

    2016-08-23

    In cancer immunotherapy, robust and efficient activation of cytotoxic CD8(+) T cell immune responses is a promising, but challenging task. Dendritic cells (DCs) are well-known professional antigen presenting cells that initiate and regulate antigen-specific cytotoxic CD8(+) T cells that kill their target cells directly as well as secrete IFN-γ, a cytokine critical in tumor rejection. Here, we employed recently established protein cage nanoparticles, encapsulin (Encap), as antigenic peptide nanocarriers by genetically incorporating the OT-1 peptide of ovalbumin (OVA) protein to the three different positions of the Encap subunit. With them, we evaluated their efficacy in activating DC-mediated antigen-specific T cell cytotoxicity and consequent melanoma tumor rejection in vivo. DCs efficiently engulfed Encap and its variants (OT-1-Encaps), which carry antigenic peptides at different positions, and properly processed them within phagosomes. Delivered OT-1 peptides were effectively presented by DCs to naïve CD8(+) T cells successfully, resulting in the proliferation of antigen-specific cytotoxic CD8(+) T cells. OT-1-Encap vaccinations in B16-OVA melanoma tumor bearing mice effectively activated OT-1 peptide specific cytotoxic CD8(+) T cells before or even after tumor generation, resulting in significant suppression of tumor growth in prophylactic as well as therapeutic treatments. A large number of cytotoxic CD8(+) T cells that actively produce both intracellular and secretory IFN-γ were observed in tumor-infiltrating lymphocytes collected from B16-OVA tumor masses originally vaccinated with OT-1-Encap-C upon tumor challenges. The approaches we describe herein may provide opportunities to develop epitope-dependent vaccination systems that stimulate and/or modulate efficient and epitope-specific cytotoxic T cell immune responses in nonpathogenic diseases.

  6. Heterogeneous breakpoints on the immunoglobulin genes are involved in fusion with the 5' region of BCL2 in B-cell tumors.

    Science.gov (United States)

    Yonetani, N; Ueda, C; Akasaka, T; Nishikori, M; Uchiyama, T; Ohno, H

    2001-09-01

    The 5' flanking region of the BCL2 gene (5'-BCL2) is a breakpoint cluster of rearrangements with immunoglobulin genes (IGs). In contrast to t(14;18)(q32;q21) affecting the 3' region of BCL2, 5'-BCL2 can fuse to not only the heavy chain gene (IGH), but also two light chain gene (IGL) loci. We report here cloning and sequencing of a total of eleven 5'-BCL2 / IGs junctional areas of B-cell tumors, which were amplified by long-distance polymerase chain reaction-based assays. The breakpoints on 5'-BCL2 were distributed from 378 to 2312 bp upstream of the translational initiation site and, reflecting the alteration of regulatory sequences of BCL2, 5'-BCL2 / IGs-positive cells showed markedly higher levels of BCL2 expression than those of t(14;18)-positive cells. In contrast, the breakpoints on the IGs were variable. Two 5'-BCL2 / IGH and two 5'-BCL2 / IGLkappa junctions occurred 5' of the joining (J) segments, suggesting operation of an erroneous variable (V) / diversity (D) / J and V / J rearrangement mechanism. However, two other 5'-BCL2 / IGH junctions affected switch regions, and the kappa-deleting element, which is located 24 kb downstream of the constant region of IGLkappa, followed the 5'-BCL2 in another case. One 5'-BCL2 / IGLkappa and two 5'-BCL2 / IGLlambda junctions involved intronic regions where the normal recombination process does not occur. In the remaining one case, the 5'-BCL2 fused 3' of a Vlambda gene that was upstream of another Vlambda / Jlambda complex carrying a non-producing configuration, indicating that the receptor editing mechanism was likely involved in this rearrangement. Our study revealed heterogeneous anatomy of the 5'-BCL2 / IGs fusion gene leading to transcriptional activation of BCL2, and suggested that the mechanisms underlying the formation of this particular oncogene / IGs recombination are not identical to those of t(14;18).

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

    Science.gov (United States)

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

    2015-11-01

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

  8. Compact fusion reactors

    CERN Document Server

    CERN. Geneva

    2015-01-01

    Fusion research is currently to a large extent focused on tokamak (ITER) and inertial confinement (NIF) research. In addition to these large international or national efforts there are private companies performing fusion research using much smaller devices than ITER or NIF. The attempt to achieve fusion energy production through relatively small and compact devices compared to tokamaks decreases the costs and building time of the reactors and this has allowed some private companies to enter the field, like EMC2, General Fusion, Helion Energy, Lawrenceville Plasma Physics and Lockheed Martin. Some of these companies are trying to demonstrate net energy production within the next few years. If they are successful their next step is to attempt to commercialize their technology. In this presentation an overview of compact fusion reactor concepts is given.

  9. The rise of developmental genetics - a historical account of the fusion of embryology and cell biology with human genetics and the emergence of the Stem Cell Initiative.

    Science.gov (United States)

    Kidson, S H; Ballo, R; Greenberg, L J

    2016-05-25

    Genetics and cell biology are very prominent areas of biological research with rapid advances being driven by a flood of theoretical, technological and informational knowledge. Big biology and small biology continue to feed off each other. In this paper, we provide a brief overview of the productive interactions that have taken place between human geneticists and cell biologists at UCT, and credit is given to the enabling environment created led by Prof. Peter Beighton. The growth of new disciplines and disciplinary mergers that have swept away division of the past to make new exciting syntheses are discussed. We show how our joint research has benefitted from worldwide advances in developmental genetics, cloning and stem cell technologies, genomics, bioinformatics and imaging. We conclude by describing the role of the UCT Stem Cell Initiative and show how we are using induced pluripotent cells to carry out disease-in-the- dish studies on retinal degeneration and fibrosis.

  10. A novel approach for targeted elimination of CSPG4-positive triple-negative breast cancer cells using a MAP tau-based fusion protein.

    Science.gov (United States)

    Amoury, Manal; Mladenov, Radoslav; Nachreiner, Thomas; Pham, Anh-Tuan; Hristodorov, Dmitrij; Di Fiore, Stefano; Helfrich, Wijnand; Pardo, Alessa; Fey, Georg; Schwenkert, Michael; Thepen, Theophilus; Kiessling, Fabian; Hussain, Ahmad F; Fischer, Rainer; Kolberg, Katharina; Barth, Stefan

    2016-08-15

    Chondroitin sulfate proteoglycan 4 (CSPG4) has been identified as a highly promising target antigen for immunotherapy of triple-negative breast cancer (TNBC). TNBC represents a highly aggressive heterogeneous group of tumors lacking expression of estrogen, progesterone and human epidermal growth factor receptor 2. TNBC is particularly prevalent among young premenopausal women. No suitable targeted therapies are currently available and therefore, novel agents for the targeted elimination of TNBC are urgently needed. Here, we present a novel cytolytic fusion protein (CFP), designated αCSPG4(scFv)-MAP, that consists of a high affinity CSPG4-specific single-chain antibody fragment (scFv) genetically fused to a functionally enhanced form of the human microtubule-associated protein (MAP) tau. Our data indicate that αCSPG4(scFv)-MAP efficiently targets CSPG4(+) TNBC-derived cell lines MDA-MB-231 and Hs 578T and potently inhibits their growth with IC50 values of ∼200 nM. Treatment with αCSPG(scFv)-MAP resulted in induction of the mitochondrial stress pathway by activation of caspase-9 as well as endonuclease G translocation to the nucleus, while induction of the caspase-3 apoptosis pathway was not detectable. Importantly, in vivo studies in mice bearing human breast cancer xenografts revealed efficient targeting to and accumulation of αCSPG4(scFv)-MAP at tumor sites resulting in prominent tumor regression. Taken together, this preclinical proof of concept study confirms the potential clinical value of αCSPG4(scFv)-MAP as a novel targeted approach for the elimination of CSPG4-positive TNBC.

  11. Protein-protein fusion catalyzed by sortase A.

    Science.gov (United States)

    Levary, David A; Parthasarathy, Ranganath; Boder, Eric T; Ackerman, Margaret E

    2011-04-06

    Chimeric proteins boast widespread use in areas ranging from cell biology to drug delivery. Post-translational protein fusion using the bacterial transpeptidase sortase A provides an attractive alternative when traditional gene fusion fails. We describe use of this enzyme for in vitro protein ligation and report the successful fusion of 10 pairs of protein domains with preserved functionality--demonstrating the robust and facile nature of this reaction.

  12. Protein-protein fusion catalyzed by sortase A.

    Directory of Open Access Journals (Sweden)

    David A Levary

    Full Text Available Chimeric proteins boast widespread use in areas ranging from cell biology to drug delivery. Post-translational protein fusion using the bacterial transpeptidase sortase A provides an attractive alternative when traditional gene fusion fails. We describe use of this enzyme for in vitro protein ligation and report the successful fusion of 10 pairs of protein domains with preserved functionality--demonstrating the robust and facile nature of this reaction.

  13. Fusion of NUP98 and the SET binding protein 1 (SETBP1) gene in a paediatric acute T cell lymphoblastic leukaemia with t(11;18)(p15;q12)

    DEFF Research Database (Denmark)

    Panagopoulos, Ioannis; Kerndrup, Gitte; Carlsen, Niels

    2007-01-01

    Three NUP98 chimaeras have previously been reported in T cell acute lymphoblastic leukaemia (T-ALL): NUP98/ADD3, NUP98/CCDC28A, and NUP98/RAP1GDS1. We report a T-ALL with t(11;18)(p15;q12) resulting in a novel NUP98 fusion. Fluorescent in situ hybridisation showed NUP98 and SET binding protein 1......(SETBP1) fusion signals; other analyses showed that exon 12 of NUP98 was fused in-frame with exon 5 of SETBP1. Nested polymerase chain reaction did not amplify the reciprocal SETBP1/NUP98, suggesting that NUP98/SETBP1 transcript is pathogenetically important. SETBP1 has previously not been implicated...

  14. Fusion transcriptome profiling provides insights into alveolar rhabdomyosarcoma.

    Science.gov (United States)

    Xie, Zhongqiu; Babiceanu, Mihaela; Kumar, Shailesh; Jia, Yuemeng; Qin, Fujun; Barr, Frederic G; Li, Hui

    2016-11-15

    Gene fusions and fusion products were thought to be unique features of neoplasia. However, more and more studies have identified fusion RNAs in normal physiology. Through RNA sequencing of 27 human noncancer tissues, a large number of fusion RNAs were found. By analyzing fusion transcriptome, we observed close clusterings between samples of same or similar tissues, supporting the feasibility of using fusion RNA profiling to reveal connections between biological samples. To put the concept into use, we selected alveolar rhabdomyosarcoma (ARMS), a myogenic pediatric cancer whose exact cell of origin is not clear. PAX3-FOXO1 (paired box gene 3 fused with forkhead box O1) fusion RNA, which is considered a hallmark of ARMS, was recently found during normal muscle cell differentiation. We performed and analyzed RNA sequencing from various time points during myogenesis and uncovered many chimeric fusion RNAs. Interestingly, we found that the fusion RNA profile of RH30, an ARMS cell line, is most similar to the myogenesis time point when PAX3-FOXO1 is expressed. In contrast, full transcriptome clustering analysis failed to uncover this connection. Strikingly, all of the 18 chimeric RNAs in RH30 cells could be detected at the same myogenic time point(s). In addition, the seven chimeric RNAs that follow the exact transient expression pattern as PAX3-FOXO1 are specific to rhabdomyosarcoma cells. Further testing with clinical samples also confirmed their specificity to rhabdomyosarcoma. These results provide further support for the link between at least some ARMSs and the PAX3-FOXO1-expressing myogenic cells and demonstrate that fusion RNA profiling can be used to investigate the etiology of fusion-gene-associated cancers.

  15. A Fusion-Inhibiting Peptide against Rift Valley Fever Virus Inhibits Multiple, Diverse Viruses

    Science.gov (United States)

    2013-09-12

    opportunities to disrupt early stages in viral replication and prevent a productive infection [11,13]. Targeting this viral entry process with inhibitory...stage of the viral replication cycle. Enveloped viruses require fusion of viral and cellular membranes for the viral genome to enter the cell cytoplasm...Rad, Hercules, CA). Cell-cell fusion assay A plasmid based cell-cell fusion assay was developed similar to the alphavirus replicon-based system

  16. Resveratrol stimulates mitochondrial fusion by a mechanism requiring mitofusin-2.

    Science.gov (United States)

    Robb, Ellen L; Moradi, Fereshteh; Maddalena, Lucas A; Valente, Andrew J F; Fonseca, Joao; Stuart, Jeffrey A

    2017-04-01

    Resveratrol (RES) is a plant-derived stilbene associated with a wide range of health benefits. Mitochondria are a key downstream target of RES, and in some cell types RES promotes mitochondrial biogenesis, altered cellular redox status, and a shift toward oxidative metabolism. Mitochondria exist as a dynamic network that continually remodels via fusion and fission processes, and the extent of fusion is related to cellular redox status and metabolism. We investigated RES's effects on mitochondrial network morphology in several cell lines using a quantitative approach to measure the extent of network fusion. 48 h continuous treatment with 10-20 μM RES stimulated mitochondrial fusion in C2C12 myoblasts, PC3 cancer cells, and mouse embryonic fibroblasts stimulated significant increases in fusion in all instances, resulting in larger and more highly branched mitochondrial networks. Mitofusin-2 (Mfn2) is a key protein facilitating mitochondrial fusion, and its expression was also stimulated by RES. Using Mfn2-null cells we demonstrated that RES's effects on mitochondrial fusion, cellular respiration rates, and cell growth are all dependent upon the presence of Mfn2. Taken together, these results demonstrate that Mfn2 and mitochondrial fusion are affected by RES in ways that appear to relate to RES's known effects on cellular metabolism and growth.

  17. Fusion Studies in Japan

    Science.gov (United States)

    Ogawa, Yuichi

    2016-05-01

    A new strategic energy plan decided by the Japanese Cabinet in 2014 strongly supports the steady promotion of nuclear fusion development activities, including the ITER project and the Broader Approach activities from the long-term viewpoint. Atomic Energy Commission (AEC) in Japan formulated the Third Phase Basic Program so as to promote an experimental fusion reactor project. In 2005 AEC has reviewed this Program, and discussed on selection and concentration among many projects of fusion reactor development. In addition to the promotion of ITER project, advanced tokamak research by JT-60SA, helical plasma experiment by LHD, FIREX project in laser fusion research and fusion engineering by IFMIF were highly prioritized. Although the basic concept is quite different between tokamak, helical and laser fusion researches, there exist a lot of common features such as plasma physics on 3-D magnetic geometry, high power heat load on plasma facing component and so on. Therefore, a synergetic scenario on fusion reactor development among various plasma confinement concepts would be important.

  18. Control of Fusion and Solubility in Fusion Systems

    CERN Document Server

    Craven, David A

    2009-01-01

    In this article, we consider the control of fusion in fusion systems, proving three previously known, non-trivial results in a new, largely elementary way. We then reprove a result of Aschbacher, that the product of two strongly closed subgroups is strongly closed; to do this, we consolidate the theory of quotients of fusion systems into a consistent theory. We move on considering p-soluble fusion systems, and prove that they are constrained, allowing us to effectively characterize fusion systems of p-soluble groups. This leads us to recast Thompson Factorization for Qd(p)-free fusion systems, and consider Thompson Factorization for more general fusion systems.

  19. Remote sensing image fusion

    CERN Document Server

    Alparone, Luciano; Baronti, Stefano; Garzelli, Andrea

    2015-01-01

    A synthesis of more than ten years of experience, Remote Sensing Image Fusion covers methods specifically designed for remote sensing imagery. The authors supply a comprehensive classification system and rigorous mathematical description of advanced and state-of-the-art methods for pansharpening of multispectral images, fusion of hyperspectral and panchromatic images, and fusion of data from heterogeneous sensors such as optical and synthetic aperture radar (SAR) images and integration of thermal and visible/near-infrared images. They also explore new trends of signal/image processing, such as

  20. Efficient activation of human T cells of both CD4 and CD8 subsets by urease-deficient recombinant Mycobacterium bovis BCG that produced a heat shock protein 70-M. tuberculosis-derived major membrane protein II fusion protein.

    Science.gov (United States)

    Mukai, Tetsu; Tsukamoto, Yumiko; Maeda, Yumi; Tamura, Toshiki; Makino, Masahiko

    2014-01-01

    For the purpose of obtaining Mycobacterium bovis bacillus Calmette-Guérin (BCG) capable of activating human naive T cells, urease-deficient BCG expressing a fusion protein composed of Mycobacterium tuberculosis-derived major membrane protein II (MMP-II) and heat shock protein 70 (HSP70) of BCG (BCG-DHTM) was produced. BCG-DHTM secreted the HSP70-MMP-II fusion protein and effectively activated human monocyte-derived dendritic cells (DCs) by inducing phenotypic changes and enhanced cytokine production. BCG-DHTM-infected DCs activated naive T cells of both CD4 and naive CD8 subsets, in an antigen (Ag)-dependent manner. The T cell activation induced by BCG-DHTM was inhibited by the pretreatment of DCs with chloroquine. The naive CD8(+) T cell activation was mediated by the transporter associated with antigen presentation (TAP) and the proteosome-dependent cytosolic cross-priming pathway. Memory CD8(+) T cells and perforin-producing effector CD8(+) T cells were efficiently produced from the naive T cell population by BCG-DHTM stimulation. Single primary infection with BCG-DHTM in C57BL/6 mice efficiently produced T cells responsive to in vitro secondary stimulation with HSP70, MMP-II, and M. tuberculosis-derived cytosolic protein and inhibited the multiplication of subsequently aerosol-challenged M. tuberculosis more efficiently than did vector control BCG. These results indicate that the introduction of MMP-II and HSP70 into urease-deficient BCG may be useful for improving BCG for control of tuberculosis.

  1. G-protein coupled receptor 56 promotes myoblast fusion through serum response factor- and nuclear factor of activated T-cell-mediated signalling but is not essential for muscle development in vivo.

    Science.gov (United States)

    Wu, Melissa P; Doyle, Jamie R; Barry, Brenda; Beauvais, Ariane; Rozkalne, Anete; Piao, Xianhua; Lawlor, Michael W; Kopin, Alan S; Walsh, Christopher A; Gussoni, Emanuela

    2013-12-01

    Mammalian muscle cell differentiation is a complex process of multiple steps for which many of the factors involved have not yet been defined. In a screen to identify the regulators of myogenic cell fusion, we found that the gene for G-protein coupled receptor 56 (GPR56) was transiently up-regulated during the early fusion of human myoblasts. Human mutations in the gene for GPR56 cause the disease bilateral frontoparietal polymicrogyria; however, the consequences of receptor dysfunction on muscle development have not been explored. Using knockout mice, we defined the role of GPR56 in skeletal muscle. GPR56(-/-) myoblasts have decreased fusion and smaller myotube sizes in culture. In addition, a loss of GPR56 expression in muscle cells results in decreases or delays in the expression of myogenic differentiation 1, myogenin and nuclear factor of activated T-cell (NFAT)c2. Our data suggest that these abnormalities result from decreased GPR56-mediated serum response element and NFAT signalling. Despite these changes, no overt differences in phenotype were identified in the muscle of GPR56 knockout mice, which presented only a mild but statistically significant elevation of serum creatine kinase compared to wild-type. In agreement with these findings, clinical data from 13 bilateral frontoparietal polymicrogyria patients revealed mild serum creatine kinase increase in only two patients. In summary, targeted disruption of GPR56 in mice results in myoblast abnormalities. The absence of a severe muscle phenotype in GPR56 knockout mice and human patients suggests that other factors may compensate for the lack of this G-protein coupled receptor during muscle development and that the motor delay observed in these patients is likely not a result of primary muscle abnormalities.

  2. Ploidy reductions in murine fusion-derived hepatocytes.

    Directory of Open Access Journals (Sweden)

    Andrew W Duncan

    2009-02-01

    Full Text Available We previously showed that fusion between hepatocytes lacking a crucial liver enzyme, fumarylacetoacetate hydrolase (FAH, and wild-type blood cells resulted in hepatocyte reprogramming. FAH expression was restored in hybrid hepatocytes and, upon in vivo expansion, ameliorated the effects of FAH deficiency. Here, we show that fusion-derived polyploid hepatocytes can undergo ploidy reductions to generate daughter cells with one-half chromosomal content. Fusion hybrids are, by definition, at least tetraploid. We demonstrate reduction to diploid chromosome content by multiple methods. First, cytogenetic analysis of fusion-derived hepatocytes reveals a population of diploid cells. Secondly, we demonstrate marker segregation using ss-galactosidase and the Y-chromosome. Approximately 2-5% of fusion-derived FAH-positive nodules were negative for one or more markers, as expected during ploidy reduction. Next, using a reporter system in which ss-galactosidase is expressed exclusively in fusion-derived hepatocytes, we identify a subpopulation of diploid cells expressing ss-galactosidase and FAH. Finally, we track marker segregation specifically in fusion-derived hepatocytes with diploid DNA content. Hemizygous markers were lost by >or=50% of Fah-positive cells. Since fusion-derived hepatocytes are minimally tetraploid, the existence of diploid hepatocytes demonstrates that fusion-derived cells can undergo ploidy reduction. Moreover, the high degree of marker loss in diploid daughter cells suggests that chromosomes/markers are lost in a non-random fashion. Thus, we propose that ploidy reductions lead to the generation of genetically diverse daughter cells with about 50% reduction in nuclear content. The generation of such daughter cells increases liver diversity, which may increase the likelihood of oncogenesis.

  3. Cold nuclear fusion

    Directory of Open Access Journals (Sweden)

    Huang Zhenqiang Huang Yuxiang

    2013-10-01

    Full Text Available In normal temperature condition, the nuclear force constraint inertial guidance method, realize the combination of deuterium and tritium, helium and lithium... And with a magnetic moment of light nuclei controlled cold nuclear collide fusion, belongs to the nuclear energy research and development in the field of applied technology "cold nuclear collide fusion". According to the similarity of the nuclear force constraint inertial guidance system, the different velocity and energy of the ion beam mixing control, developed ion speed dc transformer, it is cold nuclear fusion collide, issue of motivation and the nuclear power plant start-up fusion and power transfer system of the important equipment, so the merger to apply for a patent

  4. Fusion Revisits CERN

    CERN Multimedia

    2001-01-01

    It's going to be a hot summer at CERN. At least in the Main Building, where from 13 July to 20 August an exhibition is being hosted on nuclear fusion, the energy of the Stars. Nuclear fusion is the engine driving the stars but also a potential source of energy for mankind. The exhibition shows the different nuclear fusion techniques and research carried out on the subject in Europe. Inaugurated at CERN in 1993, following collaboration between Lausanne's CRPP-EPFL and CERN, with input from Alessandro Pascolini of Italy's INFN, this exhibition has travelled round Europe before being revamped and returning to CERN. 'Fusion, Energy of the Stars', from 13 July onwards, Main Building

  5. Laser-Driven Fusion.

    Science.gov (United States)

    Gibson, A. F.

    1980-01-01

    Discusses the present status and future prospects of laser-driven fusion. Current research (which is classified under three main headings: laser-matter interaction processes, compression, and laser development) is also presented. (HM)

  6. Fusion plasma physics

    CERN Document Server

    Stacey, Weston M

    2012-01-01

    This revised and enlarged second edition of the popular textbook and reference contains comprehensive treatments of both the established foundations of magnetic fusion plasma physics and of the newly developing areas of active research. It concludes with a look ahead to fusion power reactors of the future. The well-established topics of fusion plasma physics -- basic plasma phenomena, Coulomb scattering, drifts of charged particles in magnetic and electric fields, plasma confinement by magnetic fields, kinetic and fluid collective plasma theories, plasma equilibria and flux surface geometry, plasma waves and instabilities, classical and neoclassical transport, plasma-materials interactions, radiation, etc. -- are fully developed from first principles through to the computational models employed in modern plasma physics. The new and emerging topics of fusion plasma physics research -- fluctuation-driven plasma transport and gyrokinetic/gyrofluid computational methodology, the physics of the divertor, neutral ...

  7. Optical Fiber Fusion Splicing

    CERN Document Server

    Yablon, Andrew D

    2005-01-01

    This book is an up-to-date treatment of optical fiber fusion splicing incorporating all the recent innovations in the field. It provides a toolbox of general strategies and specific techniques that the reader can apply when optimizing fusion splices between novel fibers. It specifically addresses considerations important for fusion splicing of contemporary specialty fibers including dispersion compensating fiber, erbium-doped gain fiber, polarization maintaining fiber, and microstructured fiber. Finally, it discusses the future of optical fiber fusion splicing including silica and non-silica based optical fibers as well as the trend toward increasing automation. Whilst serving as a self-contained reference work, abundant citations from the technical literature will enable readers to readily locate primary sources.

  8. Sampling Based Average Classifier Fusion

    Directory of Open Access Journals (Sweden)

    Jian Hou

    2014-01-01

    fusion algorithms have been proposed in literature, average fusion is almost always selected as the baseline for comparison. Little is done on exploring the potential of average fusion and proposing a better baseline. In this paper we empirically investigate the behavior of soft labels and classifiers in average fusion. As a result, we find that; by proper sampling of soft labels and classifiers, the average fusion performance can be evidently improved. This result presents sampling based average fusion as a better baseline; that is, a newly proposed classifier fusion algorithm should at least perform better than this baseline in order to demonstrate its effectiveness.

  9. Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion

    Directory of Open Access Journals (Sweden)

    de la Vega Michelle

    2011-12-01

    Full Text Available Abstract Background We recently provided evidence that HIV-1 enters HeLa-derived TZM-bl and lymphoid CEMss cells by fusing with endosomes, whereas its fusion with the plasma membrane does not proceed beyond the lipid mixing step. The mechanism of restriction of HIV-1 fusion at the cell surface and/or the factors that aid the virus entry from endosomes remain unclear. Results We examined HIV-1 fusion with a panel of target cells lines and with primary CD4+ T cells. Kinetic measurements of fusion combined with time-resolved imaging of single viruses further reinforced the notion that HIV-1 enters the cells via endocytosis and fusion with endosomes. Furthermore, we attempted to deliberately redirect virus fusion to the plasma membrane, using two experimental strategies. First, the fusion reaction was synchronized by pre-incubating the viruses with cells at reduced temperature to allow CD4 and coreceptors engagement, but not the virus uptake or fusion. Subsequent shift to a physiological temperature triggered accelerated virus uptake followed by entry from endosomes, but did not permit fusion at the cell surface. Second, blocking HIV-1 endocytosis by a small-molecule dynamin inhibitor, dynasore, resulted in transfer of viral lipids to the plasma membrane without any detectable release of the viral content into the cytosol. We also found that a higher concentration of dynasore is required to block the HIV-endosome fusion compared to virus internalization. Conclusions Our results further support the notion that HIV-1 enters disparate cell types through fusion with endosomes. The block of HIV-1 fusion with the plasma membrane at a post-lipid mixing stage shows that this membrane is not conducive to fusion pore formation and/or enlargement. The ability of dynasore to interfere with the virus-endosome fusion suggests that dynamin could be involved in two distinct steps of HIV-1 entry - endocytosis and fusion within intracellular compartments.

  10. Fusion ignition research experiment

    Energy Technology Data Exchange (ETDEWEB)

    Dale Meade

    2000-07-18

    Understanding the properties of high gain (alpha-dominated) fusion plasmas in an advanced toroidal configuration is the largest remaining open issue that must be addressed to provide the scientific foundation for an attractive magnetic fusion reactor. The critical parts of this science can be obtained in a compact high field tokamak which is also likely to provide the fastest and least expensive path to understanding alpha-dominated plasmas in advanced toroidal systems.

  11. Economically competitive fusion

    Directory of Open Access Journals (Sweden)

    David J. Ward

    2008-12-01

    Full Text Available Not since the oil crisis of the 1970s has the perception that energy is a crucial and precious resource been as strong as it is today. The need for a new approach to world energy supply, driven by concerns over resources, pollution, and security, is leading to a reappraisal of fusion. Fusion has enormous potential and major safety and environmental advantages, and hence could make a large difference to energy supplies.

  12. Fusion dual-tracer SPECT-based hepatic dosimetry predicts outcome after radioembolization for a wide range of tumour cell types

    NARCIS (Netherlands)

    Lam, Marnix G. E. H.; Banerjee, Arjun; Goris, Michael L.; Iagaru, Andrei H.; Mittra, Erik S.; Louie, John D.; Sze, Daniel Y.

    2015-01-01

    Purpose Fusion dual-tracer SPECT imaging enables physiological rather than morphological voxel-based partitioning and dosimetry for Y-90 hepatic radioembolization (RE). We evaluated its prognostic value in a large heterogeneous cohort of patients with extensive hepatic malignancy. Methods A total of

  13. A chimeric LysK-lysostaphin fusion enzyme lysing Staphylococcus aureus cells: a study of both kinetics of inactivation and specifics of interaction with anionic polymers

    Science.gov (United States)

    A staphylolytic fusion protein (K-L) was created, harboring three unique lytic activities comprised of the LysK CHAP endopeptidase, and amidase domains, and the lysostaphin glycyl-glycine endopeptidase domain. To assess the potential of possible therapeutic applications, the kinetic behavior of K-L...

  14. Understanding of decreased sialylation of Fc-fusion protein in hyperosmotic recombinant Chinese hamster ovary cell culture: N-glycosylation gene expression and N-linked glycan antennary profile.

    Science.gov (United States)

    Lee, Jong Hyun; Jeong, Yeong Ran; Kim, Yeon-Gu; Lee, Gyun Min

    2017-03-07

    To understand the effects of hyperosmolality on protein glycosylation, recombinant Chinese hamster ovary (rCHO) cells producing the Fc-fusion protein were cultivated in hyperosmolar medium resulting from adding NaCl (415 mOsm/kg). The hyperosmotic culture showed increased specific Fc-fusion protein productivity (qFc ) but a decreased proportion of acidic isoforms and sialic acid content of the Fc-fusion protein. The intracellular and extracellular sialidase activities in the hyperosmotic cultures were similar to those in the control culture (314 mOsm/kg), indicating that reduced sialylation of Fc-fusion protein at hyperosmolality was not due to elevated sialidase activity. Expression of 52 N-glycosylation-related genes was assessed by the NanoString nCounter system, which provides a direct digital readout using custom-designed color-coded probes. After three days of hyperosmotic culture, nine genes (ugp, slc35a3, slc35d2, gcs1, manea, mgat2, mgat5b, b4galt3, and b4galt4) were differentially expressed over 1.5-fold of the control, and all these genes were down-regulated. N-linked glycan analysis by anion exchange and hydrophilic interaction HPLC showed that the proportion of highly sialylated (di-, tri-, tetra-) and tetra-antennary N-linked glycans was significantly decreased upon hyperosmotic culture. Addition of betaine, an osmoprotectant, to the hyperosmotic culture significantly increased the proportion of highly sialylated and tetra-antennary N-linked glycans (P ≤ 0.05), while it increased the expression of the N-glycan branching/antennary genes (mgat2 and mgat4b). Thus, decreased expression of the genes with roles in the N-glycan biosynthesis pathway correlated with reduced sialic acid content of Fc-fusion protein caused by hyperosmolar conditions. Taken together, the results obtained in this study provide a better understanding of the detrimental effects of hyperosmolality on N-glycosylation, especially sialylation, in rCHO cells. This article is protected

  15. Fusion, cold fusion, and space policy

    Energy Technology Data Exchange (ETDEWEB)

    Rotegard, D. (CST Ltd. (United States))

    1991-01-01

    This paper critiques Americal science policy through a consideration of two examples-cold fusion and asteroid mining. It points out that the failure of central planning in science and technology policy is just as marked as in more mundane activities. It highlights the current low level of debate and points out some technical issues that need to be addressed. It concludes with evidence that the alliance of flawed policy options is further lowering the level of debate. (author).

  16. EDITORIAL: Safety aspects of fusion power plants

    Science.gov (United States)

    Kolbasov, B. N.

    2007-07-01

    &E potential of fusion can be attained by prudent materials selection, judicious design choices, and integration of safety requirements into the design of the facility. To achieve this goal, S&E research is focused on understanding the behaviour of the largest sources of radioactive and hazardous materials in a fusion facility, understanding how energy sources in a fusion facility could mobilize those materials, developing integrated state-of-the-art S&E computer codes and risk tools for safety assessment, and evaluating and improving fusion facility design in terms of accident safety, worker safety, and waste disposal. There are three papers considering safety issues of the test blanket modules (TBM) producing tritium to be installed in ITER. These modules represent different concepts of demonstration fusion power facilities (DEMO). L. Boccaccini et al (Germany) analyses the possibility of jeopardizing the ITER safety under specific accidents in the European helium-cooled pebble-bed TBM, e.g. pressurization of the vacuum vessel (VV), hydrogen production from the Be-steam reaction, the possible interconnection between the port cell and VV causing air ingress. Safety analysis is also presented for Chinese TBM with a helium-cooled solid breeder to be tested in ITER by Z. Chen et al (China). Radiological inventories, afterheat, waste disposal ratings, electromagnetic characteristics, LOCA and tritium safety management are considered. An overview of a preliminary safety analysis performed for a US proposed TBM is presented by B. Merrill et al (USA). This DEMO relevant dual coolant liquid lead-lithium TBM has been explored both in the USA and EU. T. Pinna et al (Italy) summarize the six-year development of a failure rate database for fusion specific components on the basis of data coming from operating experience gained in various fusion laboratories. The activity began in 2001 with the study of the Joint European Torus vacuum and active gas handling systems. Two years later the

  17. 分泌型PTD4-Apoptin融合蛋白诱导HepG2细胞凋亡%Effect of Secretive PTD4-Apoptin Fusion Protein on Apoptosis of HepG2 Cells

    Institute of Scientific and Technical Information of China (English)

    陈庆; 杨辰苏; 陈虎; 陈道达; 龙跃平; 郑海

    2012-01-01

    Objective To investigate the effect of PTD4-Apoptin secreted by human umbilical venous endothelial cellsCHU-VECs)on apoptosis of HepG2 cells and the possible application for hepatocellular carcinoma gene therapy. Methods Recombi-nant plasmid of pSecTag2-PTD4-Apoptin was constructed and identified by restriction enzyme digestion analysis and DNA sequencing. HUVKCs were transiently transfected with pSecTag2-PTD4-Apoptin by lipofectamine and the culture supernatant was collected at 48 h after transfection. The expression and secretion of the PTD4-Apoptin fusion protein were detected by Western blot. HepG2 and L02 cells were co-cultured with the supernatant respectively. At 24 h after culture, the distribution of PTD4-Apoptin fusion protein in cells was detected by Western blot and apoptosis rate was measured by flow cytometry. Results The PTD4-Apoptin fusion protein secreted by pSecTag2-PTD4-Apoptin-transfected HUVECs could reenter adjacent untrans-fected HepG2 and L02 cells. The PTD4-Apoptin fusion protein could assemble in HepG2 nucleus and induce apoptosis of HepG2 cells(47. 4% ,P<0. 001). This effect was not detected in L02 cells. Conclusion The PTD4-Apoptin fusion protein secreted by pSecTag2-PTD4-Apoptin-transfected HUVECs can induce the apoptosis of adjacent untransfected HepG2 cells but not in L02 cells.%目的 研究分泌型的含蛋白质转导结构域的凋亡素(PTD4-Apoptin)融合基因经人脐静脉血管内皮细胞(HUVEC)表达和分泌后,对人肝癌HepG2细胞凋亡的影响,探讨其用于肝癌治疗的可能性.方法 构建PTD4-Apoptin融合基因的pSecTag2分泌型真核表达载体,并采用脂质体介导将其转染入HUVEC细胞,Western blot检测PTD4-Apoptin融合蛋白的表达及分泌,转染48 h后收集培养上清作为条件培养液,用于HepG2、L02细胞培养,共培养24 h后,采用Western blot检测细胞内和核内Apoptin的含量,流式细胞术检测细胞凋亡.结果 瞬时转染pSecTag2-PTD4-Apoptin

  18. PI(4,5)P2 regulates myoblast fusion through Arp2/3 regulator localization at the fusion site.

    Science.gov (United States)

    Bothe, Ingo; Deng, Su; Baylies, Mary

    2014-06-01

    Cell-cell fusion is a regulated process that requires merging of the opposing membranes and underlying cytoskeletons. However, the integration between membrane and cytoskeleton signaling during fusion is not known. Using Drosophila, we demonstrate that the membrane phosphoinositide PI(4,5)P2 is a crucial regulator of F-actin dynamics during myoblast fusion. PI(4,5)P2 is locally enriched and colocalizes spatially and temporally with the F-actin focus that defines the fusion site. PI(4,5)P2 enrichment depends on receptor engagement but is upstream or parallel to actin remodeling. Regulators of actin branching via Arp2/3 colocalize with PI(4,5)P2 in vivo and bind PI(4,5)P2 in vitro. Manipulation of PI(4,5)P2 availability leads to impaired fusion, with a reduction in the F-actin focus size and altered focus morphology. Mechanistically, the changes in the actin focus are due to a failure in the enrichment of actin regulators at the fusion site. Moreover, improper localization of these regulators hinders expansion of the fusion interface. Thus, PI(4,5)P2 enrichment at the fusion site encodes spatial and temporal information that regulates fusion progression through the localization of activators of actin polymerization.

  19. Fusion of Epstein-Barr virus nuclear antigen-1-derived glycine-alanine repeat to trans-dominant HIV-1 Gag increases inhibitory activities and survival of transduced cells in vivo.

    Science.gov (United States)

    Hammer, Diana; Wild, Jens; Ludwig, Christine; Asbach, Benedikt; Notka, Frank; Wagner, Ralf

    2008-06-01

    Trans-dominant human immunodeficiency virus type 1 (HIV-1) Gag derivatives have been shown to efficiently inhibit late steps of HIV-1 replication in vitro by interfering with Gag precursor assembly, thus ranking among the interesting candidates for gene therapy approaches. However, efficient antiviral activities of corresponding transgenes are likely to be counteracted in particular by cell-mediated host immune responses toward the transgene-expressing cells. To decrease this potential immunogenicity, a 24-amino acid Gly-Ala (GA) stretch derived from Epstein-Barr virus nuclear antigen-1 (EBNA1) and known to overcome proteasomal degradation was fused to a trans-dominant Gag variant (sgD1). To determine the capacity of this fusion polypeptide to repress viral replication, PM-1 cells were transduced with sgD1 and GAsgD1 transgenes, using retroviral gene transfer. Challenge of stably transfected permissive cell lines with various viral strains indicated that N-terminal GA fusion even enhanced the inhibitory properties of sgD1. Further studies revealed that the GA stretch increased protein stability by blocking proteasomal degradation of Gag proteins. Immunization of BALB/c mice with a DNA vaccine vector expressing sgD1 induced substantial Gag-specific immune responses that were, however, clearly diminished in the presence of GA. Furthermore, recognition of cells expressing the GA-fused transgene by CD8(+) T cells was drastically reduced, both in vitro and in vivo, resulting in prolonged survival of the transduced cells in recipient mice.

  20. The cytoplasmic domain of the gamete membrane fusion protein HAP2 targets the protein to the fusion site in Chlamydomonas and regulates the fusion reaction

    Science.gov (United States)

    Liu, Yanjie; Pei, Jimin; Grishin, Nick; Snell, William J.

    2015-01-01

    Cell-cell fusion between gametes is a defining step during development of eukaryotes, yet we know little about the cellular and molecular mechanisms of the gamete membrane fusion reaction. HAP2 is the sole gamete-specific protein in any system that is broadly conserved and shown by gene disruption to be essential for gamete fusion. The wide evolutionary distribution of HAP2 (also known as GCS1) indicates it was present in the last eukaryotic common ancestor and, therefore, dissecting its molecular properties should provide new insights into fundamental features of fertilization. HAP2 acts at a step after membrane adhesion, presumably directly in the merger of the lipid bilayers. Here, we use the unicellular alga Chlamydomonas to characterize contributions of key regions of HAP2 to protein location and function. We report that mutation of three strongly conserved residues in the ectodomain has no effect on targeting or fusion, although short deletions that include those residues block surface expression and fusion. Furthermore, HAP2 lacking a 237-residue segment of the cytoplasmic region is expressed at the cell surface, but fails to localize at the apical membrane patch specialized for fusion and fails to rescue fusion. Finally, we provide evidence that the ancient HAP2 contained a juxta-membrane, multi-cysteine motif in its cytoplasmic region, and that mutation of a cysteine dyad in this motif preserves protein localization, but substantially impairs HAP2 fusion activity. Thus, the ectodomain of HAP2 is essential for its surface expression, and the cytoplasmic region targets HAP2 to the site of fusion and regulates the fusion reaction. PMID:25655701

  1. Fusion proteins useful for producing pinene

    Science.gov (United States)

    Peralta-Yahya, Pamela P.; Keasling, Jay D

    2016-06-28

    The present invention provides for a modified host cell comprising a heterologous pinene synthase (PS), or enzymatically active fragment or variant thereof, and optionally a geranyl pyrophosphate synthase (GPPS), or enzymatically active fragment or variant thereof, or a fusion protein comprising: (a) a PS and (b) a GPPS linked by a linker.

  2. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.

    Science.gov (United States)

    Mitani, Yasuyuki; Vagnozzi, Ronald J; Millay, Douglas P

    2017-01-01

    Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non-muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle-specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.-Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.

  3. Disrupted dynamics of F-actin and insulin granule fusion in INS-1 832/13 beta-cells exposed to glucotoxicity: partial restoration by glucagon-like peptide 1.

    Science.gov (United States)

    Quinault, Aurore; Gausseres, Blandine; Bailbe, Danielle; Chebbah, Nella; Portha, Bernard; Movassat, Jamileh; Tourrel-Cuzin, Cecile

    2016-08-01

    Actin dynamics in pancreatic β-cells is involved in insulin exocytosis but the molecular mechanisms of this dynamics and its role in biphasic insulin secretion in pancreatic β-cells is largely unknown. Moreover, the impact of a glucotoxic environment on the sub-cortical actin network dynamics is poorly studied. In this study, we investigate the behavior of insulin granules and the subcortical actin network dynamics in INS-1 832/13 β-cells submitted to a normal or glucotoxic environment. Our results show that glucose stimulation leads to a reorganization of the subcortical actin network with a rupture of its interactions with t-SNARE proteins (Syntaxin 1A and SNAP-25), promoting insulin secretion in INS-1 832/13 β-cells. Prolonged exposure of INS-1 832/13 β-cells to high-glucose levels (glucotoxicity) leads to the densification of the cortical actin network, which prevents its reorganization under acute glucose, and diminishes the glucose-stimulated insulin secretion, as shown by the decreased number of fusion events. The most interesting in our results is the partial restoration by GLP-1 of the insulin secretion ability from high-glucose treated INS-1 832/13 cells. This improved insulin exocytosis is associated with partial restored actin dynamics and fusion events during the two phases of the secretion, with a preferential involvement of Epac2 signaling in the first phase and a rather involvement of PKA signaling in the second phase of insulin exocytosis. All these data provide some new insights into the mechanism by which current therapeutics may be improving insulin secretion.

  4. Constancy and diversity in the flavivirus fusion peptide

    Science.gov (United States)

    Seligman, Stephen J

    2008-01-01

    Background Flaviviruses include the mosquito-borne dengue, Japanese encephalitis, yellow fever and West Nile and the tick-borne encephalitis viruses. They are responsible for considerable world-wide morbidity and mortality. Viral entry is mediated by a conserved fusion peptide containing 16 amino acids located in domain II of the envelope protein E. Highly orchestrated conformational changes initiated by exposure to acidic pH accompany the fusion process and are important factors limiting amino acid changes in the fusion peptide that still permit fusion with host cell membranes in both arthropod and vertebrate hosts. The cell-fusing related agents, growing only in mosquitoes or insect cell lines, possess a different homologous peptide. Results Analysis of 46 named flaviviruses deposited in the Entrez Nucleotides database extended the constancy in the canonical fusion peptide sequences of mosquito-borne, tick-borne and viruses with no known vector to include more recently-sequenced viruses. The mosquito-borne signature amino acid, G104, was also found in flaviviruses with no known vector and with the cell-fusion related viruses. Despite the constancy in the canonical sequences in pathogenic flaviviruses, mutations were surprisingly frequent with a 27% prevalence of nonsynonymous mutations in yellow fever virus fusion peptide sequences, and 0 to 7.4% prevalence in the others. Six of seven yellow fever patients whose virus had fusion peptide mutations died. In the cell-fusing related agents, not enough sequences have been deposited to estimate reliably the prevalence of fusion peptide mutations. However, the canonical sequences homologous to the fusion peptide and the pattern of disulfide linkages in protein E differed significantly from the other flaviviruses. Conclusion The constancy of the canonical fusion peptide sequences in the arthropod-borne flaviviruses contrasts with the high prevalence of mutations in most individual viruses. The discrepancy may be the

  5. Constancy and diversity in the flavivirus fusion peptide

    Directory of Open Access Journals (Sweden)

    Seligman Stephen J

    2008-02-01

    Full Text Available Abstract Background Flaviviruses include the mosquito-borne dengue, Japanese encephalitis, yellow fever and West Nile and the tick-borne encephalitis viruses. They are responsible for considerable world-wide morbidity and mortality. Viral entry is mediated by a conserved fusion peptide containing 16 amino acids located in domain II of the envelope protein E. Highly orchestrated conformational changes initiated by exposure to acidic pH accompany the fusion process and are important factors limiting amino acid changes in the fusion peptide that still permit fusion with host cell membranes in both arthropod and vertebrate hosts. The cell-fusing related agents, growing only in mosquitoes or insect cell lines, possess a different homologous peptide. Results Analysis of 46 named flaviviruses deposited in the Entrez Nucleotides database extended the constancy in the canonical fusion peptide sequences of mosquito-borne, tick-bor