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Artykuły w czasopismach na temat "Retrovirus Induced Cell-Cell Fusion"
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Kubo, Yoshinao, Hideki Hayashi, Toshifumi Matsuyama, Hironori Sato i Naoki Yamamoto. "Retrovirus Entry by Endocytosis and Cathepsin Proteases". Advances in Virology 2012 (2012): 1–14. http://dx.doi.org/10.1155/2012/640894.
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Retroviruses include infectious agents inducing severe diseases in humans and animals. In addition, retroviruses are widely used as tools to transfer genes of interest to target cells. Understanding the entry mechanism of retroviruses contributes to developments of novel therapeutic approaches against retrovirus-induced diseases and efficient exploitation of retroviral vectors. Entry of enveloped viruses into host cell cytoplasm is achieved by fusion between the viral envelope and host cell membranes at either the cell surface or intracellular vesicles. Many animal retroviruses enter host cells through endosomes and require endosome acidification. Ecotropic murine leukemia virus entry requires cathepsin proteases activated by the endosome acidification. CD4-dependent human immunodeficiency virus (HIV) infection is thought to occur via endosomes, but endosome acidification is not necessary for the entry whereas entry of CD4-independent HIVs, which are thought to be prototypes of CD4-dependent viruses, is low pH dependent. There are several controversial results on the retroviral entry pathways. Because endocytosis and endosome acidification are complicatedly controlled by cellular mechanisms, the retrovirus entry pathways may be different in different cell lines.
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Andersen, Klaus Bahl. "Characterization of retrovirus-induced SC-1 cell fusion". Virus Research 37, nr 3 (sierpień 1995): 177–98. http://dx.doi.org/10.1016/0168-1702(95)00032-l.
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Andersen, K. B., i H. Skov. "Retrovirus-induced Cell Fusion Is Enhanced by Protease Treatment". Journal of General Virology 70, nr 7 (1.07.1989): 1921–27. http://dx.doi.org/10.1099/0022-1317-70-7-1921.
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Ellis, T. M., G. E. Wilcox i W. F. Robinson. "Characteristics of cell fusion induced by a caprine retrovirus". Archives of Virology 86, nr 3-4 (wrzesień 1985): 263–73. http://dx.doi.org/10.1007/bf01309830.
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Rein, Alan. "Murine Leukemia Viruses: Objects and Organisms". Advances in Virology 2011 (2011): 1–14. http://dx.doi.org/10.1155/2011/403419.
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Murine leukemia viruses (MLVs) are among the simplest retroviruses. Prototypical gammaretroviruses encode only the three polyproteins that will be used in the assembly of progeny virus particles. These are the Gag polyprotein, which is the structural protein of a retrovirus particle, the Pol protein, comprising the three retroviral enzymes—protease, which catalyzes the maturation of the particle, reverse transcriptase, which copies the viral RNA into DNA upon infection of a new host cell, and integrase, which inserts the DNA into the chromosomal DNA of the host cell, and the Env polyprotein, which induces the fusion of the viral membrane with that of the new host cell, initiating infection. In general, a productive MLV infection has no obvious effect upon host cells. Although gammaretroviral structure and replication follow the same broad outlines as those of other retroviruses, we point out a number of significant differences between different retroviral genera.
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Zavorotinskaya, Tatiana, i Lorraine M. Albritton. "Suppression of a Fusion Defect by Second Site Mutations in the Ecotropic Murine Leukemia Virus Surface Protein". Journal of Virology 73, nr 6 (1.06.1999): 5034–42. http://dx.doi.org/10.1128/jvi.73.6.5034-5042.1999.
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ABSTRACT Entry of ecotropic murine leukemia virus initiates when the envelope surface protein recognizes and binds to the virus receptor on host cells. The envelope transmembrane protein then mediates fusion of viral and host cell membranes and penetration into the cytoplasm. Using a genetic selection, we isolated an infectious retrovirus variant containing three changes in the surface protein—histidine 8 to arginine, glutamine 227 to arginine, and aspartate 243 to tyrosine. Single replacement of histidine 8 with arginine (H8R) resulted in almost complete loss of infectivity, even though the mutant envelope proteins were stable and efficiently incorporated into virions. Virions carrying H8R envelope were proficient at binding cells expressing receptor but failed to induce cell-cell fusion of XC cells, indicating that the histidine at position 8 plays an essential role in fusion during penetration of the host cell membrane. Thus, there is at least one domain in SU that is involved in fusion; the fusion functions do not reside exclusively in TM. In contrast, envelope with all three changes induced cell-cell fusion of XC cells and produced virions that were 10,000-fold more infectious than those containing only the H8R substitution, indicating that changes at positions 227 and 243 can suppress a fusion defect caused by loss of histidine 8 function. Moreover, the other two changes acted synergistically, indicating that both compensate for the loss of the same essential function of histidine 8. The ability of these changes to suppress this fusion defect might provide a means for overcoming postbinding defects found in targeted retroviral vectors for use in human gene therapy.
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Kinzler, Eric R., i Teresa Compton. "Characterization of Human Cytomegalovirus Glycoprotein-Induced Cell-Cell Fusion". Journal of Virology 79, nr 12 (15.06.2005): 7827–37. http://dx.doi.org/10.1128/jvi.79.12.7827-7837.2005.
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ABSTRACT Human cytomegalovirus (CMV) infection is dependent on the functions of structural glycoproteins at multiple stages of the viral life cycle. These proteins mediate the initial attachment and fusion events that occur between the viral envelope and a host cell membrane, as well as virion-independent cell-cell spread of the infection. Here we have utilized a cell-based fusion assay to identify the fusogenic glycoproteins of CMV. To deliver the glycoprotein genes to various cell lines, we constructed recombinant retroviruses encoding gB, gH, gL, and gO. Cells expressing individual CMV glycoproteins did not form multinucleated syncytia. Conversely, cells expressing gH/gL showed pronounced syncytium formation, although expression of gH or gL alone had no effect. Anti-gH neutralizing antibodies prevented syncytium formation. Coexpression of gB and/or gO with gH/gL did not yield detectably increased numbers of syncytia. For verification, these results were recapitulated in several cell lines. Additionally, we found that fusion was cell line dependent, as nonimmortalized fibroblast strains did not fuse under any conditions. Thus, the CMV gH/gL complex has inherent fusogenic activity that can be measured in certain cell lines; however, fusion in fibroblast strains may involve a more complex mechanism involving additional viral and/or cellular factors.
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Tomasson, Michael H., Ifor R. Williams, Shaoguang Li, Jeffrey Kutok, Danielle Cain, Silke Gillessen, Glenn Dranoff, Richard A. Van Etten i D. Gary Gilliland. "Induction of myeloproliferative disease in mice by tyrosine kinase fusion oncogenes does not require granulocyte-macrophage colony-stimulating factor or interleukin-3". Blood 97, nr 5 (1.03.2001): 1435–41. http://dx.doi.org/10.1182/blood.v97.5.1435.
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Tyrosine kinase fusion oncogenes that occur as a result of chromosomal translocations have been shown to activate proliferative and antiapoptotic pathways in leukemic cells, but the importance of autocrine and paracrine expression of hematopoietic cytokines in leukemia pathogenesis is not understood. Evidence that leukemic transformation may be, at least in part, cytokine dependent includes data from primary human leukemia cells, cell culture experiments, and murine models of leukemia. This report demonstrates that interleukin (IL)-3 plasma levels are elevated in myeloproliferative disease (MPD) caused by the TEL/tyrosine kinase fusions TEL/platelet-derived growth factor beta receptor (PDGFβR), TEL/Janus kinase 2 (JAK2), and TEL/neurotrophin-3 receptor (TRKC). Plasma granulocyte-macrophage colony-stimulating factor (GM-CSF) levels were elevated by TEL/PDGFβR and TEL/JAK2. However, all of the fusions tested efficiently induced MPD in mice genetically deficient for both GM-CSF and IL-3, demonstrating that these cytokines are not necessary for the development of disease in this model system. Furthermore, in experiments using normal marrow transduced with TEL/PDGFβR retrovirus mixed with marrow transduced with an enhanced green fluorescent protein (EGFP) retrovirus, the MPD induced in these mice demonstrated minimal stimulation of normal myelopoiesis by the TEL/PDGFβR-expressing cells. In contrast, recipients of mixed GM-CSF–transduced and EGFP-transduced marrow exhibited significant paracrine expansion of EGFP-expressing cells. Collectively, these data demonstrate that, although cytokine levels are elevated in murine bone marrow transplant models of leukemia using tyrosine kinase fusion oncogenes, GM-CSF and IL-3 are not required for myeloproliferation by any of the oncogenes tested.
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Sugimoto, Jun, Sehee Choi, Megan A. Sheridan, Iemasa Koh, Yoshiki Kudo i Danny J. Schust. "Could the Human Endogenous Retrovirus-Derived Syncytialization Inhibitor, Suppressyn, Limit Heterotypic Cell Fusion Events in the Decidua?" International Journal of Molecular Sciences 22, nr 19 (23.09.2021): 10259. http://dx.doi.org/10.3390/ijms221910259.
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Proper placental development relies on tightly regulated trophoblast differentiation and interaction with maternal cells. Human endogenous retroviruses (HERVs) play an integral role in modulating cell fusion events in the trophoblast cells of the developing placenta. Syncytin-1 (ERVW-1) and its receptor, solute-linked carrier family A member 5 (SLC1A5/ASCT2), promote fusion of cytotrophoblast (CTB) cells to generate the multi-nucleated syncytiotrophoblast (STB) layer which is in direct contact with maternal blood. Another HERV-derived protein known as Suppressyn (ERVH48-1/SUPYN) is implicated in anti-fusogenic events as it shares the common receptor with ERVW-1. Here, we explore primary tissue and publicly available datasets to determine the distribution of ERVW-1, ERVH48-1 and SLC1A5 expression at the maternal-fetal interface. While SLC1A5 is broadly expressed in placental and decidual cell types, ERVW-1 and ERVH48-1 are confined to trophoblast cell types. ERVH48-1 displays higher expression levels in CTB and extravillous trophoblast, than in STB, while ERVW-1 is generally highest in STB. We have demonstrated through gene targeting studies that suppressyn has the ability to prevent ERVW-1-induced fusion events in co-culture models of trophoblast cell/maternal endometrial cell interactions. These findings suggest that differential HERV expression is vital to control fusion and anti-fusogenic events in the placenta and consequently, any imbalance or dysregulation in HERV expression may contribute to adverse pregnancy outcomes.
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Caudell, David, Rachel M. Pierce, David P. Harper, Rachel L. Novak, Christopher Slape, Linda Wolff i Peter D. Aplan. "Identification of Genes That Collaborate with CALM-AF10 to Induce Leukemia by Retroviral Insertional Mutagenesis and Candidate Gene Sequencing". Blood 112, nr 11 (16.11.2008): 3787. http://dx.doi.org/10.1182/blood.v112.11.3787.3787.
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Abstract The t(10;11) translocation results in a CALM-AF10 fusion gene in a subset of patients with ALL or AML. Expression of a CALM-AF10 fusion gene in transgenic mice is leukemogenic, however, the prolonged latency period, and incomplete penetrance suggests that additional events are necessary to complement CALM-AF10 mediated leukemic transformation. We used retroviral insertional mutagenesis (RIM) to identify complementary genetic events that might collaborate with CALM-AF10 during leukemic transformation. Newborn CALM-AF10 mice were infected with a modified replication competent retrovirus (Mol4070LTR); by 7 months of age, 90% of the transgenic mice developed acute leukemia. Acute leukemia developed more rapidly in CALM-AF10 infected mice than either wild-type mice infected with retrovirus (p=0.004) or CALM-AF10 mice not infected with retrovirus (p=0.037). Ligation-mediated PCR of DNA isolated from leukemic spleens identified potential collaborating genes near the retroviral insertion sites. 262 unique integrations were identified in 40 CALM-AF10 mice. Of these, 55 integrations occurred at 20 common insertion sites, including Zeb2, Mn1, Evi1, Nf1, Ift57, Mpl, Kras, Vav1, and Gata1. Of the candidate genes identified, Zeb2 and Nf1 are of particular interest. Zeb2 encodes the transcriptional co-repressor Smad-interacting protein 1, which is a member of the TGF beta signaling pathway. Previous studies using RIM identified Zeb2 insertions only sporadically, never more than once in a single study, and in association with B-cell, T-cell, and myeloid leukemia. In this study retroviral integrations near Zeb2 were identified either by PCR or Southern blot analysis in 26% (11/42) of the CALM-AF10 mice. RIM studies often generate oligoclonal leukemias, with two or more independent leukemias arising in the same mouse. Southern blot analysis demonstrated that the Zeb2 integration was present in the dominant leukemic clone in most of these mice, and the Zeb2 transcript was overexpressed compared to wild-type spleen and bone marrow. Most leukemias that arise in CALM-AF10 mice without retroviral insertions are myeloid (17/20). However, almost all (10/11) of the CALM-AF10 mice with Zeb2 insertions developed B-lineage ALL suggesting that expression of Zeb2 strongly influences the phenotype of CALM-AF10 leukemias. The tumor suppressor gene Nf1 (Neurofibromatosis type 1) functions as a GTPase activating protein that regulates the activity of Ras proteins involved in cellular proliferation, and acts as a tumor suppressor gene in immature myeloid cells. Retroviral integrations into the Nf1 locus were identified by PCR or Southern blot in 26% (11/42) of the mice in this study. In at least two cases, Southern blot showed loss of the germline allele. The high frequency of Nf1 integrations identified in the RIM assay suggested that Ras pathway activation complemented the CALM-AF10 transgene. Interestingly, 4 other retroviral integrations were identified near Ras genes; 2 near Kras (one with a Kras activating mutation), and 2 near Rras2. Given the high frequency of Nf1 or Ras insertions in the RIM study, we sequenced Nras and Kras genes from CALM-AF10 mice not infected with retrovirus. Thus far, we have identified 3 activating mutations in 15 mice, demonstrating that spontaneous Ras mutations develop as complementary events. We have shown that retroviral infection accelerates the onset of acute leukemia, and identified genes that potentially collaborate with the CALM-AF10 fusion gene in the leukemic transformation process. Furthermore, by sequencing candidate genes, we have demonstrated that pathways (ie, Ras) identified by RIM can also be activated by spontaneous mutations and potentially collaborate with CALM-AF10 to induce leukemia.
Rozprawy doktorskie na temat "Retrovirus Induced Cell-Cell Fusion"
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Barkley, Russell. "Investigation of an Oncolytic MeV Cell-Cell Fusion Phenomenon Induced by an siRNA". Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/41531.
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Oncolytic measles virus is a promising cancer therapeutic in clinical trials which
possesses multiple characteristics that are advantageous over traditional therapies.
Currently, clinical oncolytic measles virus vectors are unmodified or express reporter
transgenes that benefit its therapeutic efficacy. The next phase in its development will
see genetically engineered vectors encoding transgenes that enhance its antineoplastic effects. To this end, preclinical research has focused on studying novel transgenes which favour viral replication, cytotoxicity, and the anti-cancer immune response. We sought to encode artificial micoRNAs targeting RIG-I as a strategy to interfere with innate immunity. Silencing RIG-I with multiple siRNAs yielded one which promotes measles virus syncytia formation through a mechanism that appears to be independent of RIG-I. The mechanism caused by the siRNA leads to enhanced measles virus cell-cell fusion and has peculiar characteristics which are not fully understood.
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Symeonides, Menelaos. "HIV-1-Induced Cell-Cell Fusion: Host Regulation And Consequences For Viral Spread". ScholarWorks @ UVM, 2016. https://scholarworks.uvm.edu/graddis/589.
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Human immunodeficiency virus type 1 (HIV-1) is a human retrovirus of the lentivirus subgroup which primarily infects T cells and macrophages, and causes acquired immune deficiency syndrome (AIDS). Since its emergence in the early 1980s, HIV-1 has caused a global pandemic which is still responsible for over one million deaths per year, primarily in sub-Saharan Africa.
HIV-1 has been the subject of intense study for over three decades, which has resulted not only in major advances in cell biology, but also in numerous drug treatments that effectively control the infection. However, cessation of treatment always results in reemergence of the infection due to the ability of HIV-1 (and other lentiviruses) to establish a persistent quiescent infection known as latency. The elimination of latently-infected cells is the primary goal of current research towards a cure for HIV-1, alongside efforts to develop vaccines, which have thus far been fruitless.
The spread of HIV-1 to susceptible target cells (which express the receptor CD4 and a co-receptor; CXCR4 or CCR5) can take place when antigen-presenting cells, such as dendritic cells, capture virus particles and then pass them on to target cells, without themselves becoming infected. Alternatively, productively infected T cells or macrophages can spread HIV-1 either by shedding virus particles to the milieu, which are then stochastically acquired by target cells, or through transient contacts between infected and uninfected cells known as virological synapses (VSs). VS-mediated cell-to-cell transmission is thought to be highly efficient due to the release of virus directly onto (or very near to) a target cell, and some evidence suggests that the VS is a privileged site which allows the virus to evade neutralizing antibodies and drugs. However, and most importantly, it is of central interest to us because the same transient cell adhesions that facilitate virus transfer can also result in the fusion of the two cells to form a syncytium, due to the presence of the viral fusogen Env and its receptor and co-receptor on either side of the VS. While T cell syncytia can be found in vivo, they remain small, and it appears that the majority of VSs resolve without fusion.
The regulation of HIV-1-induced cell-cell fusion and the fate of those syncytia are the focus of the work presented here. A family of host transmembrane proteins, the tetraspanins, which regulate cell-cell fusion in other contexts (e.g. the fusion of myoblasts to form and maintain myotubes), were found to inhibit HIV-1-induced cell-cell fusion. Our investigations have further characterized this regulation, concluding that tetraspanins allow cells to reach the fusion intermediate known as hemifusion before their ability to repress fusion takes effect. In parallel, because syncytia are nevertheless found both in infected individuals and in a humanized mouse model for HIV-1, we also became interested in whether small T cell-based syncytia were able to participate in HIV-1 spread by transmitting virus to target cells. Using a simple three dimensional in vitro culture system which closely recapitulates those in situ observations, we found that small syncytia can contact target cells and transmit virus without fusing with them. Overall, these studies further our understanding of HIV-1-induced syncytia and reveal a previously unrecognized role for these entities as active participants in HIV-1 spread.
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Hutchinson, Lloyd M. "Glycoprotein K of herpes simplex virus (HSV), role in viral egress and HSV-induced cell-cell fusion". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0016/NQ30094.pdf.
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Gerald, Schneikart. "Respiratory syncytial virus fusion protein-specific B cell repertoires induced by natural infection or vaccination". Doctoral thesis, Università di Siena, 2018. http://hdl.handle.net/11365/1050834.
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Respiratory syncytial virus (RSV) infections are the major contributor to acute lower respiratory syndrome in newborns. Infections generally result in hospitalization and sometimes in death. A vaccine is not available yet, despite decades of research. Vaccine development is hampered in consequence of a failed vaccine trial in the 1960s which entailed fatal outcomes. An alternative to direct vaccination of children is maternal vaccination for passive immunization of babies before birth. RSV infects every person repeatedly throughout life which implies that pregnant woman carry RSV-directed memory B cell (MBC) repertoires. A successful maternal vaccine would therefore elicit high titer RSV-neutralizing IgGs by reactivation of pre-existing MBCs which would protect newborns during the first months of life. RSV has two neutralizing antigens, of which the fusion protein (RSV F) is the most promising vaccine candidate. RSV F mediates the fusion process of viral and cellular membranes, wherefore it exists in a pre-fusion (pre F) or post-fusion (post F) conformation. Even though there is more interest in the pre F conformation as immunogen, the post F conformation may be equally considered as successful vaccine antigen. The pre F conformation is very metastable and readily switches to the highly stable post F conformation, which implies that a post F-based vaccine would be more cost-efficient to produce. More importantly, several neutralizing epitopes are preserved on the post F conformation and a substantial amount of RSV F-directed MBCs induced by natural infection are actually pre/post F cross-reactive. Since most of the pre/post F cross-reactive MBCs were previously shown to have higher affinities for the post F conformation, a post F-based vaccine may be ideal for reactivation and clonal expansion of MBCs which express neutralizing B cell receptors (BCRs). Every antigen, also RSV post F protein, has its own signatures within BCR repertoires because of preferential selection of BCR characteristics for B cell clonal expansions. In order to understand how RSV F protein shapes BCR repertoires, RSV F-directed BCRs were isolated from a healthy blood donor and three vaccinees who received an RSV post F vaccine. BCR repertoire analysis confirmed the assumption that the pre and post F protein have their own signatures within RSV F-directed BCR repertoires. The different characteristics indicated longer affinity maturation of pre F-reactive MBCs. Furthermore, estimation of clonal relatedness between the pre and post F-binding BCR repertoires from the healthy donor provided indications that a substantial number of the isolated BCRs are actually pre/post F cross-reactive, which confirmed a previous study. Analysis of the BCR repertoires isolated from the RSV post F-vaccinees showed that the vaccine induced a biased MBC response with preferential BCR characteristics. There were several implications that the post F-vaccine expanded primarily pre/post F cross-reactive MBCs. In contrast to the high variability of pre/post F cross-reactive BCRs induced by natural infection, the vaccine-induced MBC response indicated a skewed selection of VH4 gene family-encoded BCRs for clonal expansion and affinity maturation. More importantly, estimation of clonal relatedness revealed convergent MBC responses between the three analyzed subjects, while several MBC lineages shared stereotypic characteristics with pre F-binding BCRs or RSV-neutralizing antibodies. Some of the supposedly pre/post F cross-binding or neutralizing BCR sequences were expressed as mAbs and functionally characterized. RSV pre/post F cross-binding and neutralization activities could be demonstrated for all of the expressed mAbs. This project demonstrated the potential of ‘clonal’ grouping as novel reverse approach to screen BCR repertoires for functional antibodies.
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Villafranca, Locher Maria Cristina. "Fusion of bovine fibroblasts to mouse embryonic stem cells: a model to study nuclear reprogramming". Diss., Virginia Tech, 2018. http://hdl.handle.net/10919/82864.
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The cells from the inner cell mass (ICM) of an early embryo have the potential to differentiate into all the different cell types present in an adult organism. Cells from the ICM can be isolated and cultured in vitro, becoming embryonic stem cells (ESCs). ESCs have several properties that make them unique: they are unspecialized, can self-renew indefinitely in culture, and given the appropriate cues can differentiate into cells from all three germ layers (ecto-, meso-, and endoderm), including the germline, both in vivo and in vitro. Induced pluripotent stem cells (iPSCs) can be generated from adult, terminally differentiated somatic cells by transient exogenous expression of four transcription factors (Oct4, Sox2, Klf4, and cMyc; OSKM) present normally in ESCs. It has been shown that iPSCs are equivalent to ESCs in terms of morphology, gene expression, epigenetic signatures, in vitro proliferation capacity, and in vitro and in vivo differentiation potential. However, unlike ESCs, iPSCs can be obtained from a specific individual without the need for embryos. This makes them a promising source of pluripotent cells for regenerative medicine, tissue engineering, drug discovery, and disease modelling; additionally, in livestock species such as the bovine, they also have applications in genetic selection, production of transgenic animals for agricultural and biomedical purposes, and species conservancy. Nevertheless, ESC and iPSC lines that meet all pluripotency criteria have, to date, only been successfully produced in mice, rats, humans, and non-human primates.
In the first part of this dissertation, we attempted reprogramming of three types of bovine somatic cells: fetal fibroblasts (bFFs), adult fibroblasts (bAFs), and bone marrow-derived mesenchymal stem cells (bMSCs), using six different culture conditions adapted from recent work in mice and humans. Using basic mouse reprogramming conditions, we did not succeed in inducing formation of ESC-like colonies in bovine somatic cells. The combination of 2i/LIF plus ALK5 inhibitor II and ascorbic acid, induced formation of colony-like structures with flat morphology, that occasionally produced trophoblast-like structures. These trophoblast-like vesicles did not appear when an inhibitor of Rho-associated, coiled-coil containing protein kinase 1 (ROCK) was included in the medium. We screened for expression of exogenous OSKM vector with RT-PCR and found upregulation of OSKM vector 24h after Dox was added to the medium; however, expression was sharply decreased on day 2 after Dox induction, and was not detectable after day 3. In a separate experiment, we induced reprogramming of bFF and bAFs using medium supplemented with 50% of medium conditioned by co-culture with the bovine trophoblast CT1 line. These cells expressed both OCT4 and the OSKM vector 24h after Dox induction. However, similar to our previous observations, both markers decreased expression until no signal was detected after day 3. In summary, we were unable to produce fully reprogrammed bovine iPSCs using mouse and human protocols, and the exact cause of our lack of success is unclear. It is possible that a different method of transgene expression could play a role in reprogramming. However, these ideas would be driven by a rather empirical reasoning, extrapolating findings from other species, and not contributing in our understanding of the particular differences of pluripotecy in ungulates. Our inability to produce bovine iPSCs, combined with the only partial reprogramming observed by others, justifies the need for in depth study of bovine pluripotency mechanisms, before meaningful attempts to reprogram bovine somatic cells to plutipotency are made. Therefore, we focused on getting a better understanding of bovine nuclear reprogramming. This would allow us to rationally target the specific requirements of potential bovine pluripotent cells.
Cell fusion is a process that involves fusion of the membrane of two or more cells to form a multinucleated cell. Fusion of a somatic cell to an ESC is known to induce expression of pluripotency markers in the somatic nucleus. In the second part of this dissertation, we hypothesized that fusion of bFFs to mouse ESCs (mESCs) would induce expression of pluripotency markers in the bFF nucleus. We first optimized a cell fusion protocol based on the use of polyethylene glycol (PEG), and obtained up to 11.02% of multinucleated cells in bFFs. Next, we established a method to specifically select for multinucleated cells originated from the fusion of mESCs with bFFs (heterokaryons), using indirect immunofluorescence. With this in place, flow cytometry was used to select 200 heterokaryons which were further analyzed using RNA-seq. We found changes in bovine gene expression patterns between bFFs and heterokaryons obtained 24h after fusion. Focusing on the bovine transcriptome, heterokaryons presented upregulation of early pluripotency markers OCT4 and KLF4, as well as hypoxia response genes, contrasted with downregulation of cell cycle inhibitors such as SST. The cytokine IL6, known to increase survival of early embryos in vitro, was upregulated in heterokaryons, although its role and mechanism of action is still unclear. This indicates that the heterokaryon cell fusion model recapitulates several of the events of early reprogramming, and can therefore be used for further study of pluripotency in the bovine. The cell fusion model presented here can be used as a tool to characterize early changes in bovine somatic nuclear reprogramming, and to study the effect of different reprogramming conditions on the bovine transcriptome.Ph. D.
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Hess, Patricia M. "Role of c-Jun NH-terminal Kinase in Bcr/Abl Induced Cell Transformation: a dissertation". eScholarship@UMMS, 2003. https://escholarship.umassmed.edu/gsbs_diss/88.
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The c-Jun NH2-terminal kinase (JNK) group of kinases include ten members that are created by alternative splicing of transcripts derived from Jnk1, Jnk2 and Jnk3 genes. The JNK1 and JNK2 protein kinases are ubiquitously expressed while JNK3 is expressed in a limited number of tissues. The JNK signaling pathway is implicated in multiple physiological processes including cell transformation. There is growing evidence that JNK signaling is involved in oncogenesis. Nevertheless, the role that JNK plays in malignant transformation is still unclear. The aim of this thesis is to examine the role of JNK in malignant transformation. For this purpose, I used the Bcr/Abl oncogene as a transforming agent. Bcr/Abl is a leukemogenic oncogene that is created by reciprocal translocation between chromosome 9 and 22. The translocation breakpoint is variable and several different Bcr/Abl isoforms have been identified such as Bcr/AblP185 and Bcr/AblP210, whose expression is associated with different types of leukemia. Bcr/Abl activates the JNK signaling pathway in hematopoietic cells and increases AP-1 transcription activity. Furthermore, dominant negative approaches demonstrate that inhibition of c-Jun or JNK prevents Bcr/ Abl-induced cell transformation in vitro. These data implicate the JNK signaling pathway in Bcr/Abl transformation although the role that JNK might have in this process is unclear. Thus, I examined the importance of JNK signaling in Bcr/Abl-induced lymphoid or myeloid transformation. For this purpose I compared Bcr/AblP185- and Bcr/AblP210- induced transformation of wild-type and JNK1-deficient cells using three approaches: in vitro, in vivo and ex vivo. The results obtained with the in vitro approach suggest that both Bcr/AblP185 and Bcr/AblP210 require JNK activity to induce lymphoid transformation. While JNK1-deficiency inhibits Bcr/AblP210 oncogenic potential in lymphoid cells both in vitro and in vivo, pharmacological inhibition of JNK activity (JNK1 and/or JNK2) blocked Bcr/AblP185 induced malignant proliferation in vitro. The differential requirement for JNK observed in the two Bcr/Abl isoforms can be ascribed to the presence in Bcr/AblP210 of the Dbl domain which can activate the JNK pathway in vitro. In the case of Bcr/AblP210, JNK1 is critical for the survival of the ex vivo derived transformed lymphoblasts upon growth factor removal. This result correlates with the fact that mice reconstituted with Bcr/AblP210 transformed Jnk1-l- bone marrow showed normal malignant lymphoid expansion in the bone marrow yet they had reduced numbers of lymphoblast in the bloodstream and lacked peripheral organ infiltration. Thus JNK1 is essential for the survival of the transformed lymphoblast outside the bone marrow microenvironment in Bcr/AblP210induced lymphoid leukemia. Interestingly, while JNK1 is essential for lymphoid transformation, it is dispensable for the proliferation of transformed myeloblasts.
Taken together these results indicate that the JNK signaling pathway plays an essential role in the survival of Bcr/AblP210 lymphoblasts and that JNK-deficiency decreases the leukomogenic potential of Bcr/AblP210 in vivo. Thus, cell survival mediated by JNK may contribute to the pathogenesis of proliferative diseases.
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Schirmacher, Anastasiya. "Modification of transmembrane peptides to probe SNARE-induced membrane fusion and cross-presentation of membrane-buried epitopes". Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2020. http://hdl.handle.net/21.11130/00-1735-0000-0005-1576-F.
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Khedher, Ahmed. "Utilisation de technologies d'édition du génome afin de générer des cardiomyocytes matures à partir de cellules souches pluripotentes humaines induites CtIP Fusion to Cas9 Enhances Transgene Integration by Homology-Dependent Repair". Thesis, université Paris-Saclay, 2021. http://www.theses.fr/2021UPASL002.
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Les cardiomyocytes dérivés des cellules souches pluripotentes humaines induites (hiPSC-CMs) représentent des modèles in vitro prometteurs pour plusieurs applications scientifiques et thérapeutiques allant de la modélisation de pathologies à la découverte de médicaments et de la toxicologie prédictive à la médecine régénérative. Malgré les nombreux progrès dans ce domaine, les protocoles de différenciation actuels ne permettent pas d’atteindre le stade de maturité que l’on retrouve chez le myocarde adulte de l’Homme. En effet, certaines caractéristiques majeures des hiPSC-CMs demeurent similaires à celles de cardiomyocytes fœtaux telles que l’expression de plusieurs gènes cardiaques, l’électrophysiologie ou leur fonction contractile. En effet, des analyses transcriptomiques réalisés au sein de notre laboratoire à Sanofi ont révélé que les gènes KCNJ2 et CASQ2, impliqués respectivement dans l’électrophysiologie et la gestion du calcium, étaient sous-exprimés dans les hiPSC-CMs en comparaison aux cardiomyocytes adultes. Cette thèse avait pour objectif d’améliorer la maturation des hiPSC-CMs en utilisant des technologies d’édition du génome. Ainsi, nous avons généré des lignées stables de hiPSC-CMs qui expriment de manière inductible KCNJ2 ou CASQ2 ou les deux gènes simultanément puis nous avons examiné leurs phénotypes fonctionnels et électrophysiologiques par le biais de méthodes d’analyses complémentaires. A la suite à l’induction de l’expression de KCNJ2 et CASQ2 par la doxycycline, les hiPSC-CMs montraient des bénéfices phénotypiques tels que la diminution drastique de la fréquence des battements spontanés, une hyperpolarisation du potentiel de repos membranaire, la diminution de la durée du potentiel d’action et l’amélioration du flux de calcium transitoire. En plus de ces bénéfices attendus, l’expression concomitante de ces deux gènes a amélioré la pente de la pointe du potentiel de champ extracellulaire associée au courant sodique ainsi que la gestion du calcium. Nous avons ensuite évalué le bénéfice de l’expression de ces transgènes sur la toxicologie prédictive en testant des molécules agonistes ou antagonistes de canaux ioniques utilisées classiquement dans le cadre des essais précliniques de toxicité cardiaque. Nous avons notamment observé plus d’arythmies induites par l’E4031 avec les hiPSC-CMs exprimant conjointement KCNJ2 et CASQ2 par rapport aux cardiomyocytes contrôles. Ainsi, les hiPSC-CMs exprimant simultanément KCNJ2 et CASQ2 présentent un phénotype plus mature que les hiPSC-CMs natifs et de tels cardiomyocytes édités génétiquement peuvent être utiles pour l’évaluation de la toxicité cardiaque de nouveaux médicaments candidatsHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a very promising model for several scientific and therapeutic applications ranging from disease modeling to drug discovery, and from predictive toxicology to regenerative medicine. Despite numerous efforts, current protocols do not yet lead to a maturation phenotype equivalent to adult human myocardium. Indeed, key features of hiPSC-CMs remaining closer to fetal stages of development, such as gene expression, electrophysiology and function. Transcriptome analysis performed at Sanofi have confirmed these findings at the genome-wide level. Indeed, KCNJ2 and CASQ2 which are implicated in the two major physiological characteristics of cardiac cells, their electrophysiological behavior and calcium handling, respectively, were expressed at very low levels in hiPSC-CMs in comparison with adult cardiomyocytes. This thesis aimed to improve the maturation of hiPSC-CMs by using genome editing technologies. We generated stable hiPSC-CMs with inducible expression of KCNJ2, or CASQ2 or both genes (KCNJ2-CASQ2 hiPSC-CMs) and studied their functional and electrophysiological phenotype by several complementary methods. Upon doxycycline induction of KCNJ2 and CASQ2, KCNJ2-CASQ2 hiPSC-CMs displayed phenotypic benefits expected from previous studies of each maturation gene, including a drastic reduction of spontaneous beating, hyperpolarized resting membrane potential, shortened action potential duration and enhanced calcium transients. In addition, co-expression of the two genes enhanced Na+ spike slope of extracellular field potential and Ca2+ handling. We tested four reference drugs and observed signatures of known cardiac effects in KCNJ2-CASQ2 hiPSC-CMs, including arrhythmias induced by QT prolonging drug (E-4031), which were more easily detected than in control hiPSC-CMs. Therefore, KCNJ2-CASQ2 hiPSC-CMs exhibited a more mature phenotype than hiPSC-CMs and such genetically engineered hiPSC-CMs could be useful for testing cardiac toxicity of novel candidate drugs
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Ho, Sweet Ping Ellen. "Oncogenic progression in retrovirus-induced T-cell leukemia". Phd thesis, 1994. http://hdl.handle.net/1885/142565.
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Yang, Po Fu, i 楊博夫. "Optically-induced Cell Fusion on a Microfluidic Chip". Thesis, 2015. http://ndltd.ncl.edu.tw/handle/13098944466659735919.
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碩士國立清華大學
動力機械工程學系
103
Cell fusion is a critical course for all sort of biomedical applications including cell reprogramming, hybridoma formation, cancer immunotherapy, and tissue regeneration. It can be realized by using biological, chemical, or physical methods. However, efficiency and yields are limited by unstable cell contact and random cell pairings in traditional methods. Hence, improving cell contact and cell pairing are the two key factors to enhance efficiency and yields of cell fusion. This study therefore reported a new approach called optically-induced cell fusion (OICF) which integrates cell-pairing microstructures and optically-induced, localized electrical field to achieve precise cell fusion with high yields and high efficiency. By projecting light patterns on a photoconductive film (hydrogen-rich amorphous silicon, a-Si: H) coated on an indium-tin-oxide (ITO) glass while an alternating-current (AC) electric field was applied on the top and bottom ITO glasses, “virtual” electrodes would be constructed accordingly. In fact, this method could be used on several biomedical applications, including cell manipulation, cell separation, cell lysis and electroporation. Therefore, a locally enhanced electric field would be induced and the pairing cells could be precisely fused by the virtual electrodes. In this study, 57% cell paring rate and 87% fusion efficiency were achieved. Therefore, OICF is a promising method to succeed in cell fusion with high efficiency and high yields.
Książki na temat "Retrovirus Induced Cell-Cell Fusion"
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Shaw, Christine Anne. T cell responses induced by bacterial toxin fusion proteins. 2008.
Znajdź pełny tekst źródłaCzęści książek na temat "Retrovirus Induced Cell-Cell Fusion"
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Spear, Patricia G. "Virus-Induced Cell Fusion". W Cell Fusion, 3–32. Boston, MA: Springer US, 1987. http://dx.doi.org/10.1007/978-1-4757-9598-1_1.
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Schierenberg, Einhard. "Laser-Induced Cell Fusion". W Cell Fusion, 409–18. Boston, MA: Springer US, 1987. http://dx.doi.org/10.1007/978-1-4757-9598-1_19.
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Huang, Leaf, i Jerome Connor. "Acid-Induced Fusion of Liposomes". W Cell Fusion, 285–99. Boston, MA: Springer US, 1987. http://dx.doi.org/10.1007/978-1-4757-9598-1_13.
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Saunders, James A., i George W. Bates. "Chemically Induced Fusion of Plant Protoplasts". W Cell Fusion, 497–520. Boston, MA: Springer US, 1987. http://dx.doi.org/10.1007/978-1-4757-9598-1_25.
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Düzgüneş, Nejat, Keelung Hong, Patricia A. Baldwin, Joe Bentz, Shlomo Nir i Demetrios Papahadjopoulos. "Fusion of Phospholipid Vesicles Induced by Divalent Cations and Protons". W Cell Fusion, 241–67. Boston, MA: Springer US, 1987. http://dx.doi.org/10.1007/978-1-4757-9598-1_11.
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Boni, L. T., i S. W. Hui. "The Mechanism of Polyethylene Glycol-Induced Fusion in Model Membranes". W Cell Fusion, 301–30. Boston, MA: Springer US, 1987. http://dx.doi.org/10.1007/978-1-4757-9598-1_14.
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Do, Jeong Tae, i Hans R. Schöler. "Cell Fusion-Induced Reprogramming". W Cellular Programming and Reprogramming, 179–90. Totowa, NJ: Humana Press, 2010. http://dx.doi.org/10.1007/978-1-60761-691-7_11.
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Lanzrein, Markus, Magda Spycher-Burger i Christoph Kempf. "Semliki Forest Virus Induced Cell-Cell Fusion and Pore Formation". W Biological Membranes: Structure, Biogenesis and Dynamics, 341–48. Berlin, Heidelberg: Springer Berlin Heidelberg, 1994. http://dx.doi.org/10.1007/978-3-642-78846-8_34.
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Yoshida, Keiichi, Natsuko Kawano, Yuichiroh Harada i Kenji Miyado. "Role of CD9 in Sperm–Egg Fusion and Virus-Induced Cell Fusion in Mammals". W Sexual Reproduction in Animals and Plants, 383–91. Tokyo: Springer Japan, 2014. http://dx.doi.org/10.1007/978-4-431-54589-7_31.
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Koop, Hans-Ulrich, i Germán Spangenberg. "Electric Field-Induced Fusion and Cell Reconstitution with Preselected Single Protoplasts and Subprotoplasts of Higher Plants". W Electroporation and Electrofusion in Cell Biology, 355–66. Boston, MA: Springer US, 1989. http://dx.doi.org/10.1007/978-1-4899-2528-2_23.
Pełny tekst źródłaStreszczenia konferencji na temat "Retrovirus Induced Cell-Cell Fusion"
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Chen, Shuxun, Xiaolin Wang, Jinping Cheng, Chi-wing Kong, Shuk Han Cheng, Ronald A. Li i Dong Sun. "Artificially induced cell fusion by optical tweezers manipulation". W 2013 IEEE 13th International Conference on Nanotechnology (IEEE-NANO). IEEE, 2013. http://dx.doi.org/10.1109/nano.2013.6720868.
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Wang, Xiaolin, Shuxun Chen, Chi-wing Kong, Ronald A. Li i Dong Sun. "Automated laser-induced cell fusion based on microwell array". W 2013 IEEE International Conference on Robotics and Automation (ICRA). IEEE, 2013. http://dx.doi.org/10.1109/icra.2013.6630963.
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Yang, P. F., C. H. Wang i G. B. Lee. "Optically-induced cell fusion on microfluidic chip utilizing locally enhanced electric field". W TRANSDUCERS 2015 - 2015 18th International Solid-State Sensors, Actuators and Microsystems Conference. IEEE, 2015. http://dx.doi.org/10.1109/transducers.2015.7180913.
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Hsiao, Yu-Chun, Chih-Hung Wang, Wen-Bin Lee i Gwo-Bin Lee. "Automatic cell fusion using optically-induced dielectrophoresis and optically-induced localized electric field on a structure-free microfluidic chip". W 2017 IEEE 12th International Conference on Nano/Micro Engineered and Molecular Systems (NEMS). IEEE, 2017. http://dx.doi.org/10.1109/nems.2017.8016962.
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LeDuc, Philip R., i Michael J. Betenbaugh. "Implementation of a Pharmocokinetic Approach to a Baculovirus System for Analytic Solutions to Virus and Cell Interactions". W ASME 1997 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 1997. http://dx.doi.org/10.1115/imece1997-0282.
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Abstract The baculovirus, Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), expression system can be employed for a variety of different cellular applications. In recent years, this baculovirus system has been manipulated to serve as a recombinant system for the expression of heterologous proteins and as a possible retrovirus for gene therapy. A quantitative understanding of the cellular mechanics of virus trafficking would be useful in developing viral expression systems, understanding gene therapy and maximizing recombinant protein production. An analytic solution is presented which incorporates a pharmocokinetic system in order to analyze this problem of viral and cellular mechanics. The multiple stages of viral infection systems, specifically for the baculovirus system, include attachment, internalization, endosomal fusion, cytosol transportation, and nuclear accumulation. The effects of the rate parameters are investigated to determine the parameter sensitivity of the viral infection model in relation to accumulation of surface virus, internalized virus and nuclear virus. The concepts from this model can be used to design infection regimens for various cell lines and to analyze the inherent inefficiency of current baculovirus infecting systems. This approach can also be used to determine the effects of the rate-limiting behaviors exhibited by these types of cellular mechanics and can be further implemented to examine other types of infection applications including viral gene therapy [1].
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Kang, Jeonghan. "Abstract 354: Inhibition of Smad3 signaling with a TAT fusion peptide prevented TGF-β-induced fibronectin expression and cell migration in malignant gliomas". W Proceedings: AACR Annual Meeting 2017; April 1-5, 2017; Washington, DC. American Association for Cancer Research, 2017. http://dx.doi.org/10.1158/1538-7445.am2017-354.
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Weiler, J., KS Zänker i T. Dittmar. "PO-156 TNFα-induced cell fusion between MDA-MB435-pFDR.1 and M13SV1-Cre cells is suppressed by minocycline through inhibition of the NF-KB pathway". W Abstracts of the 25th Biennial Congress of the European Association for Cancer Research, Amsterdam, The Netherlands, 30 June – 3 July 2018. BMJ Publishing Group Ltd, 2018. http://dx.doi.org/10.1136/esmoopen-2018-eacr25.678.
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Martinez-Miguel, Bernardo, Melisa Martinez Paniagua, Sara Huerta-Yepez, Cesar Gonzalez-Bonilla, Mario Vega-Paredes, Otoniel Martinez-Maza i Benjamin Bonavida. "Abstract 2422: The trimeric engineered fusion protein with CD154 peptide mimetic (OmpC-CD154p), induced apoptotic signaling in B-NHL cell lines mediated by caspase 3 and caspase 8 activation". W Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC. American Association for Cancer Research, 2010. http://dx.doi.org/10.1158/1538-7445.am10-2422.
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Saji, Genn. "Scientific Bases of Corrosion Control for Water-Cooled Fusion Reactors Such as ITER". W 2012 20th International Conference on Nuclear Engineering and the ASME 2012 Power Conference. American Society of Mechanical Engineers, 2012. http://dx.doi.org/10.1115/icone20-power2012-55046.
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Scientific bases for corrosion control for water-cooled fusion reactors, such as ITER, are discussed in this paper. In the previous overview papers [1,2], the author has identified that ‘long cell action’ corrosion plays a pivotal role in practically all unresolved corrosion issues, irrespective of fission reactor types and operation. In trying to confirm the existence of radiation-induced ‘long-cell’ action (macro) corrosion cell in the primary cooling system of LWRs, the author attempted to theoretically reproduce the electrochemical potential differences demonstrated during experiments at the INCA Loop in Sweden and the NRI-Rez Loop in the Czech Republic [3,4]. Based on these knowledge bases, characterization of water chemistry for fusion was made through radiation- and electro-chemistry analysis following the methodology explained in the companion paper also presented at this conference [5]. Approximately 70 mV of potential difference is predicted between the high dose rate and the out-of-flux regions through the illustrative model examined in this paper, which supports the existence of the LGCA mechanism. This value is about 1/5 of the potential difference for a typical PWR. For further verification of this approach, ‘international benchmark in-pile tests’ are highly awaited. Further optimization studies as well as material testing under the influence of the LGCA mechanism should be performed to establish corrosion control.
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Inaba, Humio, i Shunichi Sato. "All-optical, efficient fusion technology of biological cells incorporating near-IR laser beam manipulation and UV laser beam processing". W The European Conference on Lasers and Electro-Optics. Washington, D.C.: Optica Publishing Group, 1994. http://dx.doi.org/10.1364/cleo_europe.1994.cwf25.
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Various kinds of biological cell fusion techniques such as based on biological, chemical, and physical methods have been developed up to now, and they are widely used as essential means for basic research and applications in biotechnology and life sciences. However, whole cell damage associated with these fusion techniques is unavoidable because stimulations like high concentration polyethylene glycol and high voltage electric field are collectively applied. These collective treatments also do not make possible a fusion of specific cells that are desired for particular purposes. As an alternative to these techniques, the laser- induced fusion scheme realizing a one-to- one fusion of desired cells in an enclosed and safe chamber, with cell damage limited to a small region irradiated by the laser pulse, has been studied and developed by some groups including us.1,2
Raporty organizacyjne na temat "Retrovirus Induced Cell-Cell Fusion"
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Davidson, Irit, Hsing-Jien Kung i Richard L. Witter. Molecular Interactions between Herpes and Retroviruses in Dually Infected Chickens and Turkeys. United States Department of Agriculture, styczeń 2002. http://dx.doi.org/10.32747/2002.7575275.bard.
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Tumors in commercial poultry are caused mainly by infection with avian herpes and retroviruses, the herpesvirus Marek's disease virus (MDV) and the retroviruses, reticuloendotheliosis (REV), lymphoid leukosis, subgroups A-I and J (ALV and ALV-J) in chickens, or Iymphoprolipherative disease (LPDV) in turkeys. Infection with one virus aggravates the clinical outcome of birds that are already infected by another oncogenic virus. As these viruses do not interfere for infection, MDV and one or more retroviruses can infect the same flock, the same bird and the same cell. While infecting the same cell, herpes and retroviruses might interact in at least three ways: a) Integration of retrovirus genomes, or genomic fragments (mainly the LTR) into MDV;b) alteration of LTR-driven expression of retroviral genes by MDV immediate- early genes, and c) by herpesvirus induced cellular transcriptional factors. The first type of molecular interaction have been demonstrated to happen efficiently in vitro by Dr. Kung, in cases multiple infection of cell cultures with MDV and REV or MDV and ALV. Moreover, Dr. Witter showed that an in vitro-created recombinant, RM1, had altered in vitro replication and in vivo biological properties. A more comprehensive characterization of RM1 was carried out in the present project. We sought to highlight whether events of such integrations occur also in the bird, in vivo. For that, we had first to determine the prevalence of dually-infected individual birds in commercial flocks, as no systematic survey has been yet reported. Surprisingly, about 25% of the commercial flocks infected with avian oncogenic viruses had a multiple virus infection and 5% of the total samples ana lysed had multiple virus sequences. Then, we aimed to evaluate and characterize biologically and molecularly the resulting recombinants, if formed, and to analyse the factors that affect these events (virus strains, type and age of birds and time interval between the infection with both viruses). The perception of retrovirus insertions into herpesviruses in vivo is not banal, as the in vivo and in vitro systems differ in the viral-target cells, lymphocytes or fibroblasts, in the MDV-replicative type, transforming or productive, and the immune system presence. We realized that previous methods employed to study in vitro created recombinant viruses were not adequate for the study of samples taken directly from the bird. Therefore, the Hot Spot-combined PCR was developed based on the molecularly known RM1 virus. Also, the PFGE that was used for tissue cultured-MDV separation was inefficient for separating MDV from organs, but useful with feather tips as a source of bird original MDV. Much attention was dedicated now to feathers, because if a recombinant virus would be formed in vivo, its biological significance would be evident by horizontal dissemination through the feathers. Major findings were: a) not only in vitro, but also in vivo MDV and retrovirus co-infections lead to LTR integrations into MDV. That was shown by the detection of chimeric molecules. These appeared in low quantities and as quasispecies, thus interfering with sequence analysis of cloned gel-purified chimeric molecules. Mainly inserts were located in the repeat long MDV fragments. In field birds chimeric molecules were detected at a lower frequency (2.5%) than in experimentally infected birds (30-50%). These could be transmitted experimentally to another birds by inoculation with chimeric molecules containing blood. Several types of chimeric molecules were formed, and same types were detected in birds infected by a second round. To reproduce viral integrations, in vivo infection trials were done with field inoculate that contained both viruses, but the chimeric molecule yield was undetectable.
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Newton, Ronald, Joseph Riov i John Cairney. Isolation and Functional Analysis of Drought-Induced Genes in Pinus. United States Department of Agriculture, wrzesień 1993. http://dx.doi.org/10.32747/1993.7568752.bard.
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Drought is a common factor limiting timber production in the U.S. and Israel. Loblolly (Pinus taeda) and alleppo pine (Pinus halepensis) seedling survival is reduced when out planted, and growth and reproduction are often hindered by periodic droughts during later stages of tree development. Molecular and gene responses to drought stress have not been characterized. The objectives were to characterize drought-induced gene clones from these pines, to determine the effects of a growth regulator on drought tolerance, ABA levels, and drought-induced gene expression in alleppo pine, and to develop procedures for loblolly pine transformation. Nearly 20 cDNA clones influenced by gradual, prolonged drought stress have been isolated. Many of these have been shown to be induced by drought stress, whereas several others are down-regulated. These are the first drought-induced genes isolated from a pine species. Two genomic clones (lp5-1 and lp3-1) have been sequenced and characterized, and each has been found to be associated with a gene family. Clone lp5 appears to code for a cell wall protein, and clone lp3 codes for a nuclear protein. The former may be associated with changing the elastic properties of the cell wall, while the latter may be involved in signal transduction and/or protection from desiccation in the nucleus. Clone lp3 is similar to a drought-induced gene from tomato and is regulated by ABA. Several DNA sequences that are specific to induction during growth-retardation in alleppo pine by uniconazole have been identified. The active DNA species is now being identified. Promoters from genomic clones, lp3 and lp5, have been sequenced. Both are functional when fused with the gus reporter gene and transferred to other plant tissues as well as responding to a simulated drought stress. Through exodeletion analysis, it has been established that the promoter ABRE element of lp3 responds to ABA and that drought-induction of lp3 expression may also involve ABA. Stable tobacco transformants carrying either the lp5 or the lp3 promoter fused to a reporter gus gene have been obtained. The lp5lgus fusion was expressed at several stages of tobacco development and differentiation including the reproductive stage. There was no difference in phenotype between the transformants and the wild type. Embryogenesis procedures were developed for slash pine, but attempts to couple this process with gene transfer and plantlet transformation were not successful. Transformation of pine using Agrobacterium appears tractable, but molecular data supporting stable integration of the Agrobacterium-transferred gene are still inconclusive.
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Gafny, Ron, A. L. N. Rao i Edna Tanne. Etiology of the Rugose Wood Disease of Grapevine and Molecular Study of the Associated Trichoviruses. United States Department of Agriculture, wrzesień 2000. http://dx.doi.org/10.32747/2000.7575269.bard.
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Rugose wood is a complex disease of grapevines, characterized by modification of the woody cylinder of affected vines. The control of rugose wood is based on the production of healthy propagation material. Detection of rugose wood in grapevines is difficult and expensive: budwood from tested plants is grafted onto sensitive Vitis indicators and the appearance of symptoms is monitored for 3 years. The etiology of rugose wood is complex and has not yet been elucidated. Several elongated clostero-like viruses are consistently found in affected vines; one of them, grapevine virus A (GVA), is closely associated with Kober stem grooving, a component of the rugose wood complex. GVA has a single-stranded RNA genome of 7349 nucleotides, excluding a polyA tail at the 3' terminus. The GVA genome includes five open reading frames (ORFs 1-5). ORF 4, which encodes for the coat protein of GVA, is the only ORF for which the function was determined experimentally. The original objectives of this research were: 1- To produce antisera to the structural and non-structural proteins of GVA and GVB and to use these antibodies to establish an effective detection method. 2- Develop full length infectious cDNA clones of GVA and GVB. 3- Study the roll of GVA and GVB in the etiology of the grapevine rugose wood disease. 4- Determine the function of Trichovirus (now called Vitivirus) encoded genes in the virus life cycle. Each of the ORFs 2, 3, 4 and 5 genes of GVA were cloned and expressed in E. coli and used to produce antisera. Both the CP (ORF 4) and the putative MP (ORF 3) were detected with their corresponding antisera in-GVA infected N. benthamiana and grapevine. The MP was first detected at an early stage of the infection, 6-12 h after inoculation, and the CP 2-3 days after inoculation. The MP could be detected in GVA-infected grapevines that tested negative for CP, both with CP antiserum and with a commercially available ELISA kit. Antisera to ORF 2 and 5 encoded proteins could react with the recombinant proteins but failed to detect both proteins in GVA infected plants. A full-length cDNA clone of grapevine virus A (GVA) was constructed downstream from the bacteriophage T7 RNA polymerase promoter. Capped in vitro transcribed RNA was infectious in N. benthamiana and N. clevelandii plants. Symptoms induced by the RNA transcripts or by the parental virus were indistinguishable. The infectivity of the in vitro-transcribed RNA was confirmed by serological detection of the virus coat and movement proteins and by observation of virions by electron microscopy. The full-length clone was modified to include a gus reporter gene and gus activity was detected in inoculated and systemic leaves of infected plants. Studies of GVA mutants suggests that the coat protein (ORF 4) is essential for cell to cell movement, the putative movement protein (ORF 3) indeed functions as a movement protein and that ORF 2 is not required for virus replication, cell to cell or systemic movement. Attempts to infect grapevines by in-vitro transcripts, by inoculation of cDNA construct in which the virus is derived by the CaMV 35S promoter or by approach grafting with infected N. benthamiana, have so far failed. Studies of the subcellular distribution of GFP fusion with each of ORF 2, 3 and 4 encoded protein showed that the CP fusion protein accumulated as a soluble cytoplasmatic protein. The ORF 2 fusion protein accumulated in cytoplasmatic aggregates. The MP-GFP fusion protein accumulated in a large number of small aggregates in the cytoplasm and could not move from cell to cell. However, in conditions that allowed movement of the fusion protein from cell to cell (expression by a PVX vector or in young immature leaves) the protein did not form cytoplasmatic aggregates but accumulated in the plasmodesmata.
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Epel, Bernard L., Roger N. Beachy, A. Katz, G. Kotlinzky, M. Erlanger, A. Yahalom, M. Erlanger i J. Szecsi. Isolation and Characterization of Plasmodesmata Components by Association with Tobacco Mosaic Virus Movement Proteins Fused with the Green Fluorescent Protein from Aequorea victoria. United States Department of Agriculture, wrzesień 1999. http://dx.doi.org/10.32747/1999.7573996.bard.
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The coordination and regulation of growth and development in multicellular organisms is dependent, in part, on the controlled short and long-distance transport of signaling molecule: In plants, symplastic communication is provided by trans-wall co-axial membranous tunnels termed plasmodesmata (Pd). Plant viruses spread cell-to-cell by altering Pd. This movement scenario necessitates a targeting mechanism that delivers the virus to a Pd and a transport mechanism to move the virion or viral nucleic acid through the Pd channel. The identity of host proteins with which MP interacts, the mechanism of the targeting of the MP to the Pd and biochemical information on how Pd are alter are questions which have been dealt with during this BARD project. The research objectives of the two labs were to continue their biochemical, cellular and molecular studies of Pd composition and function by employing infectious modified clones of TMV in which MP is fused with GFP. We examined Pd composition, and studied the intra- and intercellular targeting mechanism of MP during the infection cycle. Most of the goals we set for ourselves were met. The Israeli PI and collaborators (Oparka et al., 1999) demonstrated that Pd permeability is under developmental control, that Pd in sink tissues indiscriminately traffic proteins of sizes of up to 50 kDa and that during the sink to source transition there is a substantial decrease in Pd permeability. It was shown that companion cells in source phloem tissue export proteins which traffic in phloem and which unload in sink tissue and move cell to cell. The TAU group employing MP:GFP as a fluorescence probe for optimized the procedure for Pd isolation. At least two proteins kinases found to be associated with Pd isolated from source leaves of N. benthamiana, one being a calcium dependent protein kinase. A number of proteins were microsequenced and identified. Polyclonal antibodies were generated against proteins in a purified Pd fraction. A T-7 phage display library was created and used to "biopan" for Pd genes using these antibodies. Selected isolates are being sequenced. The TAU group also examined whether the subcellular targeting of MP:GFP was dependent on processes that occurred only in the presence of the virus or whether targeting was a property indigenous to MP. Mutant non-functional movement proteins were also employed to study partial reactions. Subcellular targeting and movement were shown to be properties indigenous to MP and that these processes do not require other viral elements. The data also suggest post-translational modification of MP is required before the MP can move cell to cell. The USA group monitored the development of the infection and local movement of TMV in N. benthamiana, using viral constructs expressing GFP either fused to the MP of TMV or expressing GFP as a free protein. The fusion protein and/or the free GFP were expressed from either the movement protein subgenomic promoter or from the subgenomic promoter of the coat protein. Observations supported the hypothesis that expression from the cp sgp is regulated differently than expression from the mp sgp (Szecsi et al., 1999). Using immunocytochemistry and electron microscopy, it was determined that paired wall-appressed bodies behind the leading edge of the fluorescent ring induced by TMV-(mp)-MP:GFP contain MP:GFP and the viral replicase. These data suggest that viral spread may be a consequence of the replication process. Observation point out that expression of proteins from the mp sgp is temporary regulated, and degradation of the proteins occurs rapidly or more slowly, depending on protein stability. It is suggested that the MP contains an external degradation signal that contributes to rapid degradation of the protein even if expressed from the constitutive cp sgp. Experiments conducted to determine whether the degradation of GFP and MP:GFP was regulated at the protein or RNA level, indicated that regulation was at the protein level. RNA accumulation in infected protoplast was not always in correlation with protein accumulation, indicating that other mechanisms together with RNA production determine the final intensity and stability of the fluorescent proteins.
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Granot, David, Richard Amasino i Avner Silber. Mutual effects of hexose phosphorylation enzymes and phosphorous on plant development. United States Department of Agriculture, styczeń 2006. http://dx.doi.org/10.32747/2006.7587223.bard.
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Research objectives 1) Analyze the combined effects of hexose phosphorylation and P level in tomato and Arabidopsis plants 2) Analyze the combined effects of hexose phosphorylation and P level in pho1 and pho2 Arabidopsis mutants 3) Clone and analyze the PHO2 gene 4) Select Arabidopsis mutants resistant to high and low P 5) Analyze the Arabidopsis mutants and clone the corresponding genes 6) Survey wild tomato species for growth characteristics at various P levels Background to the topic Hexose phosphorylating enzymes, the first enzymes of sugar metabolism, regulate key processes in plants such as photosynthesis, growth, senescence and vascular transport. We have previously discovered that hexose phosphorylating enzymes might regulate these processes as a function of phosphorous (P) concentration, and might accelerate acquisition of P, one of the most limiting nutrients in the soil. These discoveries have opened new avenues to gain fundamental knowledge about the relationship between P, sugar phosphorylation and plant development. Since both hexose phosphorylating enzymes and P levels affect plant development, their interaction is of major importance for agriculture. Due to the acceleration of senescence caused by the combined effects of hexose phosphorylation and P concentration, traits affecting P uptake may have been lost in the course of cultivation in which fertilization with relatively high P (30 mg/L) are commonly used. We therefore intended to survey wild tomato species for high P-acquisition at low P soil levels. Genetic resources with high P-acquisition will serve not only to generate a segregating population to map the trait and clone the gene, but will also provide a means to follow the trait in classical breeding programs. This approach could potentially be applicable for other crops as well. Major conclusions, solutions, achievements Our results confirm the mutual effect of hexose phosphorylating enzymes and P level on plant development. Two major aspects of this mutual effect arose. One is related to P toxicity in which HXK seems to play a major role, and the second is related to the effect of HXK on P concentration in the plant. Using tomato plants we demonstrated that high HXK activity increased leaf P concentration, and induced P toxicity when leaf P concentration increases above a certain high level. These results further support our prediction that the desired trait of high-P acquisition might have been lost in the course of cultivation and might exist in wild species. Indeed, in a survey of wild species we identified tomato species that acquired P and performed better at low P (in the irrigation water) compared to the cultivated Lycopersicon esculentum species. The connection between hexose phosphorylation and P toxicity has also been shown with the P sensitive species VerticordiaplumosaL . in which P toxicity is manifested by accelerated senescence (Silber et al., 2003). In a previous work we uncovered the phenomenon of sugar induced cell death (SICD) in yeast cells. Subsequently we showed that SICD is dependent on the rate of hexose phosphorylation as determined by Arabidopsis thaliana hexokinase. In this study we have shown that hexokinase dependent SICD has many characteristics of programmed cell death (PCD) (Granot et al., 2003). High hexokinase activity accelerates senescence (a PCD process) of tomato plants, which is further enhanced by high P. Hence, hexokinase mediated PCD might be a general phenomena. Botrytis cinerea is a non-specific, necrotrophic pathogen that attacks many plant species, including tomato. Senescing leaves are particularly susceptible to B. cinerea infection and delaying leaf senescence might reduce this susceptibility. It has been suggested that B. cinerea’s mode of action may be based on induction of precocious senescence. Using tomato plants developed in the course of the preceding BARD grant (IS 2894-97) and characterized throughout this research (Swartzberg et al., 2006), we have shown that B. cinerea indeed induces senescence and is inhibited by autoregulated production of cytokinin (Swartzberg et al., submitted). To further determine how hexokinase mediates sugar effects we have analyzed tomato plants that express Arabidopsis HXK1 (AtHXK1) grown at different P levels in the irrigation water. We found that Arabidopsis hexokinase mediates sugar signalling in tomato plants independently of hexose phosphate (Kandel-Kfir et al., submitted). To study which hexokinase is involved in sugar sensing we searched and identified two additional HXK genes in tomato plants (Kandel-Kfir et al., 2006). Tomato plants have two different hexose phosphorylating enzymes; hexokinases (HXKs) that can phosphorylate either glucose or fructose, and fructokinases (FRKs) that specifically phosphorylate fructose. To complete the search for genes encoding hexose phosphorylating enzymes we identified a forth fructokinase gene (FRK) (German et al., 2004). The intracellular localization of the four tomato HXK and four FRK enzymes has been determined using GFP fusion analysis in tobacco protoplasts (Kandel-Kfir et al., 2006; Hilla-Weissler et al., 2006). One of the HXK isozymes and one of the FRK isozymes are located within plastids. The other three HXK isozymes are associated with the mitochondria while the other three FRK isozymes are dispersed in the cytosol. We concluded that HXK and FRK are spatially separated in plant cytoplasm and accordingly might play different metabolic and perhaps signalling roles. We have started to analyze the role of the various HXK and FRK genes in plant development. So far we found that LeFRK2 is required for xylem development (German et al., 2003). Irrigation with different P levels had no effect on the phenotype of LeFRK2 antisense plants. In the course of this research we developed a rapid method for the analysis of zygosity in transgenic plants (German et al., 2003).