WorldWideScience

Sample records for baculovirus nuclear localization

  1. Baculoviruses and nucleosome management.

    Science.gov (United States)

    Volkman, Loy E

    2015-02-01

    Negatively-supercoiled-ds DNA molecules, including the genomes of baculoviruses, spontaneously wrap around cores of histones to form nucleosomes when present within eukaryotic nuclei. Hence, nucleosome management should be essential for baculovirus genome replication and temporal regulation of transcription, but this has not been documented. Nucleosome mobilization is the dominion of ATP-dependent chromatin-remodeling complexes. SWI/SNF and INO80, two of the best-studied complexes, as well as chromatin modifier TIP60, all contain actin as a subunit. Retrospective analysis of results of AcMNPV time course experiments wherein actin polymerization was blocked by cytochalasin D drug treatment implicate actin-containing chromatin modifying complexes in decatenating baculovirus genomes, shutting down host transcription, and regulating late and very late phases of viral transcription. Moreover, virus-mediated nuclear localization of actin early during infection may contribute to nucleosome management.

  2. Baculoviruses and nucleosome management

    Energy Technology Data Exchange (ETDEWEB)

    Volkman, Loy E., E-mail: lvolkman@berkeley.edu

    2015-02-15

    Negatively-supercoiled-ds DNA molecules, including the genomes of baculoviruses, spontaneously wrap around cores of histones to form nucleosomes when present within eukaryotic nuclei. Hence, nucleosome management should be essential for baculovirus genome replication and temporal regulation of transcription, but this has not been documented. Nucleosome mobilization is the dominion of ATP-dependent chromatin-remodeling complexes. SWI/SNF and INO80, two of the best-studied complexes, as well as chromatin modifier TIP60, all contain actin as a subunit. Retrospective analysis of results of AcMNPV time course experiments wherein actin polymerization was blocked by cytochalasin D drug treatment implicate actin-containing chromatin modifying complexes in decatenating baculovirus genomes, shutting down host transcription, and regulating late and very late phases of viral transcription. Moreover, virus-mediated nuclear localization of actin early during infection may contribute to nucleosome management. - Highlights: • Baculoviruses have negatively-supercoiled, circular ds DNA. • Negatively-supercoiled DNA spontaneously forms nucleosomes in the nucleus. • Nucleosomes must be mobilized for replication and transcription to proceed. • Actin-containing chromatin modifiers participate in baculovirus replication.

  3. A new mechanism for nuclear import by actin-based propulsion used by a baculovirus nucleocapsid.

    Science.gov (United States)

    Au, Shelly; Wu, Wei; Zhou, Lixin; Theilmann, David A; Panté, Nelly

    2016-08-01

    The transport of macromolecules into the nucleus is mediated by soluble cellular receptors of the importin β superfamily and requires the Ran-GTPase cycle. Several studies have provided evidence that there are exceptions to this canonical nuclear import pathway. Here, we report a new unconventional nuclear import mechanism exploited by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). We found that AcMNPV nucleocapsids entered the nucleus of digitonin-permeabilized cells in the absence of exogenous cytosol or under conditions that blocked the Ran-GTPase cycle. AcMNPV contains a protein that activates the Arp2/3 complex and induces actin polymerization at one end of the rod-shaped nucleocapsid. We show that inhibitors of Arp2/3 blocked nuclear import of nucleocapsids in semi-permeabilized cells. Nuclear import of nucleocapsids was also reconstituted in purified nuclei supplemented with G-actin and Arp2/3 under actin polymerization conditions. Thus, we propose that actin polymerization drives not only migration of baculovirus through the cytoplasm but also pushes the nucleocapsid through the nuclear pore complex to enter the cell nucleus. Our findings point to a very distinct role of actin-based motility during the baculovirus infection cycle. PMID:27284005

  4. Baculovirus infection of nondividing mammalian cells: mechanisms of entry and nuclear transport of capsids.

    NARCIS (Netherlands)

    N.D. van Loo; E. Fortunati (Elisabetta); E.M.E. Ehlert (Ehrich); M. Rabelink; F.G. Grosveld (Frank); B.J. Scholte (Bob)

    2001-01-01

    textabstractWe have studied the infection pathway of Autographa californica multinuclear polyhedrosis virus (baculovirus) in mammalian cells. By titration with a baculovirus containing a green fluorescent protein cassette, we found that several, but not all, mammalian c

  5. Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells

    International Nuclear Information System (INIS)

    The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV-gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type

  6. Local Immune Stimulation by Intravesical Instillation of Baculovirus to Enable Bladder Cancer Therapy

    OpenAIRE

    Wei Xia Ang; Ying Zhao; Timothy Kwang; Chunxiao Wu; Can Chen; Han Chong Toh; Ratha Mahendran; Kesavan Esuvaranathan; Shu Wang

    2016-01-01

    Intravesical instillation of Bacillus Calmette-Guérin is currently used as adjuvant therapy for superficial, non-muscle invasive bladder cancer (NMIBC). However, nearly 40% of patients with NMIBC will fail Bacillus Calmette-Guérin therapy. In an attempt to investigate the feasibility of using insect baculovirus-based vectors for bladder cancer therapy, we observed that intravesical instillation of baculoviruses without transgene up-regulated a set of Th1-type of cytokines and increased the su...

  7. Baculovirus RNA Polymerase: Activities, Composition, and Evolution

    Institute of Scientific and Technical Information of China (English)

    A.Lorena Passarelli

    2007-01-01

    Baculoviruses are the only nuclear replicating DNA-containing viruses that encode their own DNA-directed RNA polymerase (RNAP). The baculovirus RNAP is specific for the transcription of genes expressed after virus DNA replication. It is composed of four subunits, making it the simplest multisubunit RNAP known. Two subunits contain motifs found at the catalytic center of other RNAPs and a third has capping enzyme functions. The function of the fourth subunit is not known. Structural studies on this unique RNAP will provide new insights into the functions of this enzyme and the regulation of viral genes and may be instrumental to optimize the baculovirus gene expression system.

  8. Baculovirus DNA replication.

    NARCIS (Netherlands)

    Kool, M.

    1994-01-01

    Baculoviruses are attractive biological agents for the control of insect pests. They are highly specific for insects and cause a fatal disease (Granados and Federici, 1986). in addition, baculoviruses are successfully exploited as expression vectors for the production of heterologous proteins for va

  9. Baculovirus: Hospederos y especificidad

    Directory of Open Access Journals (Sweden)

    Juliana Gómez Valderrama

    2013-12-01

    Full Text Available Título corto: BaculovirusTítulo en ingles: Baculovirus: Hosts and specificityResumen: Los baculovirus son virus patógenos de insectos ampliamente empleados a nivel mundial como bioinsecticidas para el control de diferentes plagas de importancia agrícola y más recientemente como vectores de expresión de proteínas y vectores para terapia génica. Una de sus características principales es su alta especificidad de hospedero que incluye un rango muy estrecho de especies de insectos, que a menudo pertenecen a la misma familia. Sin embargo, es necesario entender los mecanismos involucrados en la definición del rango de hospederos de los baculovirus con el fin de evaluar la seguridad e inocuidad de su uso y determinar la posibilidad de mejorar sus propiedades para aplicaciones biotecnológicas mediante la construcción de baculovirus recombinantes con diferentes rangos de hospederos. En el presente artículo se revisarán los principales mecanismos comprendidos en la definición del rango de hospederos de los baculovirus, dentro de los que se destacan la especificidad para entrar en las células, la posibilidad de replicación del genoma viral, el control de los procesos bioquímicos y moleculares del insecto y las interacciones virus-hospedero que regulan la multiplicación del agente infeccioso, así como las perspectivas de la aplicación de este conocimiento.Palabras clave: infección viral, rango de hospederos, evolución, resistencia.Abstract: Baculoviruses are insect pathogenic viruses widely used as bioinsecticides for controlling of several agricultural important pests and more recently as protein expression vectors and gene therapy vectors. One of its main characteristics is its high host-specificity including a very narrow range of insect species, which often belong to the same family. However, to understand the mechanisms involved in the definition of baculoviruses host range is necessary in order to assess the security and safety

  10. Identification of two functional nuclear localization signals in the capsid protein of duck circovirus

    International Nuclear Information System (INIS)

    The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1–17 and 18–36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNA binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome.

  11. Improving baculovirus recombination

    OpenAIRE

    Zhao, Yuguang; Chapman, David A. G.; Jones, Ian M.

    2003-01-01

    Recombinant baculoviruses have established themselves as a favoured technology for the high-level expression of recombinant proteins. The construction of recombinant viruses, however, is a time consuming step that restricts consideration of the technology for high throughput developments. Here we use a targeted gene knockout technology to inactivate an essential viral gene that lies adjacent to the locus used for recombination. Viral DNA prepared from the knockout fails to initiate an infecti...

  12. The local community and the nuclear waste

    International Nuclear Information System (INIS)

    In this book social and political scientists discuss different aspects of the selection of a site for disposal of the Swedish nuclear waste. Special attention is given to the preliminary studies that have been performed at a few localities. The authors study the chain of events after a community is proposed for a site study. What powers are set in motion? How do different groups act in order to support or stop the study? Which is the role played by political parties, local environmentalist movements, media and experts? Why is there a forceful opposition in one community and not in another? Why does one local government invite the nuclear waste company to perform the study, while another refuses? The role of the local government has become crucial, since the nuclear waste company have chosen to perform studies only in municipalities that show a positive interest

  13. Localization of nuclear power plant technology

    International Nuclear Information System (INIS)

    Asia, and particularly China, has an enormous need for power and must deal with the practicalities of building large base load units. In China, as in other countries, there are limitations on the use of large quantities of fossil fuel. This raises the possibility of turning to nuclear power to satisfy their energy needs. Other issues tend to point to the nuclear option for these growing economies, including economic considerations, environmental concerns, energy independence and raising the technological capabilities of the country. When a country embarks on a nuclear power program with the intention of localizing the technology, a long-term commitment is necessary to achieve this objective. Localization of nuclear technology is not a new phenomenon. The nature of the industry from the early beginnings has always involved transfer of technology when a new country initiated a nuclear power construction program. In fact, most previous experiences with this localization process involved heavy governmental, political and financial support to drive the success of the program. Because of this strong governmental support, only the receiving nation's companies were generally allowed to participate in the local business operations of the technology recipient. What is new and different today is the retreat from heavy financial support by the receiving country's government. This change has created a strong emphasis on cost-effectiveness in the technology transfer process and opportunities for foreign companies to participate in local business activities. ABB is a world-wide company with two parent companies that have been very active over many years in establishing cost-justified local operations throughout the world. Today, ABB has become the largest electrical engineering company in the world with respected local operations in nearly every country. Lessons learned by ABB in their world-wide localization initiatives are being applied to the challenge of cost

  14. Overview of the baculovirus expression system.

    Science.gov (United States)

    Murphy, C I; Piwnica-Worms, H

    2001-05-01

    Baculoviruses have emerged as a popular system for overproducing recombinant proteins in eukaryotic cells. This overview unit describes the baculovirus life cycle and expression system, and also provides information on vectors and protocols for using the baculovirus expression system. PMID:18429185

  15. Local AREA networks in advanced nuclear reactors

    International Nuclear Information System (INIS)

    The report assesses Local Area Network Communications with a view to their application in advanced nuclear reactor control and protection systems. Attention is focussed on commercially available techniques and systems for achieving the high reliability and availability required. A basis for evaluating network characteristics in terms of broadband or baseband type, medium, topology, node structure and access method is established. The reliability and availability of networks is then discussed. Several commercial networks are briefly assessed and a distinction made between general purpose networks and those suitable for process control. The communications requirements of nuclear reactor control and protection systems are compared with the facilities provided by current technology

  16. Identification of two functional nuclear localization signals in the capsid protein of duck circovirus.

    Science.gov (United States)

    Xiang, Qi-Wang; Zou, Jin-Feng; Wang, Xin; Sun, Ya-Ni; Gao, Ji-Ming; Xie, Zhi-Jing; Wang, Yu; Zhu, Yan-Li; Jiang, Shi-Jin

    2013-02-01

    The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1-17 and 18-36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNA binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome. PMID:23174505

  17. The Long Road to Understanding the Baculovirus P10 Protein

    Institute of Scientific and Technical Information of China (English)

    David C. J.Carpentier; Linch A. King

    2009-01-01

    The baculovirus P 10 protein has always represented a mystery in the feld of insect virology. Like the baculovirus polyhedrin protein it is expressed at high levels very late in infection. Homologues of the Autographa californica nucleopolyhedrovirus plO gene are conserved in all Alphabaculoviruses and in other viruses of lepidopteran hosts yet is completely dispensable for virus replication and transmission. P10 is a microtubule interacting protein whose expression has been associated with the formation of a variety of complex and extensive cytoplasmic and nuclear structures. P10 has been associated with a number of roles during infection ranging from the formation of virus occlusion bodies, to affecting the rate of cellular and/or nuclear lysis during the final stages of the virus replication cycle. In this article we review recent work aimed at understanding the role of this enigmatic protein, putting them into context with recent advances in understanding of protein structure and function. We look back at a number of historical studies and observations, reanalysing their conclusions based on recent data and our own observations. The role of the P 10 protein during baculovirus replication remains elusive, however, novel avenues of investigation have been identified that will, we are sure, eventually lead to an understanding of this protein.

  18. Book review: Baculovirus Molecular Biology, Second Edition

    Science.gov (United States)

    The application of cell culture and molecular biology methodologies to the study of baculoviruses has resulted in an explosion of information on this group of insect pathogens. The quantity of the corresponding literature on baculoviruses has reached a level difficult for any one researcher to mast...

  19. Baculovirus display of functional antibody Fab fragments.

    Science.gov (United States)

    Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

    2015-08-01

    The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

  20. Baculoviruses as Vectors in Mammalian Cells

    Institute of Scientific and Technical Information of China (English)

    Chang-yong LIANG; Xin-wen CHEN

    2007-01-01

    The Baculoviridae are a large family of enveloped DNA viruses exclusively pathogenic to arthropods. Baculoviruses have been extensively used in insect cell-based recombinant protein expression system and as biological pesticides. They have been deomostrated to be safe to mammals, birds and fish. Recently, baculoviruses has been shown to transduce different mammalian cells in spite of the fact that they cannot replicate in mammalian cells (11, 73, 76). This has resulted in the development of baculoviruses as mammalian expression systems and even as vestors for gene therapy.

  1. Local competence building and public information in European nuclear territories

    International Nuclear Information System (INIS)

    Opinion polls and case study analysis show that there is a strong public demand for more participation in decision-making processes relating to the environment and nuclear issues specifically (IGNA, 2007; EC DG TREN 2008). In this regard, the implementation of transparency and participation at local level in municipalities hosting nuclear facilities is being claimed by local representatives over Europe. The pilot project 'Local Competence Building and Public Information in European Nuclear Territories' (2007-2008) was promoted by the Group of European Municipalities with Nuclear Facilities (GMF) and partly financed by the Directorate General for Energy and Transport (DG TREN). The project aims at supporting the European Commission (EC) in the process of harmonising national practices in the field of governance in nuclear territories. The objectives of this pilot project were two-fold: firstly, to identify local good practices at European level with regards to governance in the nuclear field and, secondly, to provide a methodological framework to recommend good practices and establish a set of indicators. The results of the project are briefly explained under the five dimensions of local governance on nuclear matters: access to environmental information, participation, access to justice, competence building and local development. Firstly, access to information on nuclear issues is broadly acknowledged by local representatives to be of relevant importance in increasing public knowledge on nuclear issues, especially on nuclear safety and radiation protection. Secondly, most local representatives feel that the current participatory level on nuclear issues in municipalities is too low and they would appreciate a greater involvement of local communities in decision-making processes on nuclear issues through local commissions or other mechanisms. Thirdly, according to local authorities, access to justice should be provided not only for the public but also for themselves

  2. Silencing structural and nonstructural genes in baculovirus by RNA interference.

    Science.gov (United States)

    Flores-Jasso, C Fabian; Valdes, Victor Julian; Sampieri, Alicia; Valadez-Graham, Viviana; Recillas-Targa, Felix; Vaca, Luis

    2004-06-01

    We review several aspects of RNAi and gene silencing with baculovirus. We show that the potency of RNAi in Spodoptera frugiperda (Sf21) insect cells correlates well with the efficiency of transfection of the siRNA. Using a fluorescein-labeled siRNA we found that the siRNA localized in areas surrounding the endoplasmic reticulum (ER). Both long (700 nucleotides long) and small ( approximately 25 nucleotides long) interfering RNAs were equally effective in initiating RNA interference (RNAi), and the duration of the interfering effect was indistinguishable. Even though RNAi in Sf21 cells is very effective, in vitro experiments show that these cells fragment the long dsRNA into siRNA poorly, when compared to HEK cells. Finally, we show that in vivo inhibition of baculovirus infection with dsRNA homologous to genes that are essential for baculovirus infectivity depends strongly on the amount of dsRNA used in the assays. Five hundred nanogram of dsRNA directly injected into the haemolymph of insects prevent animal death to over 95%. In control experiments, over 96% of insects not injected with dsRNA or injected with an irrelevant dsRNA died within a week. These results demonstrate the efficiency of dsRNA for in vivo prevention of a viral infection by virus that is very cytotoxic and lytic in animals.

  3. Genetic Modification of Baculovirus Expression Vectors

    Institute of Scientific and Technical Information of China (English)

    Shu-fen Li; Hua-lin Wang; Zhi-hong Hu; Fei Deng

    2012-01-01

    As a protein expression vector,the baculovirus demonstrates many advantages over other vectors.With the development of biotechnology,baculoviral vectors have been genetically modified to facilitate high level expression of heterologous proteins in both insect and mammalian cells.These modifications include utilization of different promoters and signal peptides,deletion or replacement of viral genes for increasing protein secretion,integration of polycistronic expression cassette for producing protein complexes,and baculovirus pseudotyping,promoter accommodation or surface display for enhancing mammalian cell targeting gene delivery.This review summarizes the development and the current state of art of the baculovirus expression system.Further development of baculovirus expression systems will make them even more feasible and accessible for advanced applications.

  4. Baculovirus ETL promoter acts as a shuttle promoter between insect cells and mammalian cells

    Institute of Scientific and Technical Information of China (English)

    Yu-kou LIU; Chih-chieh CHU; Tzong-yuan WU

    2006-01-01

    Aim:To identify a shuttle promoter that can mediate gene expression in both insect cells and mammalian cells to facilitate the development of a baculovirus vector-based mammalian cell gene delivery vehicle.Methods:Recombinant baculoviruses carrying the β-galactosidase reporter gene under the control of an early to late(ETL)promoter of the Autographa califomica multiple nuclear polyhedrosis virus(AcMNPV)or a cytomegalovirus immediate early promoter (CMV promoter)were constructed.COS1,HeLa,CHO-K1,hFob1.19,and MCF-7 mammalian cells were tested for the expression of β-galactosidase.Results:ETL promoter activity was higher in bone-derived hFob1.19 than in COS1,HeLa,CHOK1,or MCF-7 mammalian cells.The transient plasmid transfection assay indicated that ETL promoter activity in mammalian cells was dependent on baculovirus gene expression.Conclusion:ETL promoter activity in mammalian cells is baculovirus gene expression-dependent,and the shuttle promoter will facilitate the application of baculovirus expression vectors in mammalian cell expression systems and for gene therapy.

  5. Sumoylation of SAE2 C terminus regulates SAE nuclear localization.

    Science.gov (United States)

    Truong, Khue; Lee, Terry D; Li, Baozong; Chen, Yuan

    2012-12-14

    SUMOylation occurs predominantly in the nucleus, but non-nuclear proteins can also be SUMOylated. It is unclear how intracellular trafficking of the SUMOylation enzymes is regulated to catalyze SUMOylation in different cellular compartments. Here we report that the SAE2 subunit of human SUMO activation enzyme (SAE) underwent rapid nucleocytoplasmic shuttling and its nuclear accumulation depended on SUMO modification at the C terminus. The SUMOylation sites included three Lys residues on the bipartite nuclear localization sequence (NLS) and two Lys residues outside of but adjacent to the NLS, and their SUMOylation was catalyzed by Ubc9. Because SAE2 forms a tight heterodimer with SAE1 and it controls the trafficking of the heterodimer, this study has identified the mechanism used to localize SAE to the nucleus. Similar mechanisms are likely to exist for other proteins that depend on SUMOylation for nuclear localization.

  6. Persistence and coexistence of engineered baculoviruses

    NARCIS (Netherlands)

    Bonsall, M.B.; O'Reilly, D.R.; Cory, J.S.; Hails, R.S.

    2005-01-01

    Baculoviruses, and in particular, the nucleopolyhedroviruses infect a wide range of arthropod hosts and have the potential to be used as biopesticides. However, one of the major drawbacks with these pathogens as biocontrol agents is that they have a slow response time. Alterations to the speed of ki

  7. Pathogenesis induced by (recombinant) baculoviruses in insects.

    NARCIS (Netherlands)

    Flipsen, J.Th.M.

    1995-01-01

    Infection of insect larvae by a baculovirus leads to cessation of feeding and finally to the death of the larva. Under optimal conditions this process may take as little as five days during which the virus multiplies approximately a billion times and transforms 30% of the larval weight into viral pr

  8. A new baculovirus of cultured shrimps

    Institute of Scientific and Technical Information of China (English)

    陈细法; 陈平; 吴定虎; 黄槐; 池信才

    1997-01-01

    By means of ultrathin section, negative staining and sucrose gradient ultra-centrifugation, a new baculovirus has been discovered and purified in lymphoid organs and such tissues as muscles of the shrimps which have been spontaneously attacked by diseases and artificially infected. With a diameter of 96-112 nm, this is the thickest baculovirus of shrimps ever known. In the center is the high-density nucleus. Between the capsid and the envelope is a broad space, which is not found in any of the baculoviruses of the prawns ever reported. On the surface of the puri-fied nucleocapsid, there is a subunit of the spiral arrangement, which is also characteristic of this virus. It has not been observed and found in the epithelial cells of the livers, intestines and cheeks, which is quite different from the fact that prawn baculoviruses infect a certain epithepilial cell of the above-mentioned ones without exception. The viruses only multiplicate inside the core of target cells, which will not form occluded bodie

  9. Spatial localization in nuclear magnetic resonance spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Keevil, Stephen F [Department of Medical Physics, Guy' s and St Thomas' NHS Foundation Trust, Guy' s Hospital, London, SE1 9RT (United Kingdom); Division of Imaging Sciences, King' s College London, Guy' s Campus, London, SE1 9RT (United Kingdom)

    2006-08-21

    The ability to select a discrete region within the body for signal acquisition is a fundamental requirement of in vivo NMR spectroscopy. Ideally, it should be possible to tailor the selected volume to coincide exactly with the lesion or tissue of interest, without loss of signal from within this volume or contamination with extraneous signals. Many techniques have been developed over the past 25 years employing a combination of RF coil properties, static magnetic field gradients and pulse sequence design in an attempt to meet these goals. This review presents a comprehensive survey of these techniques, their various advantages and disadvantages, and implications for clinical applications. Particular emphasis is placed on the reliability of the techniques in terms of signal loss, contamination and the effect of nuclear relaxation and J-coupling. The survey includes techniques based on RF coil and pulse design alone, those using static magnetic field gradients, and magnetic resonance spectroscopic imaging. Although there is an emphasis on techniques currently in widespread use (PRESS, STEAM, ISIS and MRSI), the review also includes earlier techniques, in order to provide historical context, and techniques that are promising for future use in clinical and biomedical applications. (topical review)

  10. Baculovirus Insecticides in Latin America: Historical Overview, Current Status and Future Perspectives

    Directory of Open Access Journals (Sweden)

    Santiago Haase

    2015-04-01

    Full Text Available Baculoviruses are known to regulate many insect populations in nature. Their host-specificity is very high, usually restricted to a single or a few closely related insect species. They are amongst the safest pesticides, with no or negligible effects on non-target organisms, including beneficial insects, vertebrates and plants. Baculovirus-based pesticides are compatible with integrated pest management strategies and the expansion of their application will significantly reduce the risks associated with the use of synthetic chemical insecticides. Several successful baculovirus-based pest control programs have taken place in Latin American countries. Sustainable agriculture (a trend promoted by state authorities in most Latin American countries will benefit from the wider use of registered viral pesticides and new viral products that are in the process of registration and others in the applied research pipeline. The success of baculovirus-based control programs depends upon collaborative efforts among government and research institutions, growers associations, and private companies, which realize the importance of using strategies that protect human health and the environment at large. Initiatives to develop new regulations that promote the use of this type of ecological alternatives tailored to different local conditions and farming systems are underway.

  11. Nuclear power generation and co-existence with local area

    International Nuclear Information System (INIS)

    In addition to originally strong anxiety and unreliability on nuclear power generation, frequent accidents and troubles in the old Nuclear Reactor and Fuels Development Incorporation (the new Nuclear Fuel Cycle Development Incorporation), a fact that numbers of opposition group was over a half at citizen's vote conducted first in Japan and at Maki-machi in Niigata prefecture in August, 1996 on location of a nuclear power generation of the Tohoku Electric Co., Ltd., and so forth, affects some ripple effects to make difficult to form consensus on new location of a nuclear power generation. On making consensus of such location, every electric companies practise good communication with location areas, communication and other organizations, and so forth, promote positive information supplies and conversation type understanding works, and effort to acquire reliability for people of the location areas. Together with such efforts, they accumulate steadily safe operation results of the present nuclear power plants and develop nuclear PA (public acceptance) actions, local area promotion supporting countermeasures, and so forth. Here was introduced on a part of actions aiming at assistance to and co-existence with local areas promoted by every electric companies on various forms. (G.K.)

  12. Insecticidal properties of genetically engineered baculoviruses expressing an insect juvenile hormone esterase gene.

    OpenAIRE

    Eldridge, R; O'Reilly, D R; Hammock, B D; Miller, L K

    1992-01-01

    Exploring the possibility of enhancing the properties of baculoviruses as biological control agents of insect pests, we tested the effect of expressing an insect gene (jhe) encoding juvenile hormone esterase. Juvenile hormone esterase inactivates juvenile hormone, which regulates the outcome of an insect molt. A cDNA encoding the juvenile hormone esterase of Heliothis virescens was inserted into the genome of Autographa californica nuclear polyhedrosis virus such that the gene was expressed u...

  13. Production of human c-myc protein in insect cells infected with a baculovirus expression vector.

    OpenAIRE

    Miyamoto, C.; Smith, G. E.; Farrell-Towt, J; Chizzonite, R.; Summers, M D; Ju, G.

    1985-01-01

    A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived pr...

  14. Baculovirus integration with the vertebrate cells in system in vitro

    Directory of Open Access Journals (Sweden)

    Strokovskaya L. I.

    2010-11-01

    Full Text Available In this review the literature data are analyzed relative to the study of a new vector system for the cells of vertebrates, based on the insect viruses – baculoviruses. The ways and mechanisms of recombinant baculoviruses penetration into cells, the factors, which influence the effectiveness of transduction, the principles of the modification of virus display, and the reaction of the different types of cells on virus introduction are examined. The prospects of using recombinant baculoviruses in cellular engineering are discussed.

  15. The effects of the nuclear disaster at Fukushima Daiichi Nuclear Power Station on local governments

    International Nuclear Information System (INIS)

    All Japan council of local governments with atomic power stations consisted of 24 reactor site and 6 neighboring local governments to solve reactor site related problems. Nuclear disaster at Fukushima Daiichi Nuclear Power Station affected member local governments to be forced in severe conditions such as 'removal of administrative function' or 'refuge over a wide district beyond local government area', not imagined before. The council set up working group for thirteen local governments themselves to investigate this disaster and find safety and prevention of disaster measures to be deployed in nuclear administration, which published report in March 2012. This article described outline of investigation and derived problems and direction of their solution. Main items were related with communication, resident evacuation, prevention of disaster system, and management of refuge site. (T. Tanaka)

  16. State and local government regulation of nuclear waste transportation

    International Nuclear Information System (INIS)

    The Hazardous Materials Transportation Act (HMTA), authorizes the U.S. Department of Transportation (DOT) to promulgate rules governing the safe transportation in commerce of hazardous materials, including nuclear waste. The HMTA further provides that any state or local government requirement is preempted and thus invalid if it is inconsistent with a DOT requirement issued pursuant to the HMTA

  17. Efficient expression of histidine-tagged large hepatitis delta antigen in baculovirus-transduced baby hamster kidney cells

    Institute of Scientific and Technical Information of China (English)

    Ying-Wei Chiang; Jaw-Chin Wu; Kuei-Chun Wang; Chia-Wei Lai; Yao-Chi Chung; Yu-Chen Hu

    2006-01-01

    AIM: To study the baculovirus/mammalian cell system for efficient expression of functional large hepatitis delta antigen (L-HDAg).METHODS: A recombinant baculovirus expressing histidine-tagged L-HDAg (L-HDAgH) was constructed to transduce baby hamster kidney (BHK) cells by a simplified transduction protocol.RESULTS: The recombinant baculovirus transduced BHK cells with efficiencies higher than 90% as determined by flow cytometry. The expression level was significantly higher than that obtained by plasmid transfection and was further enhanced 3-fold to around 19 pg/cell by the addition of 10 mmol/L sodium butyrate. Importantly,the expressed L-HDAgH was localized to the cell nucleus and correctly isoprenylated as determined by immunofluorescence labeling and confocal microscopy.Moreover, L-HDAgH interacted with hepatitis B surface antigen to form virus-like particles.CONCLUSION: The fusion with histidine tags as well as overexpression of L-HDAgH in the baculovirus-transduced BHK cells does not impair the biological functions. Taken together, the baculovirus/mammalian cell system offers an attractive alternative for high level expression of L-HDAgH or other proteins that require extensive posttranslational modifications.

  18. Fundamentals of Baculovirus Expression and Applications.

    Science.gov (United States)

    Kost, Thomas A; Kemp, Christopher W

    2016-01-01

    In 1982 E. coli produced human insulin, the world's first recombinant DNA drug, was approved by the FDA. Since this historical event, remarkable progress has been made in developing bacterial, yeast, mammalian and insect cell protein expression systems that are used to produce recombinant proteins for both research and clinical applications. Of the available approaches, the insect cell based baculovirus expression vector system (BEVS) has proven to be a particularly adaptable system for producing a diverse collection of proteins. Along with E. coli, the system has been valuable for the production of proteins for structural studies, including adequate quantities of difficult to produce G protein-coupled receptors. BEVS has also been used for production of the human papilloma virus vaccine, Cervarix, the first FDA approved insect cell produced product and FluBlok, a vaccine based on the influenza virus hemagglutinin protein. Baculoviruses, modified to contain mammalian promoters (BacMam viruses), have proven to be efficient gene delivery vectors for mammalian cells and provide an alternative transient mammalian cell based protein expression approach to that of plasmid DNA based transfection methodologies. Here we provide an update on recent advances in baculovirus vector development with a focus on the numerous applications of these viruses in basic research and biotechnology. PMID:27165326

  19. Regulation of Nuclear Localization of Signaling Proteins by Cytokinin

    Energy Technology Data Exchange (ETDEWEB)

    Kieber, J.J.

    2010-05-01

    Cytokinins are a class of mitogenic plant hormones that play an important role in most aspects of plant development, including shoot and root growth, vascular and photomorphogenic development and leaf senescence. A model for cytokinin perception and signaling has emerged that is similar to bacterial two-component phosphorelays. In this model, binding of cytokinin to the extracellular domain of the Arabidopsis histidine kinase (AHKs) receptors induces autophosphorylation within the intracellular histidine-kinase domain. The phosphoryl group is subsequently transferred to cytosolic Arabidopsis histidine phosphotransfer proteins (AHPs), which have been suggested to translocate to the nucleus in response to cytokinin treatment, where they then transfer the phosphoryl group to nuclear-localized response regulators (Type-A and Type-B ARRs). We examined the effects of cytokinin on AHP subcellular localization in Arabidopsis and, contrary to expectations, the AHPs maintained a constant nuclear/cytosolic distribution following cytokinin treatment. Furthermore, mutation of the conserved phosphoacceptor histidine residue of the AHP, as well as disruption of multiple cytokinin signaling elements, did not affect the subcellular localization of the AHP proteins. Finally, we present data indicating that AHPs maintain a nuclear/cytosolic distribution by balancing active transport into and out of the nucleus. Our findings suggest that the current models indicating relocalization of AHP protein into the nucleus in response to cytokinin are incorrect. Rather, AHPs actively maintain a consistent nuclear/cytosolic distribution regardless of the status of the cytokinin response pathway.

  20. Nuclear materials transportation workshops: USDOE outreach to local governments

    International Nuclear Information System (INIS)

    To provide direct outreach to local governments, the Transportation Management Division of the United States Department of Energy asked the Urban Consortium and its Energy Task Force to assemble representatives for two workshops focusing on the transport of nuclear materials. The first session, for jurisdictions east of the Mississippi River, was held in New Orleans on May 5--6, 1988; the second was conducted on June 6--7, 1988 in Denver for jurisdictions to the west. Twenty local government professionals with management or operational responsibility for hazardous materials transportation within their jurisdictions were selected to attend each workshop. The discussions identified five major areas of concern to local government professionals; coordination; training; information resources; marking and placarding; and responder resources. Integrated federal, state, and local levels of government emerged as a priority coordination issue along with the need for expanded availability of training and training resources for first-reponders

  1. Two-stage baculovirus production in insect-cell bioreactors.

    NARCIS (Netherlands)

    Lier, van F.L.J.

    1995-01-01

    Baculoviruses are insect-pathogenic viruses with a narrow host range. The viruses can be an alternative to chemical insecticides. From research aimed at improving the efficacy of the viruses in insect control another application evolved: the use of the baculovirus to express foreign proteins in inse

  2. Cellular stress stimulates nuclear localization signal (NLS) independent nuclear transport of MRJ

    Energy Technology Data Exchange (ETDEWEB)

    Andrews, Joel F.; Sykora, Landon J.; Barik Letostak, Tiasha; Menezes, Mitchell E.; Mitra, Aparna [Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, AL (United States); Barik, Sailen [Center for Gene Regulation in Health and Disease, Department of Biological, Geological, and Environmental Sciences, College of Science, Cleveland State University, Cleveland, OH (United States); Shevde, Lalita A. [Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, AL (United States); Samant, Rajeev S., E-mail: rsamant@usouthal.edu [Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, AL (United States)

    2012-06-10

    HSP40 family member MRJ (DNAJB6) has been in the spot light for its relevance to Huntington's, Parkinson's diseases, limb-girdle muscular dystrophy, placental development, neural stem cells, cell cycle and malignancies such as breast cancer and melanoma. This gene has two spliced variants coding for 2 distinct proteins with significant homology. However, MRJ(L) (large variant) is predominantly localized to the nucleus whereas MRJ(S) (small variant) is predominantly cytoplasmic. Interestingly MRJ(S) translocates to the nucleus in response to heat shock. The classical heat shock proteins respond to crises (stress) by increasing the number of molecules, usually by transcriptional up-regulation. Our studies imply that a quick increase in the molar concentration of MRJ in the nuclear compartment is a novel method by which MRJ responds to stress. We found that MRJ(S) shows NLS (nuclear localization signal) independent nuclear localization in response to heat shock and hypoxia. The specificity of this response is realized due to lack of such response by MRJ(S) when challenged by other stressors, such as some cytokines or UV light. Deletion analysis has allowed us to narrow down on a 20 amino acid stretch at the C-terminal region of MRJ(S) as a potential stress sensing region. Functional studies indicated that constitutive nuclear localization of MRJ(S) promoted attributes of malignancy such as proliferation and invasiveness overall indicating distinct phenotypic characteristics of nuclear MRJ(S).

  3. Cellular stress stimulates nuclear localization signal (NLS) independent nuclear transport of MRJ

    International Nuclear Information System (INIS)

    HSP40 family member MRJ (DNAJB6) has been in the spot light for its relevance to Huntington's, Parkinson's diseases, limb-girdle muscular dystrophy, placental development, neural stem cells, cell cycle and malignancies such as breast cancer and melanoma. This gene has two spliced variants coding for 2 distinct proteins with significant homology. However, MRJ(L) (large variant) is predominantly localized to the nucleus whereas MRJ(S) (small variant) is predominantly cytoplasmic. Interestingly MRJ(S) translocates to the nucleus in response to heat shock. The classical heat shock proteins respond to crises (stress) by increasing the number of molecules, usually by transcriptional up-regulation. Our studies imply that a quick increase in the molar concentration of MRJ in the nuclear compartment is a novel method by which MRJ responds to stress. We found that MRJ(S) shows NLS (nuclear localization signal) independent nuclear localization in response to heat shock and hypoxia. The specificity of this response is realized due to lack of such response by MRJ(S) when challenged by other stressors, such as some cytokines or UV light. Deletion analysis has allowed us to narrow down on a 20 amino acid stretch at the C-terminal region of MRJ(S) as a potential stress sensing region. Functional studies indicated that constitutive nuclear localization of MRJ(S) promoted attributes of malignancy such as proliferation and invasiveness overall indicating distinct phenotypic characteristics of nuclear MRJ(S).

  4. Application of a nuclear localization signal gene in transgene mice

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Efficient gene transfer by cytoplasm co-injec- tion will offer a powerful means for transgenic animals. Using co-injection in cytoplasm, two independent gene constructs, including bovine (?-s1-casein-hG-CSF and a mammal expression vector expressing a nuclear localization signal (mNLS), were introduced into fertilized mouse eggs. The target gene construct was docked into host nucleus probably by the nuclear localization signal. Transgene mice have been obtained at 58% (29/50) of integration ratio. Expression level of the positive transgene mice was detected by Western blotting. Maximal expression of human G-CSF was estimated about 540 mg/L of milk. The expression ratio was up to 75% (9/12). The results here have important practical implications for the generation of mammary gland bioreactors and other transgene studies. Co-injection of a target gene with an expression vector of a mammal nuclear localization signal by cytoplasm appears to be a useful, efficient and easy strategy for generating transgenic animals, which may be able to substitute the routine method of pronucleus-injection of fertilized eggs.

  5. An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostelium.

    Science.gov (United States)

    Myre, Michael A; O'Day, Danton H

    2005-06-24

    Nucleomorphin is a novel nuclear calmodulin (CaM)-binding protein (CaMBP) containing an extensive DEED (glu/asp repeat) domain that regulates nuclear number. GFP-constructs of the 38 kDa NumA1 isoform localize as intranuclear patches adjacent to the inner nuclear membrane. The translocation of CaMBPs into nuclei has previously been shown by others to be mediated by both classic nuclear localization sequences (NLSs) and CaM-binding domains (CaMBDs). Here we show that NumA1 possesses a CaMBD (171EDVSRFIKGKLLQKQQKIYKDLERF195) containing both calcium-dependent-binding motifs and an IQ-like motif for calcium-independent binding. GFP-constructs containing only NumA1 residues 1-129, lacking the DEED and CaMBDs, still localized as patches at the internal periphery of nuclei thus ruling out a direct role for the CaMBD in nuclear import. These constructs contained the amino acid residues 48KKSYQDPEIIAHSRPRK64 that include both a putative bipartite and classical NLS. GFP-bipartite NLS constructs localized uniformly within nuclei but not as patches. As with previous work, removal of the DEED domain resulted in highly multinucleate cells. However as shown here, multinuclearity only occurred when the NLS was present allowing the protein to enter nuclei. Site-directed mutation analysis in which the NLS was changed to 48EF49 abolished the stability of the GFP fusion at the protein but not RNA level preventing subcellular analyses. Cells transfected with the 48EF49 construct exhibited slowed growth when compared to parental AX3 cells and other GFP-NumA1 deletion mutants. In addition to identifying an NLS that is sufficient for nuclear translocation of nucleomorphin and ruling out CaM-binding in this event, this work shows that the nuclear localization of NumA1 is crucial to its ability to regulate nuclear number in Dictyostelium. PMID:15896312

  6. Chitosan Nanoparticles for Nuclear Targeting: The Effect of Nanoparticle Size and Nuclear Localization Sequence Density.

    Science.gov (United States)

    Tammam, Salma N; Azzazy, Hassan M E; Breitinger, Hans G; Lamprecht, Alf

    2015-12-01

    Many recently discovered therapeutic proteins exert their main function in the nucleus, thus requiring both efficient uptake and correct intracellular targeting. Chitosan nanoparticles (NPs) have attracted interest as protein delivery vehicles due to their biocompatibility and ability to escape the endosomes offering high potential for nuclear delivery. Molecular entry into the nucleus occurs through the nuclear pore complexes, the efficiency of which is dependent on NP size and the presence of nuclear localization sequence (NLS). Chitosan nanoparticles of different sizes (S-NPs ≈ 25 nm; L-NP ≈ 150 nm) were formulated, and they were modified with different densities of the octapeptide NLS CPKKKRKV (S-NPs, 0.25, 0.5, 2.0 NLS/nm(2); L-NPs, 0.6, 0.9, 2 NLS/nm(2)). Unmodified and NLS-tagged NPs were evaluated for their protein loading capacity, extent of cell association, cell uptake, cell surface binding, and finally nuclear delivery efficiency in L929 fibroblasts. To avoid errors generated with cell fractionation and nuclear isolation protocols, nuclear delivery was assessed in intact cells utilizing Förster resonance energy transfer (FRET) fluorometry and microscopy. Although L-NPs showed ≈10-fold increase in protein loading per NP when compared to S-NPs, due to higher cell association and uptake S-NPs showed superior protein delivery. NLS exerts a size and density dependent effect on nanoparticle uptake and surface binding, with a general reduction in NP cell surface binding and an increase in cell uptake with the increase in NLS density (up to 8.4-fold increase in uptake of High-NLS-L-NPs (2 NLS/nm(2)) compared to unmodified L-NPs). However, for nuclear delivery, unmodified S-NPs show higher nuclear localization rates when compared to NLS modified NPs (up to 5-fold by FRET microscopy). For L-NPs an intermediate NLS density (0.9 NLS/nm(2)) seems to provide highest nuclear localization (3.7-fold increase in nuclear delivery compared to High

  7. Hormone activation of baculovirus expressed progesterone receptors.

    Science.gov (United States)

    Elliston, J F; Beekman, J M; Tsai, S Y; O'Malley, B W; Tsai, M J

    1992-03-15

    Human and chicken progesterone receptors (A form) were overproduced in a baculovirus expression system. These recombinant progesterone receptors were full-length bound progesterone specifically and were recognized by monoclonal antibodies, AB52 and PR22, specific for human and chicken progesterone receptor, respectively. In gel retardation studies, binding of recombinant human and chicken progesterone receptors to their progesterone response element (PRE) was specific and was enhanced in the presence of progesterone. Binding of human progesterone receptor to the PRE was also enhanced in the presence of the antiprogestin, RU486, but very little effect was observed in the presence of estradiol, dexamethasone, testosterone, and vitamin D. In our cell-free transcription system, human progesterone receptor induced transcription in a receptor-dependent and hormone-activable manner. Receptor-stimulated transcription required the presence of the PRE in the test template and could be specifically inhibited by excess PRE oligonucleotides. Furthermore, chicken progesterone receptor also induced in vitro transcription in a hormone-activable manner. These results demonstrate that steroid receptors overexpressed in a baculovirus expression system are functional and exhibit steroid-responsive binding and transcription. These observations support our present understanding of the mechanism of steroid receptor-regulated gene expression and provide a technological format for studies of the role of hormone and antihormone in altering gene expression. PMID:1544902

  8. Installation for accident localization at a nuclear power plant

    International Nuclear Information System (INIS)

    The installation for accident localization at a nuclear power plant with multiloop structure of the first circuit is described. The installation represents a pressurized shell inside which the reactor with the first circuit equipment are located. The whole volume under the shell is divided into autonomous boxes for one or several circulation loops. In case of accident as capacity for condensate discharge not the whole volume under pressurized shell is used, but only one of the autonomous boxes. This circumstance decreases the accident effect zone and expenditures related to the liquidation of accident consequences

  9. Ligands specify estrogen receptor alpha nuclear localization and degradation

    Directory of Open Access Journals (Sweden)

    Caze-Subra Stéphanie

    2010-12-01

    Full Text Available Abstract Background The estrogen receptor alpha (ERα is found predominately in the nucleus, both in hormone stimulated and untreated cells. Intracellular distribution of the ERα changes in the presence of agonists but the impact of different antiestrogens on the fate of ERα is a matter of debate. Results A MCF-7 cell line stably expressing GFP-tagged human ERα (SK19 cell line was created to examine the localization of ligand-bound GFP-ERα. We combined digitonin-based cell fractionation analyses with fluorescence and immuno-electron microscopy to determine the intracellular distribution of ligand-bound ERα and/or GFP-ERα. Using fluorescence- and electron microscopy we demonstrate that both endogenous ERα and GFP-ERα form numerous nuclear focal accumulations upon addition of agonist, 17β-estradiol (E2, and pure antagonists (selective estrogen regulator disruptor; SERD, ICI 182,780 or RU58,668, while in the presence of partial antagonists (selective estrogen regulator modulator; SERM, 4-hydroxytamoxifen (OHT or RU39,411, diffuse nuclear staining persisted. Digitonin based cell fractionation analyses confirmed that endogenous ERα and GFP-ERα predominantly reside in the nuclear fraction. Overall ERα protein levels were reduced after estradiol treatment. In the presence of SERMs ERα was stabilized in the nuclear soluble fraction, while in the presence of SERDs protein levels decreased drastically and the remaining ERα was largely found in a nuclear insoluble fraction. mRNA levels of ESR1 were reduced compared to untreated cells in the presence of all ligands tested, including E2. E2 and SERDs induced ERα degradation occurred in distinct nuclear foci composed of ERα and the proteasome providing a simple explanation for ERα sequestration in the nucleus. Conclusions Our results indicate that chemical structure of ligands directly affect the nuclear fate and protein turnover of the estrogen receptor alpha independently of their impact on

  10. Delivery of vaccine peptides by rapid conjugation to baculovirus particles.

    Science.gov (United States)

    Wilson, Sarah; Baird, Margaret; Ward, Vernon K

    2008-05-12

    Baculoviruses deliver strong activation signals to dendritic cells and can promote potent immune responses. These properties can be harnessed to use baculovirus as an adjuvant and carrier particle for immunogenic peptides. In this study we use a chemical linker to couple peptides to the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Intranasal delivery of baculovirus coupled with immunogenic peptides to mice elicited antigen-specific IgG1 and IgG2a antibody. Furthermore, antigen-specific IgA was detected in the lung, and an IFN-gamma response was observed upon re-stimulation with antigen. We show that chemical coupling enables the rapid modification of AcMNPV, allowing multiple epitopes to be delivered simultaneously on a self-adjuvanting carrier particle. PMID:18417258

  11. Ancient Coevolution of Baculoviruses and Their Insect Hosts

    OpenAIRE

    Elisabeth A Herniou; Olszewski, Julie A.; O'Reilly, David R.; Jenny S Cory

    2004-01-01

    If the relationships between baculoviruses and their insect hosts are subject to coevolution, this should lead to long-term evolutionary effects such as the specialization of these pathogens for their hosts. To test this hypothesis, a phylogeny of the Baculoviridae, including 39 viruses from hosts of the orders Lepidoptera, Diptera, and Hymenoptera, was reconstructed based on sequences from the genes lef-8 and ac22. The tree showed a clear division of the baculoviruses according to the order ...

  12. Baculoviruses as Vectors for Gene Therapy against Human Prostate Cancer

    OpenAIRE

    Stanbridge Lindsay J.; Dussupt Vincent; Maitland Norman J.

    2003-01-01

    Current curative strategies for prostate cancer are restricted to the primary tumour, and the effect of treatments to control metastatic disease is not sustained. Therefore, the application of gene therapy to prostate cancer is an attractive alternative. Baculoviruses are highly restricted insect viruses, which can enter, but not replicate in mammalian cells. Baculoviruses can incorporate large amounts of extra genetic material, and will express transgenes in mammalian cells when under the co...

  13. Functional analysis of a novel baculovirus envelope fusion protein

    OpenAIRE

    Westenberg, M.

    2004-01-01

    Baculoviridae are a family of large double stranded DNA viruses that are exclusively pathogenic to arthropods. Baculoviruses have been studied i) with the aim to develop alternatives to chemical pest control, ii) for their application asaneukaryotic expression system to express heterologous proteins, and recently iii) as gene delivery vehicle in gene therapy. Baculoviruses cluster into two distinct genera on the basis of the occlusion body (OB) morphology: Nucleopolyhedrovirus (NPV) and Granu...

  14. Genome Scale Transcriptomics of Baculovirus-Insect Interactions

    Directory of Open Access Journals (Sweden)

    Steven Reid

    2013-11-01

    Full Text Available Baculovirus-insect cell technologies are applied in the production of complex proteins, veterinary and human vaccines, gene delivery vectors‚ and biopesticides. Better understanding of how baculoviruses and insect cells interact would facilitate baculovirus-based production. While complete genomic sequences are available for over 58 baculovirus species, little insect genomic information is known. The release of the Bombyx mori and Plutella xylostella genomes, the accumulation of EST sequences for several Lepidopteran species, and especially the availability of two genome-scale analysis tools, namely oligonucleotide microarrays and next generation sequencing (NGS, have facilitated expression studies to generate a rich picture of insect gene responses to baculovirus infections. This review presents current knowledge on the interaction dynamics of the baculovirus-insect system‚ which is relatively well studied in relation to nucleocapsid transportation, apoptosis, and heat shock responses, but is still poorly understood regarding responses involved in pro-survival pathways, DNA damage pathways, protein degradation, translation, signaling pathways, RNAi pathways, and importantly metabolic pathways for energy, nucleotide and amino acid production. We discuss how the two genome-scale transcriptomic tools can be applied for studying such pathways and suggest that proteomics and metabolomics can produce complementary findings to transcriptomic studies.

  15. Genome scale transcriptomics of baculovirus-insect interactions.

    Science.gov (United States)

    Nguyen, Quan; Nielsen, Lars K; Reid, Steven

    2013-11-01

    Baculovirus-insect cell technologies are applied in the production of complex proteins, veterinary and human vaccines, gene delivery vectors' and biopesticides. Better understanding of how baculoviruses and insect cells interact would facilitate baculovirus-based production. While complete genomic sequences are available for over 58 baculovirus species, little insect genomic information is known. The release of the Bombyx mori and Plutella xylostella genomes, the accumulation of EST sequences for several Lepidopteran species, and especially the availability of two genome-scale analysis tools, namely oligonucleotide microarrays and next generation sequencing (NGS), have facilitated expression studies to generate a rich picture of insect gene responses to baculovirus infections. This review presents current knowledge on the interaction dynamics of the baculovirus-insect system' which is relatively well studied in relation to nucleocapsid transportation, apoptosis, and heat shock responses, but is still poorly understood regarding responses involved in pro-survival pathways, DNA damage pathways, protein degradation, translation, signaling pathways, RNAi pathways, and importantly metabolic pathways for energy, nucleotide and amino acid production. We discuss how the two genome-scale transcriptomic tools can be applied for studying such pathways and suggest that proteomics and metabolomics can produce complementary findings to transcriptomic studies.

  16. Additive effect of calreticulin and translation initiation factor eIF4E on secreted protein production in the baculovirus expression system

    NARCIS (Netherlands)

    Teng, C.Y.; Oers, van M.M.; Wu, T.Y.

    2013-01-01

    The baculovirus expression vector system is widely used for the production of recombinant proteins. However, the yield of membrane-bound or secreted proteins is relatively low when compared with intracellular or nuclear proteins. In a previous study, we had demonstrated that the co-expression of the

  17. Preparing of local contingency planning for nuclear accident of nuclear submarine at port

    International Nuclear Information System (INIS)

    Full text: The presence of a nuclear submarine in port involves a radiological risk which requires to be taken into account appropriately. The implementation of radiological monitoring and prevention measures are basic requisites to assure this visit being reasonably safer. In normal condition, the radiological monitoring of some marine environmental samples are necessary to be performed before the arrival and during the staying and after the departure of the nuclear ship. In the other side, prevention measures involve the preparedness of local contingency plans to protect the people in the port in any nuclear accidental event. The plan is designed by assuming an accident where 10% of core inventory of a 30 MW(e) nuclear power reactor are released to environment. The plan basically takes into account immediate measures inside a zone of 2,5 km around the submarine - from the normal place for anchor - being some of them the clearing of the zone from ships and people and taking the damaged submarine out at sea in order to reduce the scope of possible emissions. Likewise, previsions are made for protecting people up to 10 km in the coast. All these measures are performed by an organization commanded by the Port Captain and supported by other state institutions in land. (orig.)

  18. Nucleotide sequence and temporal expression of a baculovirus regulatory gene.

    Science.gov (United States)

    Guarino, L A; Summers, M D

    1987-07-01

    The nucleotide sequence of a trans-activating regulatory gene (IE-1) of the baculovirus Autographa californica nuclear polyhedrosis virus has been determined. This gene encodes a protein of 581 amino acids with a predicted molecular weight of 66,856. A DNA fragment containing the entire coding sequence of IE-1 was inserted downstream of an RNA promoter. Subsequent cell-free transcription and translation directed the synthesis of a single peptide with an apparent molecular weight of 70,000. Quantitative S1 nuclease analysis indicated that IE-1 was maximally synthesized during a 1-h virus adsorption period and that steady-state levels of IE-1 message were maintained during the first 24 h of infection. Northern blot hybridization indicated that several late transcripts which overlap the IE-1 gene were transcribed from both strands. The precise locations of the 5' and 3' ends of these overlapping transcripts were mapped using S1 nuclease. The overlapping transcripts were grouped in two transcriptional units. One unit was composed of IE-1 and overlapping gamma transcripts which initiated upstream of IE-1 and terminated downstream of IE-1. The other unit, transcribed from the opposite strand, consisted of gamma transcripts with coterminal 5' ends and extended 3' ends. The shorter, more abundant transcripts in this unit overlapped 30 to 40 bases of IE-1 at the 3' end, while the longer transcripts overlapped the entire IE-1 gene. Transcription of several early A. californica nuclear polyhedrosis virus genes, in addition to 39K, was shown to be trans-activated by IE-1, indicating that IE-1 may have a central role in the regulation of beta-gene expression. PMID:16789264

  19. Nuclear localization of Annexin A7 during murine brain development

    Directory of Open Access Journals (Sweden)

    Noegel Angelika A

    2005-04-01

    Full Text Available Abstract Background Annexin A7 is a member of the annexin protein family, which is characterized by its ability to interact with phospholipids in the presence of Ca2+-ions and which is thought to function in Ca2+-homeostasis. Results from mutant mice showed altered Ca2+-wave propagation in astrocytes. As the appearance and distribution of Annexin A7 during brain development has not been investigated so far, we focused on the distribution of Annexin A7 protein during mouse embryogenesis in the developing central nervous system and in the adult mouse brain. Results Annexin A7 is expressed in cells of the developing brain where a change in its subcellular localization from cytoplasm to nucleus was observed. In the adult CNS, the subcellular distribution of Annexin A7 depends on the cell type. By immunohistochemistry analysis Annexin A7 was detected in the cytosol of undifferentiated cells at embryonic days E5–E8. At E11–E15 the protein is still present in the cytosol of cells predominantly located in the ventricular germinative zone surrounding the lateral ventricle. Later on, at embryonic day E16, Annexin A7 in cells of the intermediate and marginal zone of the neopallium translocates to the nucleus. Neuronal cells of all areas in the adult brain present Annexin A7 in the nucleus, whereas glial fibrillary acidic protein (GFAP-positive astrocytes exhibit both, a cytoplasmic and nuclear staining. The presence of nuclear Annexin A7 was confirmed by extraction of the nucleoplasm from isolated nuclei obtained from neuronal and astroglial cell lines. Conclusion We have demonstrated a translocation of Annexin A7 to nuclei of cells in early murine brain development and the presence of Annexin A7 in nuclei of neuronal cells in the adult animal. The role of Annexin A7 in nuclei of differentiating and mature neuronal cells remains elusive.

  20. Expression of the human interleukin-2 receptor gamma chain in insect cells using a baculovirus expression vector.

    Science.gov (United States)

    Raivio, E; Oetken, C; Oker-Blom, C; Engberg, C; Akerman, K; Lindqvist, C

    1995-04-01

    The gene encoding the gamma-chain of the human Interleukin-2 receptor was expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding gene was inserted under the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The recombinant receptor protein was identified by immunoblotting in cell lysates, prepared from insect cells infected with the recombinant virus. At 40 h post infection the corresponding protein was detected as two major bands with apparent molecular weights of 50-60 kDa using a rabbit anti-human IL-2R gamma-receptor specific antiserum. Metabolic labelling with [35S]-methionine and SDS-PAGE analysis of the recombinant baculovirus infected insect cells verified the immunoblotting data. The expressed IL-2R gamma- protein could also be determined on the surface of infected insect cells by flow cytometer analysis. PMID:7899821

  1. AcMNPV As A Model for Baculovirus DNA Replication

    Institute of Scientific and Technical Information of China (English)

    Eric B. Carstens

    2009-01-01

    Baculoviruses were first identified as insect-specific pathogens, and it was this specificity that lead to their use as safe, target specific biological pesticides. For the past 30 years, AcMNPV has served as the subject of intense basic molecular research into the baculovirus infectious cycle including the interaction of the virus with a continuous insect cell line derived from Spodoptera frugiperda. The studies on baculoviruese have led to an in-depth understanding of the physical organization of the viral genomes including many complete genomic sequences, the time course of gene expression, and the application of this basic research to the use of baculoviruses not only as insecticides, but also as a universal eukaryotic protein expression system, and a potential vector in gene therapy. A great deal has also been discovered about the viral genes required for the replication of the baculovirus genome, while much remains to be learned about the mechanism of viral DNA replication. This report outlines the current knowledge of the factors involved in baculovirus DNA replication, using data on AcMNPV as a model for most members of the Baculoviridae.

  2. Gene Acquisition Convergence between Entomopoxviruses and Baculoviruses

    Directory of Open Access Journals (Sweden)

    Julien Thézé

    2015-04-01

    Full Text Available Organisms from diverse phylogenetic origins can thrive within the same ecological niches. They might be induced to evolve convergent adaptations in response to a similar landscape of selective pressures. Their genomes should bear the signature of this process. The study of unrelated virus lineages infecting the same host panels guarantees a clear identification of phyletically independent convergent adaptation. Here, we investigate the evolutionary history of genes in the accessory genome shared by unrelated insect large dsDNA viruses: the entomopoxviruses (EPVs, Poxviridae and the baculoviruses (BVs. EPVs and BVs have overlapping ecological niches and have independently evolved similar infection processes. They are, in theory, subjected to the same selective pressures from their host’s immune responses. Their accessory genomes might, therefore, bear analogous genomic signatures of convergent adaption and could point out key genomic mechanisms of adaptation hitherto undetected in viruses. We uncovered 32 homologous, yet independent acquisitions of genes originating from insect hosts, different eukaryotes, bacteria and viruses. We showed different evolutionary levels of gene acquisition convergence in these viruses, underlining a continuous evolutionary process. We found both recent and ancient gene acquisitions possibly involved to the adaptation to both specific and distantly related hosts. Multidirectional and multipartite gene exchange networks appear to constantly drive exogenous gene assimilations, bringing key adaptive innovations and shaping the life histories of large DNA viruses. This evolutionary process might lead to genome level adaptive convergence.

  3. Role of 'commission locale d'information' in France from the viewpoint of nuclear regulatory governance

    International Nuclear Information System (INIS)

    In France, there exist organizations called 'Commission Locale d'Information' (CLI) in all the siting areas where nuclear facilities located. Previously, the CLI organizations were established voluntarily by some local governments or nuclear utilities. Since 2006, however, the Nuclear Transparency and Safety Act has obliged the establishment of CLI in all the siting areas in conjunction with reforming the nuclear regulatory agencies. This means that the concerned local governments are officially part of nuclear safety regulation. In this study, we investigated present conditions of the CLI organizations through some interviews in France and consider their roles from the standpoint of nuclear regulatory governance. As a result, we found that the CLI plays the following roles: (1) medium of communication among concerned parties (not only between nuclear utilities and local habitants but also between the national nuclear regulatory agency and various local governments) and (2) implementing various activities in accordance with each local condition through the participation of local assembly members. In addition, we clarified that CLI's activities are supported by related institutional infrastructures, such as cost burden between central and local governments, and some other systems of citizen participation in building or expanding nuclear facilities. (author)

  4. Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding

    International Nuclear Information System (INIS)

    Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLSSV40) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLSSV40 in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLSSV40 formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLSSV40 likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLSSV40 can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells. - Highlights: • Nuclear localization signal-tagged GFP (GFP-NLS) showed clear nuclear localization. • The GFP-NLS dynamically localized not only in the nucleoplasm, but also to the nucleolus. • The nuclear localization of GFP-NLS results from transient oligomerization mediated via RNA binding. • Our NLS-tagging procedure is ideal for use in artificial sequestration of proteins in the nucleus

  5. Efficient and dynamic nuclear localization of green fluorescent protein via RNA binding

    Energy Technology Data Exchange (ETDEWEB)

    Kitamura, Akira; Nakayama, Yusaku; Kinjo, Masataka, E-mail: kinjo@sci.hokudai.ac.jp

    2015-07-31

    Classical nuclear localization signal (NLS) sequences have been used for artificial localization of green fluorescent protein (GFP) in the nucleus as a positioning marker or for measurement of the nuclear-cytoplasmic shuttling rate in living cells. However, the detailed mechanism of nuclear retention of GFP-NLS remains unclear. Here, we show that a candidate mechanism for the strong nuclear retention of GFP-NLS is via the RNA-binding ability of the NLS sequence. GFP tagged with a classical NLS derived from Simian virus 40 (GFP-NLS{sup SV40}) localized not only in the nucleoplasm, but also to the nucleolus, the nuclear subdomain in which ribosome biogenesis takes place. GFP-NLS{sup SV40} in the nucleolus was mobile, and intriguingly, the diffusion coefficient, which indicates the speed of diffusing molecules, was 1.5-fold slower than in the nucleoplasm. Fluorescence correlation spectroscopy (FCS) analysis showed that GFP-NLS{sup SV40} formed oligomers via RNA binding, the estimated molecular weight of which was larger than the limit for passive nuclear export into the cytoplasm. These findings suggest that the nuclear localization of GFP-NLS{sup SV40} likely results from oligomerization mediated via RNA binding. The analytical technique used here can be applied for elucidating the details of other nuclear localization mechanisms, including those of several types of nuclear proteins. In addition, GFP-NLS{sup SV40} can be used as an excellent marker for studying both the nucleoplasm and nucleolus in living cells. - Highlights: • Nuclear localization signal-tagged GFP (GFP-NLS) showed clear nuclear localization. • The GFP-NLS dynamically localized not only in the nucleoplasm, but also to the nucleolus. • The nuclear localization of GFP-NLS results from transient oligomerization mediated via RNA binding. • Our NLS-tagging procedure is ideal for use in artificial sequestration of proteins in the nucleus.

  6. Baculoviruses as Vectors for Gene Therapy against Human Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Lindsay J. Stanbridge

    2003-01-01

    Full Text Available Current curative strategies for prostate cancer are restricted to the primary tumour, and the effect of treatments to control metastatic disease is not sustained. Therefore, the application of gene therapy to prostate cancer is an attractive alternative. Baculoviruses are highly restricted insect viruses, which can enter, but not replicate in mammalian cells. Baculoviruses can incorporate large amounts of extra genetic material, and will express transgenes in mammalian cells when under the control of a mammalian or strong viral promoter. Successful gene delivery has been achieved both in vitro and in vivo and into both dividing and nondividing cells, which is important since prostate cancers divide relatively slowly. In addition, the envelope protein gp64 is sufficiently mutable to allow targeted transduction of particular cell types. In this review, the advantages of using baculoviruses for prostate cancer gene therapy are explored, and the mechanisms of viral entry and transgene expression are described.

  7. Characterization of a baculovirus-encoded RNA 5'-triphosphatase.

    Science.gov (United States)

    Gross, C H; Shuman, S

    1998-09-01

    Autographa californica nuclear polyhedrosis virus (AcNPV) encodes a 168-amino-acid polypeptide that contains the signature motif of the superfamily of protein phosphatases that act via a covalent cysteinyl phosphate intermediate. The sequence of the AcNPV phosphatase is similar to that of the RNA triphosphatase domain of the metazoan cellular mRNA capping enzyme. Here, we show that the purified recombinant AcNPV protein is an RNA 5'-triphosphatase that hydrolyzes the gamma-phosphate of triphosphate-terminated poly(A); it also hydrolyzes ATP to ADP and GTP to GDP. The phosphatase sediments as two discrete components in a glycerol gradient: a 9.5S oligomer and 2.5S putative monomer. The 2.5S form of the enzyme releases 32Pi from 1 microM gamma-32P-labeled triphosphate-terminated poly(A) with a turnover number of 52 min-1 and converts ATP to ADP with Vmax of 8 min-1 and Km of 25 microM ATP. The 9.5S oligomeric form of the enzyme displays an initial pre-steady-state burst of ADP and Pi formation, which is proportional to and stoichiometric with the enzyme, followed by a slower steady-state rate of product formation (approximately 1/10 of the steady-state rate of the 2.5S enzyme). We surmise that the oligomeric enzyme is subject to a rate-limiting step other than reaction chemistry and that this step is either distinct from or slower than the rate-limiting step for the 2.5S enzyme. Replacing the presumptive active site nucleophile Cys-119 by alanine abrogates RNA triphosphatase and ATPase activity. Our findings raise the possibility that baculoviruses encode enzymes that cap the 5' ends of viral transcripts synthesized at late times postinfection by a virus-encoded RNA polymerase. PMID:9696798

  8. An analysis on the market of instrumentation in nuclear power plant in China and its localization

    International Nuclear Information System (INIS)

    The Instrumentation plays a critical role in guaranteeing the safety and efficiency of the nuclear power plant. The instrumentations used in the working nuclear power plants in China, especially the Class 1E ones, are mainly imported products. This has been one of the key elements restricting the localized development of the Chinese nuclear power industry. Based on a fundamental survey of the Chinese nuclear instrumentation market, this paper introduces the definition, classification of nuclear instrumentations, states the market status, analyzes the cause of how this situation was formed, and makes some proposals on how to handle the localization job in the future. (authors)

  9. Expression of bovine vitamin K-dependent carboxylase activity in baculovirus-infected insect cells.

    Science.gov (United States)

    Roth, D A; Rehemtulla, A; Kaufman, R J; Walsh, C T; Furie, B; Furie, B C

    1993-09-15

    A vitamin K-dependent carboxylase has recently been purified from bovine liver microsomes and candidate cDNA clones have been isolated. Definitive identification of the carboxylase remains circumstantial since expression of candidate carboxylase cDNAs in mammalian cells is confounded by the presence of endogenous carboxylase activity. To overcome this problem, a recombinant strain of baculovirus (Autographa california nuclear polyhedrosis virus, AcMNPV) encoding a putative carboxylase (vbCbx/AcMNPV) was used to infect Sf9 insect cells, which we demonstrate have no endogenous carboxylase activity. Infection with vbCbx/AcMNPV conferred vitamin K-dependent carboxylase activity to Sf9 insect cells. Carboxylase activity was demonstrated to peak 2-3 days after infection with vbCbx/AcMNPV. Metabolic radiolabeling with L-[35S]methionine revealed that the 90-kDa recombinant protein is the major protein synthesized at the time of peak activity after infection. An anti-peptide antibody directed against residues 86-99 reacted with bovine liver carboxylase on Western blot analysis and immunoprecipitated recombinant carboxylase from infected Sf9 microsomal protein preparations. Since Sf9 insect cells lack endogenous vitamin K-dependent carboxylase activity, expression of carboxylase activity in Sf9 insect cells with recombinant baculovirus demonstrates that the protein encoded by this cDNA is a vitamin K-dependent gamma-glutamyl carboxylase. PMID:8378308

  10. The LEF-4 subunit of baculovirus RNA polymerase has RNA 5'-triphosphatase and ATPase activities.

    Science.gov (United States)

    Jin, J; Dong, W; Guarino, L A

    1998-12-01

    The baculovirus Autographa californica nuclear polyhedrosis virus encodes a DNA-dependent RNA polymerase that is required for transcription of viral late genes. This polymerase is composed of four equimolar subunits, LEF-8, LEF-4, LEF-9, and p47. The LEF-4 subunit has guanylyltransferase activity, suggesting that baculoviruses may encode a full complement of capping enzymes. Here we show that LEF-4 is a bifunctional enzyme that hydrolyzes the gamma phosphates of triphosphate-terminated RNA and also hydrolyzes ATP and GTP to the respective diphosphate forms. Alanine substitution of five residues previously shown to be essential for vaccinia virus RNA triphosphatase activity inactivated the triphosphatase component of LEF-4 but not the guanylyltransferase domain. Conversely, mutation of the invariant lysine in the guanylyltransferase domain abolished the guanylyltransferase activity without affecting triphosphatase function. We also investigated the effects of substituting phenylalanine for leucine at position 105, a mutation that results in a virus that is temperature sensitive for late gene expression. We found that this mutation had no significant effect on the ATPase or guanylyltransferase activity of LEF-4 but resulted in a modest decrease in RNA triphosphatase activity. PMID:9811739

  11. Construction of a host range-expanded hybrid baculovirus of BmNPV and AcNPV,and knockout of cysteinase gene for more efficient expression

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    AcNPV(Autographa californica nuclear polyhedrosis virus)and BmNPV(Bombyx mori nuclear polyhedrosis virus)are two principal insect-baculovirus expression systems,each having different characteristics.AcNPV has a wider host range and can infect a series of cell lines thus making it suitable for cell suspension culture expression,but the small size of the host insect,A.californica,makes AcNPV less suitable for large scale protein synthesis.In contrast,BmNPV can only infect the silkworm,Bornbyx rnori,which is well-known for its easy rearing and large size.These characteristics make the BmNPV system especially suitable for large-scale industrial expression.To utilize the advantages of both AcNPV and BmNPV,we tried to expand their host range through homologous recombination and successfully constructed a hybrid baculovirus of AcNPV and BmNPV,designated as HyNPV.The hybrid baculovirus can infect the hosts of both AcNPV and BmNPV.Taking the human basic fibroblast growth factor(Bfgf)gene as an application example,we constructed a recombinant,HyNPV-Bfgf.This construct is able to express the Bfgf protein both in silkworm larvae and in common-use cell lines,sf21,sf9 and High-five.Moreover,to reduce the loss of recombinant protein due to degradation by proteases that are simultaneously expressed by the baculovirus,we knocked out the cysteinase gene coding for one of the most important baculovirus proteases.This knockout mutation improves the production efficiency of the Bfgf recombinant protein.

  12. Studies of the silencing of Baculovirus DNA binding protein

    NARCIS (Netherlands)

    Quadt, I.; Lent, van J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovir

  13. Gene gymnastics: Synthetic biology for baculovirus expression vector system engineering.

    Science.gov (United States)

    Vijayachandran, Lakshmi S; Thimiri Govinda Raj, Deepak B; Edelweiss, Evelina; Gupta, Kapil; Maier, Josef; Gordeliy, Valentin; Fitzgerald, Daniel J; Berger, Imre

    2013-01-01

    Most essential activities in eukaryotic cells are catalyzed by large multiprotein assemblies containing up to ten or more interlocking subunits. The vast majority of these protein complexes are not easily accessible for high resolution studies aimed at unlocking their mechanisms, due to their low cellular abundance and high heterogeneity. Recombinant overproduction can resolve this bottleneck and baculovirus expression vector systems (BEVS) have emerged as particularly powerful tools for the provision of eukaryotic multiprotein complexes in high quality and quantity. Recently, synthetic biology approaches have begun to make their mark in improving existing BEVS reagents by de novo design of streamlined transfer plasmids and by engineering the baculovirus genome. Here we present OmniBac, comprising new custom designed reagents that further facilitate the integration of heterologous genes into the baculovirus genome for multiprotein expression. Based on comparative genome analysis and data mining, we herein present a blueprint to custom design and engineer the entire baculovirus genome for optimized production properties using a bottom-up synthetic biology approach. PMID:23328086

  14. Vaccines for viral and parasitic diseases produced with baculovirus vectors

    NARCIS (Netherlands)

    Oers, van M.M.

    2006-01-01

    The baculovirus¿insect cell expression system is an approved system for the production of viral antigens with vaccine potential for humans and animals and has been used for production of subunit vaccines against parasitic diseases as well. Many candidate subunit vaccines have been expressed in this

  15. Early localization of NPA58, a rat nuclear pore-associated protein, to the reforming nuclear envelope during mitosis

    Indian Academy of Sciences (India)

    Radhika Ganeshan; Nandini Rangaraj; Veena K Parnaik

    2001-03-01

    We have studied the mitotic reassembly of the nuclear envelope, using antibodies to nuclear marker proteins and NPA58 in F-111 rat fibroblast cells. In earlier studies we have proposed that NPA58, a 58 kDa rat nuclear protein, is involved in nuclear protein import. In this report, NPA58 is shown to be localized on the cytoplasmic face of the envelope in interphase cells, in close association with nuclear pores. In mitotic cells NPA58 is dispersed in the cytoplasm till anaphase. The targeting of NPA58 to the reforming nuclear envelope in early telophase coincides with the recruitment of a well-characterized class of nuclear pore proteins recognized by the antibody mAb 414, and occurs prior to the incorporation of lamin B1 into the envelope. Significant protein import activity is detectable only after localization of NPA58 in the newly-formed envelope. The early targeting of NPA58 is consistent with its proposed role in nuclear transport.

  16. Characterization of a nuclear localization signal in the foot-and-mouth disease virus polymerase

    International Nuclear Information System (INIS)

    We have experimentally tested whether the MRKTKLAPT sequence in FMDV 3D protein (residues 16 to 24) can act as a nuclear localization signal (NLS). Mutants with substitutions in two basic residues within this sequence, K18E and K20E, were generated. A decreased nuclear localization was observed in transiently expressed 3D and its precursor 3CD, suggesting a role of K18 and K20 in nuclear targeting. Fusion of MRKTKLAPT to the green fluorescence protein (GFP) increased the nuclear localization of GFP, which was not observed when GFP was fused to the 3D mutated sequences. These results indicate that the sequence MRKTKLAPT can be functionally considered as a NLS. When introduced in a FMDV full length RNA replacements K18E and K20E led to production of revertant viruses that replaced the acidic residues introduced (E) by K, suggesting that the presence of lysins at positions 18 and 20 of 3D is essential for virus multiplication. - Highlights: • The FMDV 3D polymerase contains a nuclear localization signal. • Replacements K18E and K20E decrease nuclear localization of 3D and its precursor 3CD. • Fusion of the MRKTKLAPT 3D motif to GFP increases the nuclear localization of GFP. • Replacements K18E and K20E abolish the ability of MRKTKLAPT to relocate GFP. • RNAs harboring replacements K18E and K20E lead to recovery of revertant FMDVs

  17. Characterization of a nuclear localization signal in the foot-and-mouth disease virus polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Sanchez-Aparicio, Maria Teresa; Rosas, Maria Flora [Centro de Biología Molecular, “Severo Ochoa” (CSIC-UAM), Cantoblanco 28049, Madrid (Spain); Sobrino, Francisco, E-mail: fsobrino@cbm.uam.es [Centro de Biología Molecular, “Severo Ochoa” (CSIC-UAM), Cantoblanco 28049, Madrid (Spain); Centro de Investigación en Sanidad Animal, INIA, Valdeolmos, 28130 Madrid (Spain)

    2013-09-15

    We have experimentally tested whether the MRKTKLAPT sequence in FMDV 3D protein (residues 16 to 24) can act as a nuclear localization signal (NLS). Mutants with substitutions in two basic residues within this sequence, K18E and K20E, were generated. A decreased nuclear localization was observed in transiently expressed 3D and its precursor 3CD, suggesting a role of K18 and K20 in nuclear targeting. Fusion of MRKTKLAPT to the green fluorescence protein (GFP) increased the nuclear localization of GFP, which was not observed when GFP was fused to the 3D mutated sequences. These results indicate that the sequence MRKTKLAPT can be functionally considered as a NLS. When introduced in a FMDV full length RNA replacements K18E and K20E led to production of revertant viruses that replaced the acidic residues introduced (E) by K, suggesting that the presence of lysins at positions 18 and 20 of 3D is essential for virus multiplication. - Highlights: • The FMDV 3D polymerase contains a nuclear localization signal. • Replacements K18E and K20E decrease nuclear localization of 3D and its precursor 3CD. • Fusion of the MRKTKLAPT 3D motif to GFP increases the nuclear localization of GFP. • Replacements K18E and K20E abolish the ability of MRKTKLAPT to relocate GFP. • RNAs harboring replacements K18E and K20E lead to recovery of revertant FMDVs.

  18. Genetic Variation in Field Populations of Baculoviruses: Mechanisms for Generating Variation and Its Potential Role in Baculovirus Epizootiology

    Institute of Scientific and Technical Information of China (English)

    Martin A. Erlandson

    2009-01-01

    Baculoviridae is a family of insect-specific DNA viruses that have been used as biological control agents for insect pest control. In most cases these baculovirus control agents are natural field isolates that have been selected based on their infectivity and virulence. The advent of molecular tools such as restriction endonucleases, targeted polymerase chain reaction and new DNA sequencing strategies have allowed for efficient detection and characterization of genotypic variants within and among geographic and temporal isolates of baculovirus species. It has become evident that multiple genotypic variants occur even within individual infected larvae. Clonal strains of baculovirus species derived either by in vitro or in vivo approaches have been shown to vary with respect to infectivity and virulence. Many of the cell culture derived plague-purified strains have deletions that interrupt egt expression leading to virus strains that kill infected hosts more quickly. As well, in vitro clones often involve larger genomic deletions with the loss of pif gene function, resulting in strains deficient for oral infectivity. There are an increasing number of baculovirus species for which complete genome sequences are available for more than one strain or field isolate. Results of comparative analysis of these strains indicated that hr regions and bro genes often mark "hot spots" of genetic variability between strains and of potential recombination events. In addition, the degree of nucleotide polymorphisms between and within strains and their role in amino acid substitutions within ORFs and changes in promoter motifs is also beginning to be appreciated. In this short review the potential mechanisms that generate and maintain this genetic diversity within baculovirus populations is discussed, as is the potential role of genetic variation in host-pathogen interactions.

  19. Atypical nuclear localization of VIP receptors in glioma cell lines and patients

    Energy Technology Data Exchange (ETDEWEB)

    Barbarin, Alice; Séité, Paule [Equipe Récepteurs, Régulations et Cellules Tumorales, Université de Poitiers, PBS bât 36, 1 rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9 (France); Godet, Julie [Laboratoire d’anatomie et de cytologie pathologiques, CHU de Poitiers, 2 rue de la Milétrie, 86000 Poitiers (France); Bensalma, Souheyla; Muller, Jean-Marc [Equipe Récepteurs, Régulations et Cellules Tumorales, Université de Poitiers, PBS bât 36, 1 rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9 (France); Chadéneau, Corinne, E-mail: corinne.chadeneau@univ-poitiers.fr [Equipe Récepteurs, Régulations et Cellules Tumorales, Université de Poitiers, PBS bât 36, 1 rue Georges Bonnet, TSA 51106, 86073 Poitiers Cedex 9 (France)

    2014-11-28

    Highlights: • The VIP receptor VPAC1 contains a putative NLS signal. • VPAC1 is predominantly nuclear in GBM cell lines but not VPAC2. • Non-nuclear VPAC1/2 protein expression is correlated with glioma grade. • Nuclear VPAC1 is observed in 50% of stage IV glioma (GBM). - Abstract: An increasing number of G protein-coupled receptors, like receptors for vasoactive intestinal peptide (VIP), are found in cell nucleus. As VIP receptors are involved in the regulation of glioma cell proliferation and migration, we investigated the expression and the nuclear localization of the VIP receptors VPAC1 and VPAC2 in this cancer. First, by applying Western blot and immunofluorescence detection in three human glioblastoma (GBM) cell lines, we observed a strong nuclear staining for the VPAC1 receptor and a weak nuclear VPAC2 receptor staining. Second, immunohistochemical staining of VPAC1 and VPAC2 on tissue microarrays (TMA) showed that the two receptors were expressed in normal brain and glioma tissues. Expression in the non-nuclear compartment of the two receptors significantly increased with the grade of the tumors. Analysis of nuclear staining revealed a significant increase of VPAC1 staining with glioma grade, with up to 50% of GBM displaying strong VPAC1 nuclear staining, whereas nuclear VPAC2 staining remained marginal. The increase in VPAC receptor expression with glioma grades and the enhanced nuclear localization of the VPAC1 receptors in GBM might be of importance for glioma progression.

  20. Nuclear localization of Merkel cell polyomavirus large T antigen in Merkel cell carcinoma

    International Nuclear Information System (INIS)

    To clarify whether mutations in the large T gene encoded by Merkel cell polyomavirus affect the expression and function of large T antigen in Merkel cell carcinoma cases, we investigated the expression of large T antigen in vitro and in vivo. Immunohistochemistry using a rabbit polyclonal antibody revealed that large T antigen was expressed in the nuclei of Merkel cell carcinoma cells with Merkel cell polyomavirus infection. Deletion mutant analyses identified an Arg-Lys-Arg-Lys sequence (amino acids 277-280) as a nuclear localization signal in large T antigen. Sequence analyses revealed that there were no mutations in the nuclear localization signal in any of the eleven Merkel cell polyomavirus strains examined. Furthermore, stop codons were not observed in the upstream of the nuclear localization signal in any of the Merkel cell carcinoma cases examined. These data suggest that the nuclear localization signal is highly conserved and functional in Merkel cell carcinoma cases.

  1. Localization of Manufacturing Capabilities in Setting Up Nuclear Power Plants

    International Nuclear Information System (INIS)

    Nuclear renaissance is now imminent and is inevitable in view of rapidly increasing global warming concerns. A steep shift towards environmentally benign sources of energy remains an unavoidable choice as continents are warming up pushing seas into human habitation and disturbing global ecology. Accordingly, Indian government in its integrated energy policy document has planned for raising nuclear power capacity to generate 63 GWe by 2030. This envisages estimated investments of US$22 billion in the next 15 to 20 years. Setting up of nuclear energy generation capacity, however, remains a painstakingly slow process primarily due to complex, multidisciplinary efforts required to crank up a reactor. A robust supply chain remains key to expediting this process. In the light of this, it is critically important to ensure supply-chain for materials and components and putting in place cost effective project management to complete the projects on time and within the budgets. In this context, the participation of industries and their preparedness to meet the challenges are necessary. This would also require investments towards up gradation of manufacturing technology, training of manpower and mobilization of resources at the construction site. The industry would also need to enhance detailing and design engineering capabilities for the plants. It is only when such capabilities have been brought up that the possibilities of time-bound setting up of nuclear plants can be realized. In this paper, various issues with regard to project cost, regulatory and licensing, technology and gestation period etc for new build plants relevant to manufacturing industry are discussed. The plans for enhancing manufacturing capabilities for the critical path items of the project schedule with viable business, ensuring returns to stakeholders and financing and investment cycle are brought out. The various steps and initiatives being taken by Bharat Forge Ltd, the flagship company of Kalyani

  2. Pharmacokinetics local model and Its application in nuclear medicine

    Institute of Scientific and Technical Information of China (English)

    曹国宪; 李卫一; 等

    1996-01-01

    The Pharmacokinetics local model for studying kinetic action of pharmaceuticals in the specific part of the body is established on the basis of the compartment model.A series of formulae is deduced,and with them the pharmacokinetic equation and several important parameters can be obtained.The local model successfully expanded the area of pharmacokinetics from the compartment model ,which is mainly dealing with kinetic action of pharmaceuticals in blood,to the local model,which can study kinetic action of pharmaceuticals in any organ,tissue or fluid.

  3. Expression from baculovirus and serological reactivity of the nucleocapsid protein of dolphin morbillivirus.

    Science.gov (United States)

    Grant, Rebecca J; Kelley, Karen L; Maruniak, James E; Garcia-Maruniak, Alejandra; Barrett, Tom; Manire, Charles A; Romero, Carlos H

    2010-07-14

    The nucleocapsid (N) protein of dolphin morbillivirus (DMV) was expressed from a baculovirus (Autographa californica nuclear polyhedrosis virus) vector and shown by SDS-PAGE and Western blot analysis to be about 57 kDa. Transmission electron microscopy revealed fully assembled nucleocapsid-like particles (NLPs) exhibiting the typical helical herringbone morphology. These NLPs were approximately 20-22 nm in diameter and varied in length from 50 to 100 nm. Purified DMV-N protein was used as antigen in an indirect ELISA (iELISA) and shown to react with rabbit and human antisera to measles virus (MV) and dog sera with antibodies to canine distemper virus (CDV). The iELISA was used for the demonstration of morbillivirus antibodies in the serum of cetaceans and manatees, showing potential as a serological tool for the mass screening of morbillivirus antibodies in marine mammals. PMID:20005643

  4. The Parasitoid Factor in the Virulence and Spread of Lepidopteran Baculoviruses

    Institute of Scientific and Technical Information of China (English)

    J. E. Cossentine

    2009-01-01

    Insect parasitoids and baculoviruses play important roles in the natural and strategic biological control of insects. The two parasites are frequent competitors within common hosts and much research has focused on the negative impact that baculoviral host infections have on parasitoids. This review summarizes the impacts that parasitoids may have on the virulence and spread of lepidopteran baculoviruses. By changing host behavior and development, parasitoids have been shown to decrease baculovirus virulence and productivity within parasitized baculovirus-susceptible hosts; however, studies of the tools used by hymenopteran parasitoids to overcome their hosts' immune systems, suggest that parasitoids may, in some cases, facilitate baculoviral infections in less susceptible hosts. Laboratory and field research have demonstrated that parasitoids can mechanically transmit bacuioviruses between insects, and in this way, increase the efficacy of the viruses. Instances of new, more virulent isolates of baculoviruses have been recorded from specifically parasitoid-targeted hosts suggesting other possible benefits from the transmission or activation of baculoviruses by parasitoids.

  5. Baculovirus as a highly efficient expression vector in insect and mammalian cells

    Institute of Scientific and Technical Information of China (English)

    Yu-chen HU

    2005-01-01

    Baculovirus has been widely used for the production of recombinant proteins in insect cells. Since the finding that baculovirus can efficiently transduce mammalian cells, the applications of baculovirus have been greatly expanded. The prospects and drawbacks of baculovirus-mediated gene expression, either in insect or in mammalian cells, are reviewed. Recent progresses in expanding the applications to studies of gene regulation, viral vector preparation, in vivo and ex vivo gene therapy studies, generation of vaccine vectors, etc are discussed and the efforts directed towards overcoming the existing bottlenecks are particularly emphasized.

  6. Local civic forum. An experimental study promoting public acceptance on nuclear energy

    International Nuclear Information System (INIS)

    In order to promote public acceptance on nuclear energy, an experimental study named 'Local Civic Forum' has been performed as one of the research activities on relationship between nuclear energy and society promoted by the 21st Century Center of Excellence Program: 'Innovative Nuclear Energy Systems for Sustainable Development of the World' conducted in Tokyo Institute of Technology (Tokyo Tech). The Local Civic Forum was designed to implement a dialogue among doctoral students of Tokyo Tech and multi-stakeholders of society. The framework of the forum looks like a stakeholder meeting or a consensus conference, however, it is different from these because no nuclear specialists directly join the dialogue. The specialists actively supported the doctoral students. The multi-stakeholders were selected from a viewpoint of social responsibility. Subjects in the Local Civic Forum were selected by the students from problems of nuclear energy. The participants of the forum discussed the selected subject based on the same eyes, and tried to get findings for hidden problems or issues on public acceptance of nuclear energy. We have made seven experiments of the forums between FY2005 and 2006. Some valuable comments were obtained from the participants to improve the scheme and management of the forum. It was found that the selection of multi-stakeholders and the facilitator of the Local Civic Forum played an important role. In this paper, the experimental results of the Local Civic Forum and the findings obtained by the participants will be presented. (author)

  7. Identification of novel nuclear localization signal within the ErbB-2 protein

    Institute of Scientific and Technical Information of China (English)

    Qiao Qiao CHEN; Xiao Ying CHEN; Yun Yun JIANG; Jing LIU

    2005-01-01

    ErbB2,a member of the receptor tyrosine kinase family,is frequently over-expressed in breast cancer.Proteolysis of the extracellular domain of ErbB2 results in constitutive activation of ErbB2 kinase.Recent study reported that ErbB2 is found in the nucleus.Here,we showed that ErbB2 is imported into the nucleus through a nuclear localization signal (NLS)-mediated mechanism.The NLS sequence KRRQQKIRKYTMRR (aa655-668) contains three clusters of basic amino acids and it is sufficient to target GFP into the nucleus.However,mutation in any basic amino acid cluster of this NLS sequence significantly affects its nuclear localization.Furthermore,it was found that this NLS is essential for the nuclear localization of ErbB2 since the intracellular domain of Erb2 lacking NLS completely abrogates its nuclear translocation.Taken together,our study identified a novel nuclear localization signal and reveals a novel mechanism underlying ErbB2 nuclear trafficking and localization.

  8. Highly efficient baculovirus-mediated multigene delivery in primary cells

    OpenAIRE

    Maysam Mansouri, Maysam; Bellon-Echeverria, Itxaso; Rizk, Aurélien; Ehsaei, Zahra; Cianciolo Cosentino, Chiara; Silva, Catarina S.; Berger, Imre

    2016-01-01

    Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including, synthetic and structural biology, cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity, thus impeding unrestricted multigene expression. We developed MultiPrime, a modular, non-cytotoxic, non-integrating, baculovirus-based vector system expediting hig...

  9. The conserved domain CR2 of Epstein-Barr virus nuclear antigen leader protein is responsible not only for nuclear matrix association but also for nuclear localization.

    Science.gov (United States)

    Yokoyama, A; Kawaguchi, Y; Kitabayashi, I; Ohki, M; Hirai, K

    2001-01-20

    There is a growing body of evidence for the importance of the nuclear matrix in various nuclear events including gene expression and DNA replication. Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is a nuclear matrix-associated protein that has been suggested to play an important role in EBV-induced transformation. To define the biological significance of the association of EBNA-LP with the nuclear matrix, we mapped the domain of EBNA-LP responsible for nuclear matrix association and investigated the functions of the EBNA-LP mutant mutagenized by substitution of alanines for the cluster of arginine residues in the mapped region. The results of the present study were as follows. (i) Transiently expressed EBNA-LP in COS-7 or BOSC23 cells was associated with the nuclear matrix, similarly to that in EBV-infected B cells. (ii) Mutational analysis of EBNA-LP revealed that a 10-amino acid segment of EBNA-LP is critical for nuclear matrix association of the protein. Interestingly, the identified region overlapped with the region CR2 of EBNA-LP conserved among a subset of primate gammaherpesviruses. The identified segment is referred to as EBNA-LP NMTS (nuclear matrix targeting signal). (iii) The EBNA-LP mutant with the arginine to alanine substitutions in NMTS was no longer localized not only to the nuclear matrix but also to the nucleus. (iv) The EBNA-LP mutant lacked its ability to coactivate EBNA-2-dependent transactivation. These results indicated that EBNA-LP needs to be localized in the nucleus and/or associated with the nuclear matrix through CR2 to elicit its function such as the coactivation of the EBNA-2-dependent transcriptional activation. PMID:11162796

  10. Nuclear localization signal in a cancer-related transcriptional regulator protein NAC1.

    Science.gov (United States)

    Okazaki, Kosuke; Nakayama, Naomi; Nariai, Yuko; Nakayama, Kentaro; Miyazaki, Kohji; Maruyama, Riruke; Kato, Hiroaki; Kosugi, Shunichi; Urano, Takeshi; Sakashita, Gyosuke

    2012-10-01

    Nucleus accumbens-associated protein 1 (NAC1) might have potential oncogenic properties and participate in regulatory networks for pluripotency. Although NAC1 is described as a transcriptional regulator, the nuclear import machinery of NAC1 remains unclear. We found, using a point mutant, that dimer formation was not committed to the nuclear localization of NAC1 and, using deletion mutants, that the amino-terminal half of NAC1 harbored a potential nuclear localization signal (NLS). Wild type, but not mutants of this region, alone was sufficient to drive the importation of green fluorescent protein (GFP) into the nucleus. Bimax1, a synthetic peptide that blocks the importin α/β pathway, impaired nuclear localization of NAC1 in cells. We also used the binding properties of importin to demonstrate that this region is an NLS. Furthermore, the transcriptional regulator function of NAC1 was dependent on its nuclear localization activity in cells. Taken together, these results show that the region with a bipartite motif constitutes a functional nuclear import sequence in NAC1 that is independent of NAC1 dimer formation. The identification of an NAC1 NLS thus clarifies the mechanism through which NAC1 translocates to the nucleus to regulate the transcription of genes involved in oncogenicity and pluripotency.

  11. Reflections on the development of local suppliers for the Argentine nuclear industry

    International Nuclear Information System (INIS)

    Argentina has given recently a new start to its nuclear power activities. Looking for background and experiences that can be useful under the new reality, the paper is a survey of the past development of local suppliers for the national nuclear industry. Based on the intention to answer the questions: Why so early it was decided to build a nuclear power plant? Why it was decided to buy it under a turnkey basis rather than developing an indigenous design? and what was the meaning of the 'opening of the technology package' at that time?, the paper describes the actions that led to the purchase of the Atucha I, Embalse and Atucha II nuclear power plants and how these decisions were implemented in order to maximize local participation and the technology transfer. It also analyzes the influence of the Argentine Nuclear Plan of the late seventies on the development of endogenous technology and describes the facts that helped to preserve until now the technological nuclear capabilities of the country in spite of the stopping of the Atucha II construction, and to create positive expectations regarding the revival of the local industry as a supplier of nuclear goods and services. (author)

  12. The bovine papillomavirus type 1 E2 transactivator and repressor proteins use different nuclear localization signals.

    Science.gov (United States)

    Skiadopoulos, M H; McBride, A A

    1996-02-01

    The E2 gene of bovine papillomavirus type 1 encodes at least three nuclear phosphoproteins that regulate viral transcription and DNA replication. All three proteins have a common C-terminal domain that has DNA-binding and dimerization activities. A basic region in this domain forms an alpha helix which makes direct contact with the DNA target. In this study, it is shown that in addition to its role in DNA binding, this basic region functions as a nuclear localization signal both in the E2 DNA-binding domain and in a heterologous protein. Deletion of this signal sequence resulted in increased accumulation of the E2 transactivator and repressor proteins in the cytoplasm, but nuclear localization was not eliminated. In the full-length transactivator protein, another signal, present in the N-terminal transactivation domain, is used for transport to the nucleus, and the C-terminal nuclear localization signal(s) are masked. The use of different nuclear localization signals could potentially allow differential regulation of the subcellular localization of the E2 transactivator and repressor proteins at some stage in the viral life cycle. PMID:8551571

  13. To the non-local theory of cold nuclear fusion

    OpenAIRE

    Alexeev, Boris V.

    2014-01-01

    In this paper, we revisit the cold fusion (CF) phenomenon using the generalized Bolzmann kinetics theory which can represent the non-local physics of this CF phenomenon. This approach can identify the conditions when the CF can take place as the soliton creation under the influence of the intensive sound waves. The vast mathematical modelling leads to affirmation that all parts of soliton move with the same velocity and with the small internal change of the pressure. The zone of the high dens...

  14. The Political Styles of Local Anti-Nuclear Waste Movements in Finland

    Energy Technology Data Exchange (ETDEWEB)

    Kojo, Matti [Univ. of Tampere (Finland). Dept. of Political Science and International Relations

    2001-07-01

    This paper aims to analyse the political styles of local anti-nuclear waste movements in Finland. The main focus is on the tension between the environmental impact assessment process (EIA) and the activity of local opposing groups. According to the EIA Act the purpose of the EIA process is to provide information and opportunities for citizens to participate in planning. It therefore aims to enhance the transparency of the decision-making process. The Finnish nuclear waste company, Posiva Oy, made great efforts locally to create opportunities for participation, but was much criticised by local activists. My questions are the following: How did these local movements participate in Posiva's EIA process? What kind of local differences were there in participating and how can these differences be explained? And finally; what did the EIA process mean to the political styles of movements? EIA meant a temporary change in political style on local level. Firstly; because it brought science into local politics very clearly. Secondly EIA with the Nuclear Energy Act framed decision-making with a timetable, which Posiva emphasised when local groups wanted to make a decision immediately. Thirdly EIA enabled the problem to be defined according to local needs but on the other hand plan level EIA separated nuclear waste issue from the use of nuclear power. Fourthly dialogue in the EIA process favoured rational discourse. Although local views were heard, the process did try to teach the people how to speak with the decision-making system, in the system's language. Thus the purpose of EIA was to make the local discussion controllable. All the local groups, the Romuvaara, Kivetty and Loviisa Movements and the Friends of the Earth in Pori region, took part in EIA dialogue but the reactions were different. The Romuvaara Movement was very active in participating and succeeded in exploiting EIA locally whereas the Loviisa Movement tried to displace the whole process. The Loviisa

  15. Large-scale production of porcine mature interleukin-18 (IL-18) in silkworms using a hybrid baculovirus expression system.

    Science.gov (United States)

    Muneta, Yoshihiro; Zhao, Hong Kun; Inumaru, Shigeki; Mori, Yasuyuki

    2003-02-01

    In this report, a hybrid baculovirus expression system, which means a hybrid virus of the Autographa californica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, was used for the large-scale production of porcine mature interleukin-18 (IL-18) in silkworms. Two recombinant hybrid baculoviruses containing cDNA of the porcine precursor IL-18 and the porcine caspase-1 were constructed and were used to infect silkworm larvae. After the co-infection of the two viruses, porcine mature IL-18 was efficiently produced in the haemolymph. The concentration of IL-18 in the haemolymph was 80-100 microg/ml, as determined by porcine IL-18 specific ELISA. This yield was twenty-times more than that of the insect cell expression system described previously. The porcine mature IL-18 produced by the silkworms strongly induced interferon-gamma (IFN-gamma) production from porcine PBMC. An insect factory system for the large-scale production of useful cytokines for livestock animals will be available in the near future. PMID:12655117

  16. Evaluation of nuclear power and renewable alternatives as portrayed in UK local press coverage title

    OpenAIRE

    Spears, R; Eiser, J R; van der Pligt, J.

    1986-01-01

    A content analysis was conducted of all UK local daily newspaper articles appearing in the first half of 1981, concerned with nuclear power or renewable alternatives. Evaluative coverage of these technologies was compared on dimensions found to characterise energy issues (economic, environmental, technological future/political, physical and psychological risks). In addition, comparisons were drawn between coverage in areas 'threatened' with the potential siting of a new nuclear power station ...

  17. New computer simulation technology of WSPEEDI for local and regional environmental assessment during nuclear emergency

    Energy Technology Data Exchange (ETDEWEB)

    Chino, Masamichi; Furuno, Akiko; Terada, Hiroaki; Kitabata, Hideyuki [Japan Atomic Energy Research Institute, Ibaraki-Ken (Japan)

    2002-07-01

    The increase of nuclear power plants in the Asian region necessitates the capability to predict long-range atmospheric dispersions of radionuclides and radiological impacts due to a nuclear accident. For this purpose, we have developed a computer-based emergency response system WSPEEDI. This paper aims to expanding the capability of WSPEEDI so that it can be applied to simultaneous multi-scale predictions of local and regional scales in the Asian region.

  18. Nuclear localization of DMP1 proteins suggests a role in intracellular signaling

    Energy Technology Data Exchange (ETDEWEB)

    Siyam, Arwa [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 (United States); Wang, Suzhen; Qin, Chunlin; Mues, Gabriele [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Stevens, Roy [Department of Endodontology, Kornberg School of Dentistry, Temple University, 3223 North Broad Street, Philadelphia, PA 19140-5007 (United States); D' Souza, Rena N. [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States); Lu, Yongbo, E-mail: ylu@bcd.tamhsc.edu [Department of Biomedical Sciences, Baylor College of Dentistry, Texas A and M Health Science Center, 3302 Gaston Ave., Dallas, TX 75246-2013 (United States)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Nuclear localization of DMP1 in various cell lines. Black-Right-Pointing-Pointer Non-synchronized cells show either nuclear or cytoplasmic localization of DMP1. Black-Right-Pointing-Pointer Nuclear DMP1 is restricted to the nucleoplasm but absent in the nucleolus. -- Abstract: Dentin matrix protein 1 (DMP1) is highly expressed in odontoblasts and osteoblasts/osteocytes and plays an essential role in tooth and bone mineralization and phosphate homeostasis. It is debatable whether DMP1, in addition to its function in the extracellular matrix, can enter the nucleus and function as a transcription factor. To better understand its function, we examined the nuclear localization of endogenous and exogenous DMP1 in C3H10T1/2 mesenchymal cells, MC3T3-E1 preosteoblast cells and 17IIA11 odontoblast-like cells. RT-PCR analyses showed the expression of endogenous Dmp1 in all three cell lines, while Western-blot analysis detected a major DMP1 protein band corresponding to the 57 kDa C-terminal fragment generated by proteolytic processing of the secreted full-length DMP1. Immunofluorescent staining demonstrated that non-synchronized cells presented two subpopulations with either nuclear or cytoplasmic localization of endogenous DMP1. In addition, cells transfected with a construct expressing HA-tagged full-length DMP1 also showed either nuclear or cytoplasmic localization of the exogenous DMP1 when examined with an antibody against the HA tag. Furthermore, nuclear DMP1 was restricted to the nucleoplasm but was absent in the nucleolus. In conclusion, these findings suggest that, apart from its role as a constituent of dentin and bone matrix, DMP1 might play a regulatory role in the nucleus.

  19. Identification of the nuclear localization and export signals of high risk HPV16 E7 oncoprotein

    International Nuclear Information System (INIS)

    The E7 oncoprotein of high risk human papillomavirus type 16 (HPV16) binds and inactivates the retinoblastoma (RB) family of proteins. Our previous studies suggested that HPV16 E7 enters the nucleus via a novel Ran-dependent pathway independent of the nuclear import receptors (Angeline, M., Merle, E., and Moroianu, J. (2003). The E7 oncoprotein of high-risk human papillomavirus type 16 enters the nucleus via a nonclassical Ran-dependent pathway. Virology 317(1), 13-23.). Here, analysis of the localization of specific E7 mutants revealed that the nuclear localization of E7 is independent of its interaction with pRB or of its phosphorylation by CKII. Fluorescence microscopy analysis of enhanced green fluorescent protein (EGFP) and 2xEGFP fusions with E7 and E7 domains in HeLa cells revealed that E7 contains a novel nuclear localization signal (NLS) in the N-terminal domain (aa 1-37). Interestingly, treatment of transfected HeLa cells with two specific nuclear export inhibitors, Leptomycin B and ratjadone, changed the localization of 2xEGFP-E738-98 from cytoplasmic to mostly nuclear. These data suggest the presence of a leucine-rich nuclear export signal (NES) and a second NLS in the C-terminal domain of E7 (aa 38-98). Mutagenesis of critical amino acids in the putative NES sequence (76IRTLEDLLM84) changed the localization of 2xEGFP-E738-98 from cytoplasmic to mostly nuclear suggesting that this is a functional NES. The presence of both NLSs and an NES suggests that HPV16 E7 shuttles between the cytoplasm and nucleus which is consistent with E7 having functions in both of these cell compartments

  20. Nuclear localization of DMP1 proteins suggests a role in intracellular signaling

    International Nuclear Information System (INIS)

    Highlights: ► Nuclear localization of DMP1 in various cell lines. ► Non-synchronized cells show either nuclear or cytoplasmic localization of DMP1. ► Nuclear DMP1 is restricted to the nucleoplasm but absent in the nucleolus. -- Abstract: Dentin matrix protein 1 (DMP1) is highly expressed in odontoblasts and osteoblasts/osteocytes and plays an essential role in tooth and bone mineralization and phosphate homeostasis. It is debatable whether DMP1, in addition to its function in the extracellular matrix, can enter the nucleus and function as a transcription factor. To better understand its function, we examined the nuclear localization of endogenous and exogenous DMP1 in C3H10T1/2 mesenchymal cells, MC3T3-E1 preosteoblast cells and 17IIA11 odontoblast-like cells. RT-PCR analyses showed the expression of endogenous Dmp1 in all three cell lines, while Western-blot analysis detected a major DMP1 protein band corresponding to the 57 kDa C-terminal fragment generated by proteolytic processing of the secreted full-length DMP1. Immunofluorescent staining demonstrated that non-synchronized cells presented two subpopulations with either nuclear or cytoplasmic localization of endogenous DMP1. In addition, cells transfected with a construct expressing HA-tagged full-length DMP1 also showed either nuclear or cytoplasmic localization of the exogenous DMP1 when examined with an antibody against the HA tag. Furthermore, nuclear DMP1 was restricted to the nucleoplasm but was absent in the nucleolus. In conclusion, these findings suggest that, apart from its role as a constituent of dentin and bone matrix, DMP1 might play a regulatory role in the nucleus.

  1. Expression of Recombinant Baculovirus Carrying Schistosoma japonicum 26 ku GST in Mammalian Cells

    Institute of Scientific and Technical Information of China (English)

    YU Guangqing; SONG Jianhua; LIU Wenqi; LONG Xiaochun; MO Hongmei; LI Yonglong; CHEN Xinwen

    2006-01-01

    In order to construct recombinant baculovirus carrying Schistosoma japonicum 26 ku glutathione S-transferase gene (Sj26), and observe the expression of Sj26 in mammalian cells, the Sj26 gene was amplified with plasmid pGEX-3X as template by PCR, and then recombined into Tvector for sequencing. Sj26 gene was inserted into the downstream of CMV promoter of donor plasmid pFBDGC, and the recombinant donor plasmid pFBDGC-Sj26 transformed into DH10Bac,then the recombinant bacmid AcCMVSj26 was isolated and transfected into Sf9 cells. The recombinant baculovirus was harvested and final titer of vAcCMVSj26 was measured. BHK cells were transducted with recombinant baculovirus in vitro. By using Western blot, the expression of 26 ku glutathione S-transferase (GST) was detected. The results showed that after enzyme digestion and sequencing, the donor plasmid was successfully constructed. PCR confirmed that pFBDGC-Sj26 and Bacmid homologous recombination occurred in E. coli. After transfection of Sf9 cells with recombinant Bacmid, recombinant baculovirus was replicated in Sf9 cells and expressed green fluorescent protein. PCR further revealed recombinant baculovirus contained Sj26. The titer of the harvested baculovirus was 1.24 × 108. Western blot demonstrated that recombinant baculovirus could express 26 ku GST in BHK cells. It was concluded that Sj26 recombinant baculovirus was successfully constructed, and the 26 ku GST was expressed in mammalian cells.

  2. Effect of spray drying processing parameters on the insecticidal activity of two encapsulated formulations of baculovirus

    Science.gov (United States)

    The aim of this work was to evaluate the effect of spray dryer processing parameters on the process yield and insecticidal activity of baculovirus to support the development of this beneficial group of microbes as biopesticides. For each of two baculoviruses [granulovirus (GV) from Pieris rapae (L....

  3. Pluronic F127 nanomicelles engineered with nuclear localized functionality for targeted drug delivery.

    Science.gov (United States)

    Li, Yong-Yong; Li, Lan; Dong, Hai-Qing; Cai, Xiao-Jun; Ren, Tian-Bin

    2013-07-01

    PKKKRKV (Pro-Lys-Lys-Lys-Arg-Lys-Val, PV7), a seven amino acid peptide, has emerged as one of the primary nuclear localization signals that can be targeted into cell nucleus via the nuclear import machinery. Taking advantage of chemical diversity and biological activities of this short peptide sequence, in this study, Pluronic F127 nanomicelles engineered with nuclear localized functionality were successfully developed for intracellular drug delivery. These nanomicelles with the size ~100 nm were self-assembled from F127 polymer that was flanked with two PV7 sequences at its both terminal ends. Hydrophobic anticancer drug doxorubicin (DOX) with inherent fluorescence was chosen as the model drug, which was found to be efficiently encapsulated into nanomicelles with the encapsulation efficiency at 72.68%. In comparison with the non-functionalized namomicelles, the microscopic observation reveals that PV7 functionalized nanomicelles display a higher cellular uptake, especially into the nucleus of HepG2 cells, due to the nuclear localization signal effects. Both cytotoxicity and apoptosis studies show that the DOX-loaded nanomicelles were more potent than drug nanomicelles without nuclear targeting functionality. It was thus concluded that PV7 functionalized nanomicelles could be a potentially alternative vehicle for nuclear targeting drug delivery.

  4. Sumoylation regulates nuclear localization of lipin-1alpha in neuronal cells.

    Directory of Open Access Journals (Sweden)

    Guang-Hui Liu

    Full Text Available Lipin-1 is a protein that has dual functions as a phosphatidic acid phosphohydrolase (PAP and a nuclear transcriptional coactivator. It remains unknown how the nuclear localization and coactivator functions of lipin-1 are regulated. Here, we show that lipin-1 (including both the alpha and beta isoforms is modified by sumoylation at two consensus sumoylation sites. We are unable to detect sumoylation of the related proteins lipin-2 and lipin-3. Lipin-1 is sumoylated at relatively high levels in brain, where lipin-1alpha is the predominant form. In cultured embryonic cortical neurons and SH-SY5Y neuronal cells, ectopically expressed lipin-1alpha is localized in both the nucleus and the cytoplasm, and the nuclear localization is abrogated by mutating the consensus sumyolation motifs. The sumoylation site mutant of lipin-1alpha loses the capacity to coactivate the transcriptional (co- activators PGC-1alpha and MEF2, consistent with its nuclear exclusion. Thus, these results show that sumoylation facilitates the nuclear localization and transcriptional coactivator behavior of lipin-1alpha that we observe in cultured neuronal cells, and suggest that lipin-1alpha may act as a sumoylation-regulated transcriptional coactivator in brain.

  5. Framing the national nuclear legacy at the local level: Implications for the future of federal facilities

    International Nuclear Information System (INIS)

    There are several major federal nuclear facilities located in small towns and rural areas of the United States. While many of these facilities were developed in the 1950s to support national defense, in the 1960s and 1970s, some of these shifted their mission to focus on national energy infrastructure. Now, many of these facilities are in a clean-up phase, and local communities are becoming increasingly engaged in influencing decisions about the future of the sites. Communicating with the public in rural communities is challenging when it involves a complicated environmental issue that could have widespread economic impacts. The local media reflect public understanding, so getting a sense of how these media frame issues can be a crucial first step to developing an effective community engagement strategy. A media content analysis of one local newspaper was completed in relation to a major federal nuclear facility. The content analysis is compared to the results of a telephone survey in the region served by the paper and the results suggest that there is a relationship between how the facility is portrayed in local media and public concern. This study has important implications for other nuclear facilities because of the role of local citizens in decision-making. - Highlights: ► Decisions about federal nuclear facilities include local citizen participation. ► Local media can play an important role in public perception of environmental risk. ► Local print media rely on a limited number in of sources for their stories. ► Effective risk communication should begin by understanding local public concerns.

  6. Characterization of the Nuclear Localization Signal of High Risk HPV16 E2 Protein

    OpenAIRE

    Klucevsek, Kristin; Wertz, Mary; Lucchi, John; Leszczynski, Anna; Moroianu, Junona

    2006-01-01

    The E2 protein of high risk human papillomavirus type 16 (HPV16) contains an amino-terminal (N) domain, a hinge (H) region and a carboxyl-terminal (C) DNA binding domain. Using enhanced green fluorescent protein (EGFP) fusions with full length E2 and E2 domains in transfection assays in HeLa cells we found that the C domain is responsible for the nuclear localization of E2 in vivo, whereas the N and H domains do not contain additional nuclear localization signals (NLSs). Deletion analysis of ...

  7. The RelA nuclear localization signal folds upon binding to IκBα

    OpenAIRE

    Cervantes, Carla F.; Bergqvist, Simon; Kjaergaard, Magnus; Kroon, Gerard; Sue, Shih-Che; Dyson, H Jane; Komives, Elizabeth A.

    2010-01-01

    The nuclear localization signal (NLS) polypeptide of RelA, the canonical NFκB family member, is responsible for regulating the nuclear localization of RelA-containing NFκB dimers. The RelA NLS polypeptide also plays a crucial role in mediating the high affinity and specificity of the interaction of RelA-containing dimers with the inhibitor IκBα, forming two helical motifs according to the published X-ray crystal structure. In order to define the nature of the interaction between the RelA NLS ...

  8. The development and localization of nuclear fuel technology for KMRR

    International Nuclear Information System (INIS)

    This project was implemented aiming at localizing the fabrication of the KMRR fuel by october 1993. The contents of this project were divided into three parts: fuel design, fuel fabrication and process criticality analysis. In the fuel design, the radial power distribution in the fuel core was modeled and formulated taking account of the neutron flux depression in the radial direction. It was also performed to model and formulate the thermal characteristics such as the thermal conductivity and specific heat of the fuel core, U3Si-Al, the swelling and the film coefficient of heat transfer between the aluminum clad and light water coolant. The two dimensional heat transfer in the finned fuel element was equated based on the general equation governing the heat transfer in materials in order to develope a computer code, TEMP2D. TEMP2D solves finite differenced equations to calculate a two dimensional fuel temperature distribution under the steady and transient states. In the fuel fabrication, the technologies of fabricating uranium silicide fuel meat were tried by using depleted uranium as a raw material. These were extended to find the problems in technologies and to establish the ways of approach. The end product, so called fuel meat, was a metallic powder compound, U3Six(1≤x≤2), dispersed in Al matrix. The fuel meat was fabricated by the horizontal extrusion technique, and powder extrusion technique. Fabrication technologies comprise five different continuous processes: melting and casting of metallic uranium with silicon and aluminum, heat treatment, chipping and crushing, pulverizing, and extrusion. In the process criticality analysis, AMPX-KENO benchmark calculation was performed and calculational error of AMPX-KENO system was established. (Author)

  9. Pluronic F127 nanomicelles engineered with nuclear localized functionality for targeted drug delivery

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yong-Yong; Li, Lan; Dong, Hai-Qing, E-mail: inano_donghq@tongji.edu.cn; Cai, Xiao-Jun; Ren, Tian-Bin, E-mail: rentianbin@yeah.net

    2013-07-01

    PKKKRKV (Pro-Lys-Lys-Lys-Arg-Lys-Val, PV7), a seven amino acid peptide, has emerged as one of the primary nuclear localization signals that can be targeted into cell nucleus via the nuclear import machinery. Taking advantage of chemical diversity and biological activities of this short peptide sequence, in this study, Pluronic F127 nanomicelles engineered with nuclear localized functionality were successfully developed for intracellular drug delivery. These nanomicelles with the size ∼ 100 nm were self-assembled from F127 polymer that was flanked with two PV7 sequences at its both terminal ends. Hydrophobic anticancer drug doxorubicin (DOX) with inherent fluorescence was chosen as the model drug, which was found to be efficiently encapsulated into nanomicelles with the encapsulation efficiency at 72.68%. In comparison with the non-functionalized namomicelles, the microscopic observation reveals that PV7 functionalized nanomicelles display a higher cellular uptake, especially into the nucleus of HepG2 cells, due to the nuclear localization signal effects. Both cytotoxicity and apoptosis studies show that the DOX-loaded nanomicelles were more potent than drug nanomicelles without nuclear targeting functionality. It was thus concluded that PV7 functionalized nanomicelles could be a potentially alternative vehicle for nuclear targeting drug delivery. - Highlights: ► A new nuclear targeted drug delivery system based on micelles is developed. ► This micellar system features a core-shell structure with the size peaked at 100 nm. ► PV7, a short peptide sequence, is adopted as a nuclear targeting ligand. ► PV7 functionalized drug loaded micelles are more potent in killing tumor cells.

  10. Mapping Signals That Are Important for Nuclear and Nucleolar Localization in MCRS2

    OpenAIRE

    Kuo, Chung-Wen; Wang, Wen-Hung; Liu, Shih-Tung

    2011-01-01

    MCRS2 is an oncoprotein that is sequestered in the nucleolus. When in the nucleolus, it promotes the transcription of the rRNA gene. MCRS2 also brings proteins into the nucleolus to change their function. This study analyzes the sequence of MCRS2 and determines that the nuclear localization signal, which has the sequence KRKK, is situated between amino acids 66 and 69. Meanwhile, MCRS2 contains a bipartite nucleolar localization signal, which comprises a KKSK motif, located between amino acid...

  11. EpCAM nuclear localization identifies aggressive Thyroid Cancer and is a marker for poor prognosis

    Directory of Open Access Journals (Sweden)

    MacMillan Christina

    2010-06-01

    Full Text Available Abstract Background Proteolytic cleavage of the extracellular domain (EpEx of Epithelial cell adhesion molecule (EpCAM and nuclear signaling by its intracellular oncogenic domain Ep-ICD has recently been implicated in increased proliferation of cancer cells. The clinical significance of Ep-ICD in human tumors remains an enigma. Methods EpEx, Ep-ICD and β-catenin immunohistochemistry using specific antibodies was conducted on 58 archived thyroid cancer (TC tissue blocks from 34 patients and correlated with survival analysis of these patients for up to 17 years. Results The anaplastic (ATC and aggressive thyroid cancers showed loss of EpEx and increased nuclear and cytoplasmic accumulation of Ep-ICD. In contrast, the low grade papillary thyroid cancers (PTC showed membranous EpEx and no detectable nuclear Ep-ICD. The ATC also showed concomitant nuclear expression of Ep-ICD and β-catenin. Kaplan-Meier Survival analysis revealed reduced overall survival (OS for TC patients showing nuclear Ep-ICD expression or loss of membranous EpEx (p Conclusion We report reciprocal loss of membrane EpEx but increased nuclear and cytoplasmic accumulation of Ep-ICD in aggressive TC; nuclear Ep-ICD correlated with poor OS of TC patients. Thus nuclear Ep-ICD localization may serve as a useful biomarker for aggressive TC and may represent a novel diagnostic, prognostic and therapeutic target for aggressive TC.

  12. Nuclear localization of the dehydrin OpsDHN1 is determined by histidine-rich motif.

    Science.gov (United States)

    Hernández-Sánchez, Itzell E; Maruri-López, Israel; Ferrando, Alejandro; Carbonell, Juan; Graether, Steffen P; Jiménez-Bremont, Juan F

    2015-01-01

    The cactus OpsDHN1 dehydrin belongs to a large family of disordered and highly hydrophilic proteins known as Late Embryogenesis Abundant (LEA) proteins, which accumulate during the late stages of embryogenesis and in response to abiotic stresses. Herein, we present the in vivo OpsDHN1 subcellular localization by N-terminal GFP translational fusion; our results revealed a cytoplasmic and nuclear localization of the GFP::OpsDHN1 protein in Nicotiana benthamiana epidermal cells. In addition, dimer assembly of OpsDHN1 in planta using a Bimolecular Fluorescence Complementation (BiFC) approach was demonstrated. In order to understand the in vivo role of the histidine-rich motif, the OpsDHN1-ΔHis version was produced and assayed for its subcellular localization and dimer capability by GFP fusion and BiFC assays, respectively. We found that deletion of the OpsDHN1 histidine-rich motif restricted its localization to cytoplasm, but did not affect dimer formation. In addition, the deletion of the S-segment in the OpsDHN1 protein affected its nuclear localization. Our data suggest that the deletion of histidine-rich motif and S-segment show similar effects, preventing OpsDHN1 from getting into the nucleus. Based on these results, the histidine-rich motif is proposed as a targeting element for OpsDHN1 nuclear localization. PMID:26442018

  13. Nuclear localization of the dehydrin OpsDHN1 is determined by histidine-rich motif

    Directory of Open Access Journals (Sweden)

    Itzell Euridice Hernández-Sánchez

    2015-09-01

    Full Text Available The cactus OpsDHN1 dehydrin belongs to a large family of disordered and highly hydrophilic proteins known as Late Embryogenesis Abundant (LEA proteins, which accumulate during the late stages of embryogenesis and in response to abiotic stresses. Herein, we present the in vivo OpsDHN1 subcellular localization by N-terminal GFP translational fusion; our results revealed a cytoplasmic and nuclear localization of the GFP::OpsDHN1 protein in Nicotiana benthamiana epidermal cells. In addition, dimer assembly of OpsDHN1 in planta using a Bimolecular Fluorescence Complementation (BiFC approach was demonstrated. In order to understand the in vivo role of the histidine-rich motif, the OpsDHN1-ΔHis version was produced and assayed for its subcellular localization and dimer capability by GFP fusion and BiFC assays, respectively. We found that deletion of the OpsDHN1 histidine-rich motif restricted its localization to cytoplasm, but did not affect dimer formation. In addition, the deletion of the S-segment in the OpsDHN1 protein affected its nuclear localization. Our data suggest that the deletion of histidine-rich motif and S-segment show similar effects, preventing OpsDHN1 from getting into the nucleus. Based on these results, the histidine rich motif is proposed as a targeting element for OpsDHN1 nuclear localization.

  14. Identification of a functional nuclear localization signal within the human USP22 protein

    International Nuclear Information System (INIS)

    Highlights: • USP22 was accumulated in nucleus. • We identified of a functional USP22 NLS. • The KRRK amino acid residues are indispensable in NLS. • The KRRK motif is conserved in USP22 homologues. - Abstract: Ubiquitin-specific processing enzyme 22 (USP22), a member of the deubiquitinase family, is over-expressed in most human cancers and has been implicated in tumorigenesis. Because it is an enzymatic subunit of the human SAGA transcriptional cofactor, USP22 deubiquitylates histone H2A and H2B in the nucleus, thus participating in gene regulation and cell-cycle progression. However, the mechanisms regulating its nuclear translocation have not yet been elucidated. It was here demonstrated that USP22 is imported into the nucleus through a mechanism mediated by nuclear localization signal (NLS). The bipartite NLS sequence KRELELLKHNPKRRKIT (aa152–168), was identified as the functional NLS for its nuclear localization. Furthermore, a short cluster of basic amino acid residues KRRK within this bipartite NLS plays the primary role in nuclear localization and is evolutionarily conserved in USP22 homologues. In the present study, a functional NLS and the minimal sequences required for the active targeting of USP22 to the nucleus were identified. These findings may provide a molecular basis for the mechanism underlying USP22 nuclear trafficking and function

  15. Identification of a functional nuclear localization signal within the human USP22 protein

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Jianjun [Key Laboratory of Jiangxi Province for the Systems Bio-Medicine, Jiujiang, Jiangxi Province 332000 (China); College of Basic Medical Science, Jiujiang University, Jiujiang, Jiangxi Province 332000 (China); Wang, Yaqin [Reproductive Medical Center, Renmin Hospital of Wuhan University, Wuhan, Hubei Province 430060 (China); Gong, Zhen [Key Laboratory of Jiangxi Province for the Systems Bio-Medicine, Jiujiang, Jiangxi Province 332000 (China); Liu, Jianyun [College of Basic Medical Science, Jiujiang University, Jiujiang, Jiangxi Province 332000 (China); Li, Weidong, E-mail: lwd626518@163.com [College of Basic Medical Science, Jiujiang University, Jiujiang, Jiangxi Province 332000 (China)

    2014-06-20

    Highlights: • USP22 was accumulated in nucleus. • We identified of a functional USP22 NLS. • The KRRK amino acid residues are indispensable in NLS. • The KRRK motif is conserved in USP22 homologues. - Abstract: Ubiquitin-specific processing enzyme 22 (USP22), a member of the deubiquitinase family, is over-expressed in most human cancers and has been implicated in tumorigenesis. Because it is an enzymatic subunit of the human SAGA transcriptional cofactor, USP22 deubiquitylates histone H2A and H2B in the nucleus, thus participating in gene regulation and cell-cycle progression. However, the mechanisms regulating its nuclear translocation have not yet been elucidated. It was here demonstrated that USP22 is imported into the nucleus through a mechanism mediated by nuclear localization signal (NLS). The bipartite NLS sequence KRELELLKHNPKRRKIT (aa152–168), was identified as the functional NLS for its nuclear localization. Furthermore, a short cluster of basic amino acid residues KRRK within this bipartite NLS plays the primary role in nuclear localization and is evolutionarily conserved in USP22 homologues. In the present study, a functional NLS and the minimal sequences required for the active targeting of USP22 to the nucleus were identified. These findings may provide a molecular basis for the mechanism underlying USP22 nuclear trafficking and function.

  16. Characterization, subcellular localization and nuclear targeting of casein kinase 2 from Zea mays

    DEFF Research Database (Denmark)

    Peracchia, G; Jensen, A B; Culiáñez-Macià, F A;

    1999-01-01

    by using in-frame fusions of the maize CK2alpha subunit to the reporter gene encoding beta-glucuronidase (GUS) which were assayed in transiently transformed onion epidermal cells. Analysis of chimeric constructs identified one region containing a nuclear localization signal (NLS) that is highly conserved...

  17. The N-terminal 33 amino acid domain of Siva-1 is sufficient for nuclear localization

    International Nuclear Information System (INIS)

    Siva-1 induces apoptosis in multiple pathological processes and plays an important role in the suppression of tumor metastasis, protein degradation, and other functions. Although many studies have demonstrated that Siva-1 functions in the cytoplasm, a few have found that Siva-1 can relocate to the nucleus. In this study, we found that the first 33 amino acid residues of Siva-1 are required for its nuclear localization. Further study demonstrated that the green fluorescent protein can be imported into the nucleus after fusion with these 33 amino acid residues. Other Siva-1 regions and domains showed less effect on Siva-1 nuclear localization. By site-mutagenesis of all of these 33 amino acid residues, we found that mutants of the first 1-18 amino acids affected Siva-1 nuclear compartmentalization but could not complete this localization independently. In summary, we demonstrated that the N-terminal 33 amino acid residues were sufficient for Siva-1 nuclear localization, but the mechanism of this translocation needs additional investigation

  18. Nuclear localization of human DNA mismatch repair protein exonuclease 1 (hEXO1)

    DEFF Research Database (Denmark)

    Knudsen, Nina Østergaard; Nielsen, Finn Cilius; Vinther, Lena;

    2007-01-01

    localization signals (NLSs) in hEXO1. Using fluorescent fusion proteins, we show that the sequence 418KRPR421, which exhibit strong homology to other monopartite NLS sequences, is responsible for correct nuclear localization of hEXO1. This NLS sequence is located in a region that is also required for hEXO1......Human exonuclease 1 (hEXO1) is implicated in DNA mismatch repair (MMR) and mutations in hEXO1 may be associated with hereditary nonpolyposis colorectal cancer (HNPCC). Since the subcellular localization of MMR proteins is essential for proper MMR function, we characterized possible nuclear...... interaction with hMLH1 and we show that defective nuclear localization of hEXO1 mutant proteins could be rescued by hMLH1 or hMSH2. Both hEXO1 and hMLH1 form complexes with the nuclear import factors importin beta/alpha1,3,7 whereas hMSH2 specifically recognizes importin beta/alpha3. Taken together, we infer...

  19. The N-terminal 33 amino acid domain of Siva-1 is sufficient for nuclear localization

    Energy Technology Data Exchange (ETDEWEB)

    Chen, J.Y.; Yang, L.X. [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Huang, Z.F. [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Key Laboratory of Tropical Diseases Control, Sun Yat-sen University, Ministry of Education in China, Guangzhou (China)

    2013-12-02

    Siva-1 induces apoptosis in multiple pathological processes and plays an important role in the suppression of tumor metastasis, protein degradation, and other functions. Although many studies have demonstrated that Siva-1 functions in the cytoplasm, a few have found that Siva-1 can relocate to the nucleus. In this study, we found that the first 33 amino acid residues of Siva-1 are required for its nuclear localization. Further study demonstrated that the green fluorescent protein can be imported into the nucleus after fusion with these 33 amino acid residues. Other Siva-1 regions and domains showed less effect on Siva-1 nuclear localization. By site-mutagenesis of all of these 33 amino acid residues, we found that mutants of the first 1-18 amino acids affected Siva-1 nuclear compartmentalization but could not complete this localization independently. In summary, we demonstrated that the N-terminal 33 amino acid residues were sufficient for Siva-1 nuclear localization, but the mechanism of this translocation needs additional investigation.

  20. Nuclear binding energies: global collective structures and local shell-model correlations

    International Nuclear Information System (INIS)

    In this contribution the global behaviour of nuclear binding energies, or alternatively two-neutron separation energies, are studied. Recent high-precision mass measurements show local deviations from an overall macroscopic behaviour, and it is shown how a consistent and simultaneously description of both can be given within the Interacting Boson Model. (orig.)

  1. Cultivation and differentiation change nuclear localization of chromosome centromeres in human mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Yana I Voldgorn

    Full Text Available Chromosome arrangement in the interphase nucleus is not accidental. Strong evidences support that nuclear localization is an important mechanism of epigenetic regulation of gene expression. The purpose of this research was to identify differences in the localization of centromeres of chromosomes 6, 12, 18 and X in human mesenchymal stem cells depending on differentiation and cultivating time. We analyzed centromere positions in more than 4000 nuclei in 19 mesenchymal stem cell cultures before and after prolonged cultivation and after differentiation into osteogenic and adipogenic directions. We found a centromere reposition of HSAX at late passages and after differentiation in osteogenic direction as well as of HSA12 and HSA18 after adipogenic differentiation. The observed changes of the nuclear structure are new nuclear characteristics of the studied cells which may reflect regulatory changes of gene expression during the studied processes.

  2. INVESTIGATION OF LATENT NUCLEAR LOCALIZATION SEQUENCE(NLS) OF STAT3

    Institute of Scientific and Technical Information of China (English)

    杨镇珲; 李惠; 叶中德; 冯健男; 沈倍奋

    2004-01-01

    Objective: To investigate the latent nuclear localization sequence (NLS) OF STAT3. Methods: Clustal X (1.81) was used to alignment the DNA binding domain of the STAT family. According to structure characters, corresponding expression plasmids were constructed via oligonucleotides designed with gene tool software. Through in vitro transfection, images were observed with laser confocal microscopy. Results: homology positions of arginine and lysine were found in DNA binding domain of the stat family. The wild type-STAT3 proteins primarily localized in the cytoplasm and translocated into the nucleus after interleukin-6 stimulation. However, the truncated mutant of DSTAT3-GFP protein was exclusively expressed in the cytoplasm. Conclusion: The potential NLS in the DNA binding domain of STAT3 is exposed to nuclear importing receptor when cells are stinulated by cytokine, which promotes the translocation of STAT3 into nuclear.

  3. Do nuclear collisions create a locally equilibrated quark-gluon plasma?

    CERN Document Server

    Romatschke, Paul

    2016-01-01

    Experimental results on azimuthal correlations in high energy nuclear collisions (nucleus-nucleus, proton-nucleus and proton-proton) seem to be well described by viscous hydrodynamics. It is often argued that this agreement implies either local thermal equilibrium or at least local isotropy. In this note, I present arguments why this is not the case. Neither local near-equilibrium nor near-isotropy are required in order for hydrodynamics to offer a successful and accurate description of experimental results. However, I predict the breakdown of hydrodynamics at momenta of order twenty times the temperature, corresponding to a smallest possible QCD liquid drop size of 0.05 fm.

  4. Expression of the mouse interleukin-2 receptor gamma chain in insect cells using a baculovirus expression vector--comparison with the human common gamma chain.

    Science.gov (United States)

    Stenroos, K; West, A; Raivio, E; Lindqvist, C

    1997-02-01

    The gene encoding the gamma-chain of the mouse Interleukin-2 receptor was expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding gene was inserted under the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The recombinant receptor protein was identified by immunoblotting in cell lysates prepared from insect cells infected with the produced recombinant virus VL1392-mIL-2R gamma. Kinetic analysis demonstrated that the corresponding protein could be detected as an approximately 50 kDa protein already at 24 h post-infection. Intrinsic labelling with [35S]-methionine/cysteine and SDS-PAGE analysis of the recombinant baculovirus infected insect cells verified the immunoblotting data. The expressed IL-2R gamma protein could also be determined on the surface of infected insect cells by flow cytometric analysis. Comparison of the molecular weights between baculovirus expressed human and mouse IL-2R gamma chains indicated differences in the glycosylation pattern despite similar numbers of N-linked glycosylation sites. PMID:9042425

  5. Nuclear localization of Rad51B is independent of BRCA2

    Energy Technology Data Exchange (ETDEWEB)

    Miller, K A; Hinz, J M; Yamada, A; Thompson, L H; Albala, J S

    2005-06-28

    Human Rad51 is critical for the maintenance of genome stability through its role in the repair of DNA double-strand breaks. Rad51B (Rad51L1/hRec2) is one of the five known paralogs of human Rad51 found in a multi-protein complex with three other Rad51 paralogs, Rad51C, Rad51D and Xrcc2. Examination of EGFP-Rad51B fusion protein in HeLa S3 cells and immunofluorescence in several human cell lines confirms the nuclear localization of Rad51B. This is the first report to detail putative interactions of a Rad51 paralog protein with BRCA2. Utilization of a BRCA2 mutant cell line, CAPAN-1 suggests that Rad51B localizes to the nucleus independent of BRCA2. Although both Rad51B and BRCA2 are clearly involved in the homologous recombinational repair pathway, Rad51B and BRCA2 do not appear to associate directly. Furthermore, mutations in the KKLK motif of Rad51B, amino acid residues 4-7, mislocalizes Rad51B to the cytoplasm suggesting that this is the nuclear localization signal for the Rad51B protein. Examination of wild-type EGFP-Rad51B fusion protein in mammalian cells deficient in Rad51C showed that Rad51B localizes to the nucleus independent of Rad51C; further suggesting that Rad51B, like Rad51C, contains its own nuclear localization signal.

  6. 1. European conference on local democracies and nuclear power plants. Conference proceedings

    International Nuclear Information System (INIS)

    The Group of European Municipalities with Nuclear Facilities (GMF) was constituted in 1999. Its basic objectives are to keep and strengthen the relations between municipalities with the aim of consolidating a European network of municipalities with common problems; and to participate in European forums on the future of nuclear energy, contributing and helping in decision making processes. The GMF wants to become a support element to the local authorities of nuclear areas in order to foster their participation in the decision making processes, working continuously in collaboration with other agents of the nuclear sector, especially with the authorities of the European Commission, assuming that its major contribution is the knowledge of the everyday territorial reality, as a fundamental element that should be born in mind in all defined policies, whether on european or national level. It is stated that the debate on the future of nuclear energy represents a new era for the municipalities with nuclear facilities across Europe and that international cooperation will open the way for a better use of nuclear energy in Europe

  7. Oscillator strengths, first-order properties, and nuclear gradients for local ADC(2)

    Energy Technology Data Exchange (ETDEWEB)

    Schütz, Martin, E-mail: martin.schuetz@chemie.uni-regensburg.de [Institute of Physical and Theoretical Chemistry, University of Regensburg, Universitätsstraße 31, D-93040 Regensburg (Germany)

    2015-06-07

    We describe theory and implementation of oscillator strengths, orbital-relaxed first-order properties, and nuclear gradients for the local algebraic diagrammatic construction scheme through second order. The formalism is derived via time-dependent linear response theory based on a second-order unitary coupled cluster model. The implementation presented here is a modification of our previously developed algorithms for Laplace transform based local time-dependent coupled cluster linear response (CC2LR); the local approximations thus are state specific and adaptive. The symmetry of the Jacobian leads to considerable simplifications relative to the local CC2LR method; as a result, a gradient evaluation is about four times less expensive. Test calculations show that in geometry optimizations, usually very similar geometries are obtained as with the local CC2LR method (provided that a second-order method is applicable). As an exemplary application, we performed geometry optimizations on the low-lying singlet states of chlorophyllide a.

  8. Bioengineered baculoviruses as new class of therapeutics using micro and nanotechnologies: principles, prospects and challenges.

    Science.gov (United States)

    Paul, Arghya; Hasan, Anwarul; Rodes, Laetitia; Sangaralingam, Mugundhine; Prakash, Satya

    2014-05-01

    Designing a safe and efficient gene delivery system is required for success of gene therapy trials. Although a wide variety of viral, non-viral and polymeric nanoparticle based careers have been widely studied, the current gene delivery vehicles are limited by their suboptimal, non-specific therapeutic efficacy and acute immunological reactions, leading to unwanted side effects. Recently, there has been a growing interest in insect-cell-originated baculoviruses as gene delivery vehicles for diverse biomedical applications. Specifically, the emergence of diverse types of surface functionalized and bioengineered baculoviruses is posed to edge over currently available gene delivery vehicles. This is primarily because baculoviruses are comparatively non-pathogenic and non-toxic as they cannot replicate in mammalian cells and do not invoke any cytopathic effect. Moreover, emerging advanced studies in this direction have demonstrated that hybridizing the baculovirus surface with different kinds of bioactive therapeutic molecules, cell-specific targeting moieties, protective polymeric grafts and nanomaterials can significantly improve the preclinical efficacy of baculoviruses. This review presents a comprehensive overview of the recent advancements in the field of bioengineering and biotherapeutics to engineer baculovirus hybrids for tailored gene therapy, and articulates in detail the potential and challenges of these strategies for clinical realization. In addition, the article illustrates the rapid evolvement of microfluidic devices as a high throughput platform for optimizing baculovirus production and treatment conditions.

  9. Nuclear localization of platelet-activating factor receptor controls retinal neovascularization

    Science.gov (United States)

    K Bhosle, Vikrant; Rivera, José Carlos; Zhou, Tianwei (Ellen); Omri, Samy; Sanchez, Melanie; Hamel, David; Zhu, Tang; Rouget, Raphael; Rabea, Areej Al; Hou, Xin; Lahaie, Isabelle; Ribeiro-da-Silva, Alfredo; Chemtob, Sylvain

    2016-01-01

    Platelet-activating factor (PAF) is a pleiotropic phospholipid with proinflammatory, procoagulant and angiogenic actions on the vasculature. We and others have reported the presence of PAF receptor (Ptafr) at intracellular sites such as the nucleus. However, mechanisms of localization and physiologic functions of intracellular Ptafr remain poorly understood. We hereby identify the importance of C-terminal motif of the receptor and uncover novel roles of Rab11a GTPase and importin-5 in nuclear translocation of Ptafr in primary human retinal microvascular endothelial cells. Nuclear localization of Ptafr is independent of exogenous PAF stimulation as well as intracellular PAF biosynthesis. Moreover, nuclear Ptafr is responsible for the upregulation of unique set of growth factors, including vascular endothelial growth factor, in vitro and ex vivo. We further corroborate the intracrine PAF signaling, resulting in angiogenesis in vivo, using Ptafr antagonists with distinct plasma membrane permeability. Collectively, our findings show that nuclear Ptafr translocates in an agonist-independent manner, and distinctive functions of Ptafr based on its cellular localization point to another dimension needed for pharmacologic selectivity of drugs. PMID:27462464

  10. EpCAM nuclear localization identifies aggressive Thyroid Cancer and is a marker for poor prognosis

    International Nuclear Information System (INIS)

    Proteolytic cleavage of the extracellular domain (EpEx) of Epithelial cell adhesion molecule (EpCAM) and nuclear signaling by its intracellular oncogenic domain Ep-ICD has recently been implicated in increased proliferation of cancer cells. The clinical significance of Ep-ICD in human tumors remains an enigma. EpEx, Ep-ICD and β-catenin immunohistochemistry using specific antibodies was conducted on 58 archived thyroid cancer (TC) tissue blocks from 34 patients and correlated with survival analysis of these patients for up to 17 years. The anaplastic (ATC) and aggressive thyroid cancers showed loss of EpEx and increased nuclear and cytoplasmic accumulation of Ep-ICD. In contrast, the low grade papillary thyroid cancers (PTC) showed membranous EpEx and no detectable nuclear Ep-ICD. The ATC also showed concomitant nuclear expression of Ep-ICD and β-catenin. Kaplan-Meier Survival analysis revealed reduced overall survival (OS) for TC patients showing nuclear Ep-ICD expression or loss of membranous EpEx (p < 0.0004), median OS = 5 months as compared to 198 months for patients who did not show nuclear Ep-ICD or demonstrated only membranous EpE. We report reciprocal loss of membrane EpEx but increased nuclear and cytoplasmic accumulation of Ep-ICD in aggressive TC; nuclear Ep-ICD correlated with poor OS of TC patients. Thus nuclear Ep-ICD localization may serve as a useful biomarker for aggressive TC and may represent a novel diagnostic, prognostic and therapeutic target for aggressive TC

  11. Regulation of RelA Subcellular Localization by a Putative Nuclear Export Signal and p50

    OpenAIRE

    Harhaj, Edward W; Sun, Shao-Cong

    1999-01-01

    Nuclear factor κB (NF-κB) represents a family of dimeric DNA binding proteins, the pleotropic form of which is a heterodimer composed of RelA and p50 subunits. The biological activity of NF-κB is controlled through its subcellular localization. Inactive NF-κB is sequestered in the cytoplasm by physical interaction with an inhibitor, IκBα. Signal-mediated IκBα degradation triggers the release and subsequent nuclear translocation of NF-κB. It remains unknown whether the NF-κB shuttling between ...

  12. Baculovirus vector-mediated transfer of NIS gene into colon tumor cells for radionuclide therapy

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM:To investigate the feasibility of radionuclide therapy of colon tumor cells by baculovirus vector-mediated transfer of the sodium/iodide symporter(NIS) gene.METHODS:A recombinant baculovirus plasmid carrying the NIS gene was constructed,and the viruses(BacNIS) were prepared using the Bac-to-Bac system.The infection efficiency in the colon cancer cell line SW1116 of a green fluorescent protein(GFP) expressing baculovirus(Bac-GFP) at different multiplicities of infection(MOI) with various concentrations o...

  13. Automated local bright feature image analysis of nuclear proteindistribution identifies changes in tissue phenotype

    Energy Technology Data Exchange (ETDEWEB)

    Knowles, David; Sudar, Damir; Bator, Carol; Bissell, Mina

    2006-02-01

    The organization of nuclear proteins is linked to cell and tissue phenotypes. When cells arrest proliferation, undergo apoptosis, or differentiate, the distribution of nuclear proteins changes. Conversely, forced alteration of the distribution of nuclear proteins modifies cell phenotype. Immunostaining and fluorescence microscopy have been critical for such findings. However, there is an increasing need for quantitative analysis of nuclear protein distribution to decipher epigenetic relationships between nuclear structure and cell phenotype, and to unravel the mechanisms linking nuclear structure and function. We have developed imaging methods to quantify the distribution of fluorescently-stained nuclear protein NuMA in different mammary phenotypes obtained using three-dimensional cell culture. Automated image segmentation of DAPI-stained nuclei was generated to isolate thousands of nuclei from three-dimensional confocal images. Prominent features of fluorescently-stained NuMA were detected using a novel local bright feature analysis technique, and their normalized spatial density calculated as a function of the distance from the nuclear perimeter to its center. The results revealed marked changes in the distribution of the density of NuMA bright features as non-neoplastic cells underwent phenotypically normal acinar morphogenesis. In contrast, we did not detect any reorganization of NuMA during the formation of tumor nodules by malignant cells. Importantly, the analysis also discriminated proliferating non-neoplastic cells from proliferating malignant cells, suggesting that these imaging methods are capable of identifying alterations linked not only to the proliferation status but also to the malignant character of cells. We believe that this quantitative analysis will have additional applications for classifying normal and pathological tissues.

  14. 'Nuclear emergency preparedness' for local residents. Support of on-site training of many kinds of places and people

    International Nuclear Information System (INIS)

    In order to support and ensure the nuclear emergency preparedness system and safety of residents in cities, towns and villages, NPO Nuclear Emergency Preparedness Support Center was established in May, 2003. 130 on-site training and education classes were held and above 2,000 participants attended to them for two years. Objects of the countermeasure of nuclear emergency preparedness in local area and residents, what is nuclear emergency for inhabitants, what is use of Table of International Nuclear Event Scale (INES)?, a use of INES, relation between INES level and the nuclear emergency preparedness system are discussed. (S.Y.)

  15. Localized corrosion of a candidate container material for high-level nuclear waste disposal

    International Nuclear Information System (INIS)

    Localized corrosion is one of the important considerations in the design of metallic containers used for the geologic disposal of high-level nuclear waste. This paper addresses the effect of environmental factors on the localized corrosion behavior of alloy 825, one of the candidate alloys for containers in the Yucca Mountain repository site. A two-level, full factorial experimental design was used to examine the main effects and interactions of chloride, sulfate, nitrate, fluoride, and temperature. This was augmented by additional experiments involving chloride and temperature at several levels. Cyclic, potentiodynamic polarization tests were used to determine the relative susceptibility of the alloy to localized corrosion. Crevice corrosion was detected at chloride levels as low as 20 ppm, and both pitting and crevice corrosion were observed at higher chloride levels. Among the environmental factors, chloride and sulfate were found to be promoters of localized corrosion, while nitrate and fluoride were inhibitors of localized corrosion. The experiments indicated that the electrochemical parameters (e.g., pitting potential, repassivation potential, or the difference between them) were not sufficient indicators of localized corrosion. Instead, the visual observation and electrochemical parameters were combined into an index, termed localized corrosion index (LCI), to quantify the extent of localized corrosion

  16. Assessment of national systems for obtaining local acceptance of nuclear waste management siting activities

    International Nuclear Information System (INIS)

    On behalf of the United States Department of Energy (DOE), International Energy Associates Limited (IEAL) of Washington, D.C. has conducted surveys and analyses of fourteen countries' plans and approaches for dealing with the problems of obtaining local siting acceptance for nuclear waste management facilities. It was determined that the following elements of the formal systems generally facilitate and/or expedite waste management siting decisions: (1) a clear-cut pro-nuclear power position on the part of the government; (2) a willingness on the part of the central government to exert (with prudence and restraint) its pre-emptive rights in nuclear matters; (3) political structures in which the heads of regional or provincial governments are appointed by the central government; (4) national laws that link reactor licensing with a detailed plan for waste management; (5) an established and stable policy with regard to reprocessing. In contrast, it was determined that the following elements of the formal system generally hinder waste management siting activities: (1) historically strong local land used veto laws; (2) the use of national referenda for making nuclear decisions; (3) requirements for public hearings. The informal approaches fall into the following five categories: (1) political: e.g. assertion of will by political leaders, activities to enlist support of local politicians, activities to broaden involvement in decision-making; (2) economic: e.g. emphasis on normal benefits, provision for additional economic benefits; (3) siting: e.g. at or near existing nuclear facilities, on government or utility property, at multiple locations to spread the political burden; (4) timing: e.g. decoupling drilling activities from ultimate repository site decision, deliberate deferral to (long-range) future; (5) education: e.g. creation of special government programmes, enlisting of media support

  17. Final disposal of nuclear waste. Local communities and the site selection process

    International Nuclear Information System (INIS)

    The purpose of this report is to give information about how the five local Swedish communities that have been involved in the plans for selecting a site for the Swedish repository for spent nuclear fuels, have planned their work. The information is primarily directed towards representatives for the local communities that in the future might get involved in the selection process. The report tries to answer questions like: Which decision stages can be foreseen? What possibilities - formal and real - exist for leaving the selection process at a later stage? What demands will arise for inquiries and for public information? What support can governmental agencies give?

  18. State and local planning procedures dealing with social and economic impacts from nuclear power plants

    International Nuclear Information System (INIS)

    The roles of state and local agencies in planning for and managing social and economic impacts of nuclear power plants are studied. In order to be effective in these roles state and local agencies must work with each other as well as the NRC. A comparative case study approach is used which analyzes six sites in three West Coast states. The case studies included plants in operation, plants under construction, and plants still in the planning stages. In contrast to some states, all three of these states have moderately centralized procedures for siting power plants, and all have strong environmental laws

  19. Study on Fusion Protein and Its gene in Baculovirus Specificity

    International Nuclear Information System (INIS)

    Baculoviruses are subdivided into two groups depending on the type of budded virus envelop fusion protein; group I utilized gp64 which include the most of nucleopolyhedroviruses (NPVs), group II utilized F protein which include the remnants of NPVs and all Granuloviruses (GVs). Recent studies reported the viral F protein coding gene as a host cellular sourced gene and may evolutionary acquired from the host genome referring to phylogeny analysis of fusion proteins. Thus, it was deduced that F protein coding gene is species- specific nucleotide sequence related to the type of the specific host and if virus could infect an unexpected host, the resulted virus may encode a vary F gene. In this regard, the present study utilized the mentioned properties of F gene in an attempt to produce a model of specific and more economic wider range granulovirus bio- pesticide able to infect both Spodoptera littoralis and Phthorimaea operculella larvae. Multiple sequence alignment and phylogeny analysis were performed on six members of group II baculovirus, novel universal PCR primers were manually designed from the conserved regions in the alignment graph, targeted to amplify species- specific sequence entire F gene open reading frame (ORF) which is useful in molecular identification of baculovirus in unknown samples. So, the PCR product of SpliGV used to prepare a specific probe for the F gene of this type of virus. Results reflected that it is possible to infect S. littoralis larvae by PhopGV if injected into larval haemocoel, the resulted virus of this infection showed by using DNA hybridization technique to be encode to F gene homologous with the F gene of Spli GV, which is revealed that the resulted virus acquired this F gene sequence from the host genome after infection. Consequently, these results may infer that if genetic aberrations occur in the host genome, this may affect in baculoviral infectivity. So, this study aimed to investigate the effect of gamma radiation at

  20. Nuclear charge-exchange excitations in localized covariant density functional theory

    CERN Document Server

    Liang, H Z; Nakatsukasa, T; Niu, Z M; Ring, P; Roca-Maza, X; Van Giai, N; Zhao, P W

    2014-01-01

    The recent progress in the studies of nuclear charge-exchange excitations with localized covariant density functional theory is briefly presented, by taking the fine structure of spin-dipole excitations in 16O as an example. It is shown that the constraints introduced by the Fock terms of the relativistic Hartree-Fock scheme into the particle-hole residual interactions are straightforward and robust.

  1. Attitudes towards risks and opinions on a repository for spent nuclear fuel in four local communities

    International Nuclear Information System (INIS)

    A questionnaire was mailed to 1500 persons in each of the four communities that are candidates for siting, or otherwise involved, of the nuclear waste repository. 80 of the non-respondents were interviewed over telephone. The questionnaire included questions about the inhabitants attitude towards locating a repository in their home community, about participating in a local referendum, about trust in experts and organisations and about knowledge in the radioactive waste issues

  2. Nuclear charge-exchange excitations in localized covariant density functional theory

    International Nuclear Information System (INIS)

    The recent progress in the studies of nuclear charge-exchange excitations with localized covariant density functional theory is briefly presented, by taking the fine structure of spin-dipole excitations in 16O as an example. It is shown that the constraints introduced by the Fock terms of the relativistic Hartree-Fock scheme into the particle-hole residual interactions are straightforward and robust. (authors)

  3. Nuclear matter properties from local chiral interactions with $\\Delta$ isobar intermediate states

    CERN Document Server

    Logoteta, Domenico; Kievsky, Alejandro

    2016-01-01

    Using two-nucleon and three-nucleon interactions derived in the framework of chiral perturbation theory (ChPT) with and without the explicit $\\Delta$ isobar contributions, we calculate the energy per particle of symmetric nuclear matter and pure neutron matter in the framework of the microscopic Brueckner-Hartree-Fock approach. In particular, we present for the first time nuclear matter calculations using the new fully local in coordinate-space two-nucleon interaction at the next-to-next-to-next-to-leading-order (N3LO) of ChPT with $\\Delta$ isobar intermediate states (N3LO$\\Delta$) recently developed by Piarulli et al. [arXiv:1606:06335]. We find that using this N3LO$\\Delta$ potential, supplemented with a local N2LO three-nucleon interaction with explicit $\\Delta$ isobar degrees of freedom, it is possible to obtain a satisfactory saturation point of symmetric nuclear matter. For this combination of two- and three-nucleon interactions we also calculate the nuclear symmetry energy and we compare our results wit...

  4. Transcription-dependent nuclear localization of DAZAP1 requires an N-terminal signal

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Yi-Tzu; Wen, Wan-Ching [Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan (China); Yen, Pauline H., E-mail: pyen@ibms.sinica.edu.tw [Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan (China)

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer DAZAP1 shuttles between the nucleus and the cytoplasm. Black-Right-Pointing-Pointer DAZAP1 accumulates in the cytoplasm when the nuclear transcription is inhibited. Black-Right-Pointing-Pointer DAZAP1's transcription-dependent nuclear localization requires N-terminal N42. Black-Right-Pointing-Pointer SLIRP binds to N42 and may be involved in the process. -- Abstract: Deleted in Azoospermia Associated Protein 1 (DAZAP1) is a ubiquitous hnRNP protein required for normal development and spermatogenesis. It resides predominantly in the nucleus and moves between the nucleus and the cytoplasm via a ZNS shuttling signal at its C-terminus. DAZAP1 accumulates in the cytoplasm when RNA polymerase II activity is inhibited by actinomycin D. Here we report the mapping of a 42-amino acid segment (N42) at the N-terminus of DAZAP1 that is both necessary and sufficient for its transcription-dependent nuclear localization. In addition, using a yeast two-hybrid system, we have identified SLIRP as a N42-binding protein which may regulate DAZAP1 subcellular localization.

  5. TRX-1 Regulates SKN-1 Nuclear Localization Cell Non-autonomously in Caenorhabditis elegans.

    Science.gov (United States)

    McCallum, Katie C; Liu, Bin; Fierro-González, Juan Carlos; Swoboda, Peter; Arur, Swathi; Miranda-Vizuete, Antonio; Garsin, Danielle A

    2016-05-01

    The Caenorhabditis elegans oxidative stress response transcription factor, SKN-1, is essential for the maintenance of redox homeostasis and is a functional ortholog of the Nrf family of transcription factors. The numerous levels of regulation that govern these transcription factors underscore their importance. Here, we add a thioredoxin, encoded by trx-1, to the expansive list of SKN-1 regulators. We report that loss of trx-1 promotes nuclear localization of intestinal SKN-1 in a redox-independent, cell non-autonomous fashion from the ASJ neurons. Furthermore, this regulation is not general to the thioredoxin family, as two other C. elegans thioredoxins, TRX-2 and TRX-3, do not play a role in this process. Moreover, TRX-1-dependent regulation requires signaling from the p38 MAPK-signaling pathway. However, while TRX-1 regulates SKN-1 nuclear localization, classical SKN-1 transcriptional activity associated with stress response remains largely unaffected. Interestingly, RNA-Seq analysis revealed that loss of trx-1 elicits a general, organism-wide down-regulation of several classes of genes; those encoding for collagens and lipid transport being most prevalent. Together, these results uncover a novel role for a thioredoxin in regulating intestinal SKN-1 nuclear localization in a cell non-autonomous manner, thereby contributing to the understanding of the processes involved in maintaining redox homeostasis throughout an organism. PMID:26920757

  6. Local fracture properties and dissimilar weld integrity in nuclear power plants

    Science.gov (United States)

    Wang, Guozhen; Wang, Haitao; Xuan, Fuzhen; Tu, Shantung; Liu, Changjun

    2013-09-01

    In this paper, the local fracture properties in a Alloy52M dissimilar metal welded joint (DMWJ) between A508 ferritic steel and 316 L stainless steel in nuclear power plants were investigated by using the single-edge notched bend (SENB) specimens, and their use in integrity assessment of DMWJ structures was analyzed. The results show that the local fracture resistance in the DMWJ is determined by local fracture mechanism, and which is mainly related to the microstructures and local strength mismatches of materials at the crack locations. The initial cracks always grow towards the materials with lower strength, and the crack path deviation is mainly controlled by the local strength mismatch. If the local fracture properties could not be used for cracks in the heat affected zones (HAZs), interface and near interface zones, the use of the fracture properties ( J-resistance curves) of base metals or weld metals following present codes will unavoidably produce non-conservative (unsafe) or excessive conservative assessment results. In most cases, the assessment results will be potentially unsafe. Therefore, it is recommended to obtain and use local mechanical and fracture properties in the integrity assessment of DMWJs.

  7. The Drosophila nucleoporin DNup88 localizes DNup214 and CRM1 on the nuclear envelope and attenuates NES-mediated nuclear export

    OpenAIRE

    Roth, Peggy; Xylourgidis, Nikos; Sabri, Nafiseh; Uv, Anne; Fornerod, Maarten; Samakovlis, Christos

    2003-01-01

    Many cellular responses rely on the control of nucleocytoplasmic transport of transcriptional regulators. The Drosophila nucleoporin Nup88 is selectively required for nuclear accumulation of Rel proteins and full activation of the innate immune response. Here, we investigate the mechanisms underlying its role in nucleocytoplasmic transport. Nuclear import of an nuclear localization signal-enhanced green fluorescent protein (NLS-EGFP) reporter is not affected in DNup88 (members only; mbo) muta...

  8. Processing of Baculovirus Late and Very Late mRNAs

    Institute of Scientific and Technical Information of China (English)

    Linda A. Guarino

    2007-01-01

    Baculoviruses encode a DNA-directed RNA polymerase that is evolutionarily divergent from cellular polymerases. This RNA polymerase is a multifunctional complex that has the ability to recognize late promoters, transcribe linked genes, and process transcripts at both the 5' and 3' ends. The LEF-4 subunit of the viral RNA polymerase is the mRNA capping enzyme, with both RNA triphosphatase and guanylyltransferase activities. Conversion to cap 1 structures is mediated by the viral enzyme MTase1 and another as yet unidentified methyltransferase. Termination is an intrinsic property of the viral RNA polymerase and occurs at oligoU rich sequences. Polyadenylation of the released transcripts is also a function of the viral RNA polymerase. Thus, although viral mRNAs resemble host messages with respect to their 5' and 3' end structures, the processing is mediated by viral enzymes and, in the case of the 3' ends, by mechanisms that differ from the host cell.

  9. Baculovirus: Molecular Insights on Their Diversity and Conservation

    Directory of Open Access Journals (Sweden)

    Solange Ana Belen Miele

    2011-01-01

    Full Text Available The Baculoviridae is a large group of insect viruses containing circular double-stranded DNA genomes of 80 to 180 kbp. In this study, genome sequences from 57 baculoviruses were analyzed to reevaluate the number and identity of core genes and to understand the distribution of the remaining coding sequences. Thirty one core genes with orthologs in all genomes were identified along with other 895 genes differing in their degrees of representation among reported genomes. Many of these latter genes are common to well-defined lineages, whereas others are unique to one or a few of the viruses. Phylogenetic analyses based on core gene sequences and the gene composition of the genomes supported the current division of the Baculoviridae into 4 genera: Alphabaculovirus, Betabaculovirus, Gammabaculovirus, and Deltabaculovirus.

  10. P220-S Dual-Purpose Insect Cell Expression Vector for Transient Transfection and Baculovirus Generation

    OpenAIRE

    Loomis, K.; ROCKWELL, C; Sternard, H.; Novy, R.

    2007-01-01

    Baculovirus-mediated expression has proven to be a robust method of generating recombinant proteins from insect cells. However, generating baculovirus recombinants using traditional techniques is time consuming and tedious. To accelerate the process of insect cell expression, EMD developed a rapid transient transfection-based approach, the InsectDirect System. This approach is well suited for the rapid generation of small to moderate amounts of recombinant protein. For situations that demand ...

  11. Optimization of canine interleukin-12 production using a baculovirus insect cell expression system

    OpenAIRE

    de Pinheiro, Cristiane Garboggini Melo; Pedrosa, Mayara de Oliveira; Teixeira, Naiara Carvalho; Ano Bom, Ana Paula Dinis; van Oers, Monique M.; Oliveira, Geraldo Gileno de Sá

    2016-01-01

    Background Interleukin-12 is an important cytokine in mediating cellular immune responses. Results Recombinant single-chain canine IL-12 was produced in a baculovirus-insect cell system with the aim of conducting further studies on modulation of immune responses in dogs. To optimize the production of recombinant canine IL-12, a classical baculovirus and a modified vector (chitinase A and v-cathepsin knockout) were used containing a native or an optimized insert of canine IL-12. The optimized ...

  12. Stable replication of the EBNA1/OriP-mediated baculovirus vector and its application to anti-HCV gene therapy

    Directory of Open Access Journals (Sweden)

    Chang Myint OO

    2009-10-01

    Full Text Available Abstract Background Hepatitis C virus (HCV is one of the main causes of liver-related morbidity and mortality. Although combined interferon-α-ribavirin therapy is effective for about 50% of the patients with HCV, better therapies are needed and preventative vaccines have yet to be developed. Short-hairpin RNAs (shRNAs inhibit gene expression by RNA interference. The application of transient shRNA expression is limited, however, due to the inability of the shRNA to replicate in mammalian cells and its inefficient transduction. The duration of transgene (shRNA expression in mammalian cells can be significantly extended using baculovirus-based shRNA-expressing vectors that contain the latent viral protein Epstein-Barr nuclear antigen 1 (EBNA1 and the origin of latent viral DNA replication (OriP sequences. These recombinant vectors contain compatible promoters and are highly effective for infecting primary hepatocyte and hepatoma cell lines, making them very useful tools for studies of hepatitis B and hepatitis C viruses. Here, we report the use of these baculovirus-based vector-derived shRNAs to inhibit core-protein expression in full-length hepatitis C virus (HCV replicon cells. Results We constructed a long-term transgene shRNA expression vector that contains the EBV EBNA1 and OriP sequences. We also designed baculovirus vector-mediated shRNAs against the highly conserved core-protein region of HCV. HCV core protein expression was inhibited by the EBNA1/OriP baculovirus vector for at least 14 days, which was considerably longer than the 3 days of inhibition produced by the wild-type baculovirus vector. Conclusion These findings indicate that we successfully constructed a long-term transgene (shRNA expression vector (Ac-EP-shRNA452 using the EBNA1/OriP system, which was propagated in Escherichia coli and converted into mammalian cells. The potential anti-HCV activity of the long-term transgene (shRNA expression vector was evaluated with the view

  13. Construction of an insecticidal baculovirus expressing insect-specific neurotoxin AaIT

    Institute of Scientific and Technical Information of China (English)

    姚斌; 庞义; 范云六; 赵荣敏; 杨应昌; 王天原

    1996-01-01

    Considering the factors which affect gene transcription, translation and the stability of mRNA, without changing the amino acid composition of the encoded polypeptide, AaIT gene encoding insect-specific neurotoxin was designed and synthesized according to bias in codon choice, overall G+C content and G + C content of bases at the third position in codons of polyhedrin genes of baculovirus and of plant genes as well. AaIT gene was fused behind a synthetic gp67 signal sequence and then recombined into the genome of Trichoplusia ni nuclear polyhedrosis virus (TnNPV) by transfer vector pSXIV VI+X3. The recombinant virus TnNPV-AalT (occ+-gal-) was screened. The results of Southern blotting and SDS-PAGE demonstrated that AaIT gene had integrated into the genome of virus and expressed. Bioassays on the 3rd-instar Trichoplusia ni larvae showed that recombinant viruses TnNPV-AalT could shorten the time of killing insect and improve the efficiency of killing agronomically important insects.

  14. RNA 5'-triphosphatase, nucleoside triphosphatase, and guanylyltransferase activities of baculovirus LEF-4 protein.

    Science.gov (United States)

    Gross, C H; Shuman, S

    1998-12-01

    Autographa californica nuclear polyhedrosis virus late and very late mRNAs are transcribed by an RNA polymerase consisting of four virus-encoded polypeptides: LEF-8, LEF-9, LEF-4, and p47. The 464-amino-acid LEF-4 subunit contains the signature motifs of GTP:RNA guanylyltransferases (capping enzymes). Here, we show that the purified recombinant LEF-4 protein catalyzes two reactions involved in RNA cap formation. LEF-4 is an RNA 5'-triphosphatase that hydrolyzes the gamma phosphate of triphosphate-terminated RNA and a guanylyltransferase that reacts with GTP to form a covalent protein-guanylate adduct. The RNA triphosphatase activity depends absolutely on a divalent cation; the cofactor requirement is satisfied by either magnesium or manganese. LEF-4 also hydrolyzes ATP to ADP and Pi (Km = 43 microM ATP; Vmax = 30 s-1) and GTP to GDP and Pi. The LEF-4 nucleoside triphosphatase (NTPase) is activated by manganese or cobalt but not by magnesium. The RNA triphosphatase and NTPase activities of baculovirus LEF-4 resemble those of the vaccinia virus and Saccharomyces cerevisiae mRNA capping enzymes. We suggest that these proteins comprise a novel family of metal-dependent triphosphatases. PMID:9811740

  15. Localization of latency-associated nuclear antigen (LANA) on mitotic chromosomes.

    Science.gov (United States)

    Rahayu, Retno; Ohsaki, Eriko; Omori, Hiroko; Ueda, Keiji

    2016-09-01

    In latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), viral gene expression is extremely limited and copy numbers of viral genomes remain constant. Latency-associated nuclear antigen (LANA) is known to have a role in maintaining viral genome copy numbers in growing cells. Several studies have shown that LANA is localized in particular regions on mitotic chromosomes, such as centromeres/pericentromeres. We independently examined the distinct localization of LANA on mitotic chromosomes during mitosis, using super-resolution laser confocal microscopy and correlative fluorescence microscopy-electron microscopy (FM-EM) analyses. We found that the majority of LANA were not localized at particular regions such as telomeres/peritelomeres, centromeres/pericentromeres, and cohesion sites, but at the bodies of condensed chromosomes. Thus, LANA may undergo various interactions with the host factors on the condensed chromosomes in order to tether the viral genome to mitotic chromosomes and realize faithful viral genome segregation during cell division. PMID:27254595

  16. The BRCA1-binding protein BRAP2 can act as a cytoplasmic retention factor for nuclear and nuclear envelope-localizing testicular proteins.

    Science.gov (United States)

    Davies, Rebecca G; Wagstaff, Kylie M; McLaughlin, Eileen A; Loveland, Kate L; Jans, David A

    2013-12-01

    Regulation of nuclear protein import is central to many cellular processes such as development, with a key mechanism being factors that retain cargoes in the cytoplasm that normally localize in the nucleus. The breast cancer antigen BRCA1-binding protein BRAP2 has been reported as a novel negative regulator of nuclear import of various nuclear localization signal (NLS)-containing viral and cellular proteins, but although implicated in differentiation pathways and highly expressed in tissues including testis, the gamut of targets for BRAP2 action in a developmental context is unknown. As a first step towards defining the BRAP2 interactome, we performed a yeast-2-hybrid screen to identify binding partners of BRAP2 in human testis. Here we report characterization for the first time of three of these: the high mobility group (HMG)-box-domain-containing chromatin component HMG20A, nuclear mitotic apparatus protein NuMA1 and synaptic nuclear envelope protein SYNE2. Co-immunoprecipitation experiments indicate association of BRAP2 with HMG20A, NuMA1, and SYNE2 in testis, underlining the physiological relevance of the interactions, with immunohistochemistry showing that where BRAP2 is co-expressed with HMG20A and NuMA1, both are present in the cytoplasm, in contrast to their nuclear localization in other testicular cell types. Importantly, quantitative confocal microscopic analysis of cultured cells indicates that ectopic expression of BRAP2 inhibits nuclear localization of HMG20A and NuMA1, and prevents nuclear envelope accumulation of SYNE2, the first report of BRAP2 altering localization of a non-nuclear protein. These results imply for the first time that BRAP2 may have an important role in modulating subcellular localization during testicular development. PMID:23707952

  17. IQGAP1 functions as a modulator of dishevelled nuclear localization in Wnt signaling.

    Directory of Open Access Journals (Sweden)

    Toshiyasu Goto

    Full Text Available Dishevelled (DVL is a central factor in the Wnt signaling pathway, which is highly conserved among various organisms. DVL plays important roles in transcriptional activation in the nucleus, but the molecular mechanisms underlying their nuclear localization remain unclear. In the present study, we identified IQGAP1 as a regulator of DVL function. In Xenopus embryos, depletion of IQGAP1 reduced Wnt-induced nuclear accumulation of DVL, and expression of Wnt target genes during early embryogenesis. The domains in DVL and IQGAP1 that mediated their interaction are also required for their nuclear localization. Endogenous expression of Wnt target genes was reduced by depletion of IQGAP1 during early embryogenesis, but notably not by depletion of other IQGAP family genes. Moreover, expression of Wnt target genes caused by depletion of endogenous IQGAP1 could be rescued by expression of wild-type IQGAP1, but not IQGAP1 deleting DVL binding region. These results provide the first evidence that IQGAP1 functions as a modulator in the canonical Wnt signaling pathway.

  18. Identification and characterization of multiple conserved nuclear localization signals within adenovirus E1A

    Energy Technology Data Exchange (ETDEWEB)

    Marshall, Kris S.; Cohen, Michael J.; Fonseca, Greg J.; Todorovic, Biljana; King, Cason R. [Department of Microbiology and Immunology, Western University, London Regional Cancer Program, London, ON, Canada N6A 4L6 (Canada); Yousef, Ahmed F. [Department of Chemical and Environmental Engineering, Masdar Institute, Abu Dhabi (United Arab Emirates); Zhang, Zhiying [College of Animal Science and Technologies, Northwest A and F University, Yangling, Shaanxi 712100 (China); Mymryk, Joe S., E-mail: jmymryk@uwo.ca [Department of Microbiology and Immunology, Western University, London Regional Cancer Program, London, ON, Canada N6A 4L6 (Canada); Department of Oncology, Western University, London Regional Cancer Program, London, ON, Canada N6A 4L6 (Canada)

    2014-04-15

    The human adenovirus 5 (HAdV-5) E1A protein has a well defined canonical nuclear localization signal (NLS) located at its C-terminus. We used a genetic assay in the yeast Saccharomyces cerevisiae to demonstrate that the canonical NLS is present and functional in the E1A proteins of each of the six HAdV species. This assay also detects a previously described non-canonical NLS within conserved region 3 and a novel active NLS within the N-terminal/conserved region 1 portion of HAdV-5 E1A. These activities were also present in the E1A proteins of each of the other five HAdV species. These results demonstrate that, despite substantial differences in primary sequence, HAdV E1A proteins are remarkably consistent in that they contain one canonical and two non-canonical NLSs. By utilizing independent mechanisms, these multiple NLSs ensure nuclear localization of E1A in the infected cell. - Highlights: • HAdV E1A uses multiple mechanisms for nuclear import. • We identified an additional non-canonical NLS in the N-terminal/CR1 portion of E1A. • The new NLS does not contact importin-alpha directly. • All NLSs are functionally conserved in the E1A proteins of all 6 HAdV species.

  19. Construction of recombinant baculoviruses expressing hemagglutinin of H5N1 avian influenza and research on the immunogenicity

    Science.gov (United States)

    Ge, Jingping; An, Qi; Gao, Dongni; Liu, Ying; Ping, Wenxiang

    2016-01-01

    Recombinant baculoviruses with different promoter and regulatory elements were constructed to enhance the expression of target protein and boost the efficacies of avian influenza vaccine. Hemagglutinin gene was cloned into the baculovirus transfer vectors driven by cytomegaloviru (CMV) and White spot syndrome virus immediate-early promoter one (WSSV ie1) promoter respectively, with different regulatory elements. The recombinant baculoviruses were directly used as vaccines to immunize specific pathogen-free chickens. The protein expression levels of recombinant baculoviruses BV-S-HA and BV-S-ITRs-HA were respectively 2.43 and 2.67 times than that of BV-S-con-HA, while the protein expression levels of BV-A-HA and BV-A-ITRs-HA were respectively 2.44 and 2.69 times than that of BV-S-con-HA. Immunoglobulin G (IgG) antibody levels induced by BV-A and BV-S series recombinant baculovirus were significantly higher than the commercialized vaccine group (P < 0.05). Among the groups with same promoter, the IgG antibody levels induced by the baculovirus containing regulatory elements were significantly higher than control group. Additionally, the immune effects induced by BV-A series recombinant baculoviruses with WSSV ie1 promoter were significantly stronger than the BV-S series recombinant baculoviruses with CMV promoter. The avian influenza vaccine prepared based on baculovirus vector can simultaneously stimulate the humoral and cellular immune responses. PMID:27063566

  20. Nuclear Enhanced X-ray Maximum Entropy Method Used to Analyze Local Distortions in Simple Structures

    DEFF Research Database (Denmark)

    Christensen, Sebastian; Bindzus, Niels; Christensen, Mogens;

    We introduce a novel method for reconstructing pseudo nuclear density distributions (NDDs): Nuclear Enhanced X-ray Maximum Entropy Method (NEXMEM). NEXMEM offers an alternative route to experimental NDDs, exploiting the superior quality of synchrotron X-ray data compared to neutron data. The method...... powder diffraction data of PbTe with known displacements of Pb. The increased resolution of NEXMEM proved essential for resolving Pb-displacement of 0.2 Å in simulated data. The figure below shows Pb in the (100) plane for calculated MEM and NEXMEM densities, as well as the reference electron density....... In addition, we have applied NEXMEM to multi-temperature synchrotron powder X-ray diffraction collected on PbX. Based on powder diffraction data, our study demonstrates that NEXMEM successfully improves the atomic resolution over standard MEM. This new tool aids our understanding of the local distortions...

  1. Intense deuterium nuclear fusion of pycnodeuterium-lumps coagulated locally within highly deuterated atom clusters

    CERN Document Server

    Yoshiaki, A; Zhang, Y C

    2002-01-01

    Embedded nano-Pd particles of 5 nm in size instantly abundant D-atoms more than 250% in the atomic ratio against Pd-atoms at room temperature when they are kept in D sub 2 gas pressurized to less than 10 atm. In such ultrahigh densities, 2-4 D-atoms can be coagulated inside each octahedral space of Pd lattice (pycnodeuterium-lump). When a stimulation energy such as latticequake causing by ultrasonic wave was supplied to those highly deuterated Pd particles, intense deuterium nuclear fusion (''solid fusion'') was generated there and both excess heat and sup 4 He gas were abundantly produced. Naturally, these facts can not be realized at all in bulk Pd. The results show that the nuclear fusion occurs without any hazardous rays in pycnodeuterium-lumps coagulated locally inside the each cell of the host metal lattice. These unit cells correspond to minimum unit of the solid fusion reactor as a ''Lattice Reactor''. (author)

  2. Ras-activated RSK1 phosphorylates EBP50 to regulate its nuclear localization and promote cell proliferation.

    Science.gov (United States)

    Lim, Hooi Cheng; Jou, Tzuu-Shuh

    2016-03-01

    Differential subcellular localization of EBP50 leads to its controversial role in cancer biology either as a tumor suppressor when it resides at the membrane periphery, or a tumor facilitator at the nucleus. However, the mechanism behind nuclear localization of EBP50 remains unclear. A RNA interference screening identified the downstream effector of the Ras-ERK cascade, RSK1, as the molecule unique for nuclear transport of EBP50. RSK1 binds to EBP50 and phosphorylates it at a conserved threonine residue at position 156 (T156) under the regulation of growth factor. Mutagenesis experiments confirmed the significance of T156 residue in nuclear localization of EBP50, cellular proliferation, and oncogenic transformation. Our study sheds light on a possible therapeutic strategy targeting at this aberrant nuclear expression of EBP50 without affecting the normal physiological function of EBP50 at other subcellular localization.

  3. Characterization of Local Wind Patterns around the Kori Nuclear Power Plant using Cluster Analysis and WRF meteorological modeling

    International Nuclear Information System (INIS)

    To accurately predict the atmospheric diffusion of radioactive effluent, detailed analysis of local wind patterns nearby nuclear power plants are necessary. In this study, the characteristics of typical local winds around the Kori Nuclear Power Plant (Kori NPP) were investigated using the cluster analysis and Weather Research and Forecasting (WRF) meteorological modeling. In this study, the local wind characteristics around the Kori NPP were analyzed using cluster analysis and WRF meteorological modeling. As a result of the cluster analysis, four wind patterns around the Kori NPP were selected. The model study indicated the possibility that the local winds in the target area can largely contribute to the atmospheric diffusion of radioactive effluents

  4. PAT1 (SLC36A1) shows nuclear localization and affect growth of smooth muscle cells from rats

    DEFF Research Database (Denmark)

    Jensen, Anne; Figueiredo-Larsen, Evan Manuel; Holm, René;

    2014-01-01

    of A7r5. These results were confirmed in primary SMCs derived from rat aorta and colon. A 3'-untranslated region of the PAT1 transcript directed the nuclear localization. Neither cellular starvation nor cell division altered the nuclear localization. In agreement, uptake studies of L-proline, a PAT1...... to induced cellular growth suggesting a role for PAT1 in regulating cellular proliferation of SMCs....

  5. Distinct effects of nuclear membrane localization on gene transcription silencing in Drosophila S2 cells and germ cells

    Institute of Scientific and Technical Information of China (English)

    Lu Sui; Yanhong Yang

    2011-01-01

    Nuclear envelope proteins have important roles in chromatin organization and signal-dependent transcriptional regulation. A previous study reported that the inner nuclear membrane protein, Otefin (Ote), was essential for germline stem cell (GSC) maintenance via interaction with Smad complex. The interaction of Otc with the Smad complex recruits the bam locus to the nuclear periphery and subsequently results in bam transcriptional silencing, revealing that nuclear peripheral localization is essential for bam gene regulation. However, it remains unknown whether the nuclear peripheral localization is sufficient for bam silencing. To address this issue, we have established a tethering system, in which the Gal4 DNA binding domain (DBD) of the Flag:Gal4 DBD:Ote △ LEM fusion protein physically interacts with the Gal4 binding sites upstream of bamP-gfp to artificially recruit the reporter gene gfp to the nuclear membrane. Our data demonstrated that the nuclear peripheral localization seemed to affect the expression of the target naked gene in S2 cells. By contrast, in Drosophila germ cells, the nuclear membrane localization was not sufficient for gene silencing.

  6. Review study 1995. Localization of the repository for spent nuclear fuel

    International Nuclear Information System (INIS)

    This report gives an overview of the studies performed by SKB pertinent to selection of a site for the Swedish repository for spent nuclear fuels, and is written for both experts in the various fields involved, decision-makers and the interested general public. The review can not comprise all detailed factors necessary for deciding the localization, but deals mainly with conditions on the land surface and can point out areas which are not well suited or less interesting as a site. It also treats several scientific, technical and social bases in different parts of the country. 120 refs, 53 figs

  7. Isotropic proton-detected local-field nuclear magnetic resonancein solids

    Energy Technology Data Exchange (ETDEWEB)

    Havlin, Robert H.; Walls, Jamie D.; Pines, Alexander

    2004-08-04

    A new nuclear magnetic resonance (NMR) method is presented which produces linear, isotropic proton-detected local-field spectra for InS spin systems in powdered samples. The method, HETeronuclear Isotropic Evolution (HETIE), refocuses the anisotropic portion of the heteronuclear dipolar coupling frequencies by evolving the system under a series of specially designed Hamiltonians and evolution pathways. The theory behind HETIE is represented along with experimental studies conducted on a powdered sample of ferrocene, demonstrating the methodology outlined in this paper. Applications of HETIE for structural determination in solid-state NMR are discussed.

  8. Essential functions of Sds22p in chromosome stability and nuclear localization of PP1.

    Science.gov (United States)

    Peggie, Mark W; MacKelvie, Sarah H; Bloecher, Andrew; Knatko, Elena V; Tatchell, Kelly; Stark, Michael J R

    2002-01-01

    Sds22p is a conserved, leucine-rich repeat protein that interacts with the catalytic subunit of protein phosphatase 1 (PP1(C)) and which has been proposed to regulate one or more functions of PP1(C) during mitosis. Here we show that Saccharomyces cerevisiae Sds22p is a largely nuclear protein, most of which is present as a sTable 1:1 complex with yeast PP1(C) (Glc7p). Temperature-sensitive (Ts(-)) S. cerevisiae sds22 mutants show profound chromosome instability at elevated growth temperatures but do not confer a cell cycle stage-specific arrest. In the sds22-6 Ts(-) mutant, nuclear Glc7p is both reduced in level and aberrantly localized at 37 degrees C and the interaction between Glc7p and Sds22p in vitro is reduced at higher temperatures, consistent with the in vivo Ts(-) growth defect. Like some glc7 mutations, sds22-6 can suppress the Ts(-) growth defect associated with ipl1-2, a loss of function mutation in a protein kinase that is known to work in opposition to PP1 on at least two nuclear substrates. This, together with reciprocal genetic interactions between GLC7 and SDS22, suggests that Sds22p functions positively with Glc7p to promote dephosphorylation of nuclear substrates required for faithful transmission of chromosomes during mitosis, and this role is at least partly mediated by effects of Sds22p on the nuclear distribution of Glc7p

  9. Nuclear import of influenza B virus nucleoprotein: Involvement of an N-terminal nuclear localization signal and a cleavage-protection motif

    Energy Technology Data Exchange (ETDEWEB)

    Wanitchang, Asawin; Narkpuk, Jaraspim; Jongkaewwattana, Anan, E-mail: anan.jon@biotec.or.th

    2013-08-15

    The nucleoprotein of influenza B virus (BNP) shares several characteristics with its influenza A virus counterpart (ANP), including localization in the host's nucleus. However, while the nuclear localization signal(s) (NLS) of ANP are well characterized, little is known about those of BNP. In this study, we showed that the fusion protein bearing the BNP N-terminus fused with GFP (N70–GFP) is exclusively nuclear, and identified a highly conserved KRXR motif spanning residues 44–47 as a putative NLS. In addition, we demonstrated that residues 3–15 of BNP, though not an NLS, are also crucial for nuclear import. Results from mutational analyses of N70–GFP and the full-length BNP suggest that this region may be required for protection of the N-terminus from proteolytic cleavage. Altogether, we propose that the N-terminal region of BNP contains the NLS and cleavage-protection motif, which together drive its nuclear localization. - Highlights: • The N-terminal region of BNP is required for nuclear accumulation. • The conserved motif at position 44–47 is a putative nuclear localization signal. • The first 15 amino acids of BNP may function as a cleavage-protection motif. • BNP may get access to the nucleus via a mechanism distinct from ANP.

  10. Nuclear import of influenza B virus nucleoprotein: Involvement of an N-terminal nuclear localization signal and a cleavage-protection motif

    International Nuclear Information System (INIS)

    The nucleoprotein of influenza B virus (BNP) shares several characteristics with its influenza A virus counterpart (ANP), including localization in the host's nucleus. However, while the nuclear localization signal(s) (NLS) of ANP are well characterized, little is known about those of BNP. In this study, we showed that the fusion protein bearing the BNP N-terminus fused with GFP (N70–GFP) is exclusively nuclear, and identified a highly conserved KRXR motif spanning residues 44–47 as a putative NLS. In addition, we demonstrated that residues 3–15 of BNP, though not an NLS, are also crucial for nuclear import. Results from mutational analyses of N70–GFP and the full-length BNP suggest that this region may be required for protection of the N-terminus from proteolytic cleavage. Altogether, we propose that the N-terminal region of BNP contains the NLS and cleavage-protection motif, which together drive its nuclear localization. - Highlights: • The N-terminal region of BNP is required for nuclear accumulation. • The conserved motif at position 44–47 is a putative nuclear localization signal. • The first 15 amino acids of BNP may function as a cleavage-protection motif. • BNP may get access to the nucleus via a mechanism distinct from ANP

  11. The effect of intermittent operation on local fission rate in the McMaster Nuclear Reactor

    International Nuclear Information System (INIS)

    The McMaster Nuclear Reactor operates on a 16-hr/day/5-day/week schedule causing cyclic loading of Xenon in the core and requiring compensation by the control systems to maintain operations. The constant control rod interaction affects local fission rates. This paper confirms the relationship between Xenon load and control rod movement and studies the relationship between local fission rate and control rod insertion. The results provide information related to analysis approximations used in depletion calculations. In addition, comparisons are made between the current MNR operation cycle and a proposed continuous operational approach. The results are further discussed in the context of proposed Molybdenum-99 production at MNR. (author)

  12. Localization of plastic deformation in copper canisters for spent nuclear fuel

    Energy Technology Data Exchange (ETDEWEB)

    Savolainen, K.; Saukkonen, T.; Haenninen, H. (Aalto Univ. School of Science and Technology, Espoo (Finland))

    2010-05-15

    In Finland and Sweden the spent nuclear fuel will be deposited in a deep repository in copper corrosion barrier canisters surrounding cast iron inserts. The 50 mm thick copper canisters will we sealed using either electron beam welding (EB) or friction stir welding (FSW) to join the tubes and the lids/bottoms. The canisters will deform in the repository conditions, e.g., due to the hydrostatic pressure. The deformation will localize to different discontinuities, such as defects and microstructural heterogeneity. This study compared the localization of plastic deformation in EB and FSW welds as well as in the base materials (both forged and extruded) using optical strain measurement methods. The results show that in the base materials the deformation occurs very uniformly over the entire gauge length. In FSW welds the deformation localizes in the weld at a line of entrapped oxide particles. In EB welds the deformation localizes to the large grains in the middle of the weld or at the steep grain size gradient between the weld and the tube material. Tensile strength is lowest in the EB welds (175 MPa as compared to 200 MPa or higher for the other samples). Elongation to fracture of the FSW welds is similar to those of the base materials, but for the EB welds it is significantly lower (appr. 40 % compared to 65 %). (orig.)

  13. Application of the counterfactual method to assess of the local economic impact of a nuclear power station

    International Nuclear Information System (INIS)

    Several studies have examined the local economic effects of nuclear power stations. However, their heterogeneity within countries and between countries indicate that one must be very cautious in drawing general conclusions about the positive and negative local economic effects of nuclear power stations over time. Using information about a specific nuclear facility in Spain and its zone of influence, we investigate the local economic impact of a nuclear facility by applying an infrequently used methodology called the “counterfactual” method. The purpose of this application of the counterfactual method is to establish what would have happened if the nuclear power plant had not been built where it was. This method permits observation of the impact of the nuclear power plant on the evolution of a set of variables whose influence is extremely important for the local area, such as the population, unemployment level, per capita income, and municipal governments’ revenues. Generalization of this method could offer the possibility of providing comparative results. However, the method must be complemented with other short-term approaches to provide a more specific analysis of the economic effects on the local actors involved and to offer incentives to policymakers to design and develop policies aimed at boosting economic activity in the area. - Highlights: • The Trillo Nuclear Power Plant plays a key role in stimulating its local environment. • The counterfactual method is estimated based on the performance of control group. • At least at the beginning of its operation the population dynamism was clear. • There is not strong evidence about the rise of employment and local public revenues. • The expenditure flow assesses the local turnover, jobs and municipal revenues

  14. Nuclear Fragile X Mental Retardation Protein is localized to Cajal bodies.

    Directory of Open Access Journals (Sweden)

    Alain Y Dury

    2013-10-01

    Full Text Available Fragile X syndrome is caused by loss of function of a single gene encoding the Fragile X Mental Retardation Protein (FMRP. This RNA-binding protein, widely expressed in mammalian tissues, is particularly abundant in neurons and is a component of messenger ribonucleoprotein (mRNP complexes present within the translational apparatus. The absence of FMRP in neurons is believed to cause translation dysregulation and defects in mRNA transport essential for local protein synthesis and for synaptic development and maturation. A prevalent model posits that FMRP is a nucleocytoplasmic shuttling protein that transports its mRNA targets from the nucleus to the translation machinery. However, it is not known which of the multiple FMRP isoforms, resulting from the numerous alternatively spliced FMR1 transcripts variants, would be involved in such a process. Using a new generation of anti-FMRP antibodies and recombinant expression, we show here that the most commonly expressed human FMRP isoforms (ISO1 and 7 do not localize to the nucleus. Instead, specific FMRP isoforms 6 and 12 (ISO6 and 12, containing a novel C-terminal domain, were the only isoforms that localized to the nuclei in cultured human cells. These isoforms localized to specific p80-coilin and SMN positive structures that were identified as Cajal bodies. The Cajal body localization signal was confined to a 17 amino acid stretch in the C-terminus of human ISO6 and is lacking in a mouse Iso6 variant. As FMRP is an RNA-binding protein, its presence in Cajal bodies suggests additional functions in nuclear post-transcriptional RNA metabolism. Supporting this hypothesis, a missense mutation (I304N, known to alter the KH2-mediated RNA binding properties of FMRP, abolishes the localization of human FMRP ISO6 to Cajal bodies. These findings open unexplored avenues in search for new insights into the pathophysiology of Fragile X Syndrome.

  15. Protein Sub-Nuclear Localization Based on Effective Fusion Representations and Dimension Reduction Algorithm LDA.

    Science.gov (United States)

    Wang, Shunfang; Liu, Shuhui

    2015-12-19

    An effective representation of a protein sequence plays a crucial role in protein sub-nuclear localization. The existing representations, such as dipeptide composition (DipC), pseudo-amino acid composition (PseAAC) and position specific scoring matrix (PSSM), are insufficient to represent protein sequence due to their single perspectives. Thus, this paper proposes two fusion feature representations of DipPSSM and PseAAPSSM to integrate PSSM with DipC and PseAAC, respectively. When constructing each fusion representation, we introduce the balance factors to value the importance of its components. The optimal values of the balance factors are sought by genetic algorithm. Due to the high dimensionality of the proposed representations, linear discriminant analysis (LDA) is used to find its important low dimensional structure, which is essential for classification and location prediction. The numerical experiments on two public datasets with KNN classifier and cross-validation tests showed that in terms of the common indexes of sensitivity, specificity, accuracy and MCC, the proposed fusing representations outperform the traditional representations in protein sub-nuclear localization, and the representation treated by LDA outperforms the untreated one.

  16. Protein Sub-Nuclear Localization Based on Effective Fusion Representations and Dimension Reduction Algorithm LDA

    Directory of Open Access Journals (Sweden)

    Shunfang Wang

    2015-12-01

    Full Text Available An effective representation of a protein sequence plays a crucial role in protein sub-nuclear localization. The existing representations, such as dipeptide composition (DipC, pseudo-amino acid composition (PseAAC and position specific scoring matrix (PSSM, are insufficient to represent protein sequence due to their single perspectives. Thus, this paper proposes two fusion feature representations of DipPSSM and PseAAPSSM to integrate PSSM with DipC and PseAAC, respectively. When constructing each fusion representation, we introduce the balance factors to value the importance of its components. The optimal values of the balance factors are sought by genetic algorithm. Due to the high dimensionality of the proposed representations, linear discriminant analysis (LDA is used to find its important low dimensional structure, which is essential for classification and location prediction. The numerical experiments on two public datasets with KNN classifier and cross-validation tests showed that in terms of the common indexes of sensitivity, specificity, accuracy and MCC, the proposed fusing representations outperform the traditional representations in protein sub-nuclear localization, and the representation treated by LDA outperforms the untreated one.

  17. A study on the feasibility and a possible form of local residents participation in nuclear safety regulation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Sang Hoon; Son, Moon Kyu [Korea Association for Nuclear Technology, Taejon (Korea, Republic of); Kim, Seong Sook [Shila Univ., Busan (Korea, Republic of)

    2003-02-15

    It is expected that the results of this study will help policy makers in the government and nuclear industry enhance the confidence of local residents and citizens in the safety of nuclear energy. It is also believed that the citizen confidence on the nuclear energy will help them implement nuclear policies more efficiently and more safely. Considering that the newly sworn-in government is to involve citizens in wide variety of policy areas, this study fits with the policy directions of the new government. Most importantly, desirable citizen participation in public decision-making is to contribute to the enhancement of the working democracy.

  18. Insecticidal activity of two proteases against Spodoptera frugiperda larvae infected with recombinant baculoviruses

    Directory of Open Access Journals (Sweden)

    Nagata Tatsuya

    2010-06-01

    Full Text Available Abstract Background Baculovirus comprise the largest group of insect viruses most studied worldwide, mainly because they efficiently kill agricutural insect pests. In this study, two recombinant baculoviruses containing the ScathL gene from Sarcophaga peregrina (vSynScathL, and the Keratinase gene from the fungus Aspergillus fumigatus (vSynKerat, were constructed. and their insecticidal properties analysed against Spodoptera frugiperda larvae. Results Bioassays of third-instar and neonate S. frugiperda larvae with vSynScathL and vSynKerat showed a decrease in the time needed to kill the infected insects when compared to the wild type virus. We have also shown that both recombinants were able to increase phenoloxidase activity in the hemolymph of S. frugiperda larvae. The expression of proteases in infected larvae resulted in destruction of internal tissues late in infection, which could be the reason for the increased viral speed of kill. Conclusions Baculoviruses and their recombinant forms constitute viable alternatives to chemical insecticides. Recombinant baculoviruses containing protease genes can be added to the list of engineered baculoviruses with great potential to be used in integrated pest management programs.

  19. Process-based modeling of the control of beet armyworm, Spodoptera exigua, with baculoviruses in greenhouse chrysanthemum

    NARCIS (Netherlands)

    Bianchi, F.J.J.A.

    2001-01-01

    This thesis describes the development of a process-based simulation model for the population dynamics of beet armyworm, Spodoptera exigua , and baculoviruses in greenhouse chrysanthemum. The model (BACSIM) has been validated for two baculoviruses with clear differences in biological characteristics,

  20. Interaction between a plasma membrane-localized ankyrin-repeat protein ITN1 and a nuclear protein RTV1

    Energy Technology Data Exchange (ETDEWEB)

    Sakamoto, Hikaru [Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri-shi, Hokkaido 093-2422 (Japan); Sakata, Keiko; Kusumi, Kensuke [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Kojima, Mikiko; Sakakibara, Hitoshi [RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045 (Japan); Iba, Koh, E-mail: koibascb@kyushu-u.org [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan)

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer ITN1, a plasma membrane ankyrin protein, interacts with a nuclear DNA-binding protein RTV1. Black-Right-Pointing-Pointer The nuclear transport of RTV1 is partially inhibited by interaction with ITN1. Black-Right-Pointing-Pointer RTV1 can promote the nuclear localization of ITN1. Black-Right-Pointing-Pointer Both overexpression of RTV1 and the lack of ITN1 increase salicylic acids sensitivity in plants. -- Abstract: The increased tolerance to NaCl 1 (ITN1) protein is a plasma membrane (PM)-localized protein involved in responses to NaCl stress in Arabidopsis. The predicted structure of ITN1 is composed of multiple transmembrane regions and an ankyrin-repeat domain that is known to mediate protein-protein interactions. To elucidate the molecular functions of ITN1, we searched for interacting partners using a yeast two-hybrid assay, and a nuclear-localized DNA-binding protein, RTV1, was identified as a candidate. Bimolecular fluorescence complementation analysis revealed that RTV1 interacted with ITN1 at the PM and nuclei in vivo. RTV1 tagged with red fluorescent protein localized to nuclei and ITN1 tagged with green fluorescent protein localized to PM; however, both proteins localized to both nuclei and the PM when co-expressed. These findings suggest that RTV1 and ITN1 regulate the subcellular localization of each other.

  1. Baculovirus-mediated expression of a Chinese scorpion neurotoxin improves insecticidal efficacy

    Institute of Scientific and Technical Information of China (English)

    FAN XiaoJun; ZHENG Bo; FU YueJun; SUN Yi; LIANG AiHua

    2008-01-01

    An Buthus martensii Karsch Insect Toxin (BmK IT) gene was inserted into the genome of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) to construct a recombinant baculovirus, AcMNPV-BmK IT. The expression of BmK IT was confirmed using RT-PCR, dot blot and SDS-PAGE analysis. Dose-lethal time responses to Spodoptera exigua larvae were compared between wild-type baculovirus AcMNPV and recombinant virus AcMNPV-BmK IT. At the concentration of 1×107 PIBs/mL, the median lethal time of recombinant baculovirus (LT50=73.6 h) on third instar S. Exigua larvae showed an improvement of 13.2% over the efficacy of wild type virus (LT50=84.8 h) during a 192 h in-fection.

  2. Fabrication and characterization of nuclear localization signal-conjugated glycol chitosan micelles for improving the nuclear delivery of doxorubicin

    Directory of Open Access Journals (Sweden)

    Zhao J

    2012-09-01

    Full Text Available Jingmou Yu,1 Xin Xie,1 Meirong Zheng,1 Ling Yu,2 Lei Zhang,1 Jianguo Zhao,1 Dengzhao Jiang,1 Xiangxin Che11Key Laboratory of Systems Biology Medicine of Jiangxi Province, College of Basic Medical Science, Jiujiang University, Jiujiang, 2Division of Nursing, 2nd Affiliated Hospital, Yichun University, Yichun, People's Republic of ChinaBackground: Supramolecular micelles as drug-delivery vehicles are generally unable to enter the nucleus of nondividing cells. In the work reported here, nuclear localization signal (NLS-modified polymeric micelles were studied with the aim of improving nuclear drug delivery.Methods: In this research, cholesterol-modified glycol chitosan (CHGC was synthesized. NLS-conjugated CHGC (NCHGC was synthesized and characterized using proton nuclear magnetic resonance spectroscopy, dynamic light scattering, and fluorescence spectroscopy. Doxorubicin (DOX, an anticancer drug with an intracellular site of action in the nucleus, was chosen as a model drug. DOX-loaded micelles were prepared by an emulsion/solvent evaporation method. The cellular uptake of different DOX formulations was analyzed by flow cytometry and confocal laser scanning microscopy. The cytotoxicity of blank micelles, free DOX, and DOX-loaded micelles in vitro was investigated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay in HeLa and HepG2 cells.Results: The degree of substitution was 5.9 cholesterol and 3.8 NLS groups per 100 sugar residues of the NCHGC conjugate. The critical aggregation concentration of the NCHGC micelles in aqueous solution was 0.0209 mg/mL. The DOX-loaded NCHGC (DNCHGC micelles were observed as being almost spherical in shape under transmission electron microscopy, and the size was determined as 248 nm by dynamic light scattering. The DOX-loading content of the DNCHGC micelles was 10.1%. The DOX-loaded micelles showed slow drug-release behavior within 72 hours in vitro. The DNCHGC micelles exhibited greater

  3. Monodisperse magnetite nanoparticles coupled with nuclear localization signal peptide for cell-nucleus targeting.

    Science.gov (United States)

    Xu, Chenjie; Xie, Jin; Kohler, Nathan; Walsh, Edward G; Chin, Y Eugene; Sun, Shouheng

    2008-03-01

    Functionalization of monodisperse superparamagnetic magnetite (Fe(3)O(4)) nanoparticles for cell specific targeting is crucial for cancer diagnostics and therapeutics. Targeted magnetic nanoparticles can be used to enhance the tissue contrast in magnetic resonance imaging (MRI), to improve the efficiency in anticancer drug delivery, and to eliminate tumor cells by magnetic fluid hyperthermia. Herein we report the nucleus-targeting Fe(3)O(4) nanoparticles functionalized with protein and nuclear localization signal (NLS) peptide. These NLS-coated nanoparticles were introduced into the HeLa cell cytoplasm and nucleus, where the particles were monodispersed and non-aggregated. The success of labeling was examined and identified by fluorescence microscopy and MRI. The work demonstrates that monodisperse magnetic nanoparticles can be readily functionalized and stabilized for potential diagnostic and therapeutic applications. PMID:18080259

  4. Local government's view on Chuetsu-oki earthquake and nuclear power plant facilities

    International Nuclear Information System (INIS)

    Niigataken Chuetsu-oki earthquake occurred in July 2007 and damaged individual's housing and properties in local residents and Kashiwazaki-Kariwa nuclear power plant facilities. The earthquake caused very strong ground motions far exceeding the assumed values in the design. The impacts were a fire breakout of the Unit 3 transformer and a release of small amount of radioactive materials to the environment at Unit 6 and 7. A joint of the driving shaft of the overhead crane of the Unit 6 reactor building was damaged. The fire of the Unit 3 transformer developed safety concerns among the public because of insufficient effectiveness of the private fire brigade, unavailability of fire control systems, and delayed notification to the external fire station, and as its consequence a lot of time needed to bring the fire under control. Delayed information dissemination gave deep concerns about the possible radioactive contamination in the environment. Based on New Regulatory Guide for Reviewing Seismic Design of Nuclear Power Reactor Facilities revised in September 2006, detail understanding of shakes and additional investigation of geological faults around the plant site were underway. Precise explanations on in-situ investigation and evaluation results to the residents would be necessary to answer their seismic safety concerns. (T. Tanaka)

  5. Nuclear binding energies: Global collective structure and local shell-model correlations

    International Nuclear Information System (INIS)

    Nuclear binding energies and two-neutron separation energies are analysed starting from the liquid-drop model and the nuclear shell model in order to describe the global trends of the above observables. We subsequently concentrate on the Interacting Boson Model (IBM) and discuss a new method in order to provide a consistent description of both, ground-state and excited-state properties. We address the artefacts that appear when crossing mid-shell using the IBM formulation and perform detailed numerical calculations for nuclei situated in the 50-82 shell. We also concentrate on local deviations from the above global trends in binding energy and two-neutron separation energies that appear in the neutron-deficient Pb region. We address possible effects on the binding energy, caused by mixing of low-lying 0+ intruder states into the ground state, using configuration mixing in the IBM framework. We also study ground-state properties using a macroscopic-microscopic model. Detailed comparisons with recent experimental data in the Pb region are amply discussed

  6. Nuclear translocation of glutathione S-transferase {pi} is mediated by a non-classical localization signal

    Energy Technology Data Exchange (ETDEWEB)

    Kawakatsu, Miho [Department of Stem Cell Biology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Goto, Shinji, E-mail: sgoto@nagasaki-u.ac.jp [Department of Stem Cell Biology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Yoshida, Takako; Urata, Yoshishige; Li, Tao-Sheng [Department of Stem Cell Biology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan)

    2011-08-12

    Highlights: {yields} Nuclear translocation of GST{pi} is abrogated by the deletion of the last 16 amino acid residues in the carboxy-terminal region, indicating that residues 195-208 of GST{pi} are required for nuclear translocation. {yields} The lack of a contiguous stretch of positively charged amino acid residues within the carboxy-terminal region of GST{pi}, suggests that the nuclear translocation of GST{pi} is mediated by a non-classical nuclear localization signal. {yields} An in vitro transport assay shows that the nuclear translocation of GST{pi} is dependent on cytosolic factors and ATP. -- Abstract: Glutathione S-transferase {pi} (GST{pi}), a member of the GST family of multifunctional enzymes, is highly expressed in human placenta and involved in the protection of cellular components against electrophilic compounds or oxidative stress. We have recently found that GST{pi} is expressed in the cytoplasm, mitochondria, and nucleus in some cancer cells, and that the nuclear expression of GST{pi} appears to correlate with resistance to anti-cancer drugs. Although the mitochondrial targeting signal of GST{pi} was previously identified in the amino-terminal region, the mechanism of nuclear translocation remains completely unknown. In this study, we find that the region of GST{pi}195-208 is critical for nuclear translocation, which is mediated by a novel and non-classical nuclear localization signal. In addition, using an in vitro transport assay, we demonstrate that the nuclear translocation of GST{pi} depends on the cytosolic extract and ATP. Although further experiments are needed to understand in depth the precise mechanism of nuclear translocation of GST{pi}, our results may help to establish more efficient anti-cancer therapy, especially with respect to resistance to anti-cancer drugs.

  7. Phosphatidic Acid Interacts with a MYB Transcription Factor and Regulates Its Nuclear Localization and Function in Arabidopsis[C][W

    Science.gov (United States)

    Yao, Hongyan; Wang, Geliang; Guo, Liang; Wang, Xuemin

    2013-01-01

    Phosphatidic acid (PA) has emerged as a class of cellular mediators involved in various cellular and physiological processes, but little is known about its mechanism of action. Here we show that PA interacts with WEREWOLF (WER), a R2R3 MYB transcription factor involved in root hair formation. The PA-interacting region is confined to the end of the R2 subdomain. The ablation of the PA binding motif has no effect on WER binding to DNA, but abolishes its nuclear localization and its function in regulating epidermal cell fate. Inhibition of PA production by phospholipase Dζ also suppresses WER’s nuclear localization, root hair formation, and elongation. These results suggest a role for PA in promoting protein nuclear localization. PMID:24368785

  8. Phosphatidic acid interacts with a MYB transcription factor and regulates its nuclear localization and function in Arabidopsis.

    Science.gov (United States)

    Yao, Hongyan; Wang, Geliang; Guo, Liang; Wang, Xuemin

    2013-12-01

    Phosphatidic acid (PA) has emerged as a class of cellular mediators involved in various cellular and physiological processes, but little is known about its mechanism of action. Here we show that PA interacts with werewolf (WER), a R2R3 MYB transcription factor involved in root hair formation. The PA-interacting region is confined to the end of the R2 subdomain. The ablation of the PA binding motif has no effect on WER binding to DNA, but abolishes its nuclear localization and its function in regulating epidermal cell fate. Inhibition of PA production by phospholipase Dζ also suppresses WER's nuclear localization, root hair formation, and elongation. These results suggest a role for PA in promoting protein nuclear localization.

  9. Centrin 2 Localizes to the Vertebrate Nuclear Pore and Plays a Role in mRNA and Protein Export▿ †

    OpenAIRE

    Resendes, Karen K.; Rasala, Beth A.; Forbes, Douglass J.

    2008-01-01

    Centrins in vertebrates have traditionally been associated with microtubule-nucleating centers such as the centrosome. Unexpectedly, we found centrin 2 to associate biochemically with nucleoporins, including the Xenopus laevis Nup107-160 complex, a critical subunit of the vertebrate nuclear pore in interphase and of the kinetochores and spindle poles in mitosis. Immunofluorescence of Xenopus cells and in vitro reconstituted nuclei indeed revealed centrin 2 localized at the nuclear pores. Use ...

  10. Activation of AMP-activated protein kinase stimulates the nuclear localization of glyceraldehyde 3-phosphate dehydrogenase in human diploid fibroblasts

    OpenAIRE

    Kwon, Hyun Jin; Rhim, Ji Heon; Jang, Ik-Soon; Kim, Go-Eun; Park, Sang Chul; Yeo, Eui-Ju

    2010-01-01

    In addition to its well-known glycolytic activity, GAPDH displays multiple functions, such as nuclear RNA export, DNA replication and repair, and apoptotic cell death. This functional diversity depends on its intracellular localization. In this study, we explored the signal transduction pathways involved in the nuclear translocation of GAPDH using confocal laser scanning microscopy of immunostained human diploid fibroblasts (HDFs). GAPDH was present mainly in the cytoplasm when cultured wi...

  11. Localization of nuclear cathepsin L and its association with disease progression and poor outcome in colorectal cancer.

    LENUS (Irish Health Repository)

    Sullivan, Shane

    2012-02-01

    Previous in vitro studies have identified a nuclear isoform of Cathepsin L. The aim of this study was to examine if nuclear Cathepsin L exists in vivo and examine its association with clinical, pathological and patient outcome data. Cellular localization (nuclear and cytoplasmic) and expression levels v of Cathespin L in 186 colorectal cancer cases using immunohistochemistry. The molecular weight and activity of nuclear and cytoplasmic Cathepsin L in vivo and in vitro were assessed by Western blotting and ELISA, respectively. Epithelial nuclear staining percentage (p = 0.04) and intensity (p = 0.006) increased with advancing tumor stage, whereas stromal cytoplasmic staining decreased (p = 0.02). Using multivariate statistical analysis, survival was inversely associated with staining intensity in the epithelial cytoplasm (p = 0.01) and stromal nuclei (p = 0.007). In different colorectal cell lines and in vivo tumors, pro- and active Cathepsin L isoforms were present in both the cytoplasm and nuclear samples, with pro-Cathepsin L at 50 kDa and active Cathepsin L at 25 kDa. Purified nuclear and cytoplasmic fractions from cell lines and tumors showed active Cathepsin L activity. The identification of nuclear Cathepsin L may play an important prognostic role in colorectal disease progression and patient outcome. Moreover, these findings suggest that altering active nuclear Cathepsin L may significantly influence disease progression.

  12. HDAC6 regulates androgen receptor hypersensitivity and nuclear localization via modulating Hsp90 acetylation in castration-resistant prostate cancer.

    Science.gov (United States)

    Ai, Junkui; Wang, Yujuan; Dar, Javid A; Liu, June; Liu, Lingqi; Nelson, Joel B; Wang, Zhou

    2009-12-01

    The development of castration-resistant prostate cancer (PCa) requires that under castration conditions, the androgen receptor (AR) remains active and thus nuclear. Heat shock protein 90 (Hsp90) plays a key role in androgen-induced and -independent nuclear localization and activation of AR. Histone deacetylase 6 (HDAC6) is implicated, but has not been proven, in regulating AR activity via modulating Hsp90 acetylation. Here, we report that knockdown of HDAC6 in C4-2 cells using short hairpin RNA impaired ligand-independent nuclear localization of endogenous AR and inhibited PSA expression and cell growth in the absence or presence of dihydrotestosterone (DHT). The dose-response curve of DHT-stimulated C4-2 colony formation was shifted by shHDAC6 such that approximately 10-fold higher concentration of DHT is required, indicating a requirement for HDAC6 in AR hypersensitivity. HDAC6 knockdown also inhibited C4-2 xenograft tumor establishment in castrated, but not in testes-intact, nude mice. Studies using HDAC6-deficient mouse embryonic fibroblasts cells showed that inhibition of AR nuclear localization by HDAC6 knockdown can be largely alleviated by expressing a deacetylation mimic Hsp90 mutant. Taken together, our studies suggest that HDAC6 regulates AR hypersensitivity and nuclear localization, mainly via modulating HSP90 acetylation. Targeting HDAC6 alone or in combination with other therapeutic approaches is a promising new strategy for prevention and/or treatment of castration-resistant PCa.

  13. Expression and localization of heterogeneous nuclear ribonucleoprotein K in mouse ovaries and preimplantation embryos.

    Science.gov (United States)

    Zhang, Ping; Wang, Ningling; Lin, Xianhua; Jin, Li; Xu, Hong; Li, Rong; Huang, Hefeng

    2016-02-26

    Heterogeneous nuclear ribonucleoprotein K (hnRNP K), an evolutionarily conserved protein, is involved in several important cellular processes that are relevant to cell proliferation, differentiation, apoptosis, and cancer development. However, details of hnRNP K expression during mammalian oogenesis and preimplantation embryo development are lacking. The present study investigates the expression and cellular localization of K protein in the mouse ovaries and preimplantation embryos using immunostaining. We demonstrate, for the first time, that hnRNP K is abundantly expressed in the nuclei of mouse oocytes in primordial, primary and secondary follicles. In germ vesicle (GV)-stage oocytes, hnRNP K accumulates in the germinal vesicle in a spot distribution manner. After germinal vesicle breakdown, speckled hnRNP K is diffusely distributed in the cytoplasm. However, after fertilization, the K protein relocates into the female and male pronucleus and persists in the blastomere nuclei. Localization of K protein in the human ovary and ovarian granulosa cell tumor (GCT) was also investigated. Overall, this study provides important morphological evidence to better understand the possible roles of hnRNP K in mammalian oogenesis and early embryo development. PMID:26850853

  14. Nuclear F-actin enhances the transcriptional activity of β-catenin by increasing its nuclear localization and binding to chromatin.

    Science.gov (United States)

    Yamazaki, Shota; Yamamoto, Koji; de Lanerolle, Primal; Harata, Masahiko

    2016-04-01

    Actin plays multiple roles both in the cytoplasm and in the nucleus. Cytoplasmic actin, in addition to its structural role in the cytoskeleton, also contributes to the subcellular localization of transcription factors by interacting with them or their partners. The transcriptional cofactor β-catenin, which acts as an intracellular transducer of canonical Wnt signaling, indirectly associates with the cytoplasmic filamentous actin (F-actin). Recently, it has been observed that F-actin is transiently formed within the nucleus in response to serum stimulation and integrin signaling, and also during gene reprogramming. Despite these earlier observations, information about the function of nuclear F-actin is poorly defined. Here, by facilitating the accumulation of nuclear actin artificially, we demonstrate that polymerizing nuclear actin enhanced the nuclear accumulation and transcriptional function of β-catenin. Our results also show that the nuclear F-actin colocalizes with β-catenin and enhances the binding of β-catenin to the downstream target genes of the Wnt/β-catenin signaling pathway, including the genes for the cell cycle regulators c-myc and cyclin D, and the OCT4 gene. Nuclear F-actin itself also associated with these genes. Since Wnt/β-catenin signaling has important roles in cell differentiation and pluripotency, our observations suggest that nuclear F-actin formed during these biological processes is involved in regulating Wnt/β-catenin signaling. PMID:26900020

  15. Development of the local stress analysis methodology for nuclear class 1 piping welded to the seal plate

    International Nuclear Information System (INIS)

    Lugs, brackets, stiffners and other attachments may be welded, bolted and studded to the outside or inside of piping and the local stress arise because of the radial thermal expansion of the piping, the dilatation of the piping due to its internal pressure, the circumferential contraction of the pipe as a results of an axial tensile force (Poisson's effect), etc., being constrained by those. So the evaluation of the local stress for the piping constrained by those. So the evaluation of the local stress for the piping constrained by the attachments in accordance with the ASME Section III NB-3651.3 is required for the nuclear class 1 piping. In this report the local stress analysis procedure for the nuclear class 1 piping welded to the seal plate was established. SInce the stress analysis for the nuclear class 1 piping require the fatigue analysis procedure was established using the commercial code ANSYS 5.1 for the structure analysis in this report. The procedure which was developed in this report can be used very effectively for the design for the seal plate and the local stress analysis of the nuclear class 1 piping welded to the seal plate. 14 figs., 5 refs. (Author) .new

  16. Functional analysis of the C-terminal region of human adenovirus E1A reveals a misidentified nuclear localization signal

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, Michael J.; King, Cason R.; Dikeakos, Jimmy D. [Department of Microbiology and Immunology, The University of Western Ontario, A4-833 London Regional Cancer Centre, 800 Commissioners Road E., London, Ontario, N6A 4L6 Canada (Canada); Mymryk, Joe S., E-mail: jmymryk@uwo.ca [Department of Microbiology and Immunology, The University of Western Ontario, A4-833 London Regional Cancer Centre, 800 Commissioners Road E., London, Ontario, N6A 4L6 Canada (Canada); Department of Oncology, The University of Western Ontario, London Regional Cancer Centre, Ontario (Canada)

    2014-11-15

    The immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285–289, as well as a second basic patch situated between residues 258 and 263 ({sup 258}RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP{sup 289}). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs. - Highlights: • Human adenovirus E1A is localized to the nucleus. • The C-terminus of E1A contains a bipartite nuclear localization signal (NLS). • This signal was previously misidentified to be a monopartite NLS. • Key basic amino acid residues within this sequence are highly conserved.

  17. A Dual Mechanism Controls Nuclear Localization in the Atypical Basic-Helix-Loop-Helix Protein PAR1 of Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Anahit Galstyan; Jordi Bou-Torrent; Irma Roig-Villanova; Jaime F. Martínez-García

    2012-01-01

    PAR1 is an atypical basic-helix-loop-helix (bHLH) protein that negatively regulates the shade avoidance syndrome in Arabidopsis thaliana acting as a transcriptional cofactor.Consistently with this function,PAR1 has to be in the nucleus to display biological activity.Previous structure-function analyses revealed that the N-terminal region of PAR1 drives the protein to the nucleus.However,truncated forms of PAR1 lacking this region still display biological activity,implying that PAR1 has additional mechanisms to localize into the nucleus.In this work,we compared the primary structure of PAR1 and various related and unrelated plant bHLH proteins,which led us to suggest that PAR1 contains a non-canonical nuclear localization signal (NLS) in the N-terminal region.By overexpressing truncated and mutated derivatives of PAR1,we have also investigated the importance of other regions of PAR1,such as the acidic and the extended HLH dimerization domains,for its nuclear localization.We found that,in the absence of the N-terminal region,a functional HLH domain is required for nuclear localization.Our results suggest the existence of a dual mechanism for PAR1 nuclear localization:(1) one mediated by the N-terminal non-consensus NLS and (2) a second one that involves interaction with other proteins via the dimerization domain.

  18. Baculovirus as a PRRSV and PCV2 bivalent vaccine vector: baculovirus virions displaying simultaneously GP5 glycoprotein of PRRSV and capsid protein of PCV2.

    Science.gov (United States)

    Xu, Xin-Gang; Wang, Zhi-Sheng; Zhang, Qi; Li, Zhao-Cai; Ding, Li; Li, Wei; Wu, Hung-Yi; Chang, Ching-Dong; Lee, Long-Huw; Tong, De-Wen; Liu, Hung-Jen

    2012-02-01

    The GP5 glycoprotein of PRRSV is the main target for inducing neutralizing antibodies and protective immunity in the natural host. The capsid (Cap) protein is the major immunogenic protein and associated with the production of PCV2-specific neutralizing antibodies. In the present study, one genetic recombinant baculovirus BacSC-Dual-GP5-Cap was constructed. This virus displays simultaneously histidine-tagged GP5 and Cap proteins with the baculovirus glycoprotein gp64 TM and CTD on the virion surface as well as the surface of the virus-infected cells. After infection, the GP5 and Cap proteins were expressed and anchored simultaneously on the plasma membrane of Sf-9 cells, as revealed by Western blot and confocal microscopy. This report demonstrated first that both GP5 and Cap proteins were displayed successfully on the viral surface, revealed by immunogold electron microscopy. Vaccination of swine with recombinant baculovirus BacSC-Dual-GP5-Cap elicited significantly higher GP5 and Cap ELISA antibody titers in swine than the control groups. Virus neutralization test also showed that serum from the BacSC-Dual-GP5-Cap treated swine had significant levels of virus neutralization titers. Lymphocyte proliferation responses could be induced in swine immunized with BacSC-Dual-GP5-Cap than the control groups. These findings demonstrate that the BacSC-Dual-GP5-Cap bivalent subunit vaccine can be a potential vaccine against PRRSV and PCV2 infections. PMID:22172969

  19. Novel baculovirus-derived p67 subunit vaccines efficacious against East Coast fever in cattle

    NARCIS (Netherlands)

    Kaba, S.A.; Musoke, A.J.; Schaap, D.; Schetters, T.; Rowlands, J.; Vermeulen, A.J.; Nene, V.; Vlak, J.M.; Oers, van M.M.

    2005-01-01

    Two novel baculovirus-derived recombinant Theileria parva p67 constructs were tested for their vaccine potential against East Coast fever. Boran calves were immunized with a his-GFP-p67 fusion protein (GFP:p67¿SS) or with GP64:p67C, a protein fusion between a C-terminal domain of p67 and the baculov

  20. Arbovirus vaccines: opportunities for the baculovirus-insect cell expression system

    NARCIS (Netherlands)

    Metz, S.W.H.; Pijlman, G.P.

    2011-01-01

    The baculovirus-insect cell expression system is a well-established technology for the production of heterologous viral (glyco)proteins in cultured cells, applicable for basic scientific research as well as for the development and production of vaccines and diagnostics. Arboviruses form an emerging

  1. Characterization of the Spodoptera exigua baculovirus genome: structural and functional analysis of a 20 KB fragment.

    NARCIS (Netherlands)

    Strien, van E.A.

    1997-01-01

    Baculoviruses are attractive, biological alternatives to chemical control agents of insect pests. These viruses are natural agents influencing the size insect populations. They are often species- specific and some of them are highly efficacious, though their speed of action cannot meet that of chemi

  2. Recombinant, catalytically inactive juvenile hormone esterase enhances efficacy of baculovirus insecticides

    NARCIS (Netherlands)

    Meer, van M.M.M.; Bonning, B.C.; Ward, V.K.; Vlak, J.M.; Hammock, B.D.

    2000-01-01

    The insecticidal efficacy of baculoviruses can be enhanced by engineering the viral genome to express proteins that disrupt the physiology of the host insect. Here we describe the development of a genetically engineered Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) which expresses

  3. Cytomegalovirus Assembly Protein Precursor and Proteinase Precursor Contain Two Nuclear Localization Signals That Mediate Their Own Nuclear Translocation and That of the Major Capsid Protein

    OpenAIRE

    Plafker, Scott M.; Gibson, Wade

    1998-01-01

    The cytomegalovirus (CMV) assembly protein precursor (pAP) interacts with the major capsid protein (MCP), and this interaction is required for nuclear translocation of the MCP, which otherwise remains in the cytoplasm of transfected cells (L. J. Wood et al., J. Virol. 71:179–190, 1997). We have interpreted this finding to indicate that the CMV MCP lacks its own nuclear localization signal (NLS) and utilizes the pAP as an NLS-bearing escort into the nucleus. The CMV pAP amino acid sequence has...

  4. EGFR Signaling Regulates Maspin/SerpinB5 Phosphorylation and Nuclear Localization in Mammary Epithelial Cells

    Science.gov (United States)

    Reina, Jeffrey; Morais Freitas, Vanessa

    2016-01-01

    Maspin (SerpinB5) is a non-inhibitory serpin (serine protease inhibitor) with very diverse biological activities including regulation of cell adhesion, migration, death, control of gene expression and oxidative stress response. Initially described as a tumor and metastasis suppressor, clinical data brought controversies to the field, as some studies reported no correlation between SerpinB5 expression and prognosis value. These data underscore the importance of understanding SerpinB5 function in a normal physiological context and the molecular mechanism involved. Several SerpinB5 phosphoforms have been detected in different cell lines, but the signaling pathways involved and the biological significance of this post-translational modification in vivo remains to be explored. In this study we investigated SerpinB5 expression, subcellular localization and phosphorylation in different stages of the mouse mammary gland development and the signaling pathway involved. Here we show that SerpinB5 is first detected in late pregnancy, reaches its highest levels in lactation and remains at constant levels during post-lactational regression (involution). Using high resolution isoelectric focusing followed but immunoblot, we found at least 8 different phosphoforms of SerpinB5 during lactation, which decreases steadily at the onset of involution. In order to investigate the signaling pathway involved in SerpinB5 phosphorylation, we took advantage of the non-transformed MCF-10A model system, as we have previously observed SerpinB5 phosphorylation in these cells. We detected basal levels of SerpinB5 phosphorylation in serum- and growth factor-starved cells, which is due to amphiregulin autocrine activity on MCF-10A cells. EGF and TGF alpha, two other EGFR ligands, promote important SerpinB5 phosphorylation. Interestingly, EGF treatment is followed by SerpinB5 nuclear accumulation. Altogether, these data indicate that SerpinB5 expression and phosphorylation are developmentally

  5. A Study of Community Leaders in a Nuclear Host Community: Local Issues, Expectations and Support and Opposition.

    Science.gov (United States)

    Bronfman, B. H.

    As part of a continuing effort to assess the social impacts on communities of energy facility planning, construction, operation, and decommissioning, a May 1977 survey of 37 community leaders in Hartsville, Tennessee (site of a nuclear power plant) establishes major local issues (past, present, and future) which leaders feel are important to…

  6. Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

    Science.gov (United States)

    López-Vidal, Javier; Gómez-Sebastián, Silvia; Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M

    2015-01-01

    Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health. PMID:26458221

  7. Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

    Directory of Open Access Journals (Sweden)

    Javier López-Vidal

    Full Text Available Vaccines based on virus-like particles (VLPs have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60 were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health.

  8. Protection against Amoebic Liver Abscess in Hamster by Intramuscular Immunization with an Autographa californica Baculovirus Driving the Expression of the Gal-Lectin LC3 Fragment

    Science.gov (United States)

    Meneses-Ruiz, Dulce María; Aguilar-Diaz, Hugo; Bobes, Raúl José; Sampieri, Alicia; Laclette, Juan Pedro; Carrero, Julio César

    2015-01-01

    In a previous study, we demonstrated that oral immunization using Autographa californica baculovirus driving the expression of the Gal-lectin LC3 fragment (AcNPV-LC3) of Entamoeba histolytica conferred protection against ALA development in hamsters. In this study, we determined the ability of AcNPV-LC3 to protect against ALA by the intramuscular route as well as the liver immune response associated with protection. Results showed that 55% of hamsters IM immunized with AcNPV-LC3 showed sterile protection against ALA, whereas other 20% showed reduction in the size and extent of abscesses, resulting in some protection in 75% of animals compared to the sham control group. Levels of protection showed a linear correlation with the development and intensity of specific antiamoeba cellular and humoral responses, evaluated in serum and spleen of hamsters, respectively. Evaluation of the Th1/Th2 cytokine patterns expressed in the liver of hamsters showed that sterile protection was associated with the production of high levels of IFNγ and IL-4. These results suggest that the baculovirus system is equally efficient by the intramuscular as well as the oral routes for ALA protection and that the Gal-lectin LC3 fragment is a highly protective antigen against hepatic amoebiasis through the local induction of IFNγ and IL-4. PMID:26090442

  9. Protection against Amoebic Liver Abscess in Hamster by Intramuscular Immunization with an Autographa californica Baculovirus Driving the Expression of the Gal-Lectin LC3 Fragment

    Directory of Open Access Journals (Sweden)

    Dulce María Meneses-Ruiz

    2015-01-01

    Full Text Available In a previous study, we demonstrated that oral immunization using Autographa californica baculovirus driving the expression of the Gal-lectin LC3 fragment (AcNPV-LC3 of Entamoeba histolytica conferred protection against ALA development in hamsters. In this study, we determined the ability of AcNPV-LC3 to protect against ALA by the intramuscular route as well as the liver immune response associated with protection. Results showed that 55% of hamsters IM immunized with AcNPV-LC3 showed sterile protection against ALA, whereas other 20% showed reduction in the size and extent of abscesses, resulting in some protection in 75% of animals compared to the sham control group. Levels of protection showed a linear correlation with the development and intensity of specific antiamoeba cellular and humoral responses, evaluated in serum and spleen of hamsters, respectively. Evaluation of the Th1/Th2 cytokine patterns expressed in the liver of hamsters showed that sterile protection was associated with the production of high levels of IFNγ and IL-4. These results suggest that the baculovirus system is equally efficient by the intramuscular as well as the oral routes for ALA protection and that the Gal-lectin LC3 fragment is a highly protective antigen against hepatic amoebiasis through the local induction of IFNγ and IL-4.

  10. Protection against Amoebic Liver Abscess in Hamster by Intramuscular Immunization with an Autographa californica Baculovirus Driving the Expression of the Gal-Lectin LC3 Fragment.

    Science.gov (United States)

    Meneses-Ruiz, Dulce María; Aguilar-Diaz, Hugo; Bobes, Raúl José; Sampieri, Alicia; Vaca, Luis; Laclette, Juan Pedro; Carrero, Julio César

    2015-01-01

    In a previous study, we demonstrated that oral immunization using Autographa californica baculovirus driving the expression of the Gal-lectin LC3 fragment (AcNPV-LC3) of Entamoeba histolytica conferred protection against ALA development in hamsters. In this study, we determined the ability of AcNPV-LC3 to protect against ALA by the intramuscular route as well as the liver immune response associated with protection. Results showed that 55% of hamsters IM immunized with AcNPV-LC3 showed sterile protection against ALA, whereas other 20% showed reduction in the size and extent of abscesses, resulting in some protection in 75% of animals compared to the sham control group. Levels of protection showed a linear correlation with the development and intensity of specific antiamoeba cellular and humoral responses, evaluated in serum and spleen of hamsters, respectively. Evaluation of the Th1/Th2 cytokine patterns expressed in the liver of hamsters showed that sterile protection was associated with the production of high levels of IFNγ and IL-4. These results suggest that the baculovirus system is equally efficient by the intramuscular as well as the oral routes for ALA protection and that the Gal-lectin LC3 fragment is a highly protective antigen against hepatic amoebiasis through the local induction of IFNγ and IL-4.

  11. New nuclear data service at CNEA: retrieval of the update libraries from a local Web-Server

    International Nuclear Information System (INIS)

    A new On-line Nuclear Data Service was implemented at National Atomic Energy Commission (CNEA) Web-Site. The information usually issued by the Nuclear Data Section of IAEA (NDS-IAEA) on CD-ROM, as well as complementary libraries periodically downloaded from the a mirror server of NDS-IAEA Service located at IPEN, Brazil are available on the new CNEA Web page. In the site, users can find numerical data on neutron, charged-particle, and photonuclear reactions, nuclear structure, and decay data, with related bibliographic information. This data server is permanently maintained and updated by CNEA staff members. This crew also offers assistance on the use and retrieval of nuclear data to local users. (author)

  12. Importance weighting of local flux measurements to improve reactivity predictions in nuclear systems

    Energy Technology Data Exchange (ETDEWEB)

    Dulla, Sandra; Hoh, Siew Sin; Nervo, Marta; Ravetto, Piero [Politecnico di Torino, Dipt. Energia (Italy)

    2015-07-15

    The reactivity monitoring is a key aspect for the safe operation of nuclear reactors, especially for subcritical source-driven systems. Various methods are available for both, off-line and on-line reactivity determination from direct measurements carried out on the reactor. Usually the methods are based on the inverse point kinetic model applied to signals from neutron detectors and results may be severely affected by space and spectral effects. Such effects need to be compensated and correction procedures have to be applied. In this work, a new approach is proposed, by using the full information from different local measurements to generate a global signal through a proper weighting of the signals provided by single neutron detectors. A weighting techique based on the use of the adjoint flux proves to be efficient in improving the prediction capability of inverse techniques. The idea is applied to the recently developed algorithm, named MAρTA, that can be used in both off-line and online modes.

  13. Improved radiological/nuclear source localization in variable NORM background: An MLEM approach with segmentation data

    Energy Technology Data Exchange (ETDEWEB)

    Penny, Robert D., E-mail: robert.d.penny@leidos.com [Leidos Inc., 10260 Campus Point Road, San Diego, CA (United States); Crowley, Tanya M.; Gardner, Barbara M.; Mandell, Myron J.; Guo, Yanlin; Haas, Eric B.; Knize, Duane J.; Kuharski, Robert A.; Ranta, Dale; Shyffer, Ryan [Leidos Inc., 10260 Campus Point Road, San Diego, CA (United States); Labov, Simon; Nelson, Karl; Seilhan, Brandon [Lawrence Livermore National Laboratory, Livermore, CA (United States); Valentine, John D. [Lawrence Berkeley National Laboratory, Berkeley, CA (United States)

    2015-06-01

    A novel approach and algorithm have been developed to rapidly detect and localize both moving and static radiological/nuclear (R/N) sources from an airborne platform. Current aerial systems with radiological sensors are limited in their ability to compensate for variable naturally occurring radioactive material (NORM) background. The proposed approach suppresses the effects of NORM background by incorporating additional information to segment the survey area into regions over which the background is likely to be uniform. The method produces pixelated Source Activity Maps (SAMs) of both target and background radionuclide activity over the survey area. The task of producing the SAMs requires (1) the development of a forward model which describes the transformation of radionuclide activity to detector measurements and (2) the solution of the associated inverse problem. The inverse problem is ill-posed as there are typically fewer measurements than unknowns. In addition the measurements are subject to Poisson statistical noise. The Maximum-Likelihood Expectation-Maximization (MLEM) algorithm is used to solve the inverse problem as it is well suited for under-determined problems corrupted by Poisson noise. A priori terrain information is incorporated to segment the reconstruction space into regions within which we constrain NORM background activity to be uniform. Descriptions of the algorithm and examples of performance with and without segmentation on simulated data are presented.

  14. Agrobacterium rhizogenes GALLS protein contains domains for ATP binding, nuclear localization, and type IV secretion.

    Science.gov (United States)

    Hodges, Larry D; Vergunst, Annette C; Neal-McKinney, Jason; den Dulk-Ras, Amke; Moyer, Deborah M; Hooykaas, Paul J J; Ream, Walt

    2006-12-01

    Agrobacterium tumefaciens and Agrobacterium rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells. Transfer of single-stranded DNA (ssDNA) and Vir proteins requires a type IV secretion system, a protein complex spanning the bacterial envelope. A. tumefaciens translocates the ssDNA-binding protein VirE2 into plant cells, where it binds single-stranded T-DNA and helps target it to the nucleus. Although some strains of A. rhizogenes lack VirE2, they are pathogenic and transfer T-DNA efficiently. Instead, these bacteria express the GALLS protein, which is essential for their virulence. The GALLS protein can complement an A. tumefaciens virE2 mutant for tumor formation, indicating that GALLS can substitute for VirE2. Unlike VirE2, GALLS contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. Both GALLS and VirE2 contain nuclear localization sequences and a C-terminal type IV secretion signal. Here we show that mutations in any of these domains abolished the ability of GALLS to substitute for VirE2. PMID:17012398

  15. Local power peaking factor estimation in nuclear fuel by artificial neural networks

    International Nuclear Information System (INIS)

    This paper presents the training of an artificial neural network (ANN) to accurately predict, in very short time, a physical parameter used in nuclear fuel reactor optimization: the local power peaking factor (LPPF) in a typical boiling water reactor (BWR) fuel lattice. The ANN training patterns are distribution of fissile and burnable poison materials in the fuel lattice and their associated LPPF. These data were obtained by modeling the fuel lattices with a neutronic simulator: the HELIOS transport code. The combination of the pin U235 enrichment and the Gd2O3 (gadolinia) concentration, inside the 10 x 10 fuel lattice array, was encoded by three different methods. However, the only encoding method that was able to give a good prediction of the LPPF was the method which added the U235 enrichment and the gadolinia concentration. The results show that the relative error in the estimation of the LPPF, obtained by the trained ANN, ranged from 0.022% to 0.045%, with respect to the HELIOS results

  16. Nuclear Accretion in Galaxies of the Local Universe: Clues from Chandra Observations

    CERN Document Server

    Pellegrini, S

    2005-01-01

    In order to find an explanation for the radiative quiescence of supermassive black holes in the local Universe, for a sample of nearby galaxies the most accurate estimates are collected for the mass of a central black hole M_BH, the nuclear X-ray luminosity L_X,nuc and the circumnuclear hot gas density and temperature, by using Chandra data. L_X,nuc varies by \\sim 3 orders of magnitude and does not show a relationship with M_BH or with the Bondi mass accretion rate \\dotM_B. L_X,nuc is always much lower than expected if \\dotM_B ends in a standard accretion disc with high radiative efficiency (this instead can be the case of the active nucleus of Cen A). Radiatively inefficient accretion as in the standard ADAF modeling may explain the low luminosities of a few cases; for others, the predicted luminosity is still too high and, in terms of Eddington-scaled quantities, it is increasingly higher than observed, for increasing \\dotM_B. Variants of the simple radiatively inefficient scenario including outflow and con...

  17. Predicting the Nuclear Localization Signals of 107 Types of HPV L1 Proteins by Bioinformatic Analysis

    Institute of Scientific and Technical Information of China (English)

    Jun Yang; Yi-Li Wang; Lü-Sheng Si

    2006-01-01

    In this study, 107 types of human papillomavirus (HPV) L1 protein sequences were obtained from available databases, and the nuclear localization signals (NLSs) of these HPV L1 proteins were analyzed and predicted by bioinformatic analysis.Out of the 107 types, the NLSs of 39 types were predicted by PredictNLS software (35 types of bipartite NLSs and 4 types of monopartite NLSs). The NLSs of the remaining HPV types were predicted according to the characteristics and the homology of the already predicted NLSs as well as the general rule of NLSs.According to the result, the NLSs of 107 types of HPV L1 proteins were classified into 15 categories. The different types of HPV L1 proteins in the same NLS category could share the similar or the same nucleocytoplasmic transport pathway.They might be used as the same target to prevent and treat different types of HPV infection. The results also showed that bioinformatic technology could be used to analyze and predict NLSs of proteins.

  18. Basic amino acid residues located in the N-terminal region of BEND3 are essential for its nuclear localization

    International Nuclear Information System (INIS)

    BEN domain-containing protein 3 (BEND3) has recently been reported to function as a heterochromatin-associated protein in transcriptional repression in the nucleus. BEND3 should have nuclear localization signals (NLSs) to localize to the nucleus in light of its molecular weight, which is higher than that allowed to pass through nuclear pore complexes. We here analyzed the subcellular localization of deletion/site-directed mutants of human BEND3 by an immunofluorescence assay in an attempt to identify the amino acids essential for its nuclear localization. We found that three basic amino acid residues located in the N-terminal region of BEND3 (BEND356–58, KRK) are essential, suggesting that these residues play a role as a functional NLS. These results provide valuable information for progressing research on BEND3. - Highlights: • BEND3 localizes to the nucleus. • The N-terminal 60 amino acids region of BEND3 contains NLS. • Amino acids located between 56 and 58 of BEND3 (KRK) are part of NLS. • KRK motif is highly conserved among BEND3 homologs

  19. Arabidopsis chromatin-associated HMGA and HMGB use different nuclear targeting signals and display highly dynamic localization within the nucleus

    DEFF Research Database (Denmark)

    Launholt, Dorte; Merkle, Thomas; Houben, Andreas;

    2006-01-01

    HMGproteins appear to be involved in the regulation of transcription and other DNA-dependent processes. We have examined the subcellular localization of Arabidopsis thaliana HMGA, HMGB1, and HMGB5, revealing that they localize to the cell nucleus. They display a speckled distribution pattern throughout the chromatin...... of interphase nuclei, whereas none of the proteins associate with condensed mitotic chromosomes. HMGA is targeted to the nucleus by a monopartite nuclear localization signal, while efficient nuclear accumulation of HMGB1/5 requires large portions of the basic N-terminal part of the proteins. The acidic C......-terminal domain interferes with nucleolar targeting of HMGB1. Fluorescence recovery after photobleaching experiments revealed that HMGA and HMGB proteins are extremely dynamic in the nucleus, indicating that they bind chromatin only transiently before moving on to the next site, thereby continuously scanning...

  20. ANCLI's white book about the local governance of nuclear activities; Livre blanc de l'ANCLI sur la gouvernance locale des activites nucleaires

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2005-05-15

    This white book is the result of the work carried out by the local commissions of information about nuclear activities (CLI) with the objective of making a common status and recommendations about 4 main topics: the clear definition of the structure and juridical status of CLI's and the acknowledgement of their competence at the local scale, the role and participation of CLI's, and of the national association of CLI's (ANCLI), in the decision processes at the national and international scale, the access of local CLI actors to expertise, and the consideration of ANCLI's opinion in the solutions proposed for the management of radioactive wastes. This last point is taken as an illustration of the previous 3 topics. (J.S.)

  1. Interleaved localized 1H/31P nuclear magnetic resonance spectroscopy of skeletal muscle

    International Nuclear Information System (INIS)

    Nuclear magnetic resonance (NMR) has been used as a spectroscopic method in physics and chemistry before it was developed to become a diagnostic imaging tool in medicine. When NMR spectroscopy is applied to human tissue, metabolism can be studied in normal physiological and pathological states in vivo. Metabolite concentrations and rates can be monitored dynamically and with localization of a defined region of interest. The 'window' which is opened for observation, i.e. which quantities are measured, depends on the nucleus used for RF excitation. Mechanisms of adenosine tri-phosphate (ATP) resynthesis, as a direct source of energy for muscle contraction, are phosphocreatine (PCr) splitting, glycolysis, beta-oxidation and, finally, oxidative phosphorylation. Whilst the dependency of these processes' fractional contribution to muscular energy supply on exercise type and duration is well known, quantitative models of the regulating mechanisms involved are still subject of current research. A large fraction of the established knowledge about metabolism is based on biochemical analysis of tissue acquired invasively (e.g. microdialysis and open-flow microperfusion) or representing averaged metabolic concentrations for the whole body (via serum metabolites or gas exchange analysis). Localized NMR spectroscopy, however, is capable of non-invasively acquiring time-resolved data from a defined volume of interest, in vivo. In contrast to the vast majority of MRS studies investigating metabolism, where spectra of a single nucleus (commonly 1H, 31P or 13C) were acquired or several MR spectra with different nuclei were measured in separate experiments, this work opens an additional 'window' on muscle metabolism by interleaved localized acquisition of 1H and 31P NMR spectra from human calf muscle in vivo, during rest, exercise and recovery, in a single experiment. Using this technique, the time courses of the concentrations of phosphocreatine, inorganic phosphate (Pi), ATP, total

  2. The directionality of the nuclear transport of the influenza A genome is driven by selective exposure of nuclear localization sequences on nucleoprotein

    Directory of Open Access Journals (Sweden)

    Panté Nelly

    2009-06-01

    Full Text Available Abstract Background Early in infection, the genome of the influenza A virus, consisting of eight complexes of RNA and proteins (termed viral ribonucleoproteins; vRNPs, enters the nucleus of infected cells for replication. Incoming vRNPs are imported into the nucleus of infected cells using at least two nuclear localization sequences on nucleoprotein (NP; NLS1 at the N terminus, and NLS2 in the middle of the protein. Progeny vRNP assembly occurs in the nucleus, and later in infection, these are exported from the nucleus to the cytoplasm. Nuclear-exported vRNPs are different from incoming vRNPs in that they are prevented from re-entering the nucleus. Why nuclear-exported vRNPs do not re-enter the nucleus is unknown. Results To test our hypothesis that the exposure of NLSs on the vRNP regulates the directionality of the nuclear transport of the influenza vRNPs, we immunolabeled the two NLSs of NP (NLS1 and NLS2 and analyzed their surface accessibility in cells infected with the influenza A virus. We found that the NLS1 epitope on NP was exposed throughout the infected cells, but the NLS2 epitope on NP was only exposed in the nucleus of the infected cells. Addition of the nuclear export inhibitor leptomycin B further revealed that NLS1 is no longer exposed in cytoplasmic NP and vRNPs that have already undergone nuclear export. Similar immunolabeling studies in the presence of leptomycin B and with cells transfected with the cDNA of NP revealed that the NLS1 on NP is hidden in nuclear exported-NP. Conclusion NLS1 mediates the nuclear import of newly-synthesized NP and incoming vRNPs. This NLS becomes hidden on nuclear-exported NP and nuclear-exported vRNPs. Thus the selective exposure of the NLS1 constitutes a critical mechanism to regulate the directionality of the nuclear transport of vRNPs during the influenza A viral life cycle.

  3. Identification of a nuclear localization motif in the serine/arginine protein kinase PSRPK of physarum polycephalum

    Directory of Open Access Journals (Sweden)

    Tian Shengli

    2009-08-01

    Full Text Available Abstract Background Serine/arginine (SR protein-specific kinases (SRPKs are conserved in a wide range of organisms, from humans to yeast. Studies showed that SRPKs can regulate the nuclear import of SR proteins in cytoplasm, and regulate the sub-localization of SR proteins in the nucleus. But no nuclear localization signal (NLS of SRPKs was found. We isolated an SRPK-like protein PSRPK (GenBank accession No. DQ140379 from Physarum polycephalum previously, and identified a NLS of PSRPK in this study. Results We carried out a thorough molecular dissection of the different domains of the PSRPK protein involved in its nuclear localization. By truncation of PSRPK protein, deletion of and single amino acid substitution in a putative NLS and transfection of mammalian cells, we observed the distribution of PSRPK fluorescent fusion protein in mammalian cells using confocal microscopy and found that the protein was mainly accumulated in the nucleus; this indicated that the motif contained a nuclear localization signal (NLS. Further investigation with truncated PSPRK peptides showed that the NLS (318PKKGDKYDKTD328 was localized in the alkaline Ω-loop of a helix-loop-helix motif (HLHM of the C-terminal conserved domain. If the 318PKKGDK322 sequence was deleted from the loop or K320 was mutated to T320, the PSRPK fluorescent fusion protein could not enter and accumulate in the nucleus. Conclusion This study demonstrated that the 318PKKGDKYDKTD328 peptides localized in the C-terminal conserved domain of PSRPK with the Ω-loop structure could play a crucial role in the NLS function of PSRPK.

  4. Role of a nuclear localization signal on the minor capsid Proteins VP2 and VP3 in BKPyV nuclear entry

    Energy Technology Data Exchange (ETDEWEB)

    Bennett, Shauna M. [Cellular and Molecular Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Zhao, Linbo [Doctoral Program in Cancer Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Bosard, Catherine [Department of Microbiology and Immunology University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Imperiale, Michael J., E-mail: imperial@umich.edu [Cellular and Molecular Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Doctoral Program in Cancer Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Department of Microbiology and Immunology University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States)

    2015-01-01

    BK Polyomavirus (BKPyV) is a ubiquitous nonenveloped human virus that can cause severe disease in immunocompromised populations. After internalization into renal proximal tubule epithelial cells, BKPyV traffics through the ER and enters the cytosol. However, it is unclear how the virus enters the nucleus. In this study, we elucidate a role for the nuclear localization signal located on the minor capsid proteins VP2 and VP3 during infection. Site-directed mutagenesis of a single lysine in the basic region of the C-terminus of the minor capsid proteins abrogated their nuclear localization, and the analogous genomic mutation reduced infectivity. Additionally, through use of the inhibitor ivermectin and knockdown of importin β1, we found that the importin α/β pathway is involved during infection. Overall these data are the first to show the significance of the NLS of the BKPyV minor capsid proteins during infection in a natural host cell. - Highlights: • Polyomaviruses must deliver their genome to the nucleus to replicate. • The minor capsid proteins have a well-conserved nuclear localization signal. • Mutation of this NLS diminishes, but does not completely inhibit, infection.

  5. Spontaneous excision of BAC vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells

    NARCIS (Netherlands)

    Pijlman, G.P.; Schijndel, van J.; Vlak, J.M.

    2003-01-01

    Repeated baculovirus infections in cultured insect cells lead to the generation of defective interfering viruses (DIs), which accumulate at the expense of the intact helper virus and compromise heterologous protein expression. In particular, Autographa californica multicapsid nucleopolyhedovirus (Ac

  6. Efficient, low-cost protein factories: expression of human adenosine deaminase in baculovirus-infected insect larvae.

    OpenAIRE

    Medin, J A; Hunt, L; Gathy, K; Evans, R K; Coleman, M S

    1990-01-01

    Human adenosine deaminase (EC 3.5.4.4), a key purine salvage enzyme essential for immune competence, has been overproduced in Spodoptera frugiperda cells and in Trichoplusia ni (cabbage looper) larvae infected with recombinant baculovirus. The coding sequence of human adenosine deaminase was recombined into a baculovirus immediately downstream from the strong polyhedrin gene promoter. Approximately 60 hr after infection of insect cells with the recombinant virus, maximal levels of intracellul...

  7. A novel bipartite nuclear localization signal guides BPM1 protein to nucleolus suggesting its Cullin3 independent function.

    Directory of Open Access Journals (Sweden)

    Dunja Leljak Levanić

    Full Text Available BPM1 belongs to the MATH-BTB family of proteins, which act as substrate-binding adaptors for the Cullin3-based E3 ubiquitin ligase. MATH-BTB proteins associate with Cullin3 via the BTB domain and with the substrate protein via the MATH domain. Few BPM1-interacting proteins with different functions are recognized, however, specific roles of BPM1, depending on its cellular localization have not been studied so far. Here, we found a novel bipartite nuclear localization signal at the C-terminus of the BPM1 protein, responsible for its nuclear and nucleolar localization and sufficient to drive the green fluorescent protein and cytoplasmic BPM4 protein into the nucleus. Co-localization analysis in live Nicotiana tabacum BY2 cells indicates a Cullin3 independent function since BPM1 localization is predominantly nucleolar and thus devoid of Cullin3. Treatment of BY2 cells with the proteasome inhibitor MG132 blocks BPM1 and Cullin3 degradation, suggesting turnover of both proteins through the ubiquitin-proteasome pathway. Possible roles of BPM1 in relation to its in vivo localization are discussed.

  8. Identification and Characterization of Nuclear Localization Signals within the Nucleocapsid Protein VP15 of White Spot Syndrome Virus

    Institute of Scientific and Technical Information of China (English)

    Li-juan LI; Hua-jun ZHANG; Cong ZHANG; Zheng-li SHI

    2009-01-01

    The nucleocapsid protein VP15 of white spot syndrome virus (WSSV) is a basic DNA-binding protein. Three canonical bipartite nuclear localization signals (NLSs), called NLS1 (aa 11-27), NLS2 (aa 33-49) and NLS3 (44-60), have been detected in this protein, using the ScanProsite computer program. To determine the nuclear localization sequence of VP15, the full-length open reading frame, or the sequence of one of the three NLSs, was fused to the green fluorescent protein (GFP) gene, and transiently expressed in insect Sf9 cells. Transfection with full-length VP15 resulted in GFP fluorescence being distributed exclusively in the nucleus. NLS 1 alone could also direct GFP to the nucleus, but less efficiently. Neither of the other two NLSs (NLS2 and 3) was functional when expressed alone, but exhibited similar activity to NLS1 when they were expressed as a fusion peptide. Furthermore, a mutated VP15, in which the two basic amino acids (11RR12) of NLSI were changed to two alanines (11AA12), caused GFP to be localized only in the cytoplasm of Sf9 cells. These results demonstrated that VP15, as a nuclear localization protein, needs cooperation between its three NLSs, and that the two residues (11RR12) of NLS1 play a key role in transporting the protein to the nucleus.

  9. Detection of yellowhead virus and Chinese baculovirus in penaeid shrimp by the Western blot technique.

    Science.gov (United States)

    Nadala, E C; Tapay, L M; Cao, S; Loh, P C

    1997-12-01

    The continuing threat posed by viral diseases in cultured shrimp calls for the development of detection technologies for monitoring the animals, especially broodstock. Two of the most highly pathogenic viruses of penaeid shrimp are the yellow-head virus (YHV) and Chinese baculovirus (CBV, also called white spot baculovirus). A Western blot (WB) protocol capable of detecting YHV and CBV in the hemolymph of infected shrimp was developed. The use of the hemolymph as material for virus detection allowed for sample collection without sacrificing the animals. This protocol was highly specific, rapid, and sensitive enough to detect the presence of the viruses before the appearance of overt symptoms. It was also useful for demonstrating the growth of both viruses in primary shrimp lymphoid cell cultures.

  10. A Mathematical Model of Baculovirus Infection on Insect Cells at Low Multiplicity of Infection

    Institute of Scientific and Technical Information of China (English)

    You-Hong ZHANG; Josée C. MERCHUK

    2004-01-01

    The expression efficiency of the insect cells-baculovirus system used for insecticidal virus production and the expression of medically useful foreign genes is closely related with the dynamics of infection. The present studies develop a model of the dynamic process of insect cell infection with baculovirus at low multiplicity of infection (MOI), which is based on the multi-infection cycles of insect cell infection at low MOI. A mathematical model for the amount of viruses released from primary infected cells and the amount of free viruses before secondary infected cells release viruses has been developed. Comparison of the simulation results with the experimental data confirms qualitatively that this model is highly reasonable before secondary infected cells release viruses. This model is considered as a base for further modeling the entire complicated infection process.

  11. Multi-region fuzzy logic controller with local PID controllers for U-tube steam generator in nuclear power plant

    OpenAIRE

    Puchalski Bartosz; Duzinkiewicz Kazimierz; Rutkowski Tomasz

    2015-01-01

    In the paper, analysis of multi-region fuzzy logic controller with local PID controllers for steam generator of pressurized water reactor (PWR) working in wide range of thermal power changes is presented. The U-tube steam generator has a nonlinear dynamics depending on thermal power transferred from coolant of the primary loop of the PWR plant. Control of water level in the steam generator conducted by a traditional PID controller which is designed for nominal power level of the nuclear react...

  12. Identification and characterization of a nuclear localization signal of TRIM28 that overlaps with the HP1 box

    International Nuclear Information System (INIS)

    Tripartite motif-containing 28 (TRIM28) is a transcription regulator, which forms a repressor complex containing heterochromatin protein 1 (HP1). Here, we report identification of a nuclear localization signal (NLS) within the 462-494 amino acid region of TRIM28 that overlaps with its HP1 binding site, HP1 box. GST-pulldown experiments revealed the interaction of the arginine-rich TRIM28 NLS with various importin α subtypes (α1, α2 and α4). In vitro transport assay demonstrated that nuclear localization of GFP-TRIM28 NLS is mediated by importin αs, in conjunction with importin β1 and Ran. Further, we demonstrated that HP1 and importin αs compete for binding to TRIM28. Together, our findings suggest that importin α has an essential role in the nuclear delivery and preferential HP1 interaction of TRIM28. - Highlights: • TRIM28 contains an NLS within the 462-494 amino acid region. • The nuclear import of TRIM28 is mediated by importin α/importin β1. • TRIM28 NLS overlaps with HP1 Box. • HP1 and importin α compete for binding to TRIM28

  13. Mapping of the nuclear localization signals in open reading frame 2 protein from porcine circovirus type 1

    Institute of Scientific and Technical Information of China (English)

    Jiangbing Shuai; Wei Wei; Lingli Jiang; Xiaoliang Li; Ning Chen; Weihuan Fang

    2008-01-01

    Porcine circovirus type 1 (PCV1) contains two major open reading frames encoding the replication-associated proteins and the major structural capsid (Cap) protein.PCV1 Cap has an N-terminus carrying several potential monopartite or bipartite nuclear localization signals (NLS).The contribution of these partially overlapping motifs to nuclear importing was identified by expression of mutated PCV1 Cap versions fused to enhanced green fluorescent protein (EGFP).The Cterminus truncated PCV1 Cap-EGFP was localized in nuclei of PK-15 cells similar to the wild-type PCV1 Cap-EGFP,whereas truncation of the N-terminus rendered the fusion protein distributed into cytoplasm,indicating that the nuclear import of PCV1 Cap was efficiently mediated by its N-terminal region.Substitutions of basic residues in stretches 9RRRR12 or the right part of 25RRPYLAHPAFRNRYRWRRK43 resulted in a diffused distribution of the fusion protein in both nuclei and cytoplasm,indicating that the two NLSs were responsible for restricted nuclear targeting of PCV1 Cap.

  14. Identification and characterization of a nuclear localization signal of TRIM28 that overlaps with the HP1 box

    Energy Technology Data Exchange (ETDEWEB)

    Moriyama, Tetsuji; Sangel, Percival [Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka 567-0085 (Japan); Yamaguchi, Hiroki [School of Medicine, Osaka University, Osaka 565-0871 (Japan); Obuse, Chikashi [Graduate School of Life Science, Hokkaido University, Sapporo 001-0021 (Japan); Miyamoto, Yoichi [Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka 567-0085 (Japan); Oka, Masahiro, E-mail: moka@nibiohn.go.jp [Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka 567-0085 (Japan); Laboratory of Biomedical Innovation, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); Yoneda, Yoshihiro, E-mail: y-yoneda@nibiohn.go.jp [National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka 567-0085 (Japan); Laboratory of Biomedical Innovation, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan)

    2015-07-03

    Tripartite motif-containing 28 (TRIM28) is a transcription regulator, which forms a repressor complex containing heterochromatin protein 1 (HP1). Here, we report identification of a nuclear localization signal (NLS) within the 462-494 amino acid region of TRIM28 that overlaps with its HP1 binding site, HP1 box. GST-pulldown experiments revealed the interaction of the arginine-rich TRIM28 NLS with various importin α subtypes (α1, α2 and α4). In vitro transport assay demonstrated that nuclear localization of GFP-TRIM28 NLS is mediated by importin αs, in conjunction with importin β1 and Ran. Further, we demonstrated that HP1 and importin αs compete for binding to TRIM28. Together, our findings suggest that importin α has an essential role in the nuclear delivery and preferential HP1 interaction of TRIM28. - Highlights: • TRIM28 contains an NLS within the 462-494 amino acid region. • The nuclear import of TRIM28 is mediated by importin α/importin β1. • TRIM28 NLS overlaps with HP1 Box. • HP1 and importin α compete for binding to TRIM28.

  15. Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System

    OpenAIRE

    López-Vidal, Javier; Gómez-Sebastián, Silvia; Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; José M Escribano

    2015-01-01

    Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previousl...

  16. A novel baculovirus vector for the production of nonfucosylated recombinant glycoproteins in insect cells

    OpenAIRE

    Mabashi-Asazuma, Hideaki; Kuo, Chu-Wei; Khoo, Kay-Hooi; Jarvis, Donald L

    2013-01-01

    Glycosylation is an important attribute of baculovirus-insect cell expression systems, but some insect cell lines produce core α1,3-fucosylated N-glycans, which are highly immunogenic and render recombinant glycoproteins unsuitable for human use. To address this problem, we exploited a bacterial enzyme, guanosine-5′-diphospho (GDP)-4-dehydro-6-deoxy-d-mannose reductase (Rmd), which consumes the GDP-l-fucose precursor. We expected this enzyme to block glycoprotein fucosylation by blocking the ...

  17. Expression and Characterization of Recombinant Serratia liquefaciens Nucleases Produced with Baculovirus-mediated Silkworm Expression System.

    Science.gov (United States)

    Iiyama, Kazuhiro; Lee, Jae Man; Tatsuke, Tuneyuki; Mon, Hiroaki; Kusakabe, Takahiro

    2016-06-01

    Baculovirus-Bombyx mori protein expression system has mainly been used for translation of eukaryotic proteins. In contrast, information pertaining to bacterial protein expression using this system is not sufficient. Therefore, recombinant nucleases from Serratia liquefaciens (rSlNucAs) were expressed in a Baculovirus-B. mori protein expression system. rSlNucAs containing the native signal peptide (rSlNucA-NSP) or silkworm 30-K signal peptide (rSlNucA-30K) at the NH2-terminus were constructed to enable secretion into the extracellular fraction. Both rSlNucA-30K and rSlNucA-NSP were successfully secreted into hemolymph of B. mori larvae. Affinity-purified rSlNucAs showed high nuclease activity. Optimum pH was 7.5 and half of maximum activity was maintained between pH 7.0 and 9.5. Optimum temperature was 35 °C. rSlNucAs showed sufficient activity in twofold-diluted radioimmunoprecipitation assay buffer and undiluted, mild lysis buffer. Genomic DNA of Escherichia coli was efficiently digested by rSlNucAs in the bacterial lysate. The results in this study suggest that rSlNucAs expressed by the Baculovirus-B. mori protein expression system will be a useful tool in molecular biology. Functional recombinant protein of bacteria was produced by Baculovirus-B. mori protein expression system. This system may be highly suitable for bacterial extracellular protein secreted via Sec pathway. PMID:27059494

  18. Down-regulation of β-catenin Nuclear Localization by Aspirin Correlates with Growth Inhibition of Jurkat Cell Line

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    In this study, we examined the effects of aspirin on the growth rates, subcellar distribution of β-catenin protein, the expression of β-catenin/TCF signaling pathway target gene cyclinD1 mRNA,and cell cycle of Jurkat cell line (Human T-acute lymphoblastic leukemia). Our results showed that the treatment with aspirin inhibited the growth of Jurkat cell line. Jurkat cells treated with 3 mmol/L of aspirin could significantly decrease nuclear localization of β-catenin, and at 5 mmol/L of aspirin,the nuclear localization of β-catenin was undetectable. QRT-PCR showed that the target gene cyclinD1 mRNA expression was gradually decreased with the dosage of aspirin. Aspirin induced G0/G1cell cycle arrest in Jurkat cells. We are led to conclude that aspirin acts through β-catenin-independent mechanisms. The effects of aspirin include down-regulation of β-catenin nuclear localization and G0/G1 cell cycle arrest, which might serve as a means of growth inhibition in aspirin-treated human Jurkat cell line.

  19. Ultra Deep Sequencing of a Baculovirus Population Reveals Widespread Genomic Variations

    Directory of Open Access Journals (Sweden)

    Aurélien Chateigner

    2015-07-01

    Full Text Available Viruses rely on widespread genetic variation and large population size for adaptation. Large DNA virus populations are thought to harbor little variation though natural populations may be polymorphic. To measure the genetic variation present in a dsDNA virus population, we deep sequenced a natural strain of the baculovirus Autographa californica multiple nucleopolyhedrovirus. With 124,221X average genome coverage of our 133,926 bp long consensus, we could detect low frequency mutations (0.025%. K-means clustering was used to classify the mutations in four categories according to their frequency in the population. We found 60 high frequency non-synonymous mutations under balancing selection distributed in all functional classes. These mutants could alter viral adaptation dynamics, either through competitive or synergistic processes. Lastly, we developed a technique for the delimitation of large deletions in next generation sequencing data. We found that large deletions occur along the entire viral genome, with hotspots located in homologous repeat regions (hrs. Present in 25.4% of the genomes, these deletion mutants presumably require functional complementation to complete their infection cycle. They might thus have a large impact on the fitness of the baculovirus population. Altogether, we found a wide breadth of genomic variation in the baculovirus population, suggesting it has high adaptive potential.

  20. Infection, transfection, and co-transfection of baculoviruses by microprojectile bombardment of larvae.

    Science.gov (United States)

    Obregón-Barboza, Verónica; Del Rincón-Castro, Ma Cristina; Cabrera-Ponce, José L; Ibarra, Jorge E

    2007-03-01

    The use of baculoviruses as expression vectors for heterologous proteins has been practically limited to the use of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). In this work, infection, transfection and co-transfection events with the baculoviruses AcMNPV and Trichoplusia ni granulovirus (TnGV) were accomplished by bombardment of T. ni first-instar larvae with microprojectiles coated with virions, viral DNA, and viral DNA and a transfer vector, respectively. A series of shooting conditions were tested until positive results were obtained. The use of 1.6 microm gold particles at 900 psi shooting pressure, 400 Torr vacuum, 7 cm distance to target, on sets of 20 first-instar larvae held in a 16 mm diameter container, proved to be the best shooting conditions. Typical infection symptoms were shown by larvae when shot with viruses or viral DNA from AcMNPV or TnGV. Co-transfected recombinant AcMNPV and TnGV were identified by the formation of occlusion bodies and GFP, respectively, in bombarded larvae. This technique opens a wide range of possibilities, not only to use an extensive number of baculoviruses as expression vectors for heterologous proteins, but also be used to infect, transfect or co-transfect a wide variety of viruses into animal cells. PMID:17184851

  1. Tropomyosin is localized in the nuclear matrix and chromosome scaffold of physarum polycephalum

    Institute of Scientific and Technical Information of China (English)

    ZENGXIANLU; XIAOGUANGWANG; 等

    1999-01-01

    The nuclei and chromosomes were isolated from plasmodia of Physarum polycephalum.The nuclear matrix and chromosome scaffold were obtained after the DNA and most of the proteins were extracted with DNase I and 2 M NaCl.SD-PAGE analyses revealed that the nuclear matrix and chromosome scaffold contained a 37 kD polypeptide which is equivalent to tropomyosin in molecular weight.Immunofluorescence observations upon slide preparations labeled with anti-tropomyosin antibody showed that the nuclear matrix and chromosome scaffold emanated bright fluorescence,suggesting the presence of the antigen in them.Immunodotting results confirmed the presence of tropomyosin in the nuclear matrix and chromosome scaffold.Immunoelectron microscopic observations further demonstrated that tropomyosin was dispersively distributed in the interphase nuclei and metaphase chromosomes.

  2. The dynamic polarization potential and dynamical non-locality in nuclear potentials: Deuteron-nucleus potential

    CERN Document Server

    Mackintosh, R S

    2016-01-01

    The consequences for direct reactions of the dynamical non-locality generated by the excitation of the target and projectile are much less studied than the effects of non-locality arising from exchange processes. Here we are concerned with the dynamical non-locality due to projectile excitation in deuteron induced reactions. The consequences of this non-locality can be studied by the comparison of deuteron induced direct reactions calculated with alternative representations of the elastic channel wave functions: (i) the elastic channel wave functions from coupled channel (CC) calculations involving specific reaction processes, and, (ii) elastic channel wave functions calculated from local potentials that exactly reproduce the elastic scattering $S$-matrix from the same CC calculations. In this work we produce the local equivalent deuteron potentials required for the study of direct reactions involving deuterons. These will enable the study of the effects of dynamical non-locality following a method previously...

  3. Troponin T nuclear localization and its role in aging skeletal muscle

    OpenAIRE

    Zhang, Tan; Birbrair, Alexander; Wang, Zhong-Min; Taylor, Jackson; Messi, María Laura; Delbono, Osvaldo

    2011-01-01

    Troponin T (TnT) is known to mediate the interaction between Tn complex and tropomyosin (Tm), which is essential for calcium-activated striated muscle contraction. This regulatory function takes place in the myoplasm, where TnT binds Tm. However, recent findings of troponin I and Tm nuclear translocation in Drosophila and mammalian cells imply other roles for the Tn–Tm complex. We hypothesized that TnT plays a nonclassical role through nuclear translocation. Immunoblotting with different anti...

  4. Nuclear factor-kappa B localization and function within intrauterine tissues from term and preterm labor and cultured fetal membranes

    Directory of Open Access Journals (Sweden)

    Kusanovic Juan P

    2010-01-01

    Full Text Available Abstract Background The objective of this study was to quantify the nuclear localization and DNA binding activity of p65, the major transactivating nuclear factor-kappa B (NF-kappaB subunit, in full-thickness fetal membranes (FM and myometrium in the absence or presence of term or preterm labor. Methods Paired full-thickness FM and myometrial samples were collected from women in the following cohorts: preterm no labor (PNL, N = 22, spontaneous preterm labor (PTL, N = 21, term no labor (TNL, N = 23, and spontaneous term labor (STL, N = 21. NF-kappaB p65 localization was assessed by immunohistochemistry, and DNA binding activity was evaluated using an enzyme-linked immunosorbent assay (ELISA-based method. Results Nuclear p65 labeling was rare in amnion and chorion, irrespective of clinical context. In decidua, nuclear p65 labeling was greater in the STL group relative to the TNL cohort, but there were no differences among the TNL, PTL, and PNL cohorts. In myometrium, diffuse p65 nuclear labeling was significantly associated with both term and preterm labor. There were no significant differences in ELISA-based p65 binding activity in amnion, choriodecidual, and myometrial specimens in the absence or presence of term labor. However, parallel experiments using cultured term fetal membranes demonstrated high levels of p65-like binding even the absence of cytokine stimulation, suggesting that this assay may be of limited value when applied to tissue specimens. Conclusions These results suggest that the decidua is an important site of NF-kappaB regulation in fetal membranes, and that mechanisms other than cytoplasmic sequestration may limit NF-kappaB activation prior to term.

  5. Functional analysis of the baculovirus 10 kilodalton protein.

    NARCIS (Netherlands)

    Oers, van M.M.

    1994-01-01

    Nuclear polyhedrosis viruses belong to the family Baculoviridae and cause fatal diseases in arthropods predominantly in insects of the order Lepidoptera . These viruses were first reported in silk worms where viral infections could have devastating effects on the production of silk. In nature these

  6. A nuclear localization of the infectious haematopoietic necrosis virus NV protein is necessary for optimal viral growth.

    Directory of Open Access Journals (Sweden)

    Myeong Kyu Choi

    Full Text Available The nonvirion (NV protein of infectious hematopoietic necrosis virus (IHNV has been previously reported to be essential for efficient growth and pathogenicity of IHNV. However, little is known about the mechanism by which the NV supports the viral growth. In this study, cellular localization of NV and its role in IHNV growth in host cells was investigated. Through transient transfection in RTG-2 cells of NV fused to green fluorescent protein (GFP, a nuclear localization of NV was demonstrated. Deletion analyses showed that the (32EGDL(35 residues were essential for nuclear localization of NV protein, and fusion of these 4 amino acids to GFP directed its transport to the nucleus. We generated a recombinant IHNV, rIHNV-NV-ΔEGDL in which the (32EGDL(35 was deleted from the NV. rIHNVs with wild-type NV (rIHNV-NV or with the NV gene replaced with GFP (rIHNV-ΔNV-GFP were used as controls. RTG-2 cells infected with rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL yielded 12- and 5-fold less infectious virion, respectively, than wild type rIHNV-infected cells at 48 h post-infection (p.i.. While treatment with poly I∶C at 24 h p.i. did not inhibit replication of wild-type rIHNVs, replication rates of rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL were inhibited by poly I∶C. In addition, both rIHNV-ΔNV and rIHNV-NV-ΔEGDL induced higher levels of expressions of both IFN1 and Mx1 than wild-type rIHNV. These data suggest that the IHNV NV may support the growth of IHNV through inhibition of the INF system and the amino acid residues of (32EGDL(35 responsible for nuclear localization are important for the inhibitory activity of NV.

  7. A nuclear localization of the infectious haematopoietic necrosis virus NV protein is necessary for optimal viral growth

    Science.gov (United States)

    Choi, M.K.; Moon, C.H.; Ko, M.S.; Lee, U.-H.; Cho, W.J.; Cha, S.J.; Do, J.W.; Heo, G.J.; Jeong, S.G.; Hahm, Y.S.; Harmache, A.; Bremont, M.; Kurath, G.; Park, J.-W.

    2011-01-01

    The nonvirion (NV) protein of infectious hematopoietic necrosis virus (IHNV) has been previously reported to be essential for efficient growth and pathogenicity of IHNV. However, little is known about the mechanism by which the NV supports the viral growth. In this study, cellular localization of NV and its role in IHNV growth in host cells was investigated. Through transient transfection in RTG-2 cells of NV fused to green fluorescent protein (GFP), a nuclear localization of NV was demonstrated. Deletion analyses showed that the 32EGDL35 residues were essential for nuclear localization of NV protein, and fusion of these 4 amino acids to GFP directed its transport to the nucleus. We generated a recombinant IHNV, rIHNV-NV-ΔEGDL in which the 32EGDL35 was deleted from the NV. rIHNVs with wild-type NV (rIHNV-NV) or with the NV gene replaced with GFP (rIHNV-ΔNV-GFP) were used as controls. RTG-2 cells infected with rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL yielded 12- and 5-fold less infectious virion, respectively, than wild type rIHNV-infected cells at 48 h post-infection (p.i.). While treatment with poly I:C at 24 h p.i. did not inhibit replication of wild-type rIHNVs, replication rates of rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL were inhibited by poly I:C. In addition, both rIHNV-ΔNV and rIHNV-NV-ΔEGDL induced higher levels of expressions of both IFN1 and Mx1 than wild-type rIHNV. These data suggest that the IHNV NV may support the growth of IHNV through inhibition of the INF system and the amino acid residues of 32EGDL35 responsible for nuclear localization are important for the inhibitory activity of NV.

  8. Dynamic nuclear polarization enhanced nuclear magnetic resonance and electron spin resonance studies of hydration and local water dynamics in micelle and vesicle assemblies.

    Science.gov (United States)

    McCarney, Evan R; Armstrong, Brandon D; Kausik, Ravinath; Han, Songi

    2008-09-16

    We present a unique analysis tool for the selective detection of local water inside soft molecular assemblies (hydrophobic cores, vesicular bilayers, and micellar structures) suspended in bulk water. Through the use of dynamic nuclear polarization (DNP), the (1)H NMR signal of water is amplified, as it interacts with stable radicals that possess approximately 658 times higher spin polarization. We utilized stable nitroxide radicals covalently attached along the hydrophobic tail of stearic acid molecules that incorporate themselves into surfactant-based micelle or vesicle structures. Here, we present a study of local water content and fluid viscosity inside oleate micelles and vesicles and Triton X-100 micelles to serve as model systems for soft molecular assemblies. This approach is unique because the amplification of the NMR signal is performed in bulk solution and under ambient conditions with site-specific spin labels that only detect the water that is directly interacting with the localized spin labels. Continuous wave (cw) electron spin resonance (ESR) analysis provides rotational dynamics of the spin-labeled molecular chain segments and local polarity parameters that can be related to hydration properties, whereas we show that DNP-enhanced (1)H NMR analysis of fluid samples directly provides translational water dynamics and permeability of the local environment probed by the spin label. Our technique therefore has the potential to become a powerful analysis tool, complementary to cw ESR, to study hydration characteristics of surfactant assemblies, lipid bilayers, or protein aggregates, where water dynamics is a key parameter of their structure and function. In this study, we find that there is significant penetration of water inside the oleate micelles with a higher average local water viscosity (approximately 1.8 cP) than in bulk water, and Triton X-100 micelles and oleate vesicle bilayers mostly exclude water while allowing for considerable surfactant chain

  9. A novel role for the nuclear localization signal in regulating hnRNP K protein stability in vivo.

    Science.gov (United States)

    Hutchins, Erica J; Belrose, Jamie L; Szaro, Ben G

    2016-09-16

    hnRNP K is a highly conserved nucleocytoplasmic shuttling protein, which associates with RNAs through synergistic binding via its three KH domains. hnRNP K is required for proper nuclear export and translational control of its mRNA targets, and these processes are controlled by hnRNP K's movement between subcellular compartments. Whereas the nuclear export and localization of hnRNP K that is associated with mRNP complexes has been well studied, the trafficking of hnRNP K that is unbound to mRNA has yet to be elucidated. To that end, we expressed an EGFP-tagged RNA binding-defective form of hnRNP K in intact Xenopus embryos, and found it was rapidly degraded in vivo. Deleting hnRNP K's nuclear localization signal (NLS), which contains two prospective ubiquitination sites, rescued the protein from degradation. These data demonstrate a novel activity for the NLS of hnRNP K in regulating the protein's stability in vivo when it is unbound to nucleic acids.

  10. Growing from a local into an international nuclear services provider - challenges and opportunities

    International Nuclear Information System (INIS)

    Apart from nuclear power plants' (NPPs) internally staffed expert resources, the support of qualified external nuclear maintenance and modification services providers (NSPs) is extremely important. The postponement of the nuclear power renaissance along with an aging workforce is pushing the industry to search for different approaches to maintain and improve the required level of expertise and quality. Operating in a domestic market with only one NPP unit, the leading Slovenian NSP has been partnering with complimentary companies to jointly satisfy the needs of the domestic, US and EU customers. By remaining competitive in quality and price and sharing resources, global experience and good practices, results in added value to all of the interested parties is achieved. In addition to supporting peak outage season shortages of qualified resources, the concept also aids in load-levelling the resource needs throughout the year. The challenges and the opportunities related to the concept are discussed in the paper. (authors)

  11. Baculovirus-mediated Expression of p35 Confers Resistance to Apoptosis in Human Embryo Kidney 293 cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Baculovirus has many advantages as vectors for gene transfer. We demonstrated that recombinant baculovirus vectors expressing p35 (Ac-CMV-p35) and eGFP (Ac-CMV-GFP) could be transduced into human kidney 293 cells efficiently. The level of transgene expression was viral dose dependent and high-level expression of the target gene could be achieved under the heterogonous promoter. MTT assay suggested that both Ac-CMV-p35 and Ac-CMV-GFP did not have cytotoxic effect on human embryo kidney 293 cells. Cell growth curve showed the Ac-CMV-p35 and Ac- CMV-GFP transduced and non-transduced cells had similar proliferation rate, so baculovirus-mediated p35expression had no adverse effect on cell proliferation. In addition, baculovirus-mediated p35 gene expression protected human embryo kidney 293 cells against apoptosis induced by various apoptosis inducers such as Actinomycin D, UV or serum-free media. These results suggested that the baculovirus vector mediated p35 gene expression was functional and it could be widely used in molecular research and even gene therapy.

  12. Localization of a bacterial group II intron-encoded protein in eukaryotic nuclear splicing-related cell compartments.

    Directory of Open Access Journals (Sweden)

    Rafael Nisa-Martínez

    Full Text Available Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.

  13. Casein kinase 2 (CK2) phosphorylates the deubiquitylase OTUB1 at Ser16 to trigger its nuclear localization.

    Science.gov (United States)

    Herhaus, Lina; Perez-Oliva, Ana B; Cozza, Giorgio; Gourlay, Robert; Weidlich, Simone; Campbell, David G; Pinna, Lorenzo A; Sapkota, Gopal P

    2015-01-01

    The deubiquitylating enzyme OTUB1 is present in all tissues and targets many substrates, in both the cytosol and nucleus. We found that casein kinase 2 (CK2) phosphorylated OTUB1 at Ser(16) to promote its nuclear accumulation in cells. Pharmacological inhibition or genetic ablation of CK2 blocked the phosphorylation of OTUB1 at Ser(16), causing its nuclear exclusion in various cell types. Whereas we detected unphosphorylated OTUB1 mainly in the cytosol, we detected Ser(16)-phosphorylated OTUB1 only in the nucleus. In vitro, Ser(16)-phosphorylated OTUB1 and nonphosphorylated OTUB1 exhibited similar catalytic activity, bound K63-linked ubiquitin chains, and interacted with the E2 enzyme UBE2N. CK2-mediated phosphorylation and subsequent nuclear localization of OTUB1 promoted the formation of 53BP1 (p53-binding protein 1) DNA repair foci in the nucleus of osteosarcoma cells exposed to ionizing radiation. Our findings indicate that the activity of CK2 is necessary for the nuclear translocation and subsequent function of OTUB1 in DNA damage repair. PMID:25872870

  14. Mutation of the rice XA21 predicted nuclear localization sequence does not affect resistance to Xanthomonas oryzae pv. oryzae

    Science.gov (United States)

    Ho, Yuen Ting

    2016-01-01

    Background The rice receptor kinase XA21 confers robust resistance to the bacterial pathogen Xanthomonas oryzaepv. oryzae(Xoo). We previously reported that XA21 is cleaved in transgenic plants overexpressing XA21 with a GFP tag (Ubi-XA21-GFP) and that the released C-terminal domain is localized to the nucleus. XA21 carries a predicted nuclear localization sequence (NLS) that directs the C-terminal domain to the nucleus in transient assays, whereas alanine substitutions in the NLS disrupt the nuclear localization. Methods To determine if the predicted NLS is required for XA21-mediated immunity in planta, we generated transgenic plants overexpressing an XA21 variant carrying the NLS with the same alanine substitutions (Ubi-XA21nls-GFP). Results Ubi-XA21nls-GFP plants displayed slightly longer lesion lengths, higher Xoobacterial populations after inoculation and lower levels of reactive oxygen species production compared with the Ubi-XA21-GFP control plants. However, the Ubi-XA21nls-GFP plants express lower levels of protein than that observed in Ubi-XA21-GFP. Discussion These results demonstrate that the predicted NLS is not required for XA21-mediated immunity.

  15. Multi-region fuzzy logic controller with local PID controllers for U-tube steam generator in nuclear power plant

    Directory of Open Access Journals (Sweden)

    Puchalski Bartosz

    2015-12-01

    Full Text Available In the paper, analysis of multi-region fuzzy logic controller with local PID controllers for steam generator of pressurized water reactor (PWR working in wide range of thermal power changes is presented. The U-tube steam generator has a nonlinear dynamics depending on thermal power transferred from coolant of the primary loop of the PWR plant. Control of water level in the steam generator conducted by a traditional PID controller which is designed for nominal power level of the nuclear reactor operates insufficiently well in wide range of operational conditions, especially at the low thermal power level. Thus the steam generator is often controlled manually by operators. Incorrect water level in the steam generator may lead to accidental shutdown of the nuclear reactor and consequently financial losses. In the paper a comparison of proposed multi region fuzzy logic controller and traditional PID controllers designed only for nominal condition is presented. The gains of the local PID controllers have been derived by solving appropriate optimization tasks with the cost function in a form of integrated squared error (ISE criterion. In both cases, a model of steam generator which is readily available in literature was used for control algorithms synthesis purposes. The proposed multi-region fuzzy logic controller and traditional PID controller were subjected to broad-based simulation tests in rapid prototyping software - Matlab/Simulink. These tests proved the advantage of multi-region fuzzy logic controller with local PID controllers over its traditional counterpart.

  16. The estrogen receptor alpha nuclear localization sequence is critical for fulvestrant-induced degradation of the receptor.

    Science.gov (United States)

    Casa, Angelo J; Hochbaum, Daniel; Sreekumar, Sreeja; Oesterreich, Steffi; Lee, Adrian V

    2015-11-01

    Fulvestrant, a selective estrogen receptor down-regulator (SERD) is a pure competitive antagonist of estrogen receptor alpha (ERα). Fulvestrant binds ERα and reduces the receptor's half-life by increasing protein turnover, however, its mechanism of action is not fully understood. In this study, we show that removal of the ERα nuclear localization sequence (ERΔNLS) resulted in a predominantly cytoplasmic ERα that was degraded in response to 17-β-estradiol (E2) but was resistant to degradation by fulvestrant. ERΔNLS bound the ligands and exhibited receptor interaction similar to ERα, indicating that the lack of degradation was not due to disruption of these processes. Forcing ERΔNLS into the nucleus with a heterologous SV40-NLS did not restore degradation, suggesting that the NLS domain itself, and not merely receptor localization, is critical for fulvestrant-induced ERα degradation. Indeed, cloning of the endogenous ERα NLS onto the N-terminus of ERΔNLS significantly restored both its nuclear localization and turnover in response to fulvestrant. Moreover, mutation of the sumoylation targets K266 and K268 within the NLS impaired fulvestrant-induced ERα degradation. In conclusion, our study provides evidence for the unique role of the ERα NLS in fulvestrant-induced degradation of the receptor.

  17. Localization of phosphorylated TrkA in carrier vesicles involved in its nuclear translocation in U251 cell line

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A number of transmembrane receptors are targeted to the nucleus and convincingly localized therein. However, what remains a conundrum is how these cell-surface receptors end up in the nucleus. In this study, we reported that the transmembrane receptor phosphorylated TrkA was located in a series of carrier vesicles, including ring-like vesicles near the plasma membrane, large core vesicles and small dense core vesicles around the nuclei, as well as in the nucleus in human glioma cell line U251 using immunocytochemistry and immunofluorescence staining. Meanwhile, we also showed that small dense core vesicles budded from large core vesicles, and interacted with the nuclear envelope. Accordingly, our results suggested that such a series of membrane compartments might be involved in the pathway of nuclear translocation of the transmembrane receptor TrkA.

  18. Localization of phosphorylated TrkA in carrier vesicles involved in its nuclear translocation in U251 cell line

    Institute of Scientific and Technical Information of China (English)

    GONG AiHua; ZHANG ZhiJian; XIAO DeSheng; YANG Yong; WANG YongZhong; CHEN YongChang

    2007-01-01

    A number of transmembrane receptors are targeted to the nucleus and convincingly localized therein.However, what remains a conundrum is how these cell-surface receptors end up in the nucleus. In this study, we reported that the transmembrane receptor phosphorylated TrkA was located in a series of carrier vesicles, including ring-like vesicles near the plasma membrane, large core vesicles and small dense core vesicles around the nuclei, as well as in the nucleus in human glioma cell line U251 using immunocytochemistry and immunofluorescence staining. Meanwhile, we also showed that small dense core vesicles budded from large core vesicles, and interacted with the nuclear envelope. Accordingly,our results suggested that such a series of membrane compartments might be involved in the pathway of nuclear translocation of the transmembrane receptor TrkA.

  19. Calmodulin Involvement in Stress-Activated Nuclear Localization of Albumin in JB6 Epithelial Cells.

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Thomas J.; Negash, Sewite; Smallwood, Heather S.; Ramos, Kenneth S.; Thrall, Brian D.; Squier, Thomas C.

    2004-06-15

    We report that in response to oxidative stress, albumin is translocated to the nucleus where it binds in concert with known transcription factors to an antioxidant response element (ARE), which controls the expression of glutathione-S-transferase and other antioxidant enzymes, functioning to mediate adaptive cellular responses. To investigate the mechanisms underlying this adaptive cell response, we have identified linkages between calcium signaling and the nuclear translocation of albumin in JB6 epithelial cells. Under resting conditions, albumin and the calcium regulatory protein, calmodulin (CaM), co-immunoprecipitate using antibodies against either protein, indicating a tight association. Calcium activation of CaM disrupts the association between CaM and albumin, suggesting that transient increases in cytosolic calcium levels function to mobilize intracellular albumin to facilitate its translocation into the nucleus. Likewise, nuclear translocation of albumin is induced by exposure of cells to hydrogen peroxide or a phorbol ester, indicating a functional linkage between reactive oxygen species, calcium, and PKC-signaling pathways. Inclusion of an antioxidant enzyme (i.e., superoxide dismutase) blocks nuclear translocation, suggesting that the oxidation of sensitive proteins functions to coordinate the adaptive cellular response. These results suggest that elevated calcium transients, and associated increases in reactive oxygen species, contribute to adaptive cellular responses through the mobilization and nuclear translocation of cellular albumin to mediate the transcriptional regulation of antioxidant responsive elements.

  20. Analysis of nucleolar morphology and protein localization as an indicator of nuclear reprogramming

    DEFF Research Database (Denmark)

    Østrup, Olga; Pedersen, Hanne Skovsgaard; Holm, Hanne M.;

    2015-01-01

    to cloning by somatic cell nuclear transfer. However, when cells are reprogrammed by less fundamental means, as for example treatment by Xenopus extract or expression of pluripotency genes, more subtle nucleolar modulations can also be noted. The monitoring and understanding of the reprogramming...

  1. Localization of influenza virus proteins to nuclear dot 10 structures in influenza virus-infected cells

    International Nuclear Information System (INIS)

    We studied influenza virus M1 protein by generating HeLa and MDCK cell lines that express M1 genetically fused to green fluorescent protein (GFP). GFP-M1 was incorporated into virions produced by influenza virus infected MDCK cells expressing the fusion protein indicating that the fusion protein is at least partially functional. Following infection of either HeLa or MDCK cells with influenza A virus (but not influenza B virus), GFP-M1 redistributes from its cytosolic/nuclear location and accumulates in nuclear dots. Immunofluorescence revealed that the nuclear dots represent nuclear dot 10 (ND10) structures. The colocalization of authentic M1, as well as NS1 and NS2 protein, with ND10 was confirmed by immunofluorescence following in situ isolation of ND10. These findings demonstrate a previously unappreciated involvement of influenza virus with ND10, a structure involved in cellular responses to immune cytokines as well as the replication of a rapidly increasing list of viruses

  2. Localization of nuclear retained mRNAs in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Thomsen, Rune; Libri, Domenico; Boulay, Jocelyne;

    2003-01-01

    In the yeast Saccharomyces cerevisiae, a common conditional phenotype associated with deletion or mutation of genes encoding mRNA export factors is the rapid accumulation of mRNAs in intranuclear foci, suggested to be near transcription sites. The nuclear RNA exosome has been implicated in retain...

  3. Participation of local citizens groups in the Swedish nuclear waste process

    Energy Technology Data Exchange (ETDEWEB)

    Holmstrand, O. [The Waste Network, Lerum (Sweden)

    1999-12-01

    The Waste Network's conclusions and views on the nuclear waste issue are summarised in the following points: The management of the nuclear waste is not solved. In order to minimise the amount of waste, the further operating of nuclear reactors should be questioned. The choice of method should be made before the siting. The deadlock to the KBS method must come to an end. The choice of method must be based on clearly expressed functional conditions formulated in advance. The siting must be based on considerations to environment and security, not political acceptance. The pilot studies in municipalities must cease and should be replaced by a clear and understandable sieving process at a national scale. An independent authority must control and supervise the EIA process instead of the nuclear industry. A well performed EIA process is the necessary condition to give the choice of method and site enough legitimacy and acceptance. Environmental organisations being the representatives of the public must be given reasonable conditions and resources to take part in the EIA process to handle the question.

  4. Nuclear-localized Calcineurin Homologous Protein CHP1 Interacts with Upstream Binding Factor and Inhibits Ribosomal RNA Synthesis*

    OpenAIRE

    Jiménez-Vidal, Maite; Srivastava, Jyoti; Putney, Luanna K; Barber, Diane L.

    2010-01-01

    Calcineurin homologous protein 1 (CHP1) is a widely expressed, 22-kDa myristoylated EF-hand Ca2+-binding protein that shares a high degree of similarity with the regulatory B subunit of calcineurin (65%) and with calmodulin (59%). CHP1 localizes to the plasma membrane, the Golgi apparatus, and the nucleus and functions to regulate trafficking of early secretory vesicles, activation of T cells, and expression and transport of the Na-H exchanger NHE1. Although CHP1 contains nuclear export signa...

  5. Review study 1995. Localization of the repository for spent nuclear fuel; Oeversiktsstudie 95. Lokalisering av djupfoervar foer anvaent kaernbraensle

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-10-01

    This report gives an overview of the studies performed by SKB pertinent to selection of a site for the Swedish repository for spent nuclear fuels, and is written for both experts in the various fields involved, decision-makers and the interested general public. The review can not comprise all detailed factors necessary for deciding the localization, but deals mainly with conditions on the land surface and can point out areas which are not well suited or less interesting as a site. It also treats several scientific, technical and social bases in different parts of the country. 120 refs, 53 figs.

  6. Characterization of amino acid residues within the N-terminal region of Ubc9 that play a role in Ubc9 nuclear localization

    International Nuclear Information System (INIS)

    As the sole E2 enzyme for SUMOylation, Ubc9 is predominantly nuclear. However, the underlying mechanisms of Ubc9 nuclear localization are still not well understood. Here we show that RNAi-depletion of Imp13, an importin known to mediate Ubc9 nuclear import, reduces both Ubc9 nuclear accumulation and global SUMOylation. Furthermore, Ubc9-R13A or Ubc9-H20D mutation previously shown to interrupt the interaction of Ubc9 with nucleus-enriched SUMOs reduces the nuclear enrichment of Ubc9, suggesting that the interaction of Ubc9 with the nuclear SUMOs may enhance Ubc9 nuclear retention. Moreover, Ubc9-R17E mutation, which is known to disrupt the interaction of Ubc9 with both SUMOs and Imp13, causes a greater decrease in Ubc9 nuclear accumulation than Ubc9-R13A or Ubc9-H20D mutation. Lastly, Ubc9-K74A/S89D mutations that perturb the interaction of Ubc9 with nucleus-enriched SUMOylation-consensus motifs has no effect on Ubc9 nuclear localization. Altogether, our results have elucidated that the amino acid residues within the N-terminal region of Ubc9 play a pivotal role in regulation of Ubc9 nuclear localization. - Highlights: • Imp13-mediated nuclear import of Ubc9 is critical for global SUMOylation. • Ubc9 mutations disrupting Ubc9-SUMO interaction decrease Ubc9 nuclear accumulation. • N-terminal amino acid residues of Ubc9 are critical for Ubc9 nuclear enrichment

  7. Characterization of amino acid residues within the N-terminal region of Ubc9 that play a role in Ubc9 nuclear localization

    Energy Technology Data Exchange (ETDEWEB)

    Sekhri, Palak [Department of Biological Sciences, Wayne State University, 5947 Gullen Mall, Detroit, MI 48202 (United States); Tao, Tao [School of Life Sciences, Xiamen University, Xiamen (China); Kaplan, Feige [Department of Human Genetics, McGill University, Montreal (Canada); Zhang, Xiang-Dong, E-mail: xzhang@wayne.edu [Department of Biological Sciences, Wayne State University, 5947 Gullen Mall, Detroit, MI 48202 (United States)

    2015-02-27

    As the sole E2 enzyme for SUMOylation, Ubc9 is predominantly nuclear. However, the underlying mechanisms of Ubc9 nuclear localization are still not well understood. Here we show that RNAi-depletion of Imp13, an importin known to mediate Ubc9 nuclear import, reduces both Ubc9 nuclear accumulation and global SUMOylation. Furthermore, Ubc9-R13A or Ubc9-H20D mutation previously shown to interrupt the interaction of Ubc9 with nucleus-enriched SUMOs reduces the nuclear enrichment of Ubc9, suggesting that the interaction of Ubc9 with the nuclear SUMOs may enhance Ubc9 nuclear retention. Moreover, Ubc9-R17E mutation, which is known to disrupt the interaction of Ubc9 with both SUMOs and Imp13, causes a greater decrease in Ubc9 nuclear accumulation than Ubc9-R13A or Ubc9-H20D mutation. Lastly, Ubc9-K74A/S89D mutations that perturb the interaction of Ubc9 with nucleus-enriched SUMOylation-consensus motifs has no effect on Ubc9 nuclear localization. Altogether, our results have elucidated that the amino acid residues within the N-terminal region of Ubc9 play a pivotal role in regulation of Ubc9 nuclear localization. - Highlights: • Imp13-mediated nuclear import of Ubc9 is critical for global SUMOylation. • Ubc9 mutations disrupting Ubc9-SUMO interaction decrease Ubc9 nuclear accumulation. • N-terminal amino acid residues of Ubc9 are critical for Ubc9 nuclear enrichment.

  8. Aberrant nuclear localization of β-catenin without genetic alterations in β-catenin or Axin genes in esophageal cancer

    Directory of Open Access Journals (Sweden)

    Shinoda Noriyuki

    2007-02-01

    Full Text Available Abstract Background β-catenin is a multifunctional protein involved in two apparently independent processes: cell-cell adhesion and signal transduction. β-catenin is involved in Wnt signaling pathway that regulates cellular differentiation and proliferation. In this study, we investigated the expression pattern of β-catenin and cyclin D1 using immunohistochemistry and searched for mutations in exon 3 of the β-catenin gene and Axin gene in esophageal squamous cell carcinoma. Materials and methods Samples were obtained from 50 esophageal cancer patients. Immunohistochemical staining for β-catenin and cyclin D1 was done. Mutational analyses of the exon3 of the β-catenin gene and Axin gene were performed on tumors with nuclear β-catenin expression. Results Four (8% esophageal cancer tissues showed high nuclear β-catenin staining. Overexpression of cyclin D1 was observed in 27 out of 50 (54% patients. All four cases that showed nuclear β-catenin staining overexpressed cyclin D1. No relationship was observed between the expression pattern of β-catenin and cyclin D1 and age, sex, tumor size, stage, differentiation grade, lymph node metastasis, response to chemotherapy, or survival. No mutational change was found in β-catenin exon 3 in the four cases with nuclear β-catenin staining. Sequencing analysis of the Axin cDNA revealed only a splicing variant (108 bp deletion, position 2302–2409 which was present in the paired normal mucosa. Conclusion A fraction of esophageal squamous cell carcinomas have abnormal nuclear accumulation of β-catenin accompanied with increased cyclin D1 expression. Mutations in β-catenin or axin genes are not responsible for this abnormal localization of β-catenin.

  9. Localized Beampipe Heating due to $e^-$ Capture and Nuclear Excitation in Heavy Ion Colliders

    OpenAIRE

    Klein, Spencer R.

    2000-01-01

    At heavy ion colliders, two major sources of beam loss are expected to be $e^+e^-$ production, where the $e^-$ is bound to one of the nuclei, and photonuclear excitation and decay via neutron emission. Both processes alter the ions charged to mass ratio by well defined amounts, creating beams of particles with altered magnetic rigidity. These beams will deposit their energy in a localized region of the accelerator, causing localized heating, The size of the target region depends on the collid...

  10. Phenomenon of Local Natural Circulation in a Circuit of Nuclear Power Plant

    International Nuclear Information System (INIS)

    The report presents results of experimental research performed at the BN-350 reactor facility, which have allowed identifying a local natural circulation phenomenon arising in some sections of heat removal loops. The nature of this phenomenon and features of its interaction with total-circuit natural circulation is described. In particular, specific features and degree of possible influence of local natural circulation on operational modes of reactor facility are analyzed. (author)

  11. Does nuclear power-related facility siting always lower the local property Values? Comparative analysis among the sites in Japan

    International Nuclear Information System (INIS)

    Yamane et al. (2011) carried out hedonic house rent analyses for several nuclear power plant sites in Japan, compared the result for each plant, and obtained some empirical results indicating that the local residents' marginal willingness-to-pay (MWTP) for living near the plant was not necessarily positive and that the MWTP was correlated with operation years of the plants and some of the host communities' attributes (i.e., population density, financial condition and public service improvement). However, these results may suffer from biases and inefficiency in estimating hedonic functions, caused by spatial dependency: spatial autoregression and spatial autocorrelation. In this paper, we introduce spatial econometric techniques to settle this problem. As a revised result, it is indicated that the local residents' MWTP is correlated with past accidents in the plants, education service improvement in the host communities and so on. (author)

  12. Nuclear

    International Nuclear Information System (INIS)

    This document proposes a presentation and discussion of the main notions, issues, principles, or characteristics related to nuclear energy: radioactivity (presence in the environment, explanation, measurement, periods and activities, low doses, applications), fuel cycle (front end, mining and ore concentration, refining and conversion, fuel fabrication, in the reactor, back end with reprocessing and recycling, transport), the future of the thorium-based fuel cycle (motivations, benefits and drawbacks), nuclear reactors (principles of fission reactors, reactor types, PWR reactors, BWR, heavy-water reactor, high temperature reactor of HTR, future reactors), nuclear wastes (classification, packaging and storage, legal aspects, vitrification, choice of a deep storage option, quantities and costs, foreign practices), radioactive releases of nuclear installations (main released radio-elements, radioactive releases by nuclear reactors and by La Hague plant, gaseous and liquid effluents, impact of releases, regulation), the OSPAR Convention, management and safety of nuclear activities (from control to quality insurance, to quality management and to sustainable development), national safety bodies (mission, means, organisation and activities of ASN, IRSN, HCTISN), international bodies, nuclear and medicine (applications of radioactivity, medical imagery, radiotherapy, doses in nuclear medicine, implementation, the accident in Epinal), nuclear and R and D (past R and D programmes and expenses, main actors in France and present funding, main R and D axis, international cooperation)

  13. Signal peptide peptidase-mediated nuclear localization of heme oxygenase-1 promotes cancer cell proliferation and invasion independent of its enzymatic activity.

    Science.gov (United States)

    Hsu, F-F; Yeh, C-T; Sun, Y-J; Chiang, M-T; Lan, W-M; Li, F-A; Lee, W-H; Chau, L-Y

    2015-04-30

    Heme oxygenase-1 (HO-1) is a heme-degrading enzyme anchored in the endoplasmic reticulum by a carboxyl-terminal transmembrane segment (TMS). HO-1 is highly expressed in various cancers and its nuclear localization is associated with the progression of some cancers. Nevertheless, the mechanism underlying HO-1 nuclear translocation and its pathological significance remain elusive. Here we show that the signal peptide peptidase (SPP) catalyzes the intramembrane cleavage of HO-1. Coexpression of HO-1 with wild-type SPP, but not a dominant-negative SPP, promoted the nuclear localization of HO-1 in cells. Mass spectrometry analysis of cytosolic HO-1 isolated from HeLa cells overexpressing HO-1 and SPP revealed two adjacent intramembrane cleavage sites located after S275 and F276 within the TMS. Mutations of S275F276 to A275L276 significantly hindered SPP-mediated HO-1 cleavage and nuclear localization. Nuclear HO-1 was detected in A549 and DU145 cancer cell lines expressing high levels of endogenous HO-1 and SPP. SPP knockdown or inhibition significantly reduced nuclear HO-1 localization in A549 and DU145 cells. The positive nuclear HO-1 stain was also evident in lung cancer tissues expressing high levels of HO-1 and SPP. Overexpression of a truncated HO-1 (t-HO-1) lacking the TMS in HeLa and H1299 cells promoted cell proliferation and migration/invasion. The effect of t-HO-1 was not affected by a mutation in the catalytic site. However, blockade of t-HO-1 nuclear localization abolished t-HO-1-mediated effect. The tumorigenic effect of t-HO-1 was also demonstrated in the mouse model. These findings disclose that SPP-mediated intramembrane cleavage of HO-1 promotes HO-1 nuclear localization and cancer progression independent of HO-1 enzymatic activity.

  14. Construction of Recombinant Baculovirus Surface-Displayed the Capsid Protein of Porcine Circovirus Type 2%表面展示猪圆环病毒2型衣壳蛋白的重组杆状病毒的构建

    Institute of Scientific and Technical Information of China (English)

    程晓亮; 林文耀; 叶煜; 陈筱薇; 严常燕; 廖明; 樊惠英

    2011-01-01

    利用PCR方法扩增猪圆环病毒2型核定位信号区缺失的Cap蛋白基因,将其亚克隆入杆状病毒表面展示质粒pBACsurf-1的gp64信号肽和gp64成熟蛋白之间.将此融合片段亚克隆到质粒pcDNA3.1(+),获得重组质粒pcDNA3.1-gp64-dCap.然后将含有CMV启动子,gp64及dCap的基因片段克隆到杆状病毒转移质粒pFastBac-V,得到重组质粒pFastBac-dCap-V.然后将此转化DH10Bac感受态细胞,获得的重组穿梭质粒Bacmid-dCap-V,经脂质体转染St9细胞,获得重组杆状病毒Ac-dCap-V.该重组病毒感染Sf9细胞后可以产生典型的细胞病变,转导哺乳动物细胞后经间接免疫荧光试验证明,该重组杆状病毒成功转导哺乳动物细胞并表达dCap蛋白.%The open capsid(Cap)protein gene without nuclear localization signal (NLS) of porcine circo-virus 2(PCV2)was amplified by PCR and sub-cloned into pBACsurf-1 between the upstream gp64 signal sequence and downstresm gp64 mature domain. The fusion gene containing dCap and gp64 was inserted into pcDNA3.1 ( + ) to construct the recombinant plasmid pcDNA3. L-gp64-dCap. A fragment of the CMV promoter-gp64-dCap fusion gene cassette was excised from pcDNA3. L-gp64-dCap and inserted into the baculovirus transfer vector pFastBac-V to construct the recombinant plasmid pFastBac-dCap-V. It was followed by the transformation of plasmid into Escherichia coli DHlOBac competent cells to obtain the recombinant shuttle vector Bacmid-dCap-V. Eventually, the recombinant Bacmid was transfected into Sf9 cells to produce the recombinant baculovirus Ac-dCap-V using the LipofectamineTM 2000. The Sf9 cells produced cytopathic after infection of recombinant baculoviruses Ac-dCap-V. Indirect immunofluoresent assay demonstrated that the Ac-dCap-V efficiently transducted the mammalian cells in vitro and the dCap protein was expressed successfully.

  15. Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: Implications for glycosylation and CD4 binding

    International Nuclear Information System (INIS)

    Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4

  16. Adipogenesis stimulates the nuclear localization of EWS with an increase in its O-GlcNAc glycosylation in 3T3-L1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Qiang; Kamemura, Kazuo, E-mail: k_kamemura@nagahama-i-bio.ac.jp

    2014-07-18

    Highlights: • The majority of EWS localizes stably in the cytosol in 3T3-L1 preadipocytes. • Adipogenic stimuli induce the nuclear localization of EWS. • Adipogenesis promotes O-GlcNAcylation of EWS. • O-GlcNAcylation stimulates the recruitment of EWS to the nuclear periphery. - Abstract: Although the Ewing sarcoma (EWS) proto-oncoprotein is found in the nucleus and cytosol and is associated with the cell membrane, the regulatory mechanisms of its subcellular localization are still unclear. Here we found that adipogenic stimuli induce the nuclear localization of EWS in 3T3-L1 cells. Tyrosine phosphorylation in the C-terminal PY-nuclear localization signal of EWS was negative throughout adipogenesis. Instead, an adipogenesis-dependent increase in O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation of EWS was observed. Pharmacological inactivation of O-GlcNAcase in preadipocytes promoted perinuclear localization of EWS. Our findings suggest that the nuclear localization of EWS is partly regulated by the glycosylation.

  17. Monodisperse Magnetite Nanoparticles Coupled with Nuclear Localization Signal Peptide for Cell-Nucleus Targeting

    OpenAIRE

    Xu, Chenjie; Xie, Jin; Kohler, Nathan; Walsh, Edward G; Chin, Y. Eugene; Sun, Shouheng

    2008-01-01

    Functionalization of monodisperse superparamagnetic magnetite (Fe3O4) nanoparticles for cell specific targeting is crucial for cancer diagnostics and therapeutics. Targeted magnetic nanoparticles can be used to enhance the tissue contrast in magnetic resonance imaging (MRI), to improve the efficiency in anticancer drug delivery, and to eliminate tumor cells by magnetic fluid hyperthermia. Herein we report the nucleus-targeting Fe3O4 nanoparticles functionalized with protein and nuclear locali...

  18. Interstitial tissue-specific gene expression in mouse testis by intra-tunica albuguineal injection of recombinant baculovirus

    Institute of Scientific and Technical Information of China (English)

    Hyun Jung Park; Won Young Lee; Jin Hoi Kim; Jae Hwan Kim; Hun Jong Jung; Nam Hyung Kim; Bo Kyung Kim; Hyuk Song

    2009-01-01

    The purpose of this study is to establish a gene delivery system for interstitial tissue-specific protein expression in mice testes using modified recombinant baculovirus. Green fluorescent protein (GFP)-expressing recombinant bacuiovirus (GFP-baculovirus), in which the insect cell-specific polyhedron promoter was replaced by the cytomegalovirus (CMV)-IE promoter, was used to transfect testicular cells in vitro, and for intra-tunica albuguineai injection of the interstitial tissue of the testis. GFP expression was monitored in frozen testes sections by fluorescence microscopy. Expression of GFP in testicular tissues was also assessed by reverse transcription polymerase chain reaction (RT-PCR), and protein expression was assessed by Western blot. Testicular cells in vitro were infected efficiently by modified recombinant GFP-baculovirus. Intra-tunica albuguineal injection of GFP-baculovirus into the mouse testis resulted in a high level of GFP expression in the interstitial tissues. RT-PCR analysis clearly showed GFP gene expression in the testis, particularly interstitial tissues. Intra-tunica albuguineal injection of a modified baculovirus that encoded recombinant rat insulin-like growth factor binding protein (IGFBP)-5 resulted in an increase in IGFBP-5 in testis and semen. In conclusion, we have developed an efficient delivery system for gene expression in vivo in testicular cells, particularly cells of the interstitial tissue using intra-tunica albuguineal injection of a modified recombinant baculovirus. This method will be particularly relevant for application that requires gene delivery and protein expression in the testicular cells of the outer seminiferous tubule of the testis.

  19. Construction and immunogenicity of recombinant pseudotype baculovirus expressing the capsid protein of porcine circovirus type 2 in mice.

    Science.gov (United States)

    Fan, Huiying; Pan, Yongfei; Fang, Liurong; Wang, Dang; Wang, Shengping; Jiang, Yunbo; Chen, Huanchun; Xiao, Shaobo

    2008-06-01

    Baculovirus has emerged recently as a novel and attractive gene delivery vehicle for mammalian cells. Porcine circovirus type 2 (PCV2) is known to be associated with post-weaning multisystemic wasting syndrome (PMWS), an emerging swine disease which results in tremendous economic losses. In this study, baculovirus pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) was used as a vector to express capsid (Cap) protein, the most important immunogen of PCV2, under the transcriptional control of cytomegalovirus immediate early (CMV-IE) enhancer/promoter. The resultant recombinant baculovirus (BV-G-ORF2) efficiently transduced and expressed the Cap protein in mammalian cells, as demonstrated by Western blot and flow cytometric analyses. After direct vaccination with 1x10(8) or 1x10(9)plaque forming units (PFU)/mouse of BV-G-ORF2, significant PCV2-specific ELISA antibodies, neutralizing antibodies, as well as cellular immune responses could be induced in mice. BV-G-ORF2 exhibited better immunogenicity than a DNA vaccine encoding the Cap protein, even at a dose of 1x10(8)PFU/mouse. Taken together, the improved immunogenicity of BV-G-ORF2, together with the unique advantages of pseudotype baculovirus, including easy manipulation, simple scale-up, lack of toxicity, and no pre-existing antibody against baculovirus in the hosts, indicate that pseudotype baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop a new generation of vaccines against PCV2 infection. PMID:18394722

  20. Construction of recombinant baculovirus Ac-CMV-hSox9 for gene therapy of intervertebral disc degeneration

    Institute of Scientific and Technical Information of China (English)

    LIU Xiao-yun; YANG Shu-hua; LIANG Chang-yong; SONG Jian-hua; LI Kang-hua; CHEN Xin-wen

    2007-01-01

    Objective: To construct the recombinant baculovirus Ac-cytomegalovirus (CMV)-hSox9 for gene therapy of intervertebral disc degeneration. Methods: Bac-to-Bac system was used for the construction of baculovirus Ac-CMV-hSox9. The cDNA of hSox9 was first cloned into a plasmid vector under the control of CMV promotor to generate the donor plasmid pFastBacDul-green fluorescene protein (GFP)-CMV (pFGC)-hSox9.The resultant plasmid was transformed into DH10Bac cells and then the transformation mixture was spread on Luria-Bertani (LB) agarose culture medium containing isopropyl-β-D-thiogalactoside (IPTG), X-gal, gentamicin, kanamycin and tetracycline.The white colonies were selected and cultured for amplification, and the hSox9Bacmid DNA was extracted. After verification, recombinant baculovirus Ac-CMV-hSox9 was obtained through transfecting Sf 21 cells.The expression of hSox9 gene in the intervertebral disc cells in rabbits was determined by Western blotting and immunohistochemical staining.Results: Polymerase chain reaction (PCR) confirmed the presence of hSox9 gene in the recombinant baculovirus and the Sf 21 cells transfected by the baculovirus showed the expression of fluorescence protein.Western blotting and immunohistochemical staining analysis indicated that exogenous hSox9 gene was expressed in the disc cells.Conclusions: The successful construction of the recombinant baculovirus Ac-CMV-hSox9 and the confirmation of the target gene expression provides a novel expression vector system for basic research and clinical treatment of intervertebral degenerative disc disease.

  1. Local stakeholder involvement in the perspective of nuclear waste management: lessons form the Cowam network

    International Nuclear Information System (INIS)

    The management of high level radioactive waste is nowadays recognised as a complex decision-making process entailing technical, environmental, ethical, social, political and economic dimensions where no solution can be reached solely on the basis of technical considerations. While this issue is acknowledged as a problem for the community as a whole, waste management remains a global problem looking for a local solution. Starting from this view, COWAM network (Community Waste Management), developed under the Fifth Framework Programme of the European Commission, addressed the following objectives: 1) To empower local actors through a networking process; 2) To gather and discuss the available experiences of decision-making processes at the local level within their national context in Europe; 3) To set up an arena for balanced exchanges between local actors, NGOs, regulators and implementers; 4) To promote new approaches to decision-making in national contexts in Europe. COWAM network comprises 230 delegates from 10 European countries, involving in priority local communities and NGOs. The emphasis put on the local participation enabled members of COWAM network to overcome distrust and to build common lessons and views beyond usual stakeholder positions. Through the analysis of case studies different issues were identified, among them two relate more specifically to: 1) Expertise what is the purpose of expertise on environmental impact in the decision-making process? How is this expertise linked with other scientific and non scientific issues? What is the role of stakeholders in expertise? 2) Environmental quality in the long term and sustainable development how is the impact of radioactive waste management facilities on the environment in the long term taken into account? how is this associated with the sustainable development of the hosting community? How are local stakeholders involved in these issues and what is the expected benefit from their participation? (author)

  2. On-site storage of high level nuclear waste: Attitudes and perceptions of local residents

    International Nuclear Information System (INIS)

    No public policy issue has been as difficult as high-level nuclear waste. Debates continue regarding Yucca Mountain as a disposal site, and - more generally - the appropriateness of geologic disposal and the need to act quickly. Previous research has focused on possible social, political, and economic consequences of a facility in Nevada. Impacts have been predicted to be potentially large and to emanate mainly from stigmatization of the region due to increased perceptions of risk. Analogous impacts from leaving waste at power plants have been either ignored or assumed to be negligible. This paper presents survey results on attitudes of residents in three countries where nuclear waste is currently stored. Topics include perceived risk, knowledge of nuclear waste and radiation, and impacts on jobs, tourism, and housing values from leaving waste on site. Results are similar to what has been reported for Nevada; the public is concerned about possible adverse effects from on-site storage of waste. 24 refs., 7 figs., 5 tabs

  3. Baculovirus Coinfection Strategy for Improved Galactosylation of Recombinant Glycoprotein Produced by Insect Cell Culture

    Science.gov (United States)

    Ney, Yap Wei; Rahman, Badarulhisam Abdul; Aziz, Azila Abdul

    Baculovirus Expression Vector System (BEVS) is widely used for the production of recombinant glycoproteins, but it is not ideal for pharmaceutical glycoprotein production due to incomplete glycosylation. The factors that ensure successful glycosylation are the presence of sufficient amount of glycosyltransferases, sugar nucleotides as the substrate donor and the recombinant protein as the substrate acceptor. In this study, we analyzed the galactosylation process by the introduction of ß-1,4galactosyltransferase (ß-1,4GalT) as the glycosyltransferase of interest and uridine-5`-diphosphogalactose (UDP-Gal) as the substrate donor. Recombinant human transferrin (rhTf) as a model protein was used as the substrate acceptor. Insect cell lines have been reported to produce a small amount of ß-1,4GalT and thus insufficient for effective galactosylation. In this study, we developed a method to produce galactosylated rhTf and optimized the expression of rhTf with better N-glycan quality. Recombinant ß-1,4GalT was introduced during protein expression by the coinfection of the BEVS with baculovirus carrying bovine ß-1,4GalT. To evaluate the extent of galactosylation by the coinfection strategy, a binding assay was established. In this binding assay, glycoprotein acceptor was absorbed onto ELISA plate surface. A lectin known as Ricinus communis agglutinin-I (RCA-I) labeled with peroxidase, was added and allowed to recognize Gal ß1>4GlcNAc group on the N-glycan of the glycoprotein, followed by appropriate color reaction measurements. Coexpression between rhTf and ß-1,4GalT did not show encouraging results due to the reduction of UDP-Gal upon baculovirus infection. This interesting finding suggested that the introduction of ß-1,4GalT alone was not sufficient for successful galactosylation. Alternatively, post harvest glycosylation method strategy seems to be a promising technique in the improvement of glycoprotein quality.

  4. Pre-operative localization of parathyroid adenomas: A comparison of power and colour Doppler ultrasonography with nuclear medicine scintigraphy

    Energy Technology Data Exchange (ETDEWEB)

    Scheiner, Jac D.; Dupuy, Damian E.; Monchik, John M.; Noto, Richard B.; Cronan, John J

    2001-12-01

    AIM: To compare power and colour Doppler ultrasonography (US) with nuclear medicine scintigraphy (NM) in the preoperative localization of parathyroid adenomas in patients with primary hyperparathyroidism (PHPT). MATERIALS AND METHODS: Thirty-one patients with biochemical evidence of PHPT underwent pre-operative US and NM for parathyroid adenoma localization. Both studies were interpreted independently without prior knowledge of the other study's findings. All patients had surgical removal of the parathyroid adenoma utilizing standard neck exploration or minimally invasive unilateral surgical techniques with rapid serum assay of circulating parathyroid hormone levels. RESULTS: All patients had single parathyroid adenomas at surgery. Prospective sensitivities for US, NM and both studies combined were 65%, 68%, and 74%, respectively, with a positive predictive value of 100% each. The adenoma was localized by only one imaging modality in 16% of cases. CONCLUSIONS: US and NM provide complementary roles in the pre-operative localization of parathyroid adenomas in patients with PHPT. Scheiner, J.D.et al. (2001)

  5. Localized form of Fock terms in nuclear covariant density functional theory

    CERN Document Server

    Liang, Haozhao; Ring, Peter; Roca-Maza, Xavier; Meng, Jie

    2012-01-01

    In most of the successful versions of covariant density functional theory in nuclei, the Fock terms are not included explicitly, which leads to local functionals and forms the basis of their widespread applicability at present. However, it has serious consequences for the description of Gamow-Teller resonances (GTR) and spin-dipole resonances (SDR) which can only be cured by adding further phenomenological parameters. Relativistic Hartree-Fock models do not suffer from these problems. They can successfully describe the GTR and SDR as well as the isovector part of the Dirac effective mass without any additional parameters. However, they are non-local and require considerable numerical efforts. By the zero-range reduction and the Fierz transformation, a new method is proposed to take into account the Fock terms in local functionals, which retains the simplicity of conventional models and provides proper descriptions of the spin-isospin channels and the Dirac masses.

  6. Interaction of nucleosome assembly proteins abolishes nuclear localization of DGK{zeta} by attenuating its association with importins

    Energy Technology Data Exchange (ETDEWEB)

    Okada, Masashi; Hozumi, Yasukazu [Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585 (Japan); Ichimura, Tohru [Department of Chemistry, Graduate School of Sciences and Engineering, Tokyo Metropolitan University, Hachioji 192-0397 (Japan); Tanaka, Toshiaki; Hasegawa, Hiroshi; Yamamoto, Masakazu; Takahashi, Nobuya [Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585 (Japan); Iseki, Ken [Department of Emergency and Critical Care Medicine, Yamagata University School of Medicine, Yamagata 990-9585 (Japan); Yagisawa, Hitoshi [Laboratory of Biological Signaling, Graduate School of Life Science, University of Hyogo, Hyogo 678-1297 (Japan); Shinkawa, Takashi; Isobe, Toshiaki [Department of Chemistry, Graduate School of Sciences and Engineering, Tokyo Metropolitan University, Hachioji 192-0397 (Japan); Goto, Kaoru, E-mail: kgoto@med.id.yamagata-u.ac.jp [Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585 (Japan)

    2011-12-10

    Diacylglycerol kinase (DGK) is involved in the regulation of lipid-mediated signal transduction through the metabolism of a second messenger diacylglycerol. Of the DGK family, DGK{zeta}, which contains a nuclear localization signal, localizes mainly to the nucleus but translocates to the cytoplasm under pathological conditions. However, the detailed mechanism of translocation and its functional significance remain unclear. To elucidate these issues, we used a proteomic approach to search for protein targets that interact with DGK{zeta}. Results show that nucleosome assembly protein (NAP) 1-like 1 (NAP1L1) and NAP1-like 4 (NAP1L4) are identified as novel DGK{zeta} binding partners. NAP1Ls constitutively shuttle between the nucleus and the cytoplasm in transfected HEK293 cells. The molecular interaction of DGK{zeta} and NAP1Ls prohibits nuclear import of DGK{zeta} because binding of NAP1Ls to DGK{zeta} blocks import carrier proteins, Qip1 and NPI1, to interact with DGK{zeta}, leading to cytoplasmic tethering of DGK{zeta}. In addition, overexpression of NAP1Ls exerts a protective effect against doxorubicin-induced cytotoxicity. These findings suggest that NAP1Ls are involved in a novel molecular basis for the regulation of nucleocytoplasmic shuttling of DGK{zeta} and provide a clue to examine functional significance of its translocation under pathological conditions.

  7. Hypercholesterolemia Increases the Production of Leukotriene B4 in Neutrophils by Enhancing the Nuclear Localization of 5-Lipoxygenase

    Directory of Open Access Journals (Sweden)

    Xiao-Feng Lai

    2014-11-01

    Full Text Available Aims: Neutrophils can synthesize leukotriene B4 (LTB4 by activating the 5-lipoxygenase (5-LOsignaling pathway. LTB4 is a pro-inflammatory mediator associated with the etiology and progression of atherosclerosis. It can increase function and number of neutrophils in an autocrine manner. Since hypercholesterolemia is associated with an increase in the number and function of neutrophils, we hypothesized that this effect could be mediated through increased production of LTB4 in neutrophils. Methods/Results: Hypercholesterolemia was modeled in Wistar rats by feeding them with a high cholesterol diet. The induction of hypercholesterolemia caused an increase in the plasma levels of LTB4, following lipopolysaccharide stimulation. This effect was recapitulated in vitro, both in the presence and absence of stimulation with the activator of 5-LO, A23187. Neutrophils in hypercholesterolemia rats expressed similar total levels of 5-LO as control rats, but displayed increased nuclear localization of 5-LO, as well as elevated levels of phosphorylated 5-LO and ERK1/2. In vitro, MβCD/cholesterol complexes enriched cholesterol in neutrophils, resulted in similar changes in 5-LO/LTB4. In addition, these alterations could be inhibited with the ERK inhibitor PD98059. Conclusion: Hypercholesterolemia increases LTB4 production in neutrophils by increasing the nuclear localization of 5-LO, which is the result of its phosphorylation by activated ERK1/2.

  8. Interaction of Sp1 zinc finger with transport factor in the nuclear localization of transcription factor Sp1

    Energy Technology Data Exchange (ETDEWEB)

    Ito, Tatsuo [Department of Medicinal Biotechnology, Institute for Medicinal Research, Graduate School of Pharmaceutical Sciences, The University of Tokushima, 1-78 Sho-machi, Tokushima 770-8505 (Japan); Kitamura, Haruka; Uwatoko, Chisana; Azumano, Makiko [Department of Molecular Biophysical Chemistry, Faculty of Pharmaceutical Sciences, Doshisha Women' s University, Kodo, Kyotanabe City, Kyoto 610-0395 (Japan); Itoh, Kohji, E-mail: kitoh@ph.tokushima-u.ac.jp [Department of Medicinal Biotechnology, Institute for Medicinal Research, Graduate School of Pharmaceutical Sciences, The University of Tokushima, 1-78 Sho-machi, Tokushima 770-8505 (Japan); Kuwahara, Jun, E-mail: jkuwahar@dwc.doshisha.ac.jp [Department of Molecular Biophysical Chemistry, Faculty of Pharmaceutical Sciences, Doshisha Women' s University, Kodo, Kyotanabe City, Kyoto 610-0395 (Japan)

    2010-12-10

    Research highlights: {yields} Sp1 zinc fingers themselves interact with importin {alpha}. {yields} Sp1 zinc finger domains play an essential role as a nuclear localization signal. {yields} Sp1 can be transported into the nucleus in an importin-dependent manner. -- Abstract: Transcription factor Sp1 is localized in the nucleus and regulates the expression of many cellular genes, but the nuclear transport mechanism of Sp1 is not well understood. In this study, we revealed that GST-fused Sp1 protein bound to endogenous importin {alpha} in HeLa cells via the Sp1 zinc finger domains, which comprise the DNA binding domain of Sp1. It was found that the Sp1 zinc finger domains directly interacted with a wide range of importin {alpha} including the armadillo (arm) repeat domain and the C-terminal acidic domain. Furthermore, it turned out that all three zinc fingers of Sp1 are essential for binding to importin {alpha}. Taken together, these results suggest that the Sp1 zinc finger domains play an essential role as a NLS and Sp1 can be transported into the nucleus in an importin-dependent manner even though it possesses no classical NLSs.

  9. Retinoic acid exerts dual regulatory actions on the expression and nuclear localization of interferon regulatory factor-1.

    Science.gov (United States)

    Luo, Xin M; Ross, A Catharine

    2006-05-01

    Interferon regulatory factor-1 (IRF-1), a transcription factor and tumor suppressor involved in cell growth regulation and immune responses, has been shown to be induced by all-trans retinoic acid (ATRA). However, the factors controlling the cellular location and activity of IRF-1 are not well understood. In this study, we examined the expression of IRF-1 and its nuclear localization, DNA-binding activity, and target gene expression in human mammary epithelial MCF10A cells, a model of breast epithelial cell differentiation and carcinogenesis. Following initial treatment with ATRA, IRF-1 mRNA and protein were induced within 2 hrs, reached a peak (>30-fold induction) at 8 hrs, and declined afterwards. IRF-1 protein was predominantly cytoplasmic during this treatment. Although a second dose of ATRA or Am580 (a related retinoid selective for retinoic acid receptor-alpha [RARalpha]), given 16 hrs after the first dose, restimulated IRF-1 mRNA and protein levels to a similar level to that obtained by the first dose, IRF-1 was predominantly concentrated in the nucleus after restimulation. ATRA and Am580 also increased nuclear RARalpha, whereas retinoid X receptor-alpha (RXRalpha)--a dimerization partner for RARalpha, was localized to the nucleus upon second exposure to ATRA. However, ATRA and Am580 did not regulate the expression or activation of signal transducer and activator of transcription-1 (STAT-1), a transcription factor capable of inducing the expression of IRF-1, indicating an STAT-1-independent mechanism of regulation by ATRA and Am580. The increase in nuclear IRF-1 after retinoid restimulation was accompanied by enhanced binding to an IRF-E DNA response element, and elevated expression of an IRF-1 target gene, 2',5'-oligoadenylate synthetase-2. The dual effect of retinoids in increasing IRF-1 mRNA and protein and in augmenting the nuclear localization of IRF-1 protein may be essential for maximizing the tumor suppressor activity and the immunosurveillance

  10. Nuclear localization of phosphorylated c-Myc protein in human tumor cells.

    Directory of Open Access Journals (Sweden)

    C. Soldani

    2010-05-01

    Full Text Available Using immunocytochemical techniques at light and electron microscopy, we analysed the distribution of phosphorylated c-Myc in actively proliferating human HeLa cells. The distribution pattern of c-Myc was also compared with those of other ribonucleoprotein (RNP-containing components (PANA, hnRNP-core proteins, fibrillarin or RNP-associated nuclear proteins (SC-35 splicing factor. Our results provide the first evidence that phosphorylated c-Myc accumulates in the nucleus of tumor cells, where it colocalizes with fibrillarin, both in the nucleolus and in extranucleolar structures.

  11. Sizewell nuclear power station: investigation of radiation exposure pathways from liquid effluents. Local habits survey 1981

    International Nuclear Information System (INIS)

    A habits and consumption survey to review radiation exposure pathways due to liquid effluents released from the CEGB Sizewell site is described. It is relevant to both the Sizewell A and proposed Sizewell B nuclear power stations. The main objectives are to provide input data to a radiological assessment by means of identifying critical groups and to provide data for guidance in a review of environmental monitoring programmes. The way in which data for the different pathways should be combined in order to aid the subsequent radiological assessment is discussed. Recommendations are made for adjustments to the present monitoring programmes. (U.K.)

  12. Functional characterization of nuclear localization and export signals in hepatitis C virus proteins and their role in the membranous web.

    Directory of Open Access Journals (Sweden)

    Aviad Levin

    Full Text Available The hepatitis C virus (HCV is a positive strand RNA virus of the Flavivirus family that replicates in the cytoplasm of infected hepatocytes. Previously, several nuclear localization signals (NLS and nuclear export signals (NES have been identified in HCV proteins, however, there is little evidence that these proteins travel into the nucleus during infection. We have recently shown that nuclear pore complex (NPC proteins (termed nucleoporins or Nups are present in the membranous web and are required during HCV infection. In this study, we identify a total of 11 NLS and NES sequences in various HCV proteins. We show direct interactions between HCV proteins and importin α5 (IPOA5/kapα1, importin β3 (IPO5/kap β3, and exportin 1 (XPO1/CRM1 both in-vitro and in cell culture. These interactions can be disrupted using peptides containing the specific NLS or NES sequences of HCV proteins. Moreover, using a synchronized infection system, we show that these peptides inhibit HCV infection during distinct phases of the HCV life cycle. The inhibitory effects of these peptides place them in two groups. The first group binds IPOA5 and inhibits infection during the replication stage of HCV life cycle. The second group binds IPO5 and is active during both early replication and early assembly. This work delineates the entire life cycle of HCV and the active involvement of NLS sequences during HCV replication and assembly. Given the abundance of NLS sequences within HCV proteins, our previous finding that Nups play a role in HCV infection, and the relocation of the NLS double-GFP reporter in HCV infected cells, this work supports our previous hypothesis that NPC-like structures and nuclear transport factors function in the membranous web to create an environment conducive to viral replication.

  13. Evaluación de aislamientos de baculovirus para el control de Spodoptera frugiperda (Smith, 1797 (LEP.: NOCTUIDAE, plaga clave del maíz en el noroeste argentino Evaluation of baculovirus strains to control the fall armyworm, Spodoptera frugiperda (Smith, 1797 (LEP.: NOCTUIDAE, a key corn pest in North Western Argentina

    Directory of Open Access Journals (Sweden)

    Marta G. Yasem de Romero

    for Spodoptera frugiperda control, a key pest affecting corn crops in North Western Argentina. These insecticides, however, frequently show low effectiveness. The baculoviruses are a biological alternative to control the fall armyworm. The objective of this research was to assess Nucleopolyhedrovirus native and foreign strains as regards their effectiveness in controlling S. frugiperda. The results showed that S. frugiperda larvae death rate rose with increasing viral concentrations, while viral susceptibility of larvae decreased with insect age. LC50 (lethal concentration for 50% of the tested sample of 7.6 x 10(4 and 4.5 x 10(5 polyhedra/ml for three and five day-old larvae, respectively, were determined for the nuclear polyhedrosis virus (NPV isolated in Leales (Tucumán, Argentina. Similar control levels were determined for the NPV isolated in Oliveros (Santa Fe, Argentina, with lethal viral concentrations (LC50 of 8.6 x 10(4 and 4.0 x 10(5 polyhedra/ml, respectively. The Brazilian isolate was characterized by LC50 levels of 5.9 x 10(5 and 1.5 x 10(6 polyhedral/ml for three-day-old and five-day-old larvae, respectively. The local isolate (VPNSf -Tucumán showed the most lethal effect on the native population of young S. frugiperda (three to five-day-old larvae, with an average lifespan of six days at LC50 levels, while the Oliveros and Brazil isolates showed an average lifespan of seven and nine days, respectively. The VPNSf - Leales isolate was therefore selected as the object of study for this research. Moreover, being a native isolate, it was considered the best alternative from the environmental impact standpoint.

  14. Effect of local and large-scale environments on nuclear activity and star formation

    CERN Document Server

    Argudo-Fernández, M; Sabater, J; Puertas, S Duarte; Verley, S; Yang, X

    2016-01-01

    Active galactic nuclei (AGN) is one of the main drivers for transition from star-forming disk to passive spheroidal galaxies. However, the role of large-scale environment versus one-on-one interactions in triggering different types of AGN is still uncertain. We present a statistical study of the prevalence of the nuclear activity in isolated galaxies and physically bound isolated pairs. For the purpose of this study we considered optically and radio selected nuclear activity types. We aim to assess the effect of one-on-one interaction on the fraction of AGN and the role of their large-scale environment. To study the effect of one-on-one interaction on the fraction of AGN in isolated galaxy pairs, we compare with a sample of isolated galaxies homogeneously selected under the same isolation criterion. We examine the effect of the large-scale environment by comparing with control samples of single galaxies and galaxy pairs. In general we found no difference in the prevalence of optical AGN for the considered sam...

  15. A localized navigation algorithm for radiation evasion for nuclear facilities: Optimizing the “Radiation Evasion” criterion: Part I

    International Nuclear Information System (INIS)

    Highlights: ► A new navigation algorithm for radiation evasion around nuclear facilities. ► An optimization criteria minimized under algorithm operation. ► A man-borne device guiding the occupational worker towards paths that warrant least radiation × time products. ► Benefits of using localized navigation as opposed to global navigation schemas. ► A path discrimination function for finding the navigational paths exhibiting the least amounts of radiation. -- Abstract: In this paper, we introduce a navigation algorithm having general utility for occupational workers at nuclear facilities and places where radiation poses serious health hazards. This novel algorithm leverages the use of localized information for its operation. Therefore, the need for central processing and decision resources is avoided, since information processing and the ensuing decision-making are done aboard a man-borne device. To acquire the information needed for path planning in radiation avoidance, a well-designed and distributed wireless sensory infrastructure is needed. This will automatically benefit from the most recent trends in technology developments in both sensor networks and wireless communication. When used to navigate based on local radiation information, the algorithm will behave more reliably when accidents happen, since no long-haul communication links are required for information exchange. In essence, the proposed algorithm is designed to leverage nearest neighbor information coming in through the sensory network overhead, to compute successful navigational paths from one point to another. The proposed algorithm is tested under the “Radiation Evasion” criterion. It is also tested for the case when more information, beyond nearest neighbors, is made available; here, we test its operation for different numbers of step look-ahead. We verify algorithm performance by means of simulations, whereby navigational paths are calculated for different radiation fields

  16. Lysine 271 but not lysine 210 of XRCC4 is required for the nuclear localization of XRCC4 and DNA ligase IV

    Energy Technology Data Exchange (ETDEWEB)

    Fukuchi, Mikoto; Wanotayan, Rujira; Liu, Sicheng; Imamichi, Shoji; Sharma, Mukesh Kumar; Matsumoto, Yoshihisa, E-mail: yoshim@nr.titech.ac.jp

    2015-06-12

    XRCC4 and DNA Ligase IV (LIG4) cooperate to join two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). However, it is not fully understood how these proteins are localized to the nucleus. Here we created XRCC4{sup K271R} mutant, as Lys271 lies within the putative nuclear localization signal (NLS), and XRCC4{sup K210R} mutant, as Lys210 was reported to undergo SUMOylation, implicated in the nuclear localization of XRCC4. Wild-type and mutated XRCC4 with EGFP tag were introduced into HeLa cell, in which endogenous XRCC4 had been knocked down using siRNA directed to 3′-untranslated region, and tested for the nuclear localization function by fluorescence microscopy. XRCC4{sup K271R} was defective in the nuclear localization of itself and LIG4, whereas XRCC4{sup K210R} was competent for the nuclear localization with LIG4. To examine DSB repair function, wild-type and mutated XRCC4 were introduced into XRCC4-deficient M10. M10-XRCC4{sup K271R}, but not M10-XRCC4{sup K210R}, showed significantly reduced surviving fraction after 2 Gy γ-ray irradiation as compared to M10-XRCC4{sup WT}. The number of γ-H2AX foci remaining 2 h after 2 Gy γ-ray irradiation was significantly greater in M10-XRCC4{sup K271R} than in M10-XRCC4{sup WT}, whereas it was only marginally increased in M10-XRCC4{sup K210R} as compared to M10-XRCC4{sup WT}. The present results collectively indicated that Lys271, but not Lys210, of XRCC4 is required for the nuclear localization of XRCC4 and LIG4 and that the nuclear localizing ability is essential for DSB repair function of XRCC4. - Highlights: • XRCC4{sup K271R} is defective in the nuclear localization of itself and LIG4. • XRCC4{sup K210R} is competent for the nuclear localization of itself and LIG4. • XRCC4{sup K271R} is deficient in DSB repair function. • XRCC4{sup K210R} is mostly normal in DSB repair function.

  17. Nuclear localization of Obox4 is dependent on its homeobox domain

    OpenAIRE

    Park, Geon Tae; Lee, Kyung-Ah

    2013-01-01

    Objective Oocyte-specific homeobox 4 (Obox4) is preferentially expressed in oocytes and plays an important role in the completion of meiosis of oocytes. However, the Obox4 expression pattern has not been reported yet. In this study, we investigated the subcellular localization of Obox4 using a green fluorescent protein (GFP) fusion expression system. Methods Three regions of Obox4 were divided and fused to the GFP expression vector. The partly deleted homeodomain (HD) regions of Obox4 were al...

  18. The human papillomavirus (HPV) E6 oncoproteins promotes nuclear localization of active caspase 8

    International Nuclear Information System (INIS)

    The HPV-16 E6 and E6⁎ proteins have been shown previously to be capable of regulating caspase 8 activity. We now show that the capacity of E6 to interact with caspase 8 is common to diverse HPV types, being also seen with HPV-11 E6, HPV-18 E6 and HPV-18 E6⁎. Unlike most E6-interacting partners, caspase 8 does not appear to be a major proteasomal target of E6, but instead E6 appears able to stimulate caspase 8 activation, without affecting the overall apoptotic activity. This would appear to be mediated in part by the ability of the HPV E6 oncoproteins to recruit active caspase 8 to the nucleus. - Highlights: • Multiple HPV E6 oncoproteins interact with the caspase 8 DED domain. • HPV E6 stimulates activation of caspase 8. • HPV E6 promotes nuclear accumulation of caspase 8

  19. The human papillomavirus (HPV) E6 oncoproteins promotes nuclear localization of active caspase 8

    Energy Technology Data Exchange (ETDEWEB)

    Manzo-Merino, Joaquin [Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología, México/Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México. Av. San Fernando No. 22, Col. Sección XVI, Tlalpan 14080 (Mexico); Massimi, Paola [International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34149 Trieste (Italy); Lizano, Marcela, E-mail: lizanosoberon@gmail.com [Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología, México/Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México. Av. San Fernando No. 22, Col. Sección XVI, Tlalpan 14080 (Mexico); Banks, Lawrence, E-mail: banks@icgeb.org [International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34149 Trieste (Italy)

    2014-02-15

    The HPV-16 E6 and E6{sup ⁎} proteins have been shown previously to be capable of regulating caspase 8 activity. We now show that the capacity of E6 to interact with caspase 8 is common to diverse HPV types, being also seen with HPV-11 E6, HPV-18 E6 and HPV-18 E6{sup ⁎}. Unlike most E6-interacting partners, caspase 8 does not appear to be a major proteasomal target of E6, but instead E6 appears able to stimulate caspase 8 activation, without affecting the overall apoptotic activity. This would appear to be mediated in part by the ability of the HPV E6 oncoproteins to recruit active caspase 8 to the nucleus. - Highlights: • Multiple HPV E6 oncoproteins interact with the caspase 8 DED domain. • HPV E6 stimulates activation of caspase 8. • HPV E6 promotes nuclear accumulation of caspase 8.

  20. Study of community leaders in a nuclear host community: local issues, expectations and support and opposition

    International Nuclear Information System (INIS)

    A study of community leaders was undertaken in Hartsville, Tennessee, site of the TVA Hartsville Nuclear Power Plant currently under construction. Leaders were found to be extremely supportive of the plant and of TVA's efforts to mitigate impacts expected to result from construction. Like their citizen counterparts, leaders expect economic benefits and some growth-related disruption to occur as a result of the plant, while environmental impacts are seen as extremely unlikely to occur. Plant-related issues, such as housing availability and traffic congestion, dominate leaders' thinking about current issues. These issues are expected to continue to be important in the future, and new issues dealing with growth and planning, employment and taxation are expected to arise

  1. An efficient method of constructing homologous recom binant baculovirus with PCR-amplified fragments

    Institute of Scientific and Technical Information of China (English)

    HOU; Songwang; (侯松旺); CHEN; Xinwen; (陈新文); WANG; Hanzhong; (王汉中); HU; Zhihong; (胡志红)

    2003-01-01

    This paper describes a rapid method of constructing homologous recombinant baculovirus in E. coli with PCR-amplified fragments. By using this method, the traditional steps of constructing transfer vector are omitted. The method is based on phage λ red system which can promote the recombination between the homologous fragments with the length above 36 bp. Taking HaSNPV as an example, this paper describes the rapid recombination process by using chloramphenicol resistance gene (CmR) to replace orf135 in HaSNPV genome. A pair of primers with length of 60 bp was synthesized, in which 40 bp was homologous to the each end sequence of orf135, and the rest 20 bp was homologous to the each end sequence of CmR. By using these primers, a linear fragment containing the complete CmR gene between 40 bp of homologous arms of orf135 was generated by PCR with the plasmid pKD3 which contains CmR as the template. By transforming the linear fragment into the E. coli containing the bacterial artificial chromosome of HaSNPV and with the help of a plasmid expressing λ recombinase, the recombinants on which the homologue replacement had taken place were selected by chloramphenicol resistance. This method greatly shortens the process of constructing recombinant baculovirus since the process was performed in E. coli and does not need to construct transfer vectors. It can be further used for gene replacement and gene deletion of other large viral genomes.

  2. Characterization of a non-occluded baculovirus-like agent pathogenic to penaeid shrimp.

    Science.gov (United States)

    Nadala, E C; Tapay, L M; Loh, P C

    1998-07-30

    A non-occluded baculovirus-like agent recently isolated by this laboratory from moribund Penaeus japonicus shrimps obtained from China and named Chinese baculovirus (CBV) was purified and some of its properties characterized. Under the electron microscope, negatively stained virus particles were rod-shaped, enveloped, and measured 322 to 378 nm in length and 130 to 159 nm in diameter. The nucleoprotein core exhibited a unique striated structure and measured 316 to 350 nm in length and 65 to 66 nm in diameter. The striations appear to be the result of the stacking of ring-like structures. These rings consisted of 2 rows of 12 to 14 globular subunits. Each globular subunit measured approximately 10 nm in diameter. SDS-PAGE gels of purified virus preparations showed, among several, 4 prominent protein bands with approximate molecular weights of 19, 23.5, 27.5 and 75 kDa. The structural viral proteins were identified by western blot analysis using polyclonal hyperimmune serum made against purified CBV. The 19, 27.5, and 75 kDa structural proteins were determined to be non-glycosylated components associated with the viral envelope. The 23.5 kDa protein, also non-glycosylated, was identified with the capsid structure. Viral genomic DNA digested with Hind III restriction endonuclease revealed at least 29 different fragments with a conservatively estimated total size of at least 183 kb.

  3. The construction and preliminary analysis of a Tn5 transposon based random mutant library of baculovirus

    Institute of Scientific and Technical Information of China (English)

    Li Hui; Zhao Minglei; Yin Juan; Zhong Jiang

    2006-01-01

    A transposon-based random mutation library of AcMNPV,the type species of baculovirus,was constructed using a Tn5 transposon.The green fluorescence protein gene under the control of the Drosophila hsp70 promoter was inserted into the transposon for easy tracking in insect cells.In vitro transposition was carried out using the transposon and AcMNPV genomic DNA to allow the random insertion of the transposon into the virus genome.The transposed genome was then used to transfect Sf21 insect cells,and a library of mutant viruses capable of expressing green fluorescence protein was obtained.Two mutant viruses,B9F and Li6A were isolated,and the sites of transposon insertion were determined to be within the coding regions of the 94k and p10 genes,respectively.Both genes were determined to be nonessential in viral replication and infection.This technique will be very useful in the functional study of baculovirus genes.

  4. Expression of biologically active recombinant equine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae.

    Science.gov (United States)

    Wu, Donglai; Murakami, Kenji; Liu, Nihong; Inoshima, Yasuo; Yokoyama, Takashi; Kokuho, Takehiro; Inumaru, Shigeki; Matsumura, Tomio; Kondo, Takashi; Nakano, Katsushige; Sentsui, Hiroshi

    2002-10-21

    The full-length equine interferon-gamma (eIFN-gamma) cDNA, including the secretion signal peptide coding region, was recloned into baculovirus transfer vector pAcYM1. This vector was co-transfected with Autographa californica nuclear polyhedrosis virus DNA or hybrid nuclear polyhedrosis virus DNA into Spodoptera frugiperda cells. The recombinant viruses, named AcEIFN-gamma and HyEIFN-gamma, were then recovered. Recombinant eIFN-gamma (reIFN-gamma) was accumulated in the culture fluid of the AcEIFN-gamma or HyEIFN-gamma infected Tricoplusia ni -derived cell line, BTI TN 5B1-4, and hemolymph of HyEIFN-gamma infected silkworm larvae. These reIFN-gamma forms were shown to be 14, 16, 18 and 20kDa proteins, and glycosylated as confirmed by SDS-PAGE and tunicamycin treatment. Both reIFN-gamma proteins, showed high-level biological activities to vesicular stomatitis virus by cytopathic effect reduction assay, and MHC class II antigen induction on the equine fetal kidney-78 cell line. PMID:12445800

  5. Production of biologically active recombinant bovine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae.

    Science.gov (United States)

    Murakami, K; Uchiyama, A; Kokuho, T; Mori, Y; Sentsui, H; Yada, T; Tanigawa, M; Kuwano, A; Nagaya, H; Ishiyama, S; Kaki, H; Yokomizo, Y; Inumaru, S

    2001-01-01

    The full-length bovine interferon-gamma (bIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into baculovirus transfer vectors pAcYM1 and pBm050. These vectors were co-transfected with Autographa californica nuclear polyhedrosis virus (AcNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV) DNA into Spodoptera frugiperda cells (SF21AE) and Bombyx mori cells (BmN), respectively. The recombinant viruses, named AcBIFN-gamma and BmBIFN-gamma, were then recovered. Recombinant bIFN-gamma (rbIFN-gamma) was accumulated in the culture fluid of AcBIFN-gamma-infected Trichoplusia ni cells and BmBIFN-gamma-infected silkworm larvae. These rbIFN-gamma forms were shown to be glycosylated 20 and 22 kDa proteins as confirmed by SDS-PAGE and tunicamycin treatment. These products were sensitive to cystein proteinase. Both rbIFN-gamma proteins, showed high-level biological activities by plaque reduction assay using vesicular stomatitis virus, and MHC class II antigen induction on bovine macrophage cells. PMID:11145838

  6. Local decision-making facing issues of national interest experiences from the swedish siting process for a spent nuclear fuel repository

    International Nuclear Information System (INIS)

    It is common knowledge that there are difficulties in convincing the general public and their democratically elected representatives that final disposal of spent nuclear fuel can be made in safe way. Special problems for the decision-makers are created by the demands put on today's generations to make a responsible risk assessment in a area with genuine uncertainties and characterised by any expressions of lack of confidence in social institutions. The current Swedish process for siting a deep repository for spent nuclear fuel has evolved during a period of many years, through inputs by the industry, Government, regulatory authorities and concerned municipalities. It is clear that the nuclear industry, represented by the Swedish Nuclear Fuel and Waste Management CO (SKB), has the full responsibility to find a solution to the waste management problem and to implement the solution - and to for this under the supervision of Government and regulating authorities. But, given the strong tradition of local self-government, the concerned municipalities, the local population in this process. this is simply the following fact: For people who have engaged themselves in local politics - and are prepared to take their responsibility for the well-being and development of their local community - the issue of a possible nuclear repository in the neighbourhood is difficult to handle. A relevant question is: Why should the nation as a whole expect these locally elected representatives to feel a responsibility for an issue of national importance? (author)

  7. Characterization of the recombinant proteins of porcine circovirus type2 field isolate expressed in the baculovirus system.

    Science.gov (United States)

    Kim, Yuna; Kim, Jinhyun; Kang, Kyoungsoo; Lyoo, Young S

    2002-03-01

    Porcine circovirus (PCV) type2 was isolated using primary porcine kidney cells from lymph node of piglets with typical PMWS. The presence of the virus was identified by PCR using primers specific to PCV type2. The ORFs 1 and 2 were amplified by PCR using primers corresponding to the target genes of the PCV type 2. Cloned genes were inserted into the baculovirus expression vector and PCV recombinant proteins were expressed using baculovirus expression system. Recombinant protein expression was determined by indirect immunofluorescent assay (IFA) and immunoblotting using polyclonal antiserum to PCV. ORF1 gene expressed two proteins with approximately 17 kDa and 31 kDa proteins in the baculovirus system. Recombinant protein of the ORF2 was similar to that of the native virus except minor bands with different molecular weight were detected. Recombinant protein expressed in the baculovirus system showed at least two glycosylation sites based on the tunicamycin treatment. Recombinant protein of the ORF2 assembled virus-like particle in recombinant virus infected insect cells. PMID:14614268

  8. Modelling biological control with wild-type and genetically modified baculoviruses in the Helicoverpa armigera-cotton system

    NARCIS (Netherlands)

    Sun, X.; Werf, van der W.; Bianchi, F.J.J.A.; Hu, Z.; Vlak, J.M.

    2006-01-01

    A comprehensive model was developed to simulate virus epizootics in a stage structured insect population and analyse scenarios for the biological control of cotton bollworm (CBW), Helicoverpa armigera, in cotton, using wild-type or genetically modified baculoviruses. In simulations on dosage and tim

  9. Evaluation of the control of beet armyworm, Spodoptera exigua, with baculoviruses in greenhouse using a process-based simulation model

    NARCIS (Netherlands)

    Bianchi, F.J.J.A.; Vlak, J.M.; Werf, van der W.

    2002-01-01

    Scenario studies were carried out with a process-based model for control of wild-type and genetically modified baculoviruses in populations of Spodoptera exigua in glasshouse chrysanthemum (BACSIM). These scenario studies were used to evaluate the effectiveness of different spraying regimes, concent

  10. Behaviour of wild-type and genetically modified baculoviruses in the Helicoverpa armigera - cotton system: a simulation approach

    NARCIS (Netherlands)

    Sun, X.

    2005-01-01

    Keywords:   Helicoverpa armigera , baculovirus, genetic modification, cotton,transmissionBiological control of beet armyworm, Spodoptera exigua, with baculoviruses in greenhouses : development of a comprehensive process-based model

    NARCIS (Netherlands)

    Bianchi, F.J.J.A.; Vlak, J.M.; Rabbinge, R.; Werf, van der W.

    2002-01-01

    We describe the development of a comprehensive process-based model simulating the epizootiology and agronomic efficacy of baculoviruses used for biological control of beet armyworm, Spodoptera exigua, in greenhouse chrysanthemum. The model is built to help understand, evaluate, and predict the effec

  11. Fasciola hepatica procathepsin L3 protein expressed by a baculovirus recombinant can partly protect rats against fasciolosis

    NARCIS (Netherlands)

    Reszka, N.; Cornelissen, J.B.W.J.; Harmsen, M.M.; Bree, de J.; Boersma, W.J.A.; Rijsewijk, F.A.M.

    2005-01-01

    Fasciola hepatica juveniles express immunodominant cathepsin L proteins, which are mainly found in their immature, procathepsin form. A gene encoding such a procathepsin L (FheCL3) was expressed by a baculovirus recombinant and by Saccharomyces cerevisiae. The glycosylated FheCL3 proteins obtained b

  12. The feasibility of using a baculovirus vector to deliver the sodium-iodide symporter gene as a reporter

    Energy Technology Data Exchange (ETDEWEB)

    Zhou Xiang; Li Biao; Wang Jun; Yin Hongyan [Department of Nuclear Medicine, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025 (China); Zhang Yifan [Department of Nuclear Medicine, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025 (China)], E-mail: zhangyifan1992@yahoo.com.cn

    2010-04-15

    Purpose: To evaluate the efficiency of baculovirus vectors in transducing FTC-133 cells and to examine the feasibility of using baculovirus vectors for the delivery of the sodium-iodide symporter (NIS) gene as a reporter through co-transduction to monitor the expression of the target gene. Method: Two recombinant baculoviruses were constructed to express NIS and green fluorescent protein (GFP) respectively. FTC-133, 8050C, SW1116, A549 cells, were infected with Bac-GFP. The infection efficiency of Bac-GFP and the intensity of fluorescence, in either the presence or absence of sodium butyrate, were monitored by flow cytometry. The iodine uptake by FTC-133 cells infected with Bac-NIS was measured using a {gamma} counter. FTC-133 cells were infected with a mixture of equal amounts of Bac-NIS and Bac-GFP at different setting of multiplicity of infection (MOI). The changes of GFP fluorescence intensity and iodine uptake were monitored 24 h after infection in the coinfected cells. Results: We have successfully constructed recombinant baculoviruses carrying NIS and GFP under the control of the cytomegalovirus IE-1 promoter. We found that transduced efficiency of baculovirus in 8505C, SW1116, A549 cells are low in absence of sodium butyrate. Yet Bac-GFP infects FTC-133 cells at a high efficiency, 77.67%, 85.57% and 93.23% with MOI of 100, 200 and 400, respectively. The fluorescence intensity of the Bac-GFP infected tumor cells correlated positively with the MOI of the virus. Sodium butyrate induction increased both the infection efficiency and the fluorescence intensity, but increase of infection efficiency was insignificant in FTC-133 cells. Reporter gene (GFP) expression in FTC-133 is stable within 7 days after infection. The radioactivity incorporated by the tumor cells infected with Bac-NIS correlated positively with the MOI of Bac-NIS as well. In tumor cells co-infected with Bac-NIS and Bac-GFP, the amount of radioactivity incorporated significantly correlated with

  13. Structure of Importin-α from a Filamentous Fungus in Complex with a Classical Nuclear Localization Signal.

    Directory of Open Access Journals (Sweden)

    Natalia E Bernardes

    Full Text Available Neurospora crassa is a filamentous fungus that has been extensively studied as a model organism for eukaryotic biology, providing fundamental insights into cellular processes such as cell signaling, growth and differentiation. To advance in the study of this multicellular organism, an understanding of the specific mechanisms for protein transport into the cell nucleus is essential. Importin-α (Imp-α is the receptor for cargo proteins that contain specific nuclear localization signals (NLSs that play a key role in the classical nuclear import pathway. Structures of Imp-α from different organisms (yeast, rice, mouse, and human have been determined, revealing that this receptor possesses a conserved structural scaffold. However, recent studies have demonstrated that the Impα mechanism of action may vary significantly for different organisms or for different isoforms from the same organism. Therefore, structural, functional, and biophysical characterization of different Impα proteins is necessary to understand the selectivity of nuclear transport. Here, we determined the first crystal structure of an Impα from a filamentous fungus which is also the highest resolution Impα structure already solved to date (1.75 Å. In addition, we performed calorimetric analysis to determine the affinity and thermodynamic parameters of the interaction between Imp-α and the classical SV40 NLS peptide. The comparison of these data with previous studies on Impα proteins led us to demonstrate that N. crassa Imp-α possess specific features that are distinct from mammalian Imp-α but exhibit important similarities to rice Imp-α, particularly at the minor NLS binding site.

  14. Reconstruction of local fallout composition and gamma-ray exposure in a village contaminated by the first USSR nuclear test in the Semipalatinsk nuclear test site in Kazakhstan.

    Science.gov (United States)

    Imanaka, Tetsuji; Yamamoto, Masayoshi; Kawai, Kenta; Sakaguchi, Aya; Hoshi, Masaharu; Chaizhunusova, Nailya; Apsalikov, Kazbek

    2010-11-01

    After the disintegration of the USSR in end of 1991, it became possible for foreign scientists to visit Kazakhstan, in order to investigate the radiological consequences of nuclear explosions that had been conducted at the Semipalatinsk nuclear test site (SNTS). Since the first visit in 1994, our group has been continuing expeditions for soil sampling at various areas around SNTS. The current level of local fallout at SNTS was studied through γ-spectrometry for (137)Cs as well as α-spectrometry for (239,240)Pu. Average values of soil inventory from wide areas around SNTS were 3,500 and 3,700 Bq m(-2) for (137)Cs and (239,240)Pu, respectively, as of January 1, 2000. The average level of (137)Cs is comparable to that in Japan due to global fallout, while the level of (239,240)Pu is several tens of times larger than that in Japan. Areas of strong contamination were found along the trajectories of radioactive fallout, information on which was declassified after the collapse of the USSR. Our recent efforts of soil sampling were concentrated on the area around the Dolon village heavily affected by the radioactive plume from the first USSR atomic bomb test in 1949 and located 110 km east from ground zero of the explosion. Using soil inventory data, retrospective dosimetry was attempted by reconstructing γ-ray exposure from fission product nuclides deposited on the ground. Adopting representative parameters for the initial (137)Cs deposition (13 kBq m(-2)), the refractory/volatile deposition ratio (3.8) and the plume arrival time after explosion (2.5 h), an absorbed dose in air of 600 mGy was obtained for the 1-year cumulative dose in Dolon village, due to the first bomb test in 1949. Considering possible ranges of the parameters, 350 and 910 mGy were estimated for high and low cases of γ-ray dose in air, respectively. It was encouraging that the deduced value was consistent with other estimations using thermal luminescence and archived monitoring data. The present

  15. Age determination of the nuclear stellar population of Active Galactic Nuclei using Locally Weighted Regression

    CERN Document Server

    Estrada-Piedra, T; Terlevich, R J; Fuentes, O; Terlevich, E; Estrada-Piedra, Trilce; Torres-Papaqui, Juan Pablo; Terlevich, Roberto; Fuentes, Olac; Terlevich, Elena

    2003-01-01

    We present a new technique to segregate old and young stellar populations in galactic spectra using machine learning methods. We used an ensemble of classifiers, each classifier in the ensemble specializes in young or old populations and was trained with locally weighted regression and tested using ten-fold cross-validation. Since the relevant information concentrates in certain regions of the spectra we used the method of sequential floating backward selection offline for feature selection. The application to Seyfert galaxies proved that this technique is very insensitive to the dilution by the Active Galactic Nucleus (AGN) continuum. Comparing with exhaustive search we concluded that both methods are similar in terms of accuracy but the machine learning method is faster by about two orders of magnitude.

  16. A new theraphosid spider toxin causes early insect cell death by necrosis when expressed in vitro during recombinant baculovirus infection.

    Directory of Open Access Journals (Sweden)

    Daniel Mendes Pereira Ardisson-Araújo

    Full Text Available Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3 has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV expression. Five different forms of Ba3 were assessed; (1 the full-length sequence, (2 the pro-peptide and mature region, (3 only the mature region, and the mature region fused to an (4 insect or a (5 virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect

  17. A new theraphosid spider toxin causes early insect cell death by necrosis when expressed in vitro during recombinant baculovirus infection.

    Science.gov (United States)

    Ardisson-Araújo, Daniel Mendes Pereira; Morgado, Fabrício Da Silva; Schwartz, Elisabeth Ferroni; Corzo, Gerardo; Ribeiro, Bergmann Morais

    2013-01-01

    Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3) has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV) expression. Five different forms of Ba3 were assessed; (1) the full-length sequence, (2) the pro-peptide and mature region, (3) only the mature region, and the mature region fused to an (4) insect or a (5) virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect cells. PMID

  18. Residues R199H200 of prototype foamy virus transactivator Bel1 contribute to its binding with LTR and IP promoters but not its nuclear localization

    International Nuclear Information System (INIS)

    Prototype foamy virus encodes a transactivator called Bel1 that enhances viral gene transcription and is essential for PFV replication. Nuclear localization of Bel1 has been reported to rely on two proximal basic motifs R199H200 and R221R222R223 that likely function together as a bipartite nuclear localization signal. In this study, we report that mutating R221R222R223, but not R199H200, relocates Bel1 from the nucleus to the cytoplasm, suggesting an essential role for R221R222R223 in the nuclear localization of Bel1. Although not affecting the nuclear localization of Bel1, mutating R199H200 disables Bel1 from transactivating PFV promoters. Results of EMSA reveal that the R199H200 residues are vital for the binding of Bel1 to viral promoter DNA. Moreover, mutating R199H200 in Bel1 impairs PFV replication to a much greater extent than mutating R221R222R223. Collectively, our findings suggest that R199H200 directly participate in Bel1 binding to viral promoter DNA and are indispensible for Bel1 transactivation activity. - Highlights: • The R221R222R223 residues are essential for the nuclear localization of Bel1. • Although not affecting the nuclear localization of Bel1, mutating R199H200 disables Bel1 from transactivating PFV promoters. • The R199H200 residues directly participate in Bel1 binding to viral promoter DNA. • Mutating R199H200 in Bel1 impairs PFV replication to a much greater extent than mutating R221R222R223

  19. Estrogen promotes fat mass and obesity-associated protein nuclear localization and enhances endometrial cancer cell proliferation via the mTOR signaling pathway.

    Science.gov (United States)

    Zhu, Yaping; Shen, Jiaqi; Gao, Liyan; Feng, Youji

    2016-04-01

    Extensive exposure to estrogen is generally acknowledged as a risk factor for endometrial cancer. Given that the accumulation of adipocytes also contributes to the increased production of estrogen, in the present study, we evaluated the expression of the fat mass and obesity-associated (FTO) gene in endometrial tumor tissues and further explored the mechanism of how estrogen facilitates FTO nuclear localization and promotes endometrial cancer cell proliferation. Immunohistochemical (IHC) staining assay was used to detect the FTO expression in endometrial tumor samples. Western blotting was performed to investigate the mechanism of estrogen-induced FTO nuclear localization. siRNA was used to knock down ERα and further explore its role in FTO nuclear localization. MTT assay was carried out to determine cell proliferation. We found that FTO was overexpressed in endometrial carcinoma tissues and served as a poor prognostic marker. Additionally, estrogen induced FTO nuclear accumulation via the mTOR signaling pathway and the nuclear localization was ERα-dependent, which contributed to enhanced proliferative activity. Therefore, the present study provides new insight into the mechanisms of estrogen-induced proliferation, implying the possibility of using FTO as a potential therapeutic target for the treatment of endometrial cancer.

  1. Identification of a novel nuclear localization signal and speckle-targeting sequence of tuftelin-interacting protein 11, a splicing factor involved in spliceosome disassembly

    Energy Technology Data Exchange (ETDEWEB)

    Tannukit, Sissada [Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA Rm103, Los Angeles, CA 90033-1004 (United States); Crabb, Tara L.; Hertel, Klemens J. [Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, CA 92697-4025 (United States); Wen, Xin [Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA Rm103, Los Angeles, CA 90033-1004 (United States); Jans, David A. [Department of Biochemistry and Molecular Biology, Nuclear Signalling Laboratory, Monash University, Clayton, Victoria 3800 (Australia); Paine, Michael L., E-mail: paine@usc.edu [Center for Craniofacial Molecular Biology, University of Southern California, 2250 Alcazar Street, CSA Rm103, Los Angeles, CA 90033-1004 (United States)

    2009-12-18

    Tuftelin-interacting protein 11 (TFIP11) is a protein component of the spliceosome complex that promotes the release of the lariat-intron during late-stage splicing through a direct recruitment and interaction with DHX15/PRP43. Expression of TFIP11 is essential for cell and organismal survival. TFIP11 contains a G-patch domain, a signature motif of RNA-processing proteins that is responsible for TFIP11-DHX15 interactions. No other functional domains within TFIP11 have been described. TFIP11 is localized to distinct speckled regions within the cell nucleus, although excluded from the nucleolus. In this study sequential C-terminal deletions and mutational analyses have identified two novel protein elements in mouse TFIP11. The first domain covers amino acids 701-706 (VKDKFN) and is an atypical nuclear localization signal (NLS). The second domain is contained within amino acids 711-735 and defines TFIP11's distinct speckled nuclear localization. The identification of a novel TFIP11 nuclear speckle-targeting sequence (TFIP11-STS) suggests that this domain directly interacts with additional spliceosomal components. These data help define the mechanism of nuclear/nuclear speckle localization of the splicing factor TFIP11, with implications for it's function.

  2. Local fracture behavior in an alloy 52M dissimilar metal welded joint in nuclear power plant

    International Nuclear Information System (INIS)

    Background: Dissimilar metal welded joints (DMWJs) were indicated to be vulnerable components in the primary systems, an accurate structural integrity assessment for such DMWJ is needed. Purpose: Understand the fracture performance of the welded joint containing defects, determine if appropriate to assess the defects in the weld joint with the present codes. Methods: The fracture tests of the initial crack located in the different positions of Alloy 52M DMWJ specimens were carried out by using single-edge notched bend (SENB) method. Results: Different crack resistance and crack propagation path were obtained for the SENB specimens, and the cracks always deviate to the materials with lower strength. Conclusions: The crack path deviation is mainly controlled by strength mismatch, rather than toughness mismatch. The J-resistance curves with larger crack path deviation only reflect the apparent fracture resistance along the crack growth region, rather than the intrinsic fracture resistance of the initial crack-tip region material., Without considering the local fracture resistance properties of heat affected zone (HAZ), interface region and internal affected region (IAZ), following present assessment codes will unavoidably produce non-conservative or excessive conservative assessment results. In most cases, the assessment results will be potentially non-conservative. (authors)

  3. Systematic determination of replication activity type highlights interconnections between replication, chromatin structure and nuclear localization.

    Directory of Open Access Journals (Sweden)

    Shlomit Farkash-Amar

    associated with compact chromatin and are located significantly closer to the nuclear envelope. Supplementary material is available. Raw and processed data were deposited in Geo (GSE17236.

  4. Characterization of the immune responses elicited by baculovirus-based vector vaccines against influenza virus hemagglutinin

    Institute of Scientific and Technical Information of China (English)

    Zhi-peng HU; Juan YIN; Yuan-yuan ZHANG; Shu-ya JIA; Zuo-jia CHEN; Jiang ZHONG

    2012-01-01

    Aim:To compare the specific immune responses elicited by different baculovirus vectors in immunized mice.Methods:We constructed and characterized two recombinant baculoviruses carrying the expression cassette for the H5N1 influenza virus hemagglutinin (HA) gene driven by either an insect cell promoter (vAc-HA) or a dual-promoter active both in insect and mammalian cells (vAc-HA-DUAL).Virus without the HA gene (vAc-EGFP) was used as a control.These viruses were used to immunize mice subcutaneously and intraperitoneally.The production of total and specific antibodies was determined by ELISA and competitive ELISA.Cytokine production by the spleen cells of immunized mice was studied using the ELISPOT assay.Results:Both the vAc-HA and vAc-HA-DUAL vectors expressed HA proteins in insect Sf9 cells,and HA antigen was present in progeny virions.The vAc-HA-DUAL vector also mediated HA expression in virus-transduced mammalian cell lines (BHK and A547).Both vAo-HA and vAc-HA-DUAL exhibited higher transduction efficiencies than vAc-EGFP in mammalian cells,as shown by the expression of the reporter gene egfp.Additionally,both vAc-HA and vAc-HA-DUAL induced high levels of HA-specific antibody production in immunized mice; vAc-HA-DUAL was more efficient in inducing IFN-Y and IL-2 upon stimulation with specific antigen,whereas vAc-HA was more efficient in inducing IL-4 and IL-6.Conclusion:Baculovirus vectors elicited efficient,specific immune responses in immunized mice.The vector displaying the HA antigen on the virion surface (vAc-HA) elicited a Th2-biased immune response,whereas the vector displaying HA and mediating HA expression in the cell (vAc-HA-DUAL) elicited a Th1-biased immune response.

  5. The R35 residue of the influenza A virus NS1 protein has minimal effects on nuclear localization but alters virus replication through disrupting protein dimerization

    International Nuclear Information System (INIS)

    The influenza A virus NS1 protein has a nuclear localization sequence (NLS) in the amino terminal region. This NLS overlaps sequences that are important for RNA binding as well as protein dimerization. To assess the significance of the NS1 NLS on influenza virus replication, the NLS amino acids were individually mutated to alanines and recombinant viruses encoding these mutations were rescued. Viruses containing NS1 proteins with mutations at R37, R38 and K41 displayed minimal changes in replication or NS1 protein nuclear localization. Recombinant viruses encoding NS1 R35A were not recovered but viruses containing second site mutations at position D39 in addition to the R35A mutation were isolated. The mutations at position 39 were shown to partially restore NS1 protein dimerization but had minimal effects on nuclear localization. These data indicate that the amino acids in the NS1 NLS region play a more important role in protein dimerization compared to nuclear localization. - Highlights: • Mutations were introduced into influenza NS1 NLS1. • NS1 R37A, R38A, K41A viruses had minimal changes in replication and NS1 localization. • Viruses from NS1 R35A rescue all contained additional mutations at D39. • NS1 R35A D39X mutations recover dimerization lost in NS1 R35A mutations. • These results reaffirm the importance of dimerization for NS1 protein function

  6. Cytoplasmic localization of NPM in myeloid leukemias is dictated by gain-of-function mutations that create a functional nuclear export signal.

    Science.gov (United States)

    Mariano, A R; Colombo, E; Luzi, L; Martinelli, P; Volorio, S; Bernard, L; Meani, N; Bergomas, R; Alcalay, M; Pelicci, P G

    2006-07-20

    Nucleophosmin (NPM) is a nucleus-cytoplasmic shuttling protein that is implicated in centrosome duplication, cell cycle progression and stress response. At the steady state, NPM localizes mainly in the nucleolus, where it forms a complex with different cellular proteins. One-third of acute myeloid leukemias (AML) are characterized by aberrant cytoplasmic localization of NPM, due to mutations within its last coding exon (exon 12) that cause a frameshift and the formation of novel C-termini. We report here our investigations on the molecular basis for the aberrant localization of mutated NPM. Alignment of the C-terminus of the various NPM mutants revealed the obligatory presence of four amino-acid residues that match a CRM1-dependent nuclear export signal (NES). Single alanine-substitutions at these sites provoked nuclear re-localization, while fusion of the mutated C-terminus to a heterologous nuclear protein induced CRM1-dependent cytoplasmic localization. Molecular characterization of one exceptional AML carrying cytoplasmic NPM and germ line exon 12 revealed a somatic mutation in the splicing donor site of exon 9 that caused the formation of a functional NES. It appears, therefore, that AMLs are frequently characterized by gain-of-function mutations of NPM that create functional NES, suggesting that alterations of nuclear export might represent a general mechanism of leukemogenesis and a novel target for therapeutic intervention.

  7. The R35 residue of the influenza A virus NS1 protein has minimal effects on nuclear localization but alters virus replication through disrupting protein dimerization

    Energy Technology Data Exchange (ETDEWEB)

    Lalime, Erin N.; Pekosz, Andrew, E-mail: apekosz@jhsph.edu

    2014-06-15

    The influenza A virus NS1 protein has a nuclear localization sequence (NLS) in the amino terminal region. This NLS overlaps sequences that are important for RNA binding as well as protein dimerization. To assess the significance of the NS1 NLS on influenza virus replication, the NLS amino acids were individually mutated to alanines and recombinant viruses encoding these mutations were rescued. Viruses containing NS1 proteins with mutations at R37, R38 and K41 displayed minimal changes in replication or NS1 protein nuclear localization. Recombinant viruses encoding NS1 R35A were not recovered but viruses containing second site mutations at position D39 in addition to the R35A mutation were isolated. The mutations at position 39 were shown to partially restore NS1 protein dimerization but had minimal effects on nuclear localization. These data indicate that the amino acids in the NS1 NLS region play a more important role in protein dimerization compared to nuclear localization. - Highlights: • Mutations were introduced into influenza NS1 NLS1. • NS1 R37A, R38A, K41A viruses had minimal changes in replication and NS1 localization. • Viruses from NS1 R35A rescue all contained additional mutations at D39. • NS1 R35A D39X mutations recover dimerization lost in NS1 R35A mutations. • These results reaffirm the importance of dimerization for NS1 protein function.

  8. Construction and characteristics of a transformed lepidopteran cell clone expressing baculovirus p35

    Institute of Scientific and Technical Information of China (English)

    ZHENG Guiling; LI Changyou; LI Guoxun; WANG Ping; Robert R. Granados

    2005-01-01

    A transformed cell line was constructed from Mythimna separata cells Ms7311 by lipofection method. TMs7311 cells were generated using a double selection technique involving a selection in the antibiotic Zeocin, followed by a second round of selection by exhibiting cell characterization. A cell clone expressing p35 was obtained with high level of AcMNPV and recombinant proteins. Compared with wild type Ms7311 cells, the cell clone showed increased resistance to Actinamycin D-induced apoptosis and a profound resistance to nutrient development (PBS). When the cell clone was infected with recombinant baculoviruses expressing secreted alkaline phosphatase (SEAP) and β-galactosi- dase, expression of the recombinant proteins from TMs7311 cells exceeded that from parental Ms7311 cells. Production of budded virus and occlusion body was significantly higher than that from parental cells Ms7311.

  9. Production of CCHF Virus-Like Particle by a Baculovirus-Insect Cell Expression System

    Institute of Scientific and Technical Information of China (English)

    Zhao-rui Zhou; Man-li Wang; Fei Deng; Tian-xian Li; Zhi-hong Hu; Hua-fin Wang

    2011-01-01

    Crimean-Congo Haemorrhagic Fever Virus(CCHFV)is a tick-born virus of the Nairovirus genus within the Bunyaviridae family,which is widespread and causes,high fatality. The nucleocapsid of CCHFV is comprised of N proteins that are encoded by the S segment. In this research,the N protein of CCHFV was expressed in insect cells using a recombinant baculovirus. Under an electron microscope,Virus-Like Particles (VLPs)with various size and morphology were observed in cytoplasmic vesicles in the infected cells.Sucrose-gradient purification of the cell lysate indicated that the VLPs were mainly located in the upper fraction after ultracentrifugation,which was confirmed by Western blot analysis and immuno-electron microscopy(IEM).

  10. Efficient production of type 2 porcine circovirus-like particles by a recombinant baculovirus.

    Science.gov (United States)

    Liu, Lan-Jun; Suzuki, Takako; Tsunemitsu, Hiroshi; Kataoka, Michiyo; Ngata, Noriyo; Takeda, Naokazu; Wakita, Takaji; Miyamura, Tatsuo; Li, Tian-Cheng

    2008-01-01

    The capsid protein of PCV2 was expressed by using a recombinant baculovirus with insect Tn5 cells. A large amount of 28-kDa protein was released into the culture medium and self-assembled into PCV2-like particles (PCV2-LPs) with a buoyant density of 1.365 g/cm(3) and a diameter of 20 nm. PCV2-LPs were efficiently expressed, yielding 1 mg of purified particles per 10(7) Tn5 cells. The PCV2-LPs have antigenicity similar to that of authentic PCV2 particles, allowing us to develop a method for sensitively detecting PCV2-specific IgG antibodies. In addition, the PCV2-LPs appeared to be the most promising PCV2 vaccine candidate, by virtue of their potent immunogenicity. PMID:18998045

  11. Identification of a single-nucleocapsid baculovirus isolated from Clanis bilineata tsingtauica (Lepidoptera: Sphingidae).

    Science.gov (United States)

    Wang, Liqun; Yi, Jianping; Zhu, Shanying; Li, Bing; Chen, Yan; Shen, Weide; Wang, Wenbing

    2008-01-01

    A nucleopolyhedrovirus isolated from infected larvae of Clanis bilineata tsingtauica was characterized. Electron microscopical studies on the ultrastructure of C. bilineata nucleopolyhedrovirus (ClbiSNPV) occlusion bodies (OBs) showed several virions (up to 16) with a single nucleocapsid packaged within a single viral envelope. The diameter of the OBs was 0.77-1.7 mum with a mean of 1.13 +/- 0.19 mum. The complete sequence of the ClbiSNPV polyhedrin (polh) gene contained 741 nucleotides, predicting a protein of 246 amino acids. Phylogenetic analyses using the complete sequence of the polh genes indicated that ClbiSNPV clusters with Group II NPVs. This is the first record of a baculovirus from C. bilineata. PMID:18584114

  12. Construction of recombinant baculovirus vaccines for Newcastle disease virus and an assessment of their immunogenicity.

    Science.gov (United States)

    Ge, Jingping; Liu, Ying; Jin, Liying; Gao, Dongni; Bai, Chengle; Ping, Wenxiang

    2016-08-10

    Newcastle disease (ND) is a lethal avian infectious disease caused by Newcastle disease virus (NDV) which poses a substantial threat to China's poultry industry. Conventional live vaccines against NDV are available, but they can revert to virulent strains and do not protect against mutant strains of the virus. Therefore, there is a critical unmet need for a novel vaccine that is safe, efficacious, and cost effective. Here, we designed novel recombinant baculovirus vaccines expressing the NDV F or HN genes. To optimize antigen expression, we tested the incorporation of multiple regulatory elements including: (1) truncated vesicular stomatitis virus G protein (VSV-GED), (2) woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), (3) inverted terminal repeats (ITRs) of adeno-associated virus (AAV Serotype II), and (4) the cytomegalovirus (CMV) promoter. To test the in vivo efficacy of the viruses, we vaccinated chickens with each construct and characterized the cellular and humoral immune response to challenge with virulent NDV (F48E9). All of the vaccine constructs provided some level of protection (62.5-100% protection). The F-series of vaccines provided a greater degree of protection (87.5-100%) than the HN-series (62.5-87.5%). While all of the vaccines elicited a robust cellular and humoral response subtle differences in efficacy were observed. The combination of the WPRE and VSV-GED regulatory elements enhanced the immune response and increased antigen expression. The ITRs effectively increased the length of time IFN-γ, IL-2, and IL-4 were expressed in the plasma. The F-series elicited higher titers of neutralizing antibody and NDV-specific IgG. The baculovirus system is a promising platform for NDV vaccine development that combines the immunostimulatory benefits of a recombinant virus vector with the non-replicating benefits of a DNA vaccine. PMID:27015979

  13. Troponin T3 regulates nuclear localization of the calcium channel Cavβ1a subunit in skeletal muscle

    International Nuclear Information System (INIS)

    The voltage-gated calcium channel (Cav) β1a subunit (Cavβ1a) plays an important role in excitation–contraction coupling (ECC), a process in the myoplasm that leads to muscle-force generation. Recently, we discovered that the Cavβ1a subunit travels to the nucleus of skeletal muscle cells where it helps to regulate gene transcription. To determine how it travels to the nucleus, we performed a yeast two-hybrid screening of the mouse fast skeletal muscle cDNA library and identified an interaction with troponin T3 (TnT3), which we subsequently confirmed by co-immunoprecipitation and co-localization assays in mouse skeletal muscle in vivo and in cultured C2C12 muscle cells. Interacting domains were mapped to the leucine zipper domain in TnT3 COOH-terminus (160–244 aa) and Cavβ1a NH2-terminus (1–99 aa), respectively. The double fluorescence assay in C2C12 cells co-expressing TnT3/DsRed and Cavβ1a/YFP shows that TnT3 facilitates Cavβ1a nuclear recruitment, suggesting that the two proteins play a heretofore unknown role during early muscle differentiation in addition to their classical role in ECC regulation. - Highlights: • Previously, we demonstrated that Cavβ1a is a gene transcription regulator. • Here, we show that TnT3 interacts with Cavβ1a. • We mapped TnT3 and Cavβ1a interaction domain. • TnT3 facilitates Cavβ1a nuclear enrichment. • The two proteins play a heretofore unknown role during early muscle differentiation

  14. Cervix carcinoma is associated with an up-regulation and nuclear localization of the dual-specificity protein phosphatase VHR

    International Nuclear Information System (INIS)

    The 21-kDa Vaccinia virus VH1-related (VHR) dual-specific protein phosphatase (encoded by the DUSP3 gene) plays a critical role in cell cycle progression and is itself regulated during the cell cycle. We have previously demonstrated using RNA interference that cells lacking VHR arrest in the G1 and G2 phases of the cell cycle and show signs of beginning of cell senescence. In this report, we evaluated successfully the expression levels of VHR protein in 62 hysterectomy or conization specimens showing the various (pre) neoplastic cervical epithelial lesions and 35 additional cases of hysterectomy performed for non-cervical pathologies, from patients under 50 years of age. We used a tissue microarray and IHC technique to evaluate the expression of the VHR phosphatase. Immunofluorescence staining under confocal microscopy, Western blotting and RT-PCR methods were used to investigate the localization and expression levels of VHR. We report that VHR is upregulated in (pre) neoplastic lesions (squamous intraepithelial lesions; SILs) of the uterine cervix mainly in high grade SIL (H-SIL) compared to normal exocervix. In the invasive cancer, VHR is also highly expressed with nuclear localization in the majority of cells compared to normal tissue where VHR is always in the cytoplasm. We also report that this phosphatase is highly expressed in several cervix cancer cell lines such as HeLa, SiHa, CaSki, C33 and HT3 compared to primary keratinocytes. The immunofluorescence technique under confocal microscopy shows that VHR has a cytoplasmic localization in primary keratinocytes, while it localizes in both cytoplasm and nucleus of the cancer cell lines investigated. We report that the up-regulation of this phosphatase is mainly due to its post-translational stabilization in the cancer cell lines compared to primary keratinocytes rather than increases in the transcription of DUSP3 locus. These results together suggest that VHR can be considered as a new marker for cancer

  15. Overcoming inefficient secretion of recombinant VEGF-C in baculovirus expression vector system by simple purification of the protein from cell lysate.

    Science.gov (United States)

    Klaus, Tomasz; Kulesza, Małgorzata; Bzowska, Monika; Wyroba, Barbara; Kilarski, Witold W; Bereta, Joanna

    2015-06-01

    The first reports about successfully expressed recombinant proteins with the use of a baculovirus vector were published over 30years ago. Despite the long time of refining this expression system, early problems with the production of baculovirus-derived secretory proteins are still not satisfactorily solved. The high expression level driven by baculoviral promoters often does not result in the desired yield of secreted recombinant proteins, which frequently accumulate inside insect cells and are only partially processed. During our attempts to produce vascular endothelial growth factor C (VEGF-C) with the use of a baculovirus vector we also faced an inefficient secretion of the recombinant protein to culture medium. We were not able to improve the outcome and obtain an acceptable concentration of VEGF-C in the medium by changing the culture conditions or utilizing different signal peptides. However, as a significant amount of native VEGF-C was detected inside the baculovirus-infected cells, we developed a simple method to purify recombinant, glycosylated VEGF-C from a lysate of the cells. The presented results indicate that the lack of a secretory protein in the insect cell culture medium after baculovirus infection does not necessarily signify failure in the production of the protein. As demonstrated by us and contrary to generally accepted views, the lysate of baculovirus-infected cells may constitute a valuable source of the biologically active, secretory protein.

  16. Internal ribosome entry site of Rhopalosiphum padi virus is functional in mammalian cells and has cryptic promoter activity in baculovirus-in fected Sf21 cells

    Institute of Scientific and Technical Information of China (English)

    Yi-jane WU; Chao-yi TENG; Yu-jie CHEN; Seng-chi CHEN; Ying-ju CHEN; Yi-ting LIN; Tzong-yuan WU

    2008-01-01

    Aim: To substantiate the in vitro translational studies of a cross-kingdom, inter- nal ribosome entry site (IRES), the 5"untranslated region of the Rhopalosiphum padi virus (RhPV), can function in mammalian cells and act as a shuttle IRES between insect cells and mammalian cells. Methods: Cytomegalovirus (CMV) promoter-based bicistronic mammalian cell expression vectors, either in plasmids or baculovirus vectors, were generated. Plasmid transient transfection and baculovirus transduction assays were performed to test whether the RhPV IRES can mediate translation activity in versatile mammalian cell lines. Results: Both plasmids and recombinant baculoviruses containing the CMV promoter and the RhPV IRES can mediate bicistronic gene expression in mammalian cells. However, in the CMV promoter containing recombinant baculovirus-infected insect Sf21 cells, only the second cistron gene expression was observed. Northern blot analysis and a promoterless assay demonstrated that the RhPV IRES exhibited cryptic promoter activity in baculovirus-infected insect cells. Conclusion: RhPV IRES can mediate gene expression in both insect cells and mammalian cells, and this characteristic of the RhPV IRES will facilitate the development of a bicistronic baculovirus gene therapy vectors.

  17. The LSD1-type zinc finger motifs of Pisum sativa LSD1 are a novel nuclear localization signal and interact with importin alpha.

    Directory of Open Access Journals (Sweden)

    Shanping He

    Full Text Available BACKGROUND: Genetic studies of the Arabidopsis mutant lsd1 highlight the important role of LSD1 in the negative regulation of plant programmed cell death (PCD. Arabidopsis thaliana LSD1 (AtLSD1 contains three LSD1-type zinc finger motifs, which are involved in the protein-protein interaction. METHODOLOGY/PRINCIPAL FINDINGS: To further understand the function of LSD1, we have analyzed cellular localization and functional localization domains of Pisum sativa LSD1 (PsLSD1, which is a homolog of AtLSD1. Subcellular localization analysis of green fluorescent protein (GFP-tagged PsLSD1 indicates that PsLSD1 is localized in the nucleus. Using a series of GFP-tagged PsLSD1 deletion mutants, we found that the three LSD1-type zinc finger motifs of PsLSD1 alone can target GFP to the nucleus, whereas deletion of the three zinc finger motifs or any individual zinc finger motif causes PsLSD1 to lose its nuclear localization, indicating that the three zinc finger motifs are necessary and sufficient for its nuclear localization. Moreover, site-directed mutagenesis analysis of GFP-tagged PsLSD1 indicates that tertiary structure and basic amino acids of each zinc finger motif are necessary for PsLSD1 nuclear localization. In addition, yeast two-hybrid, pull-down, and BiFC assays demonstrate that the three zinc finger motifs of PsLSD1 directly bind to importin α in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate that the LSD1-type zinc finger motifs of PsLSD1 are a novel nuclear localization signal and directly bind to importin α, and suggest that the nuclear import of LSD1 may rely on the interaction between its zinc finger motifs and importin α. Moreover, the nuclear localization of PsLSD1 suggests that LSD1 may function as a transcription regulator involved in negatively regulating PCD.

  18. AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells.

    Science.gov (United States)

    Lorenzato, Annalisa; Biolatti, Marta; Delogu, Giuseppe; Capobianco, Giampiero; Farace, Cristiano; Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco; Madeddu, Roberto; Olivero, Martina; Di Renzo, Maria Flavia

    2013-10-15

    The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. PMID:23948303

  19. Nuclear localization of CPI-17, a protein phosphatase-1 inhibitor protein, affects histone H3 phosphorylation and corresponds to proliferation of cancer and smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Eto, Masumi, E-mail: masumi.eto@jefferson.edu [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Kirkbride, Jason A.; Chugh, Rishika; Karikari, Nana Kofi [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Kim, Jee In [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Cardiovascular Research Institute, Kyungpook National University School of Medicine, Daegu 700-422 (Korea, Republic of)

    2013-04-26

    Highlights: •Non-canonical roles of the myosin phosphatase inhibitor (CPI-17) were studied. •CPI-17 is localized in the nucleus of hyperplastic cancer and smooth muscle cells. •CPI-17 Ser12 phosphorylation may regulate the nuclear import. •CPI-17 regulates histone H3 phosphorylation and cell proliferation. •The nuclear CPI-17-PP1 axis plays a proliferative role in cells. -- Abstract: CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17 kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentrated in nuclei of human pancreatic cancer (Panc1) cells. Nuclear accumulation of CPI-17 was also detected in the proliferating vascular SM cell culture and cells at neointima of rat vascular injury model. The N-terminal 21-residue tail domain of CPI-17 was necessary for the nuclear localization. Phospho-mimetic Asp-substitution of CPI-17 at Ser12 attenuated the nuclear import. CPI-17 phosphorylated at Ser12 was not localized at nuclei, suggesting a suppressive role of Ser12 phosphorylation in the nuclear import. Activated CPI-17 bound to all three isoforms of PP1 catalytic subunit in Panc1 nuclear extracts. CPI-17 knockdown in Panc1 resulted in dephosphorylation of histone H3 at Thr3, Ser10 and Thr11, whereas it had no effects on the phosphorylation of myosin light chain and merlin, the known targets of MLCP. In parallel, CPI-17 knockdown suppressed Panc1 proliferation. We propose that CPI-17 accumulated in the nucleus through the N-terminal tail targets multiple PP1 signaling pathways regulating cell proliferation.

  20. Nuclear localization of CPI-17, a protein phosphatase-1 inhibitor protein, affects histone H3 phosphorylation and corresponds to proliferation of cancer and smooth muscle cells

    International Nuclear Information System (INIS)

    Highlights: •Non-canonical roles of the myosin phosphatase inhibitor (CPI-17) were studied. •CPI-17 is localized in the nucleus of hyperplastic cancer and smooth muscle cells. •CPI-17 Ser12 phosphorylation may regulate the nuclear import. •CPI-17 regulates histone H3 phosphorylation and cell proliferation. •The nuclear CPI-17-PP1 axis plays a proliferative role in cells. -- Abstract: CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17 kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentrated in nuclei of human pancreatic cancer (Panc1) cells. Nuclear accumulation of CPI-17 was also detected in the proliferating vascular SM cell culture and cells at neointima of rat vascular injury model. The N-terminal 21-residue tail domain of CPI-17 was necessary for the nuclear localization. Phospho-mimetic Asp-substitution of CPI-17 at Ser12 attenuated the nuclear import. CPI-17 phosphorylated at Ser12 was not localized at nuclei, suggesting a suppressive role of Ser12 phosphorylation in the nuclear import. Activated CPI-17 bound to all three isoforms of PP1 catalytic subunit in Panc1 nuclear extracts. CPI-17 knockdown in Panc1 resulted in dephosphorylation of histone H3 at Thr3, Ser10 and Thr11, whereas it had no effects on the phosphorylation of myosin light chain and merlin, the known targets of MLCP. In parallel, CPI-17 knockdown suppressed Panc1 proliferation. We propose that CPI-17 accumulated in the nucleus through the N-terminal tail targets multiple PP1 signaling pathways regulating cell proliferation

  1. A unique sequence in the N-terminal regulatory region controls the nuclear localization of KLF8 by cooperating with the C-terminal zinc-fingers

    Institute of Scientific and Technical Information of China (English)

    Tina S Mehta; Heng Lu; Xianhui Wang; Alison M Urvalek; Kim-Hang H Nguyen; Farah Monzur; Jojo D Hammond; Jameson Q Ma; Jihe Zhao

    2009-01-01

    Kruppel-like factor 8 (KLF8) transcription factor plays a critical role in cell cycle progression, oncogenic trans-formation, epithelial to mesenchymal transition and invasion. However, its nuclear localization signal(s) (NLS) has not been identified. KLF8 shares with other KLFs monopartite NLSs (mNLS) and C2H2 zinc fingers (ZFs), both of which have been shown to be the NLSs for some other KLFs. In this report, using PCR-directed mutagenesis and immunofluorescent microscopy, we show that disruption of the mNLSs, deletion of any single ZF, or mutation of the Zn2+-binding or DNA-contacting motifs did not affect the nuclear localization of KLF8. Deletion of>1.5 ZFs from C-terminus, however, caused cytoplasmic accumulation of KLF8. Surprisingly, deletion of amino acid (aa) 151-200 re-gion almost eliminated KLF8 from the nucleus. S165A, K171E or K171R mutation, or treatment with PKC inhibitor led to partial cytoplasmic accumulation. Co-immunoprecipitation demonstrated that KLF8 interacted with importin-β and this interaction required the ZF motif. Deletion of aa 1-150 or 201-261 region alone did not alter the nuclear lo-calization. BrdU incorporation and cyclin D1 promoter luciferase assays showed that the KLF8 mutants defective in nuclear localization could not promote DNA synthesis or cyclin D1 promoter activation as the wild-type KLF8 did. Taken together, these results suggest that KLF8 has two NLSs, one surrounding S165 and K171 and the other being two tandem ZFs, which are critical for the regulation of KLF8 nuclear localization and its cellular functions.

  2. A localized corrosion assessment of 6% molybdenum stainless steel condenser tubing at the Calvert Cliffs Nuclear Power Plant

    International Nuclear Information System (INIS)

    A condition assessment was performed on the Calvert Cliffs Nuclear Power Plant Unit 1 Main Steam Condenser 6% molybdenum stainless steel tubing. This work consisted of eddy current (EC) examination, in-situ visual examination of tubes, and destructive analysis of pulled tubes. The assessment objectives were as follows: to determine the tubing degradation mechanism, to render the condenser leak-tight, to identify methods of improving condenser life management, and to develop a basis for projecting the remaining condenser life. Once the corrosion mechanism was identified, seawater corrosion tests were performed on UNS N08366 and N08367 tubing using vinyl sleeves, Type 316 stainless compression fittings, and epoxy paint as crevice formers. Based on eddy current examination results, greater than 95% of the 49,500 tubes in the condenser had no detectable degradation. Over 1,200 tubes were plugged with either eddy current indications measuring greater than 60% wall loss or with obstructions. One waterbox (11A) contained 52% of all eddy current indications with over half of these in one section (B). Destructive analysis of pulled tubes revealed crevice corrosion, a form of localized corrosion that initiated under deposits. This type of corrosion occurred most often and had the deepest penetration in the weld seam. This was attributed to molybdenum and chromium segregation in the as-welded tubes and to deleterious secondary phases formed during weld solidification

  3. A Fungal Effector With Host Nuclear Localization and DNA-Binding Properties Is Required for Maize Anthracnose Development.

    Science.gov (United States)

    Vargas, Walter A; Sanz-Martín, José M; Rech, Gabriel E; Armijos-Jaramillo, Vinicio D; Rivera, Lina P; Echeverria, María Mercedes; Díaz-Mínguez, José M; Thon, Michael R; Sukno, Serenella A

    2016-02-01

    Plant pathogens have the capacity to manipulate the host immune system through the secretion of effectors. We identified 27 putative effector proteins encoded in the genome of the maize anthracnose pathogen Colletotrichum graminicola that are likely to target the host's nucleus, as they simultaneously contain sequence signatures for secretion and nuclear localization. We functionally characterized one protein, identified as CgEP1. This protein is synthesized during the early stages of disease development and is necessary for anthracnose development in maize leaves, stems, and roots. Genetic, molecular, and biochemical studies confirmed that this effector targets the host's nucleus and defines a novel class of double-stranded DNA-binding protein. We show that CgEP1 arose from a gene duplication in an ancestor of a lineage of monocot-infecting Colletotrichum spp. and has undergone an intense evolution process, with evidence for episodes of positive selection. We detected CgEP1 homologs in several species of a grass-infecting lineage of Colletotrichum spp., suggesting that its function may be conserved across a large number of anthracnose pathogens. Our results demonstrate that effectors targeted to the host nucleus may be key elements for disease development and aid in the understanding of the genetic basis of anthracnose development in maize plants.

  4. Absence of PTHrP nuclear localization and carboxyl terminus sequences leads to abnormal brain development and function.

    Directory of Open Access Journals (Sweden)

    Zhen Gu

    Full Text Available We assessed whether the nuclear localization sequences (NLS and C terminus of parathyroid hormone-related protein (PTHrP play critical roles in brain development and function. We used histology, immunohistochemistry, histomorphometry, Western blots and electrophysiological recordings to compare the proliferation and differentiation of neural stem cells, neuronal hippocampal synaptic transmission, and brain phenotypes including shape and structures, in Pthrp knock-in mice, which express PTHrP (1-84, a truncated form of the protein that is missing the NLS and the C-terminal region of the protein, and their wild-type littermates. Results showed that Pthrp knock-in mice display abnormal brain shape and structures; decreased neural cell proliferative capacity and increased apoptosis associated with up-regulation of cyclin dependent kinase inhibitors p16, p21, p27 and p53 and down-regulation of the Bmi-1 oncogene; delayed neural cell differentiation; and impaired hippocampal synaptic transmission and plasticity. These findings provide in vivo experimental evidence that the NLS and C-terminus of PTHrP are essential not only for the regulation of neural cell proliferation and differentiation, but also for the maintenance of normal neuronal synaptic transmission and plasticity.

  5. Beyond defense-in-depth: cost and funding of state and local government radiological emergency response plans and preparedness in support of commercial nuclear power stations

    International Nuclear Information System (INIS)

    Inadequate, sporadic, uncertain and frustrating are words local, state and Federal officials use to describe the current hodgepodge funding approach to State and local government radiological emergency response plans and preparedeness in support of commercial nuclear power stations. The creation of a Radiological Emergency Response Plans and Preparedness Fund for State and Local Government is offered as a preferred solution. Monies for the Fund could be derived from a one time Fee of $1 million levied on the operator of each nuclear power station. Every five years, adjustments could be made in the Fee to assure full recovery of costs because of inflation, revised criteria and other cost related factors. Any surplus would be refunded to the utilities. Any state that has obtained NRC concurrence or is in the process could be reimbursed for previous expenditures up to two years prior to NRC concurrence. Concurrence in all state and local government plans is the objective of the funding program. The Fund should be administered by the Nuclear Regulatory Commission. The report also discusses actions by Federal and state agencies and points to long range considerations, such as a training institute, including transportation and non-commercial and other fixed nuclear facilities, where preparedness could be enhanced by a coherent funding mechanism. All recommendations are based on an inquiry by the Office of state Programs, NRC, into the historical and future costs and funding of radiological emergency response plans and preparedness at the state and local government levels and are derived from discussions with many local, State and Federal officials

  6. Pathogen Persistence in the Environment and Insect-Baculovirus Interactions: Disease-Density Thresholds, Epidemic Burnout and Insect Outbreaks

    OpenAIRE

    Fuller, Emma; Elderd, Bret D.; Dwyer, Greg

    2012-01-01

    Classical epidemic theory focuses on directly transmitted pathogens, but many pathogens are instead transmitted when hosts encounter infectious particles. Theory has shown that for such diseases pathogen persistence time in the environment can strongly affect disease dynamics, but estimates of persistence time, and consequently tests of the theory, are extremely rare. We consider the consequences of persistence time for the dynamics of the gypsy moth baculovirus, a pathogen transmitted when l...

  7. Novel strategies to exploit existing natural infections: synergisms between baculoviruses and other toxins. Research Project Final Report

    OpenAIRE

    Hesketh, Helen; Hails, Rosemary

    2007-01-01

    EXECUTIVE SUMMARY Baculoviruses are a well studied group of insect viruses which have known advantages as biological control agents of insect pests in agriculture and forestry; strains used are very specific to the insect species they can infect, they can be formulated and sprayed conventionally and they are easily broken down by UV thereby leaving minimal non-toxic residues. They are the major group of viruses which infect insects but they do not replicate in vertebrates, plants or other ...

  8. IRES mediated expression of viral 3C protease for enhancing the yield of FMDV empty capsids using baculovirus system.

    Science.gov (United States)

    Vivek Srinivas, V M; Basagoudanavar, Suresh H; Hosamani, Madhusudan

    2016-03-01

    For expression of FMDV empty capsids, high protease activity associated with 3C co-expressed with P1 polyprotein has been reported to adversely affect the yields of capsids. Limiting the levels of 3Cpro relative to P1-2A polypeptide is thus critical to enhance the yields. In this study, FMDV internal ribosome entry site (IRES) sequence which serves as an alternative to the CAP-dependent translation initiation mechanism, was used for controlled translation of 3C protease. Baculovirus expressing bicistronic cDNA cassette containing two open reading frames-FMDV capsid gene (P1-2A) and 3Cpro intervened by IRES was prepared. Analysis of the expression in insect cells infected with baculovirus showed increased accumulation of processed capsids. Recombinant capsids showed higher immunoreactivity similar to the whole virus antigen, when reacted with polyclonal antibodies against the purified whole virus 146S particles. Thus, inclusion of the IRES upstream of 3Cpro facilitated reduced expression of the protease in baculovirus expression system, without causing significant proteolysis, thereby contributing to improved yields of the processed capsid antigens. PMID:26775685

  9. Introduction of temperature-sensitive helper and donor plasmids into Bac-to-Bac baculovirus expression systems

    Institute of Scientific and Technical Information of China (English)

    Zhihong; Huang; Ao; Li; Mengjia; Pan; Wenbi; Wu; Meijin; Yuan; Kai; Yang

    2015-01-01

    In the baculovirus shuttle vector(bacmid) system, a helper plasmid and a donor plasmid are employed to insert heterologous genes into a cloned baculovirus genome via Tn7 transposition in Escherichia coli. The helper and donor plasmids are usually cotransfected with constructed bacmids into insect cells, which will lead to integration of these plasmids into the viral genome,and hence to the production of defective virions. In this study, to facilitate the preparation of plasmid-free recombinant bacmids, we modified a set of helper and donor plasmids by replacing their replication origins with that of a temperature-sensitive(ts) plasmid, p SIM6. Using the resulting ts helper plasmid p MON7124 ts and the ts donor plasmid p FB1ts-PH-GFP, a recombinant bacmid,b Ac WT-PG(-), was constructed, and the transposition efficiency was found to be 33.1%. The plasmids were then removed by culturing at 37 °C. For b Ac WT-PG(-), the infectious progeny virus titer and the protein expression level under the control of the polyhedrin promoter were similar to those of a bacmid constructed with unmodified helper and donor plasmids. These ts plasmids will be useful for obtaining plasmid-free bacmids for both heterologous protein production and fundamental studies of baculovirus biology.

  10. AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells

    International Nuclear Information System (INIS)

    The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. - highlights: • CSE1L is a key player in nucleocytoplasmic traffic by forming complex with Ran. • AKT phosphorylates RanBP3 that regulates the nucleocytoplasmic gradient of Ran. • The activated oncogenic AKT drives the nuclear accumulation of CSE1L. • CSE1L in the nucleus up-regulates genes conveying pro-oncogenic signals. • CSE1L might contribute to tumor progression driven by the activated oncogenic AKT

  11. AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Lorenzato, Annalisa; Biolatti, Marta [Department of Oncology, University of Torino School of Medicine, Torino (Italy); Institute for Cancer Research at Candiolo, Candiolo, Torino (Italy); Delogu, Giuseppe [Department of Biomedical Sciences-Histology, University of Sassari, Sassari (Italy); Capobianco, Giampiero [Department of Surgical, Microsurgical and Medical Sciences, University of Sassari, Sassari (Italy); Farace, Cristiano [Department of Biomedical Sciences-Histology, University of Sassari, Sassari (Italy); Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco [Department of Surgical, Microsurgical and Medical Sciences, University of Sassari, Sassari (Italy); Madeddu, Roberto [Department of Biomedical Sciences-Histology, University of Sassari, Sassari (Italy); National Institute of Biostructures and Biosystems, Rome (Italy); Olivero, Martina [Department of Oncology, University of Torino School of Medicine, Torino (Italy); Institute for Cancer Research at Candiolo, Candiolo, Torino (Italy); Di Renzo, Maria Flavia, E-mail: mariaflavia.direnzo@unito.it [Department of Oncology, University of Torino School of Medicine, Torino (Italy); Institute for Cancer Research at Candiolo, Candiolo, Torino (Italy)

    2013-10-15

    The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. - highlights: • CSE1L is a key player in nucleocytoplasmic traffic by forming complex with Ran. • AKT phosphorylates RanBP3 that regulates the nucleocytoplasmic gradient of Ran. • The activated oncogenic AKT drives the nuclear accumulation of CSE1L. • CSE1L in the nucleus up-regulates genes conveying pro-oncogenic signals. • CSE1L might contribute to tumor progression driven by the activated oncogenic AKT.

  12. Effects of the nuclear localization of the N(pro) protein of classical swine fever virus on its virulence in pigs.

    Science.gov (United States)

    Li, Yongfeng; Shen, Liang; Sun, Yuan; Wang, Xiao; Li, Chao; Huang, Junhua; Chen, Jianing; Li, Lianfeng; Zhao, Bibo; Luo, Yuzi; Li, Su; Qiu, Hua-Ji

    2014-12-01

    The N(pro) protein of classical swine fever virus (CSFV) is localized in the cytoplasm and nucleus. However, it is unknown whether the nuclear localization of N(pro) correlates with the virulence of CSFV in the host. Previously, we showed that the N(pro) protein fused with interferon regulatory factor 3 (IRF3) was present only in the cytoplasm. Here, we generated and evaluated a recombinant CSFV vSM-IRF3 harboring the IRF3 gene inserted into the N(pro) gene of the highly virulent CSFV Shimen strain. Compared to the even nuclear and cytoplasmic distribution of the enhanced green fluorescent protein (EGFP)-N(pro) fusion expressed by the recombinant CSFV EGFP-CSFV, vSM-IRF3 expressed an IRF3-N(pro) fusion protein that only was localized in the cytoplasm. vSM-IRF3 was markedly attenuated in vitro and in vivo, and the inoculated pigs were completely protected from lethal CSFV challenge, whereas the parental virus as well as EGFP-CSFV exhibited a typical virulent phenotype. Taken together, the nuclear localization of N(pro) plays a significant role in the CSFV replication and virulence.

  13. Introduction of the anti-apoptotic baculovirus p35 gene in passion fruit induces herbicide tolerance, reduced bacterial lesions, but does not inhibits passion fruit woodiness disease progress induced by cowpea aphid-borne mosaic virus (CABMV).

    Science.gov (United States)

    de Freitas, Daniele Scandiucci; Coelho, Marly C Felipe; Souza, Manoel T; Marques, Abi; Ribeiro, E Bergmann Morais

    2007-01-01

    The introduction of anti-apoptotic genes into plants leads to resistance to environmental stress and broad-spectrum disease resistance. The anti-apoptotic gene (p35) from a baculovirus was introduced into the genome of passion fruit plants by biobalistics. Eleven regenerated plants showed the presence of the p35 gene by PCR and/or dot blot hybridization. Transcriptional analysis of regenerated plants showed the presence of specific p35 transcripts in 9 of them. Regenerated plants containing the p35 gene were inoculated with the cowpea aphid-borne mosaic virus (CABMV), the bacterium Xanthomonas axonopodis pv passiflorae, and the herbicide, glufosinate, (Syngenta). None of the plants showed resistance to CABMV. Regenerated plants (p35+) showed less than half of local lesions showed by non-transgenic plants when inoculated with X. axonopodis and some p35+ plants showed increased tolerance to the glufosinate herbicide when compared to non-transgenic plants. PMID:17016672

  14. Identification and fine mapping of nuclear and nucleolar localization signals within the human ribosomal protein S17.

    Directory of Open Access Journals (Sweden)

    Scott P Kenney

    Full Text Available Human ribosomal protein S17 (RPS17 is mutated in Diamond-Blackfan Anemia (DBA, a bone marrow disorder that fails to produce sufficient red blood cells leading to anemia. Recently, an RPS17 protein sequence was also found to be naturally inserted in the genome of hepatitis E virus (HEV from patients chronically-infected by HEV. The role of RPS17 in HEV replication and pathogenesis remains unknown due to the lack of knowledge about how RPS17 functions at a molecular level. Understanding the biological function of RPS17 is critical for elucidating its role in virus infection and DBA disease processes. In this study we probed the subcellular distribution of normal and mutant RPS17 proteins in a human liver cell line (Huh7. RPS17 was primarily detected within the nucleus, and more specifically within the nucleoli. Using a transient expression system in which RPS17 or truncations were expressed as fusions with enhanced yellow fluorescent protein (eYFP, we were able to identify and map, for the first time, two separate nuclear localization signals (NLSs, one to the first 13 amino acids of the amino-terminus of RPS17 and the other within amino acids 30-60. Additionally, we mapped amino acid sequences required for nucleolar accumulation of RPS17 to amino acids 60-70. Amino acids 60-70 possess a di-RG motif that may be necessary for nucleolar retention of RPS17. The results from this study enhance our knowledge of RSP17 and will facilitate future mechanistic studies about the roles of RSP17 in hepatitis E and DBA disease processes.

  15. G-actin guides p53 nuclear transport: potential contribution of monomeric actin in altered localization of mutant p53

    Science.gov (United States)

    Saha, Taniya; Guha, Deblina; Manna, Argha; Panda, Abir Kumar; Bhat, Jyotsna; Chatterjee, Subhrangsu; Sa, Gaurisankar

    2016-01-01

    p53 preserves genomic integrity by restricting anomaly at the gene level. Till date, limited information is available for cytosol to nuclear shuttling of p53; except microtubule-based trafficking route, which utilizes minus-end directed motor dynein. The present study suggests that monomeric actin (G-actin) guides p53 traffic towards the nucleus. Histidine-tag pull-down assay using purified p53(1–393)-His and G-actin confirms direct physical association between p53 and monomeric G-actin. Co-immunoprecipitation data supports the same. Confocal imaging explores intense perinuclear colocalization between p53 and G-actin. To address atomistic details of the complex, constraint-based docked model of p53:G-actin complex was generated based on crystal structures. MD simulation reveals that p53 DNA-binding domain arrests very well the G-actin protein. Docking benchmark studies have been carried out for a known crystal structure, 1YCS (complex between p53DBD and BP2), which validates the docking protocol we adopted. Co-immunoprecipitation study using “hot-spot” p53 mutants suggested reduced G-actin association with cancer-associated p53 conformational mutants (R175H and R249S). Considering these findings, we hypothesized that point mutation in p53 structure, which diminishes p53:G-actin complexation results in mutant p53 altered subcellular localization. Our model suggests p53Arg249 form polar-contact with Arg357 of G-actin, which upon mutation, destabilizes p53:G-actin interaction and results in cytoplasmic retention of p53R249S. PMID:27601274

  16. G-actin guides p53 nuclear transport: potential contribution of monomeric actin in altered localization of mutant p53.

    Science.gov (United States)

    Saha, Taniya; Guha, Deblina; Manna, Argha; Panda, Abir Kumar; Bhat, Jyotsna; Chatterjee, Subhrangsu; Sa, Gaurisankar

    2016-01-01

    p53 preserves genomic integrity by restricting anomaly at the gene level. Till date, limited information is available for cytosol to nuclear shuttling of p53; except microtubule-based trafficking route, which utilizes minus-end directed motor dynein. The present study suggests that monomeric actin (G-actin) guides p53 traffic towards the nucleus. Histidine-tag pull-down assay using purified p53(1-393)-His and G-actin confirms direct physical association between p53 and monomeric G-actin. Co-immunoprecipitation data supports the same. Confocal imaging explores intense perinuclear colocalization between p53 and G-actin. To address atomistic details of the complex, constraint-based docked model of p53:G-actin complex was generated based on crystal structures. MD simulation reveals that p53 DNA-binding domain arrests very well the G-actin protein. Docking benchmark studies have been carried out for a known crystal structure, 1YCS (complex between p53DBD and BP2), which validates the docking protocol we adopted. Co-immunoprecipitation study using "hot-spot" p53 mutants suggested reduced G-actin association with cancer-associated p53 conformational mutants (R175H and R249S). Considering these findings, we hypothesized that point mutation in p53 structure, which diminishes p53:G-actin complexation results in mutant p53 altered subcellular localization. Our model suggests p53Arg249 form polar-contact with Arg357 of G-actin, which upon mutation, destabilizes p53:G-actin interaction and results in cytoplasmic retention of p53R249S. PMID:27601274

  17. Nuclear-localized AtHSPR links abscisic acid-dependent salt tolerance and antioxidant defense in Arabidopsis.

    Science.gov (United States)

    Yang, Tao; Zhang, Liang; Hao, Hongyan; Zhang, Peng; Zhu, Haowei; Cheng, Wei; Wang, Yongli; Wang, Xinyu; Wang, Chongying

    2015-12-01

    Salt stress from soil or irrigation water limits plant growth. A T-DNA insertion mutant in C24, named athspr (Arabidopsis thaliana heat shock protein-related), showed several phenotypes, including reduced organ size and enhanced sensitivity to environmental cues. The athspr mutant is severely impaired under salinity levels at which wild-type (WT) plants grow normally. AtHSPR encodes a nuclear-localized protein with ATPase activity, and its expression was enhanced by high salinity and abscisic acid (ABA). Overexpression (OE) of AtHSPR significantly enhanced tolerance to salt stress by increasing the activities of the antioxidant system and by maintaining K(+) /Na(+) homeostasis. Quantitative RT-PCR analyses showed that OE of AtHSPR increased the expression of ABA/stress-responsive, salt overly sensitive (SOS)-related and antioxidant-related genes. In addition, ABA content was reduced in athspr plants with or without salt stress, and exogenous ABA restored WT-like salt tolerance to athspr plants. athspr exhibited increased leaf stomatal density and stomatal index, slower ABA-induced stomatal closure and reduced drought tolerance relative to the WT. AtHSPR OE enhanced drought tolerance by reducing leaf water loss and stomatal aperture. Transcript profiling in athspr showed a differential salt-stress response for genes involved in accumulation of reactive oxygen species (ROS), ABA signaling, cell death, stress response and photosynthesis. Taken together, our results suggested that AtHSPR is involved in salt tolerance in Arabidopsis through modulation of ROS levels, ABA-dependent stomatal closure, photosynthesis and K(+) /Na(+) homeostasis. PMID:26603028

  18. Collaboration of local governments and experts responding to the increase of the environmental radiation level secondary to the nuclear accident: a unique activity to relieve residents' anxiety.

    Science.gov (United States)

    Fujii, H; Iimoto, T; Tsuzuki, T; Iiizumi, S; Someya, S; Hamamichi, S; Kessler, M M

    2015-11-01

    After the Fukushima nuclear power plant accident, 'hot spots' were found in Tokatsu area in Chiba prefecture. Although ambient radiation dose in this area was too low to harm residents' health, local residents were particularly worried about possible adverse effects from exposure to radiation. To avoid unnecessary panic reactions in the public, local governments in Tokatsu area collaborated with radiation specialists and conducted activities to provide local residents with accurate information on health effects from radiation. In addition to these activities, the authors offered one-to-one consultations with a radiologist for parents of small children and expecting mothers. They herein report this unique attempt, focusing on parents' anxiety and the age of their children. Taken together, this unique collaborative activity between local government and experts would be one of the procedures to relieve residents' anxiety.

  19. Novel nuclear localization and potential function of insulin-like growth factor-1 receptor/insulin receptor hybrid in corneal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Yu-Chieh Wu

    Full Text Available BACKGROUND: Type I insulin-like growth factor receptor (IGF-1R and insulin receptor (INSR are highly homologous molecules, which can heterodimerize to form an IGF-1R/INSR hybrid (Hybrid-R. The presence and biological significance of the Hybrid-R in human corneal epithelium has not yet been established. In addition, while nuclear localization of IGF-1R was recently reported in cancer cells and human corneal epithelial cells, the function and profile of nuclear IGF-1R is unknown. In this study, we characterized the nuclear localization and function of the Hybrid-R and the role of IGF-1/IGF-1R and Hybrid-R signaling in the human corneal epithelium. METHODOLOGY/PRINCIPLE FINDINGS: IGF-1-mediated signaling and cell growth were examined in a human telomerized corneal epithelial (hTCEpi cell line using co-immunoprecipitation, immunoblotting and cell proliferation assays. The presence of Hybrid-R in hTCEpi and primary cultured human corneal epithelial cells was confirmed by immunofluorescence and reciprocal immunoprecipitation of whole cell lysates. We found that IGF-1 stimulated Akt and promoted cell growth through IGF-1R activation, which was independent of the Hybrid-R. The presence of Hybrid-R, but not IGF-1R/IGF-1R, was detected in nuclear extracts. Knockdown of INSR by small interfering RNA resulted in depletion of the INSR/INSR and preferential formation of Hybrid-R. Chromatin-immunoprecipitation sequencing assay with anti-IGF-1R or anti-INSR was subsequently performed to identify potential genomic targets responsible for critical homeostatic regulatory pathways. CONCLUSION/SIGNIFICANCE: In contrast to previous reports on nuclear localized IGF-1R, this is the first report identifying the nuclear localization of Hybrid-R in an epithelial cell line. The identification of a nuclear Hybrid-R and novel genomic targets suggests that IGF-1R traffics to the nucleus as an IGF-1R/INSR heterotetrameric complex to regulate corneal epithelial homeostatic

  20. Nuclear localization of P-glycoprotein is responsible for protection of the nucleus from doxorubicin in the resistant LoVo cell line.

    Science.gov (United States)

    Szaflarski, Witold; Sujka-Kordowska, Patrycja; Januchowski, Radosław; Wojtowicz, Karolina; Andrzejewska, Małgorzata; Nowicki, Michał; Zabel, Maciej

    2013-07-01

    The high expression of P-glycoprotein (P-gp) belongs to one of the most important factors causing multidrug-resistant (MDR) of cancer cells. P-gp is primarily associated with plasma membrane; however, small fraction of that protein is present in the nuclear envelope. Such phenomenon is observed in cancer cells and may result in the selection of MDR cells as the secondary tumor and/or resistant metastasis that significantly shorten patient survival rate. Here, we confirmed nuclear localization of P-gp in resistant LoVo cells and demonstrated its impact on doxorubicin efflux from the nucleus to cytoplasm. Furthermore, we showed that P-gp located at the nuclear envelope might have a different glycoside chain when compared to the form located in the cytoplasm. It suggests that the glycoside chain plays a role in the intracellular trafficking of P-gp and may decide about the destination place in the cell. PMID:23602050

  1. Nuclear localization of P-glycoprotein is responsible for protection of the nucleus from doxorubicin in the resistant LoVo cell line.

    Science.gov (United States)

    Szaflarski, Witold; Sujka-Kordowska, Patrycja; Januchowski, Radosław; Wojtowicz, Karolina; Andrzejewska, Małgorzata; Nowicki, Michał; Zabel, Maciej

    2013-07-01

    The high expression of P-glycoprotein (P-gp) belongs to one of the most important factors causing multidrug-resistant (MDR) of cancer cells. P-gp is primarily associated with plasma membrane; however, small fraction of that protein is present in the nuclear envelope. Such phenomenon is observed in cancer cells and may result in the selection of MDR cells as the secondary tumor and/or resistant metastasis that significantly shorten patient survival rate. Here, we confirmed nuclear localization of P-gp in resistant LoVo cells and demonstrated its impact on doxorubicin efflux from the nucleus to cytoplasm. Furthermore, we showed that P-gp located at the nuclear envelope might have a different glycoside chain when compared to the form located in the cytoplasm. It suggests that the glycoside chain plays a role in the intracellular trafficking of P-gp and may decide about the destination place in the cell.

  2. Coordinate regulation/localization of the carbohydrate responsive binding protein (ChREBP) by two nuclear export signal sites: Discovery of a new leucine-rich nuclear export signal site

    International Nuclear Information System (INIS)

    Carbohydrate response element binding protein (ChREBP) is responsible for conversion of dietary carbohydrate to storage fat in liver by coordinating expression of the enzymes that channel glycolytic pyruvate into lipogenesis. The activation of ChREBP in response to high glucose is nuclear localization and transcription, and the inactivation of ChREBP under low glucose involves export from the nucleus to the cytosol. Here we report a new nuclear export signal site ('NES1') of ChREBP. Together these signals provide ChREBP with two NES sequences, both the previously reported NES2 and now the new NES1 coordinate to interact together with CRM1 (exportin) for nuclear export of the carbohydrate response element binding protein.

  3. A case study of economic incentives and local citizens' attitudes toward hosting a nuclear power plant in Japan: Impacts of the Fukushima accident

    International Nuclear Information System (INIS)

    The attitude of local communities near a nuclear power plant (NPP) is a key factor in nuclear policy decision making in Japan. This case study compared local citizens' attitudes in 2010 and 2011 toward the benefits and drawbacks of hosting Kashiwazaki–Kariwa NPP. The Fukushima accident occurred in this period. After the accident, benefit recognition of utility bill refunds clearly declined, while that of public facilities did not, suggesting the influence of a bribery effect. The negative shift of attitudes about hosting the NPP after the accident was more modest in Kariwa Village, which saw a large expansion of social welfare programs, than in the other two areas, which lacked such a budget expansion. Policy implications of these results regarding the provision of economic incentives in NPP host areas after the Fukushima accident were discussed. - Highlights: • The Fukushima accident shocked Japan's nuclear policy. • Citizens' attitudes toward incentives of hosting a nuclear power plant surveyed. • More citizens thought negatively about incentives after the Fukushima accident. • The bribery effect, mode and amount of incentives affected citizens' attitudes

  4. Effects of temperature and shear force on infectivity of the baculovirus Autographa californica M nucleopolyhedrovirus.

    Science.gov (United States)

    Michalsky, Ronald; Pfromm, Peter H; Czermak, Peter; Sorensen, Christopher M; Passarelli, A Lorena

    2008-11-01

    Virus stability and infectivity during stressful conditions was assessed to establish guidelines for future virus filtration experiments and to contribute to the body of knowledge on a widely used virus. A recombinant baculovirus of Autographa californica M nucleopolyhedrovirus (AcMNPV), vHSGFP, was incubated at 15-65 degrees C. A 2-log decrease in virus infectivity occurred after virus incubation above 45 degrees C. The activation energy of virus deactivation was circa 108 kJ/mol. Dynamic light scattering revealed an increase in apparent virus particle size from 150+/-19 to 249+/-13 nm at 55 degrees C. Protein and DNA concentrations in solution correlated well with virus aggregation as temperature was increased. Infectivity of vHSGFP stored for 5 months at 4 degrees C or exposed to shear stress from stirring (100 rpm, 1.02x10(-5) psi) and pumping (50-250 ml/min, 1.45x10(-5) to 7.25x10(-5) psi) did not change with time. Unlike temperature variations, cold storage and shear stress appeared to have little impact on infectivity.

  5. Baculovirus-mediated promoter assay and transcriptional analysis of white spot syndrome virus orf427 gene

    Directory of Open Access Journals (Sweden)

    Yu Li

    2005-08-01

    Full Text Available Abstract Background White spot syndrome virus (WSSV is an important pathogen of the penaeid shrimp with high mortalities. In previous reports, Orf427 of WSSV is characterized as one of the three major latency-associated genes of WSSV. Here, we were interested to analyze the promoter of orf427 and its expression during viral pathogenesis. Results in situ hybridization revealed that orf427 was transcribed in all the infected tissues during viral lytic infection and the translational product can be detected from the infected shrimp. A time-course RT-PCR analysis indicated that transcriptional products of orf427 could only be detected after 6 h post virus inoculation. Furthermore, a baculovirus-mediated promoter analysis indicated that the promoter of orf427 failed to express the EGFP reporter gene in both insect SF9 cells and primary shrimp cells. Conclusion Our data suggested that latency-related orf427 might not play an important role in activating virus replication from latent phase due to its late transcription during the lytic infection.

  6. 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.

  7. Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses

    Energy Technology Data Exchange (ETDEWEB)

    Bejerman, Nicolás, E-mail: n.bejerman@uq.edu.au [Instituto de Patología Vegetal (IPAVE), Centro de Investigaciones Agropecuarias (CIAP), Instituto Nacional de Tecnología Agropecuaria INTA, Camino a 60 Cuadras k 5,5, Córdoba X5020ICA (Argentina); Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, QLD 4072 (Australia); Giolitti, Fabián; Breuil, Soledad de; Trucco, Verónica; Nome, Claudia; Lenardon, Sergio [Instituto de Patología Vegetal (IPAVE), Centro de Investigaciones Agropecuarias (CIAP), Instituto Nacional de Tecnología Agropecuaria INTA, Camino a 60 Cuadras k 5,5, Córdoba X5020ICA (Argentina); Dietzgen, Ralf G. [Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, QLD 4072 (Australia)

    2015-09-15

    Summary: We have determined the full-length 14,491-nucleotide genome sequence of a new plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3′-N-P-P3-M-G-P6-L-5′. The ORFs are separated by conserved intergenic regions and the genome coding region is flanked by complementary 3′ leader and 5′ trailer sequences. Phylogenetic analysis of the nucleoprotein amino acid sequence indicated that this alfalfa-infecting rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins accumulated in the cell periphery, but unexpectedly P protein was localized exclusively in the nucleus. ADV P protein was shown to have a homotypic, and heterotypic nuclear interactions with N, P3 and M proteins by bimolecular fluorescence complementation. ADV appears unique in that it combines properties of both cytoplasmic and nuclear plant rhabdoviruses. - Highlights: • The complete genome of alfalfa dwarf virus is obtained. • An integrated localization and interaction map for ADV is determined. • ADV has a genome sequence similarity and evolutionary links with cytorhabdoviruses. • ADV protein localization and interaction data show an association with the nucleus. • ADV combines properties of both cytoplasmic and nuclear plant rhabdoviruses.

  8. Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP): ROLE OF AMP AS AN ALLOSTERIC INHIBITOR.

    Science.gov (United States)

    Sato, Shogo; Jung, Hunmin; Nakagawa, Tsutomu; Pawlosky, Robert; Takeshima, Tomomi; Lee, Wan-Ru; Sakiyama, Haruhiko; Laxman, Sunil; Wynn, R Max; Tu, Benjamin P; MacMillan, John B; De Brabander, Jef K; Veech, Richard L; Uyeda, Kosaku

    2016-05-13

    The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis. PMID:26984404

  9. Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses

    International Nuclear Information System (INIS)

    Summary: We have determined the full-length 14,491-nucleotide genome sequence of a new plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3′-N-P-P3-M-G-P6-L-5′. The ORFs are separated by conserved intergenic regions and the genome coding region is flanked by complementary 3′ leader and 5′ trailer sequences. Phylogenetic analysis of the nucleoprotein amino acid sequence indicated that this alfalfa-infecting rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins accumulated in the cell periphery, but unexpectedly P protein was localized exclusively in the nucleus. ADV P protein was shown to have a homotypic, and heterotypic nuclear interactions with N, P3 and M proteins by bimolecular fluorescence complementation. ADV appears unique in that it combines properties of both cytoplasmic and nuclear plant rhabdoviruses. - Highlights: • The complete genome of alfalfa dwarf virus is obtained. • An integrated localization and interaction map for ADV is determined. • ADV has a genome sequence similarity and evolutionary links with cytorhabdoviruses. • ADV protein localization and interaction data show an association with the nucleus. • ADV combines properties of both cytoplasmic and nuclear plant rhabdoviruses

  10. Intracellular distribution of an integral nuclear pore membrane protein fused to green fluorescent protein--localization of a targeting domain.

    Science.gov (United States)

    Söderqvist, H; Imreh, G; Kihlmark, M; Linnman, C; Ringertz, N; Hallberg, E

    1997-12-15

    The 121-kDa pore membrane protein (POM121) is a bitopic integral membrane protein specifically located in the pore membrane domain of the nuclear envelope with its short N-terminal tail exposed on the luminal side and its major C-terminal portion adjoining the nuclear pore complex. In order to locate a signal for targeting of POM121 to the nuclear pores, we overexpressed selected regions of POM121 alone or fused to the green fluorescent protein (GFP) in transiently transfected COS-1 cells or in a stably transfected neuroblastoma cell line. Microscopic analysis of the GFP fluorescence or immunostaining was used to determine the intracellular distribution of the overexpressed proteins. The endofluorescent GFP tag had no effect on the distribution of POM121, since the chimerical POM121-GFP fusion protein was correctly targeted to the nuclear pores of both COS-1 cells and neuroblastoma cells. Based on the differentiated intracellular sorting of the POM121 variants, we conclude that the first 128 amino acids of POM121 contains signals for targeting to the continuous endoplasmic reticulum/nuclear envelope membrane system but not specifically to the nuclear pores and that a specific nuclear pore targeting signal is located between amino acids 129 and 618 in the endoplasmically exposed portion of POM121. PMID:9461306

  11. A viral nuclear noncoding RNA binds re-localized poly(A binding protein and is required for late KSHV gene expression.

    Directory of Open Access Journals (Sweden)

    Sumit Borah

    2011-10-01

    Full Text Available During the lytic phase of infection, the gamma herpesvirus Kaposi's Sarcoma-Associated Herpesvirus (KSHV expresses a highly abundant, 1.1 kb nuclear noncoding RNA of unknown function. We observe that this polyadenylated nuclear (PAN RNA avidly binds host poly(A-binding protein C1 (PABPC1, which normally functions in the cytoplasm to bind the poly(A tails of mRNAs, regulating mRNA stability and translation efficiency. During the lytic phase of KSHV infection, PABPC1 is re-localized to the nucleus as a consequence of expression of the viral shutoff exonuclease (SOX protein; SOX also mediates the host shutoff effect in which host mRNAs are downregulated while viral mRNAs are selectively expressed. We show that whereas PAN RNA is not required for the host shutoff effect or for PABPC1 re-localization, SOX strongly upregulates the levels of PAN RNA in transient transfection experiments. This upregulation is destroyed by the same SOX mutation that ablates the host shutoff effect and PABPC1 nuclear re-localization or by removal of the poly(A tail of PAN. In cells induced into the KSHV lytic phase, depletion of PAN RNA using RNase H-targeting antisense oligonucleotides reveals that it is necessary for the production of late viral proteins from mRNAs that are themselves polyadenylated. Our results add to the repertoire of functions ascribed to long noncoding RNAs and suggest a mechanism of action for nuclear noncoding RNAs in gamma herpesvirus infection.

  12. Collaboration of local government and experts responding to increase in environmental radiation level due to the nuclear disaster: focusing on their activities and latest radiological discussion.

    Science.gov (United States)

    Iimoto, T; Nunokawa, J; Fujii, H; Takashima, R; Hashimoto, M; Fukuhara, T; Yajima, T; Matsuzawa, H; Kurosawa, K; Yanagawa, Y; Someya, S

    2015-11-01

    Activities were introduced in Kashiwa city in the Tokyo metropolitan area to correspond to the elevated environmental radiation level after the disaster of the Fukushima Daiichi nuclear power plant. These were based on a strong cooperation between local governments and experts. Ambient dose rate and radioactivity of foodstuff produced inside of the city have been monitored. Representative ambient dose rates around living environments have almost already become their original levels of the pre-accident because of the decontamination activity, natural washout and effective half-lives of radioactivity. The internal annual dose due to radioactive cesium under the policy of 'Local Production for Local Consumption' is estimated as extremely low comparing the variation range due to natural radioactivity. Systematic survey around a retention basin has been started. All of these latest monitoring data would be one of the core information for the policy making as well as a cost-benefit discussion and risk communication.

  13. Collaboration of local government and experts responding to increase in environmental radiation level due to the nuclear disaster: focusing on their activities and latest radiological discussion

    International Nuclear Information System (INIS)

    Activities were introduced in Kashiwa city in the Tokyo metropolitan area to correspond to the elevated environmental radiation level after the disaster of the Fukushima Daiichi nuclear power plant. These were based on a strong cooperation between local governments and experts. Ambient dose rate and radioactivity of foodstuff produced inside of the city have been monitored. Representative ambient dose rates around living environments have almost already become their original levels of the pre-accident because of the decontamination activity, natural washout and effective half-lives of radioactivity. The internal annual dose due to radioactive cesium under the policy of 'Local Production for Local Consumption' is estimated as extremely low comparing the variation range due to natural radioactivity. Systematic survey around a retention basin has been started. All of these latest monitoring data would be one of the core information for the policy making as well as a cost-benefit discussion and risk communication. (authors)

  14. Local fallout from nuclear test detonations. Volume 2. Compilation of fallout patterns and related test data. Supplement. Foreign nuclear tests. Sanitized

    Energy Technology Data Exchange (ETDEWEB)

    Morgenthau, M.; Showers, R.L.

    1964-10-01

    The available fallout patterns and related test data for nuclear weapon tests conducted by the United Kingdom, the Republic of France, and the Union of Soviet Socialist Republics, are included in this supplement to NDL-TR-34. The related test data for the British and French tests include: date and time of detonation, location of test site, total yield, fission yield, type of burst and placement, height of burst, cloud-top and -bottom heights, crater data, and wind information up to nuclear cloud-top height. No fallout patterns are available for any of the Soviet detonations. The list of Soviet detonations, which is as comprehensive as possible, contains the chronological order of the detonations, date, yield, type of burst and location of test site.

  15. A study on the institutional design concerning role-sharing between national and local governments about nuclear safety regulation in Japan

    International Nuclear Information System (INIS)

    In this paper, we consider the concept and the concrete direction of the institutional design concerning role-sharing between national and local governments with respect to nuclear safety regulation, and propose the following four institutional options: 1) strengthen the regulatory body's independence and clarify its accountability, 2) establish a local information committee in reference to the French CLI (Commission Locale d'Information), 3) institutionalize the environmental monitoring of local governments, and 4) institutionalize consultation among the regulatory body and local governments. Options 2, 3, and 4 are designed on the assumption that option 1 is implemented, because it is thought to be essential for fundamental improvements to enhance the regulatory body's ability as well as to reinforce bridges between safety regulation and society. Each option has some advantages and disadvantages, so there is room for further research into achieving reforms. However, it is believed that these institutional options can lead to the development of a trusting relationship between organizations responsible for safety regulation and siting areas and to the reduction in consensus-building costs in local areas. (author)

  16. 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.

  17. 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

  18. Production of mink enteritis parvovirus empty capsids by expression in a baculovirus vector system: a recombinant vaccine for mink enteritis parvovirus in mink

    DEFF Research Database (Denmark)

    Christensen, J; Alexandersen, Søren; Bloch, B.;

    1994-01-01

    The VP-2 gene of mink enteritis parvovirus (MEV) was amplified by the polymerase chain reaction using MEV DNA isolated from the faeces of a naturally infected mink. Subsequently the VP-2 gene was cloned into a baculovirus expression vector. Recombinant baculo-viruses were isolated and the MEV VP-2...... protein was able to form parvovirus-like particles, which had haemagglutinating properties comparable with the wild-type MEV. The cloned VP-2 gene was sequenced and only five nucleotide differences were found after alignment with the known sequences of the MEV type 1 and type 2 isolates. Surprisingly...

  19. Development of a quantal assay in primary shrimp cell culture for yellow head baculovirus (YBV) of penaeid shrimp.

    Science.gov (United States)

    Lu, Y; Tapay, L M; Loh, P C; Brock, J A; Gose, R

    1995-03-01

    A 50% tissue culture infectious dose assay (TCID50) using primary culture of shrimp lymphoid organ (Oka) cells was developed for the quantitative titration of yellow-head baculovirus (YBV), a newly isolated virus of penaeid shrimp. The assay protocol includes the use of Primaria-grade 96-well tissue culture plates to grow the primary lymphoid organ cells of penaeid shrimp. A 15% gill suspension from YBV-infected shrimp was determined to have an infectious virus titer of 5 x 10(5.75) TCID50/ml. This report represents the first convenient assay protocol using cell culture derived from penaeid shrimp to titer a shrimp virus.

  20. The hepatitis E virus ORF3 protein regulates the expression of liver-specific genes by modulating localization of hepatocyte nuclear factor 4.

    Directory of Open Access Journals (Sweden)

    Vivek Chandra

    Full Text Available The hepatitis E virus (HEV is a small RNA virus and the cause of acute viral hepatitis E. The open reading frame 3 protein (pORF3 of HEV appears to be a pleiotropic regulatory protein that helps in the establishment, propagation and progression of viral infection. However, the global cellular effects of this protein remain to be explored. In the absence of traditional in vitro viral infection systems or efficient replicon systems, we made an adenovirus based ORF3 protein expression system to study its effects on host cell gene expression. We infected Huh7 hepatoma cells with recombinant adenoviruses expressing pORF3 and performed microarray-based gene expression analyses. Several genes down regulated in pORF3-expressing cells were found to be under regulation of the liver-enriched hepatocyte nuclear factor 4 (HNF4, which regulates hepatocyte-specific gene expression. While HNF4 localizes to the nucleus, its phosphorylation results in impaired nuclear localization of HNF4. Here we report that pORF3 increases HNF4 phosphorylation through the ERK and Akt kinases, which results in impaired nuclear translocation of HNF4 and subsequently the down modulation of HNF4-responsive genes in pORF3-expressing cells. We propose that modulation of several hepatocyte specific genes by pORF3 will create an environment favorable for viral replication and pathogenesis.

  1. Methods in laboratory investigation. Autoradiographic demonstration of the specific binding and nuclear localization of 3H-dexamethasone in adult mouse lung.

    Science.gov (United States)

    Beer, D G; Cunha, G R; Malkinson, A M

    1983-12-01

    This report describes the first autoradiographic demonstration of specific nuclear localization of 3H-dexamethasone in different cell types of the lung. Adult mouse lung tissue was incubated in vitro for 90 minutes with 17 nM 3H-dexamethasone in the presence or absence of various nonradioactive steroids. After extensive washing to remove any nonspecifically bound ligand, the specimens were processed for autoradiography using the thaw-mount method. In the absence of competing steroids, silver grains were localized in the nuclei of alveolar type II cells, bronchiolar and arteriolar smooth muscle cells, fibroblasts, and endothelial cells of the pulmonary vasculature. No significant nuclear concentration of label was observed in the bronchiolar epithelium, however. The specificity of 3H-dexamethasone labeling was demonstrated by incubating 17 nM 3H-dexamethasone with a 600-fold excess of either unlabeled dexamethasone, estrogen, dihydrotestosterone, or progesterone. These autoradiographic binding and steroid competition studies were confirmed by quantifying with liquid scintillation counting the specific 3H-dexamethasone binding in nuclear and cytosolic fractions prepared from lung tissues that had undergone identical incubation and washing procedures as those for autoradiography. These results demonstrate that many cell types in adult lung are targets for glucocorticoids and may respond to physiologic concentrations of this hormone.

  2. Protein Expression in Insect and Mammalian Cells Using Baculoviruses in Wave Bioreactors.

    Science.gov (United States)

    Kadwell, Sue H; Overton, Laurie K

    2016-01-01

    Many types of disposable bioreactors for protein expression in insect and mammalian cells are now available. They differ in design, capacity, and sensor options, with many selections available for either rocking platform, orbitally shaken, pneumatically mixed, or stirred-tank bioreactors lined with an integral disposable bag (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). WAVE Bioreactors™ were among the first disposable systems to be developed (Singh, Cytotechnology 30:149-158, 1999). Since their commercialization in 1999, Wave Bioreactors have become routinely used in many laboratories due to their ease of operation, limited utility requirements, and protein expression levels comparability to traditional stirred-tank bioreactors. Wave Bioreactors are designed to use a presterilized Cellbag™, which is attached to a rocking platform and inflated with filtered air provided by the bioreactor unit. The Cellbag can be filled with medium and cells and maintained at a set temperature. The rocking motion, which is adjusted through angle and rock speed settings, provides mixing of oxygen (and CO2, which is used to control pH in mammalian cell cultures) from the headspace created in the inflated Cellbag with the cell culture medium and cells. This rocking motion can be adjusted to prevent cell shear damage. Dissolved oxygen and pH can be monitored during scale-up, and samples can be easily removed to monitor other parameters. Insect and mammalian cells grow very well in Wave Bioreactors (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). Combining Wave Bioreactor cell growth capabilities with recombinant baculoviruses engineered for insect or mammalian cell expression has proven to be a powerful tool for rapid production of a wide range of proteins.

  3. Measurement of 2-dimensional local instantaneous liquid film thickness around simulated nuclear fuel rod by ultrasonic transmission technique

    International Nuclear Information System (INIS)

    An accurate knowledge of hydro-dynamic behavior of a liquid film flow on nuclear fuel rods is indispensable for analysis of the CHF under postulated loss-of-coolant-accidents in boiling water reactors. This work is concerned with a new development of ultrasonic transmission technique for film flow measurements. The technique adopted a rotating reflector, capable of measuring time-dependent spatial distribution of liquid film thickness around a simulated nuclear fuel rod. The scanning time is currently 4 ms for reconstruction of one image of the circumferential film thickness distribution. (orig.)

  4. Development and Implementation of a High-Throughput High-Content Screening Assay to Identify Inhibitors of Androgen Receptor Nuclear Localization in Castration-Resistant Prostate Cancer Cells.

    Science.gov (United States)

    Johnston, Paul A; Nguyen, Minh M; Dar, Javid A; Ai, Junkui; Wang, Yujuan; Masoodi, Khalid Z; Shun, Tongying; Shinde, Sunita; Camarco, Daniel P; Hua, Yun; Huryn, Donna M; Wilson, Gabriela Mustata; Lazo, John S; Nelson, Joel B; Wipf, Peter; Wang, Zhou

    2016-05-01

    Patients with castration-resistant prostate cancer (CRPC) can be treated with abiraterone, a potent inhibitor of androgen synthesis, or enzalutamide, a second-generation androgen receptor (AR) antagonist, both targeting AR signaling. However, most patients relapse after several months of therapy and a majority of patients with relapsed CRPC tumors express the AR target gene prostate-specific antigen (PSA), suggesting that AR signaling is reactivated and can be targeted again to inhibit the relapsed tumors. Novel small molecules capable of inhibiting AR function may lead to urgently needed therapies for patients resistant to abiraterone, enzalutamide, and/or other previously approved antiandrogen therapies. Here, we describe a high-throughput high-content screening (HCS) campaign to identify small-molecule inhibitors of AR nuclear localization in the C4-2 CRPC cell line stably transfected with GFP-AR-GFP (2GFP-AR). The implementation of this HCS assay to screen a National Institutes of Health library of 219,055 compounds led to the discovery of 3 small molecules capable of inhibiting AR nuclear localization and function in C4-2 cells, demonstrating the feasibility of using this cell-based phenotypic assay to identify small molecules targeting the subcellular localization of AR. Furthermore, the three hit compounds provide opportunities to develop novel AR drugs with potential for therapeutic intervention in CRPC patients who have relapsed after treatment with antiandrogens, such as abiraterone and/or enzalutamide. PMID:27187604

  5. Qualification of the indentation test for the local characterization of nuclear facility materials. Final report; Qualifizierung des Eindruckversuchs zur lokalen Charakterisierung kerntechnischer Werkstoffe. Abschlussbericht

    Energy Technology Data Exchange (ETDEWEB)

    Tandler, Martin; Seifert, Thomas; Schlesinger, Michael; Mohrmann, Ralf; Kilgus, Normen; Venugopal, Ravula

    2007-12-21

    With the aid of the registrating indentation test, the project intends to characterise the operational changes in the local material properties of nuclear materials by a quasi-nondestructive indentation test. The focus was on the materials 22NiMoCr3-7 and X6CrNiNb18-10, both of which are widely used in nuclear engineering. As the accuracy of the method depends on experimental influencing factors like surface treatment, intrinsic stresses, or material anisotropy, these influences are to be quantified and will be considered in the evaluation of the material characteristics. The influencing parameters will be investigated experimentally and numerically by FE simulations so that their influence can be distinguished from the actual material behaviour. (orig.)

  6. Local consequences and reactions to the closing down of nuclear reactors in Sweden, a KSO report, 1999

    International Nuclear Information System (INIS)

    The Swedish Government decision to close down the Barsebaeck-1 NPP resulted in a studi funded by the Organisation of Municipalities with Nuclear Operation (KSO). It was meant to evaluate the scenario if all nuclear reactors in Sweden were closed down by year 2005 or if the phase out took place in an orderly manner after 40 years i.e. between 2012 and 2025. Most important consequence would be the loss of approximately 8000 jobs mostly in five municipalities concerned. This should also be regarded from a qualitative viewpoint, namely as a significant loss of labour opportunities for highly educated personnel. The estimates show in addition, a dramatic increase in migration from nuclear power plant municipalities of particularly younger population. In conclusion it is mentioned that Sweden still has 11 nuclear reactors in operation supplying 50% of the electricity used in the country. If the government would demand immediate closure of all reactors, Sweden would be a dark and cold country at least during the six months of winter

  7. The 14 october 1999 nuclear accident simulation drill at Nogent-sur-Seine NPP role of the local information committee (C.L.I.)

    International Nuclear Information System (INIS)

    The EDF nuclear power plant at Nogent-sur-Seine consists in two 1 300 MWe pressurised water reactor units. It is located 120 km south-east of Paris. The Local Information Committee, which brigs 120 members together, was created when the plant started operating in 1988. The 14 October 1999 nuclear accident simulation drill, the sequence of which started from an incident in the virtual Unit 3, was intended to testing the reacting capacities of the local and national emergency PCs (command post). The main objective was to actually implement the evacuation of a 500 inhabitants village near the plant in order to manage their rallying in a reception centre about 15 kilometers from there. The aim was to assess the speed and accuracy of the alert to the population, the requisitioning of human and practical resources, the affected area cordoning off, the evacuation of two schools, the activation of the reception centre with firemen, doctors and finally direct work with the media. The CLI was involved from the initial stages in the 6 preparatory meetings and, on the day of the drill, put observers in the strategic places. After the drill it was invited to give its opinion during feedback meetings organised by authorities, both the Aube Prefecture in Troyes and the Nuclear Installation Safety Directorate in Paris. Our CLI had prepared in 1997 reactor cards 'fiches reflexes' for local representatives, which in the light of this drill, could be improved. CLI participation to the rewriting - at national level - PPI (offsite emergency plans) is all the more active since it is done in the light of this drill. Il will be the same with the reissue of our public information brochure dealing with security instructions. In a more general way, it appears that such a drill would have been completely excluded only ten years ago. (author)

  8. Nuclear Localization of the Autism Candidate Gene Neurobeachin and Functional Interaction with the NOTCH1 Intracellular Domain Indicate a Role in Regulating Transcription.

    Directory of Open Access Journals (Sweden)

    Krizia Tuand

    Full Text Available Neurobeachin (NBEA is an autism spectrum disorders (ASD candidate gene. NBEA deficiency affects regulated secretion, receptor trafficking, synaptic architecture and protein kinase A (PKA-mediated phosphorylation. NBEA is a large multidomain scaffolding protein. From N- to C-terminus, NBEA has a concanavalin A-like lectin domain flanked by armadillo repeats (ACA, an A-kinase anchoring protein domain that can bind to PKA, a domain of unknown function (DUF1088 and a BEACH domain, preceded by a pleckstrin homology-like domain and followed by WD40 repeats (PBW. Although most of these domains mediate protein-protein interactions, no interaction screen has yet been performed.Yeast two-hybrid screens with the ACA and PBW domain modules of NBEA gave a list of interaction partners, which were analyzed for Gene Ontology (GO enrichment. Neuro-2a cells were used for confocal microscopy and nuclear extraction analysis. NOTCH-mediated transcription was studied with luciferase reporter assays and qRT-PCR, combined with NBEA knockdown or overexpression.Both domain modules showed a GO enrichment for the nucleus. PBW almost exclusively interacted with transcription regulators, while ACA interacted with a number of PKA substrates. NBEA was partially localized in the nucleus of Neuro-2a cells, albeit much less than in the cytoplasm. A nuclear localization signal was found in the DUF1088 domain, which was shown to contribute to the nuclear localization of an EGFP-DPBW fusion protein. Yeast two-hybrid identified the Notch1 intracellular domain as a physical interactor of the PBW domain and a role for NBEA as a negative regulator in Notch-mediated transcription was demonstrated.Defining novel interaction partners of conserved NBEA domain modules identified a role for NBEA as transcriptional regulator in the nucleus. The physical interaction of NBEA with NOTCH1 is most relevant for ASD pathogenesis because NOTCH signaling is essential for neural development.

  9. SU-E-J-61: Electrodynamics and Nano-Scale Fluid Dynamics in Protein Localization of Nuclear Pore Complexes

    Energy Technology Data Exchange (ETDEWEB)

    Cunningham, J; Gatenby, R [Moffitt Cancer Research Institute, Tampa, FL (United States)

    2014-06-01

    Purpose: To develop a simulation to catalyze a reevaluation of common assumptions about 3 dimensional diffusive processes and help cell biologists gain a more nuanced, intuitive understanding of the true physical hurdles of protein signaling cascades. Furthermore, to discuss the possibility of intracellular electrodynamics as a critical, unrecognized component of cellular biology and protein dynamics that is necessary for optimal information flow from the cell membrane to the nucleus. Methods: The Unity 3D gaming physics engine was used to build an accurate virtual scale model of the cytoplasm within a few hundred nanometers of the nuclear membrane. A cloud of simulated pERK proteins is controlled by the physics simulation, where diffusion is based on experimentally measured values and the electrodynamics are based on theoretical nano-fluid dynamics. The trajectories of pERK within the cytoplasm and through the 1250 nuclear pores on the nuclear surface is recorded and analyzed. Results: The simulation quickly demonstrates that pERKs moving solely by diffusion will rarely locate and come within capture distance of a nuclear pore. The addition of intracellular electrodynamics between charges on the nuclear pore complexes and on pERKs increases the number of successful translocations by allowing the electro-physical attractive effects to draw in pERKs from the cytoplasm. The effects of changes in intracellular shielding ion concentrations allowed for estimation of the “capture radius” under varying conditions. Conclusion: The simulation allows a shift in perspective that is paramount in attempting to communicate the scale and dynamics of intracellular protein cascade mechanics. This work has allowed researchers to more fully understand the parameters involved in intracellular electrodynamics, such as shielding anion concentration and protein charge. As these effects are still far below the spatial resolution of currently available measurement technology this

  10. The complete genome of a baculovirus isolated from an insect of medical interest: Lonomia obliqua (Lepidoptera: Saturniidae).

    Science.gov (United States)

    Aragão-Silva, C W; Andrade, M S; Ardisson-Araújo, D M P; Fernandes, J E A; Morgado, F S; Báo, S N; Moraes, R H P; Wolff, J L C; Melo, F L; Ribeiro, B M

    2016-01-01

    Lonomia obliqua (Lepidoptera: Saturniidae) is a species of medical importance due to the severity of reactions caused by accidental contact with the caterpillar bristles. Several natural pathogens have been identified in L. obliqua, and among them the baculovirus Lonomia obliqua multiple nucleopolyhedrovirus (LoobMNPV). The complete genome of LoobMNPV was sequenced and shown to have 120,022 bp long with 134 putative open reading frames (ORFs). Phylogenetic analysis of the LoobMNPV genome showed that it belongs to Alphabaculovirus group I (lepidopteran-infective NPV). A total of 12 unique ORFs were identified with no homologs in other sequenced baculovirus genomes. One of these, the predicted protein encoded by loob035, showed significant identity to an eukaryotic transcription terminator factor (TTF2) from the Lepidoptera Danaus plexippus, suggesting an independent acquisition through horizontal gene transfer. Homologs of cathepsin and chitinase genes, which are involved in host integument liquefaction and viral spread, were not found in this genome. As L. obliqua presents a gregarious behavior during the larvae stage the impact of this deletion might be neglectable. PMID:27282807

  11. Baculovirus expression of beak and feather disease virus (BFDV) capsid protein capable of self-assembly and haemagglutination.

    Science.gov (United States)

    Stewart, Meredith E; Bonne, Nicolai; Shearer, Patrick; Khalesi, Bahman; Sharp, Margaret; Raidal, Shane

    2007-05-01

    Beak and feather disease virus (BFDV) is a common avian circovirus infection of wild Psittaciformes and is a recognised threat to endangered psittacine species. Currently, there is a requirement to develop BFDV antigen for diagnostic purposes and since efforts to propagate BFDV in vitro have so far been unsuccessful the entire coding region of BFDV ORF C1 was expressed in Sf9 insect cells using a baculovirus expression system. The entire coding region of BFDV ORF C1, the presumptive capsid, was expressed in Sf9 insect cells using baculovirus expression system. Electron microscopic examination of negatively stained material demonstrated that the recombinant protein self-assembled to produce virus-like particles (VLPs) thus confirming that ORF C1 is likely to be the sole determinant for capsid construction in vivo. BFDV VLPs also possessed haemagglutinating activity which provides further evidence that self-assembled BFDV VLPs retain receptor mediated biological activity and that the determinants for BFDV haemagglutination activity rely solely on the capsid protein. The recombinant protein reacted with anti-BFDV sera from naturally immune parrots and cockatoo and from chickens experimentally inoculated with native BFDV in both Western blots and haemagglutination inhibition (HI) assay. BFDV VLPs were also a suitable replacement antigen for serological detection of BFDV antibody by HI. PMID:17218022

  12. Development and evaluation of baculovirus-expressed Chikungunya virus E1 envelope proteins for serodiagnosis of Chikungunya infection.

    Science.gov (United States)

    Kumar, Pankaj; Pok, Kwoon-Yong; Tan, Li-Kiang; Angela, Chow; Leo, Yee-Sin; Ng, Lee-Ching

    2014-09-01

    Population-based serosurveillance studies provide critical estimates on community-level immunity and the potential for future outbreaks. Currently, serological assays, such as IgG enzyme-linked immunosorbent assays (ELISAs) and indirect immunofluorescence tests (IIFT) based on the inactivated whole virus are used to determine past Chikungunya virus (CHIKV) infection. However, these commercially available tests have variable sensitivities. To develop and evaluate recombinant based CHIKV-specific IgG antibody capture ELISAs (GAC-ELISAs), baculoviruses carrying wild-type (E1-A226, named WT) or mutant (E1-A226V, named MUT) E1 envelope protein genes of CHIKV were generated. The seroreactivity of recombinant CHIKV WT and MUT envelope proteins were determined using residual blood, collected from CHIKV-confirmed patients. The sensitivities of both recombinant CHIKV envelope proteins were 83.0% as measured by GAC-ELISAs. The specificities of both recombinant proteins were 87.8%. These GAC-ELISAs were also able to detect the persistence of anti-CHIKV IgG antibodies up to 6 months after the disease onset, together with rise in sensitivities with increasing time. These results suggest that the baculovirus purified recombinant CHIKV envelope proteins react with anti-CHIKV IgG antibodies and may be useful in population-based seroprevalence surveys. In addition, these GAC-ELISAs offer good diagnostic value to determine the recent/past CHIKV infection status in non-endemic populations.

  13. Analysis of recombinant, multivalent dengue virus containing envelope (E proteins from serotypes-1, -3 and -4 and expressed in baculovirus

    Directory of Open Access Journals (Sweden)

    Fedik A. Rantam

    2015-01-01

    Full Text Available Dengue virus has four serotypes that cause a public health problem in Indonesia. Currently, there is no preventative vaccine for this disease, but some model vaccines are in development. The envelop (E protein genes from three isolates of dengue virus (DENV-1, -3 and -4 were isolated, cloned into Escherichia coli and then sub-cloned into a baculovirus vector before co-transfection into Sf9 cells. Recombinant E genes were inserted between the Smal and Sacl sites of the plasmid, adjacent to the baculoviral structural gene, polyhedrin. The sequence of recombinant E gene was relatively stable with 97–98% homology, although there were amino acid substitutions in some regions. The recombinant protein was more antigenic when exposed to polyclonal sera from infected humans than sera from immunized mice, but its binding to monoclonal antibodies IgG1a and IgG2b was stronger than other isotopes, including IgM, IgG and Ig1b. Recombinant E protein induced cellular immune responses in immunized mice, as demonstrated by lymphocyte secretion of IL-3. This study indicates that recombinant E protein expressed in a baculovirus system can induce humoral and cellular immune responses.

  14. Nuclear Quadrupole Hyperfine Structure in HC14N/H14NC and DC15N/D15NC Isomerization: A Diagnostic Tool for Characterizing Vibrational Localization

    CERN Document Server

    Wong, Bryan M

    2010-01-01

    Large-amplitude molecular motions which occur during isomerization can cause significant changes in electronic structure. These variations in electronic properties can be used to identify vibrationally-excited eigenstates which are localized along the potential energy surface. This work demonstrates that nuclear quadrupole hyperfine interactions can be used as a diagnostic marker of progress along the isomerization path in both the HC14N/H14NC and DC15N/D15NC chemical systems. Ab initio calculations at the CCSD(T)/cc-pCVQZ level indicate that the hyperfine interaction is extremely sensitive to the chemical bonding of the quadrupolar 14N nucleus and can therefore be used to determine in which potential well the vibrational wavefunction is localized. A natural bonding orbital analysis along the isomerization path further demonstrates that hyperfine interactions arise from the asphericity of the electron density at the quadrupolar nucleus. Using the CCSD(T) potential surface, the quadrupole coupling constants of...

  15. Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system.

    Science.gov (United States)

    Maghodia, Ajay B; Geisler, Christoph; Jarvis, Donald L

    2016-06-01

    Cell lines derived from the fall armyworm, Spodoptera frugiperda (Sf), are widely used as hosts for recombinant protein production in the baculovirus-insect cell system (BICS). However, it was recently discovered that these cell lines are contaminated with a virus, now known as Sf-rhabdovirus [1]. The detection of this adventitious agent raised a potential safety issue that could adversely impact the BICS as a commercial recombinant protein production platform. Thus, we examined the properties of Sf-RVN, an Sf-rhabdovirus-negative Sf cell line, as a potential alternative host. Nested RT-PCR assays showed Sf-RVN cells had no detectable Sf-rhabdovirus over the course of 60 passages in continuous culture. The general properties of Sf-RVN cells, including their average growth rates, diameters, morphologies, and viabilities after baculovirus infection, were virtually identical to those of Sf9 cells. Baculovirus-infected Sf-RVN and Sf9 cells produced equivalent levels of three recombinant proteins, including an intracellular prokaryotic protein and two secreted eukaryotic glycoproteins, and provided similar N-glycosylation patterns. In fact, except for the absence of Sf-rhabdovirus, the only difference between Sf-RVN and Sf9 cells was SF-RVN produced higher levels of infectious baculovirus progeny. These results show Sf-RVN cells can be used as improved, alternative hosts to circumvent the potential safety hazard associated with the use of Sf-rhabdovirus-contaminated Sf cells for recombinant protein manufacturing with the BICS.

  16. Enhanced recombinant protein production and differential expression of molecular chaperones in sf-caspase-1-repressed stable cells after baculovirus infection

    Directory of Open Access Journals (Sweden)

    Lai Yiu-Kay

    2012-11-01

    Full Text Available Abstract Background There are few studies that have examined the potential of RNA inference (RNAi to increase protein production in the baculovirus expression vector system (BEVS. Spodoptera frugiperda (fall armyworm (Sf-caspase-1-repressed stable cells exhibit resistance to apoptosis and enhancement of recombinant protein production. However, the mechanism of recombinant protein augmentation in baculovirus-infected Caspase-repressed insect cells has not been elucidated. Results In the current study, we utilized RNAi-mediated Sf-caspase-1-repressed stable cells to clarify how the resistance to apoptosis can enhance both intracellular (firefly luciferase and extracellular (secreted alkaline phosphatase [SEAP] recombinant protein production in BEVS. Since the expression of molecular chaperones is strongly associated with the maximal production of exogenous proteins in BEVS, the differential expression of molecular chaperones in baculovirus-infected stable cells was also analyzed in this study. Conclusion The data indicated that the retention of expression of molecular chaperones in baculovirus-infected Sf-caspase-1-repressed stable cells give the higher recombinant protein accumulation.

  17. 杆状病毒表面展示系统的发展及应用%Development and application of Baculovirus surface display system

    Institute of Scientific and Technical Information of China (English)

    郑浩; 徐亮亮; 韦磊; 孙京臣

    2014-01-01

    杆状病毒表面展示系统(Baculovirus Surface Display,BSD)是近年来发展起来的一种崭新的膜展示技术。其主要原理是通过基因工程的方法,将杆状病毒表面囊膜蛋白基因与所要展示的目的蛋白基因进行融合,形成重组杆状病毒。重组杆状病毒的囊膜蛋白在表达同时,目的蛋白也随即进行表达,并与囊膜蛋白共同运输至病毒囊膜处,特异的与锚定部位结合,展示在杆状病毒表面。该系统由于具有较强的可控性和较高的展示效率,目前已经被广泛应用于基因转导与基因治疗、较复杂的真核蛋白展示、新型疫苗研发以及单克隆抗体的制备等领域。本文对杆状病毒表面展示系统的研究现状进行了阐述和总结,并对其发展和应用前景做出了展望。%Successful examples in Baculovirus Surface Display system (BSD) have recently been achieved by the recombinant DNA technique via recombinant DNA technology. The main principle is to combine the bac-ulovirus surface envelope protein genes and the target genes and to express a fusion protein via the recombinant baculovirus. The envelope protein of recombinant baculovirus is expressed, meanwhile, the target protein is expressed as well. The target genes are expressed and transported to virus envelope with the envelope protein, then specifically anchored and displayed on the baculovirus surface. Due to the strong controllability and high efficiency, BSD has been widely used in gene transduction, gene therapy, complex eukaryotic proteins dis-play, new vaccine development and the preparation of monoclonal antibodies, etc. In this paper, the re-search status quo of baculovirus surface display system is reviewed, including the development and application prospect.

  18. The Site Selected. The Local Decision-Making Regarding the Siting of the Spent Nuclear Fuel Repository in Olkiluoto

    International Nuclear Information System (INIS)

    In May 1999 Posiva, the company responsible for the final disposal of spent nuclear fuel in Finland, suggested that the Finnish Government considers only Olkiluoto in Eurajoki in its application of a decision in principle to be a final disposal site. In January 2000 the municipal council of Eurajoki made a positive statement on the decision in principle. The Government made the decision in principle in Dec 2000, and the Parliament ratified the decision in May 2001. The paper is focused on the decision making of Eurajoki municipality regarding the siting of the spent nuclear fuel repository. The paper shows how the interaction between the representatives of the candidate municipality and the nuclear energy industry was the crucial factor in the decision-making. Eurajoki serves as an example, in where the parties reached an agreement of the compensations for the final disposal repository. The negotiations between the Eurajoki municipality and the nuclear energy industry in reaching a positive decision are analysed from the beginning of the 1980s. The main emphasis is however on the years 1996-99, when the nuclear energy industry negotiated with the municipality on the compensation for the final disposal repository. The loss of income was an important reason why some of the councillors of Eurajoki were interested in having the final disposal repository in Olkiluoto. The industry's problem on the other hand was to safeguard the final disposal site. From the TVO's angle Olkiluoto was a potential final disposal site for example for its limited need for transport and for the existing infrastructure. The company used the financial benefits of the project as its trump card. The attitude of Eurajoki municipality to the final disposal of spent nuclear fuel turned positive with the Olkiluoto vision in December 1998, when still five years earlier the municipal council was prepared to act and prevent the final disposal. The future image presented by the municipality now matched

  19. SCF Ubiquitin Ligase F-box Protein Fbx15 Controls Nuclear Co-repressor Localization, Stress Response and Virulence of the Human Pathogen Aspergillus fumigatus

    Science.gov (United States)

    Jöhnk, Bastian; Bayram, Özgür; Heinekamp, Thorsten; Mattern, Derek J.; Brakhage, Axel A.; Jacobsen, Ilse D.; Valerius, Oliver; Braus, Gerhard H.

    2016-01-01

    F-box proteins share the F-box domain to connect substrates of E3 SCF ubiquitin RING ligases through the adaptor Skp1/A to Cul1/A scaffolds. F-box protein Fbx15 is part of the general stress response of the human pathogenic mold Aspergillus fumigatus. Oxidative stress induces a transient peak of fbx15 expression, resulting in 3x elevated Fbx15 protein levels. During non-stress conditions Fbx15 is phosphorylated and F-box mediated interaction with SkpA preferentially happens in smaller subpopulations in the cytoplasm. The F-box of Fbx15 is required for an appropriate oxidative stress response, which results in rapid dephosphorylation of Fbx15 and a shift of the cellular interaction with SkpA to the nucleus. Fbx15 binds SsnF/Ssn6 as part of the RcoA/Tup1-SsnF/Ssn6 co-repressor and is required for its correct nuclear localization. Dephosphorylated Fbx15 prevents SsnF/Ssn6 nuclear localization and results in the derepression of gliotoxin gene expression. fbx15 deletion mutants are unable to infect immunocompromised mice in a model for invasive aspergillosis. Fbx15 has a novel dual molecular function by controlling transcriptional repression and being part of SCF E3 ubiquitin ligases, which is essential for stress response, gliotoxin production and virulence in the opportunistic human pathogen A. fumigatus. PMID:27649508

  20. Expression and localization of nuclear proteins in autosomal-dominant Emery-Dreifuss muscular dystrophy with LMNA R377H mutation

    Directory of Open Access Journals (Sweden)

    Ewald Andrea

    2004-03-01

    Full Text Available Abstract Background The autosomal dominant form of Emery-Dreifuss muscular dystrophy (AD-EDMD is caused by mutations in the gene encoding for the lamins A and C (LMNA. Lamins are intermediate filament proteins which form the nuclear lamina underlying the inner nuclear membrane. We have studied the expression and the localization of nuclear envelope proteins in three different cell types and muscle tissue of an AD-EDMD patient carrying a point mutation R377H in the lamin A/C gene. Results Lymphoblastoid cells, skin fibroblasts, primary myoblasts and muscle thin sections were studied by immunocytochemistry and electron microscopy. Cellular levels of A-type lamins were reduced compared to control cells. In contrast, the amount of emerin and lamin B appeared unaltered. Cell synchronization experiments showed that the reduction of the cellular level of A-type lamin was due to instability of lamin A. By electron microscopy, we identified a proportion of nuclei with morphological alterations in lymphoblastoid cells, fibroblasts and mature muscle fibres. Immunofluorescence microscopy showed that a major population of the lamin B receptor (LBR, an inner nuclear membrane protein, was recovered in the cytoplasm in association with the ER. In addition, the intranuclear organization of the active form of RNA polymerase II was markedly different in cells of this AD-EDMD patient. This aberrant intranuclear distribution was specifically observed in muscle cells where the pathology of EDMD predominates. Conclusions From our results we conclude: Firstly, that structural alterations of the nuclei which are found only in a minor fraction of lymphoblastoid cells and mature muscle fibres are not sufficient to explain the clinical pathology of EDMD; Secondly, that wild type lamin A is required not only for the retention of LBR in the inner nuclear membrane but also for a correct localization of the transcriptionally active RNA pol II in muscle cells. We speculate that

  1. Nuclear localization of lymphocyte-specific protein tyrosine kinase (Lck) and its role in regulating LIM domain only 2 (Lmo2) gene

    Energy Technology Data Exchange (ETDEWEB)

    Venkitachalam, Srividya; Chueh, Fu-Yu [Department of Microbiology and Immunology, H. M. Bligh Cancer Research Laboratories, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064 (United States); Yu, Chao-Lan, E-mail: chaolan.yu@rosalindfranklin.edu [Department of Microbiology and Immunology, H. M. Bligh Cancer Research Laboratories, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064 (United States)

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer Lmo2 expression is elevated in Lck-transformed cells. Black-Right-Pointing-Pointer Both endogenous and exogenous Lck localize in the nucleus. Black-Right-Pointing-Pointer Nuclear Lck is active in Lck-transformed cells. Black-Right-Pointing-Pointer Lck binds to the promoter region of Lmo2 gene in vivo. Black-Right-Pointing-Pointer In contrast to JAK2, Lck does not increase histone H3 phosphorylation on Tyr 41. -- Abstract: LIM domain only protein 2 (Lmo2) is a transcription factor that plays a critical role in the development of T-acute lymphoblastic leukemia (T-ALL). A previous report established a link between Lmo2 expression and the nuclear presence of oncogenic Janus kinase 2 (JAK2), a non-receptor protein tyrosine kinase. The oncogenic JAK2 kinase phosphorylates histone H3 on Tyr 41 that leads to the relief of Lmo2 promoter repression and subsequent gene expression. Similar to JAK2, constitutive activation of lymphocyte-specific protein tyrosine kinase (Lck) has been implicated in lymphoid malignancies. However, it is not known whether oncogenic Lck regulates Lmo2 expression through a similar mechanism. We show here that Lmo2 expression is significantly elevated in T cell leukemia LSTRA overexpressing active Lck kinase and in HEK 293 cells expressing oncogenic Y505FLck kinase. Nuclear localization of active Lck kinase was confirmed in both Lck-transformed cells by subcellular fractionation and immunofluorescence microscopy. More importantly, in contrast to oncogenic JAK2, oncogenic Lck kinase does not result in significant increase in histone H3 phosphorylation on Tyr 41. Instead, chromatin immunoprecipitation experiment shows that oncogenic Y505FLck kinase binds to the Lmo2 promoter in vivo. This result raises the possibility that oncogenic Lck may activate Lmo2 promoter through direct interaction.

  2. Study of the influence of central nuclear power plants at Valdaliga (Civitavecchia) on the chemical composition of local rain

    International Nuclear Information System (INIS)

    Data from a wet deposition sampling over the area collocated nearby Civitavecchia power plants are here presented and discussed. In order to establish the possible influence of the power plants emission on the chemical composition of the rain collected, the experimental data have been correlated with synoptic and local meteorological conditions. (author)

  3. Efficient Subcellular Targeting to the Cell Nucleus of Quantum Dots Densely Decorated with a Nuclear Localization Sequence Peptide.

    Science.gov (United States)

    Maity, Amit Ranjan; Stepensky, David

    2016-01-27

    Organelle-targeted drug delivery can enhance the efficiency of the intracellularly acting drugs and reduce their toxicity. We generated core-shell type CdSe-ZnS quantum dots (QDs) densely decorated with NLS peptidic targeting residues using a 3-stage decoration approach and investigated their endocytosis and nuclear targeting efficiencies. The diameter of the generated QDs increased following the individual decoration stages (16.3, 18.9, and 21.9 nm), the ζ-potential became less negative (-33.2, -17.5, and -11.9 mV), and characteristic changes appeared in the FTIR spectra following decoration with the linker and NLS peptides. Quantitative analysis of the last decoration stage revealed that 37.9% and 33.2% of the alkyne-modified NLS groups that were added to the reaction mix became covalently attached or adsorbed to the QDs surface, respectively. These numbers correspond to 63.6 and 55.7 peptides conjugated or adsorbed to a single QD (the surface density of 42 and 37 conjugated and adsorbed peptides per 1000 nm(2) of the QDs surface), which is higher than in the majority of previous studies that reported decoration efficiencies of formulations intended for nuclear-targeted drug delivery. QDs decorated with NLS peptides undergo more efficient endocytosis, as compared to other investigated QDs formulations, and accumulated to a higher extent in the cell nucleus or in close vicinity to it (11.9%, 14.6%, and 56.1% of the QDs endocytosed by an average cell for the QD-COOH, QD-azide, and QD-NLS formulations, respectively). We conclude that dense decoration of QDs with NLS residues increased their endocytosis and led to their nuclear targeting (preferential accumulation in the cells nuclei or in close vicinity to them). The experimental system and research tools that were used in this study allow quantitative investigation of the mechanisms that govern the QDs nuclear targeting and their dependence on the formulation properties. These findings will contribute to the

  4. Deletion of the nuclear localization sequences and C-terminus of PTHrP impairs embryonic mammary development but also inhibits PTHrP production.

    Directory of Open Access Journals (Sweden)

    Kata Boras-Granic

    Full Text Available Parathyroid hormone-related protein (PTHrP can be secreted from cells and interact with its receptor, the Type 1 PTH/PTHrP Receptor (PTHR1 in an autocrine, paracrine or endocrine fashion. PTHrP can also remain inside cells and be transported into the nucleus, where its functions are unclear, although recent experiments suggest that it may broadly regulate cell survival and senescence. Disruption of either the PTHrP or PTHR1 gene results in many abnormalities including a failure of embryonic mammary gland development in mice and in humans. In order to examine the potential functions of nuclear PTHrP in the breast, we examined mammary gland development in PTHrP (1-84 knock-in mice, which express a mutant form of PTHrP that lacks the C-terminus and nuclear localization signals and which can be secreted but cannot enter the nucleus. Interestingly, we found that PTHrP (1-84 knock-in mice had defects in mammary mesenchyme differentiation and mammary duct outgrowth that were nearly identical to those previously described in PTHrP-/- and PTHR1-/- mice. However, the mammary buds in PTHrP (1-84 knock-in mice had severe reductions in mutant PTHrP mRNA levels, suggesting that the developmental defects were due to insufficient production of PTHrP by mammary epithelial cells and not loss of PTHrP nuclear function. Examination of the effects of nuclear PTHrP in the mammary gland in vivo will require the development of alternative animal models.

  5. Localization of nucleoporin Tpr to the nuclear pore complex is essential for Tpr mediated regulation of the export of unspliced RNA.

    Science.gov (United States)

    Rajanala, Kalpana; Nandicoori, Vinay Kumar

    2012-01-01

    Nucleoporin Tpr is a component of the nuclear pore complex (NPC) that localizes exclusively to intranuclear filaments. Tpr functions as a scaffolding element in the nuclear phase of the NPC and plays a role in mitotic spindle checkpoint signalling. Export of intron-containing mRNA in Mason Pfizer Monkey Virus is regulated by direct interaction of cellular proteins with the cis-acting Constitutive Transport Element (CTE). In mammalian cells, the transport of Gag/Pol-CTE reporter construct is not very efficient, suggesting a regulatory mechanism to retain this unspliced RNA. Here we report that the knockdown of Tpr in mammalian cells leads to a drastic enhancement in the levels of Gag proteins (p24) in the cytoplasm, which is rescued by siRNA resistant Tpr. Tpr's role in the retention of unspliced RNA is independent of the functions of Sam68 and Tap/Nxf1 proteins, which are reported to promote CTE dependent export. Further, we investigated the possible role for nucleoporins that are known to function in nucleocytoplasmic transport in modulating unspliced RNA export. Results show that depletion of Nup153, a nucleoporin required for NPC anchoring of Tpr, plays a role in regulating the export, while depletion of other FG repeat-containing nucleoporins did not alter the unspliced RNA export. Results suggest that Tpr and Nup153 both regulate the export of unspliced RNA and they are most likely functioning through the same pathway. Importantly, we find that localization of Tpr to the NPC is necessary for Tpr mediated regulation of unspliced RNA export. Collectively, the data indicates that perinuclear localization of Tpr at the nucleopore complex is crucial for regulating intron containing mRNA export by directly or indirectly participating in the processing and degradation of aberrant mRNA transcripts.

  6. Initial Evaluation of the Heat-Affected Zone, Local Embrittlement Phenomenon as it Applies to Nuclear Reactor Vessels

    Energy Technology Data Exchange (ETDEWEB)

    McCabe, D.E.

    1999-09-01

    The objective of this project was to determine if the local brittle zone (LBZ) problem, encountered in the testing of the heat-affected zone (HAZ) part of welds in offshore platform construction, can also be found in reactor pressure vessel (RPV) welds. Both structures have multipass welds and grain coarsening along the fusion line. Literature was obtained that described the metallurgical evidence and the type of research work performed on offshore structure welds.

  7. The prenylation status of a novel plant calmodulin directs plasma membrane or nuclear localization of the protein.

    Science.gov (United States)

    Rodríguez-Concepción, M; Yalovsky, S; Zik, M; Fromm, H; Gruissem, W

    1999-04-01

    Post-translational attachment of isoprenyl groups to conserved cysteine residues at the C-terminus of a number of regulatory proteins is important for their function and subcellular localization. We have identified a novel calmodulin, CaM53, with an extended C-terminal basic domain and a CTIL CaaX-box motif which are required for efficient prenylation of the protein in vitro and in vivo. Ectopic expression of wild-type CaM53 or a non-prenylated mutant protein in plants causes distinct morphological changes. Prenylated CaM53 associates with the plasma membrane, but the non-prenylated mutant protein localizes to the nucleus, indicating a dual role for the C-terminal domain. The subcellular localization of CaM53 can be altered by a block in isoprenoid biosynthesis or sugar depletion, suggesting that CaM53 activates different targets in response to metabolic changes. Thus, prenylation of CaM53 appears to be a novel mechanism by which plant cells can coordinate Ca2+ signaling with changes in metabolic activities.

  8. Concentrations of Radiocesium in Local Foods Collected in Kawauchi Village after the Accident at the Fukushima Dai-ichi Nuclear Power Station

    Science.gov (United States)

    Orita, Makiko; Nakashima, Kanami; Hayashida, Naomi; Endo, Yuuko; Yamashita, Shunichi; Takamura, Noboru

    2016-06-01

    We evaluated the current concentrations of radiocesium in local foods collected in Kawauchi Village, which is located less than 30 km from Fukushima Daiichi Nuclear Power Station, to minimize public anxiety regarding internal radiation exposure through the consumption of locally produced foods after the 2011 Fukushima accident. The number of samples exceeding the regulatory radiocesium limit (100 Bq/kg for general foods) was five out of 4,080 vegetables (0.1%), 652 of 1,986 (32.8%) among edible wild plants and fungi, and eight of 647 (1.2%) in fruits. Our study confirmed that the internal radiation doses of ingesting these foods are acceptably low compared to the public dose limit, ranging from 24.4 to 42.7 μSv for males and from 21.7 to 43.4 μSv for females, although the potential for radiation exposure still exists. Long-term comprehensive follow-up should take place to clarify trends in radiocesium concentrations in local foods and the committed effective doses found in Fukushima-area residents. By constructing a system that allows residents to access information on radiocesium concentration in foods, a risk communication model between specialists and residents could be developed in the recovery phase after the Fukushima accident.

  9. Concentrations of Radiocesium in Local Foods Collected in Kawauchi Village after the Accident at the Fukushima Dai-ichi Nuclear Power Station.

    Science.gov (United States)

    Orita, Makiko; Nakashima, Kanami; Hayashida, Naomi; Endo, Yuuko; Yamashita, Shunichi; Takamura, Noboru

    2016-01-01

    We evaluated the current concentrations of radiocesium in local foods collected in Kawauchi Village, which is located less than 30 km from Fukushima Daiichi Nuclear Power Station, to minimize public anxiety regarding internal radiation exposure through the consumption of locally produced foods after the 2011 Fukushima accident. The number of samples exceeding the regulatory radiocesium limit (100 Bq/kg for general foods) was five out of 4,080 vegetables (0.1%), 652 of 1,986 (32.8%) among edible wild plants and fungi, and eight of 647 (1.2%) in fruits. Our study confirmed that the internal radiation doses of ingesting these foods are acceptably low compared to the public dose limit, ranging from 24.4 to 42.7 μSv for males and from 21.7 to 43.4 μSv for females, although the potential for radiation exposure still exists. Long-term comprehensive follow-up should take place to clarify trends in radiocesium concentrations in local foods and the committed effective doses found in Fukushima-area residents. By constructing a system that allows residents to access information on radiocesium concentration in foods, a risk communication model between specialists and residents could be developed in the recovery phase after the Fukushima accident. PMID:27334847

  10. A Deoxynivalenol-Activated Methionyl-tRNA Synthetase Gene from Wheat Encodes a Nuclear Localized Protein and Protects Plants Against Fusarium Pathogens and Mycotoxins.

    Science.gov (United States)

    Zuo, Dong-Yun; Yi, Shu-Yuan; Liu, Rong-Jing; Qu, Bo; Huang, Tao; He, Wei-Jie; Li, Cheng; Li, He-Ping; Liao, Yu-Cai

    2016-06-01

    Fusarium graminearum is the fungal pathogen that causes globally important diseases of cereals and produces mycotoxins such as deoxynivalenol (DON). Owing to the dearth of available sources of resistance to Fusarium pathogens, characterization of novel genes that confer resistance to mycotoxins and mycotoxin-producing fungi is vitally important for breeding resistant crop varieties. In this study, a wheat methionyl-tRNA synthetase (TaMetRS) gene was identified from suspension cell cultures treated with DON. It shares conserved aminoacylation catalytic and tRNA anticodon binding domains with human MetRS and with the only previously characterized plant MetRS, suggesting that it functions in aminoacylation in the cytoplasm. However, the TaMetRS comprises a typical nuclear localization signal and cellular localization studies with a TaMetRS::GFP fusion protein showed that TaMetRS is localized in the nucleus. Expression of TaMetRS was activated by DON treatment and by infection with a DON-producing F. graminearum strain in wheat spikes. No such activation was observed following infection with a non-DON-producing F. graminearum strain. Expression of TaMetRS in Arabidopsis plants conferred significant resistance to DON and F. graminearum. These results indicated that this DON-activated TaMetRS gene may encode a novel type of MetRS in plants that has a role in defense and detoxification.

  11. Concentrations of Radiocesium in Local Foods Collected in Kawauchi Village after the Accident at the Fukushima Dai-ichi Nuclear Power Station

    Science.gov (United States)

    Orita, Makiko; Nakashima, Kanami; Hayashida, Naomi; Endo, Yuuko; Yamashita, Shunichi; Takamura, Noboru

    2016-01-01

    We evaluated the current concentrations of radiocesium in local foods collected in Kawauchi Village, which is located less than 30 km from Fukushima Daiichi Nuclear Power Station, to minimize public anxiety regarding internal radiation exposure through the consumption of locally produced foods after the 2011 Fukushima accident. The number of samples exceeding the regulatory radiocesium limit (100 Bq/kg for general foods) was five out of 4,080 vegetables (0.1%), 652 of 1,986 (32.8%) among edible wild plants and fungi, and eight of 647 (1.2%) in fruits. Our study confirmed that the internal radiation doses of ingesting these foods are acceptably low compared to the public dose limit, ranging from 24.4 to 42.7 μSv for males and from 21.7 to 43.4 μSv for females, although the potential for radiation exposure still exists. Long-term comprehensive follow-up should take place to clarify trends in radiocesium concentrations in local foods and the committed effective doses found in Fukushima-area residents. By constructing a system that allows residents to access information on radiocesium concentration in foods, a risk communication model between specialists and residents could be developed in the recovery phase after the Fukushima accident. PMID:27334847

  12. A Deoxynivalenol-Activated Methionyl-tRNA Synthetase Gene from Wheat Encodes a Nuclear Localized Protein and Protects Plants Against Fusarium Pathogens and Mycotoxins.

    Science.gov (United States)

    Zuo, Dong-Yun; Yi, Shu-Yuan; Liu, Rong-Jing; Qu, Bo; Huang, Tao; He, Wei-Jie; Li, Cheng; Li, He-Ping; Liao, Yu-Cai

    2016-06-01

    Fusarium graminearum is the fungal pathogen that causes globally important diseases of cereals and produces mycotoxins such as deoxynivalenol (DON). Owing to the dearth of available sources of resistance to Fusarium pathogens, characterization of novel genes that confer resistance to mycotoxins and mycotoxin-producing fungi is vitally important for breeding resistant crop varieties. In this study, a wheat methionyl-tRNA synthetase (TaMetRS) gene was identified from suspension cell cultures treated with DON. It shares conserved aminoacylation catalytic and tRNA anticodon binding domains with human MetRS and with the only previously characterized plant MetRS, suggesting that it functions in aminoacylation in the cytoplasm. However, the TaMetRS comprises a typical nuclear localization signal and cellular localization studies with a TaMetRS::GFP fusion protein showed that TaMetRS is localized in the nucleus. Expression of TaMetRS was activated by DON treatment and by infection with a DON-producing F. graminearum strain in wheat spikes. No such activation was observed following infection with a non-DON-producing F. graminearum strain. Expression of TaMetRS in Arabidopsis plants conferred significant resistance to DON and F. graminearum. These results indicated that this DON-activated TaMetRS gene may encode a novel type of MetRS in plants that has a role in defense and detoxification. PMID:26882849

  13. 16. national conference of the presidents of the local commissions of information (C.L.I.) around nuclear facilities

    International Nuclear Information System (INIS)

    This conference contained two plenary sessions. The first one concerned the epidemiology with the following subjects: the epidemiology, its contributions and its limits; the epidemiological surveillance of cancers in France: role of registers within the device; the aggregates of leukaemia near the nuclear installations; perception and management of the risk; the point of view of the scientific committee of the A.N.C.L.I.; Followed a work in a workshop on the subjects relative to the functioning of the C.L.I. (Organization, relations, information) then the second plenary session with as subjects: organization and functioning of C.L.I., relations and links with A.N.C.L.I., information received and diffused by the C.L.I. (N.C.)

  14. Present state and some problem of health services for residents of local government neighboring Fukushima Daiichi Nuclear Power Station

    International Nuclear Information System (INIS)

    After nuclear disaster, residents of 8 towns and villages of Futaba county within Alert area moved to the west and further removed far. Public offices were also moved. On March 12 health services were started to accept refugee residents at the first refuge. From the end of March to the first part of April they moved to second refuge at specified hotel or inn prepared for a long-term refugee life and then they removed from second refuge to temporary dwelling or loan house. Some of them moved to temporary dwelling at Iwaki city and totaled to more than 20000. New organization of the Fukushima prefecture was established in Iwaki city to start health services for refugee including mental cares. (T. Tanaka)

  15. Nuclear APC.

    Science.gov (United States)

    Neufeld, Kristi L

    2009-01-01

    Mutational inactivation of the tumor suppressor gene APC (Adenomatous polyposis coli) is thought to be an initiating step in the progression of the vast majority ofcolorectal cancers. Attempts to understand APC function have revealed more than a dozen binding partners as well as several subcellular localizations including at cell-cell junctions, associated with microtubules at the leading edge of migrating cells, at the apical membrane, in the cytoplasm and in the nucleus. The present chapter focuses on APC localization and functions in the nucleus. APC contains two classical nuclear localization signals, with a third domain that can enhance nuclear import. Along with two sets of nuclear export signals, the nuclear localization signals enable the large APC protein to shuttle between the nucleus and cytoplasm. Nuclear APC can oppose beta-catenin-mediated transcription. This down-regulation of nuclear beta-catenin activity by APC most likely involves nuclear sequestration of beta-catenin from the transcription complex as well as interaction of APC with transcription corepressor CtBP. Additional nuclear binding partners for APC include transcription factor activator protein AP-2alpha, nuclear export factor Crm1, protein tyrosine phosphatase PTP-BL and perhaps DNA itself. Interaction of APC with polymerase beta and PCNA, suggests a role for APC in DNA repair. The observation that increases in the cytoplasmic distribution of APC correlate with colon cancer progression suggests that disruption of these nuclear functions of APC plays an important role in cancer progression. APC prevalence in the cytoplasm of quiescent cells points to a potential function for nuclear APC in control of cell proliferation. Clear definition of APC's nuclear function(s) will expand the possibilities for early colorectal cancer diagnostics and therapeutics targeted to APC. PMID:19928349

  16. Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system.

    Science.gov (United States)

    Silva, Haroldo Cid da; Pestana, Cristiane Pinheiro; Galler, Ricardo; Medeiros, Marco Alberto

    2016-08-01

    The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS. PMID:27581123

  17. The C-terminal region of Rad52 is essential for Rad52 nuclear and nucleolar localization, and accumulation at DNA damage sites immediately after irradiation

    International Nuclear Information System (INIS)

    Highlights: •Rad52 might play a key role in the repair of DSB immediately after irradiation. •EYFP-Rad52 accumulates rapidly at DSB sites and colocalizes with Ku80. •Accumulation of Rad52 at DSB sites is independent of the core NHEJ factors. •Localization and recruitment of Rad52 to DSB sites are dependent on the Rad52 CTR. •Basic amino acids in Rad52 CTR are highly conserved among vertebrate species. -- Abstract: Rad52 plays essential roles in homologous recombination (HR) and repair of DNA double-strand breaks (DSBs) in Saccharomyces cerevisiae. However, in vertebrates, knockouts of the Rad52 gene show no hypersensitivity to agents that induce DSBs. Rad52 localizes in the nucleus and forms foci at a late stage following irradiation. Ku70 and Ku80, which play an essential role in nonhomologous DNA-end-joining (NHEJ), are essential for the accumulation of other core NHEJ factors, e.g., XRCC4, and a HR-related factor, e.g., BRCA1. Here, we show that the subcellular localization of EYFP-Rad52(1–418) changes dynamically during the cell cycle. In addition, EYFP-Rad52(1–418) accumulates rapidly at microirradiated sites and colocalizes with the DSB sensor protein Ku80. Moreover, the accumulation of EYFP-Rad52(1–418) at DSB sites is independent of the core NHEJ factors, i.e., Ku80 and XRCC4. Furthermore, we observed that EYFP-Rad52(1–418) localizes in nucleoli in CHO-K1 cells and XRCC4-deficient cells, but not in Ku80-deficient cells. We also found that Rad52 nuclear localization, nucleolar localization, and accumulation at DSB sites are dependent on eight amino acids (411–418) at the end of the C-terminal region of Rad52 (Rad52 CTR). Furthermore, basic amino acids on Rad52 CTR are highly conserved among mammalian, avian, and fish homologues, suggesting that Rad52 CTR is important for the regulation and function of Rad52 in vertebrates. These findings also suggest that the mechanism underlying the regulation of subcellular localization of Rad52 is

  18. Using double-stranded RNA to prevent in vitro and in vivo viral infections by recombinant baculovirus.

    Science.gov (United States)

    Valdes, Victor Julian; Sampieri, Alicia; Sepulveda, Jorge; Vaca, Luis

    2003-05-23

    Introduction of double-stranded RNA (dsRNA) into a wide variety of cells and organisms results in post-transcriptional depletion of the homologue endogenous mRNA. This well-preserved phenomenon known as RNA interference (RNAi) is present in evolutionarily diverse organisms such as plants, fungi, insects, metazoans, and mammals. Because the identification of the targeted mRNA by the RNAi machinery depends upon Watson-Crick base-pairing interactions, RNAi can be exquisitely specific. We took advantage of this powerful and flexible technique to demonstrate that selective silencing of genes essential for viral propagation prevents in vitro and in vivo viral infection. Using the baculovirus Autographa californica, a rapidly replicating and highly cytolytic double-stranded DNA virus that infects many different insect species, we show for the first time that introduction of dsRNA from gp64 and ie1, two genes essential for baculovirus propagation, results in prevention of viral infection in vitro and in vivo. This is the first report demonstrating the use of RNAi to inhibit a viral infection in animals. This inhibition was specific, because dsRNA from the polyhedrin promoter (used as control) or unrelated dsRNAs did not affect the time course of viral infection. The most relevant consequences from the present study are: 1) RNAi offers a rapid and efficient way to interfere with viral genes to assess the role of specific proteins in viral function and 2) using RNAi to interfere with viral genes essential for cell infection may provide a powerful therapeutic tool for the treatment of viral infections.

  19. Developmental and adult expression patterns of the G-protein-coupled receptor GPR88 in the rat: Establishment of a dual nuclear-cytoplasmic localization.

    Science.gov (United States)

    Massart, Renaud; Mignon, Virginie; Stanic, Jennifer; Munoz-Tello, Paola; Becker, Jerôme A J; Kieffer, Brigitte L; Darmon, Michèle; Sokoloff, Pierre; Diaz, Jorge

    2016-10-01

    GPR88 is a neuronal cerebral orphan G-protein-coupled receptor (GPCR) that has been linked to various psychiatric disorders. However, no extensive description of its localization has been provided so far. Here, we investigate the spatiotemporal expression of the GPR88 in prenatal and postnatal rat tissues by using in situ hybridization and immunohistochemistry. GPR88 protein was initially detected at embryonic day 16 (E16) in the striatal primordium. From E16-E20 to adulthood, the highest expression levels of both protein and mRNA were observed in striatum, olfactory tubercle, nucleus accumbens, amygdala, and neocortex, whereas in spinal cord, pons, and medulla GPR88 expression remains discrete. We observed an intracellular redistribution of GPR88 during cortical lamination. In the cortical plate of the developing cortex, GPR88 presents a classical GPCR plasma membrane/cytoplasmic localization that shifts, on the day of birth, to nuclei of neurons progressively settling in layers V to II. This intranuclear localization remains throughout adulthood and was also detected in monkey and human cortex as well as in the amygdala and hypothalamus of rats. Apart from the central nervous system, GPR88 was transiently expressed at high levels in peripheral tissues, including adrenal cortex (E16-E21) and cochlear ganglia (E19-P3), and also at moderate levels in retina (E18-E19) and spleen (E21-P7). The description of the GPR88 anatomical expression pattern may provide precious functional insights into this novel receptor. Furthermore, the GRP88 nuclear localization suggests nonclassical GPCR modes of action of the protein that could be relevant for cortical development and psychiatric disorders. J. Comp. Neurol. 524:2776-2802, 2016. © 2016 Wiley Periodicals, Inc. PMID:26918661

  20. Hsp10 nuclear localization and changes in lung cells response to cigarette smoke suggest novel roles for this chaperonin.

    Science.gov (United States)

    Corrao, Simona; Anzalone, Rita; Lo Iacono, Melania; Corsello, Tiziana; Di Stefano, Antonino; D'Anna, Silvestro Ennio; Balbi, Bruno; Carone, Mauro; Sala, Anna; Corona, Davide; Timperio, Anna Maria; Zolla, Lello; Farina, Felicia; de Macario, Everly Conway; Macario, Alberto J L; Cappello, Francesco; La Rocca, Giampiero

    2014-10-01

    Heat-shock protein (Hsp)10 is the co-chaperone for Hsp60 inside mitochondria, but it also resides outside the organelle. Variations in its levels and intracellular distribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD). Here, we show that Hsp10 in COPD undergoes changes at the molecular and subcellular levels in bronchial cells from human specimens and derived cell lines, intact or subjected to stress induced by cigarette smoke extract (CSE). Noteworthy findings are: (i) Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers; (ii) human bronchial epithelial (16HBE) and lung fibroblast (HFL-1) cells, in vitro, showed Hsp10 in the nucleus, before and after CSE exposure; (iii) CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts; and (iv) Hsp10 nuclear levels increased after CSE stimulation in HFL-1, indicating cytosol to nucleus migration, and although Hsp10 did not bind DNA, it bound a DNA-associated protein.

  1. Hsp10 nuclear localization and changes in lung cells response to cigarette smoke suggest novel roles for this chaperonin

    Science.gov (United States)

    Corrao, Simona; Anzalone, Rita; Lo Iacono, Melania; Corsello, Tiziana; Di Stefano, Antonino; D'Anna, Silvestro Ennio; Balbi, Bruno; Carone, Mauro; Sala, Anna; Corona, Davide; Timperio, Anna Maria; Zolla, Lello; Farina, Felicia; Conway de Macario, Everly; Macario, Alberto J. L.; Cappello, Francesco; La Rocca, Giampiero

    2014-01-01

    Heat-shock protein (Hsp)10 is the co-chaperone for Hsp60 inside mitochondria, but it also resides outside the organelle. Variations in its levels and intracellular distribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD). Here, we show that Hsp10 in COPD undergoes changes at the molecular and subcellular levels in bronchial cells from human specimens and derived cell lines, intact or subjected to stress induced by cigarette smoke extract (CSE). Noteworthy findings are: (i) Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers; (ii) human bronchial epithelial (16HBE) and lung fibroblast (HFL-1) cells, in vitro, showed Hsp10 in the nucleus, before and after CSE exposure; (iii) CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts; and (iv) Hsp10 nuclear levels increased after CSE stimulation in HFL-1, indicating cytosol to nucleus migration, and although Hsp10 did not bind DNA, it bound a DNA-associated protein. PMID:25355063

  2. Distance matters. Assessing socioeconomic impacts of the Dukovany nuclear power plant in the Czech Republic: Local perceptions and statistical evidence

    Directory of Open Access Journals (Sweden)

    Frantál Bohumil

    2016-03-01

    Full Text Available The effect of geographical distance on the extent of socioeconomic impacts of the Dukovany nuclear power plant in the Czech Republic is assessed by combining two different research approaches. First, we survey how people living in municipalities in the vicinity of the power plant perceive impacts on their personal quality of life. Second, we explore the effects of the power plant on regional development by analysing long-term statistical data about the unemployment rate, the share of workers in the energy sector and overall job opportunities in the respective municipalities. The results indicate that the power plant has had significant positive impacts on surrounding communities both as perceived by residents and as evidenced by the statistical data. The level of impacts is, however, significantly influenced by the spatial and social distances of communities and individuals from the power plant. The perception of positive impacts correlates with geographical proximity to the power plant, while the hypothetical distance where positive effects on the quality of life are no longer perceived was estimated at about 15 km. Positive effects are also more likely to be reported by highly educated, young and middle-aged and economically active persons, whose work is connected to the power plant.

  3. The 21.5-kDa isoform of myelin basic protein has a non-traditional PY-nuclear-localization signal

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Graham S.T.; Seymour, Lauren V. [Molecular and Cellular Biology, University of Guelph, Guelph, Ontario (Canada); Boggs, Joan M. [Molecular Structure and Function, Research Institute, Hospital for Sick Children, and Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario (Canada); Harauz, George, E-mail: gharauz@uoguelph.ca [Molecular and Cellular Biology, University of Guelph, Guelph, Ontario (Canada)

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Full-length 21.5-kDa MBP isoform is translocated to the nucleus. Black-Right-Pointing-Pointer We hypothesized that the exon-II-encoded sequence contained the NLS. Black-Right-Pointing-Pointer We mutated this sequence in RFP-tagged constructs and transfected N19-cells. Black-Right-Pointing-Pointer Abolition of two key positively-charged residues resulted in loss of nuclear-trafficking. Black-Right-Pointing-Pointer The 21.5-kDa isoform of classic MBP contains a non-traditional PY-NLS. -- Abstract: The predominant 18.5-kDa classic myelin basic protein (MBP) is mainly responsible for compaction of the myelin sheath in the central nervous system, but is multifunctional, having numerous interactions with Ca{sup 2+}-calmodulin, actin, tubulin, and SH3-domains, and can tether these proteins to a lipid membrane in vitro. The full-length 21.5-kDa MBP isoform has an additional 26 residues encoded by exon-II of the classic gene, which causes it to be trafficked to the nucleus of oligodendrocytes (OLGs). We have performed site-directed mutagenesis of selected residues within this segment in red fluorescent protein (RFP)-tagged constructs, which were then transfected into the immortalized N19-OLG cell line to view protein localization using epifluorescence microscopy. We found that 21.5-kDa MBP contains two non-traditional PY-nuclear-localization signals, and that arginine and lysine residues within these motifs were involved in subcellular trafficking of this protein to the nucleus, where it may have functional roles during myelinogenesis.

  4. Structural and energetic basis of ALS-causing mutations in the atypical proline-tyrosine nuclear localization signal of the Fused in Sarcoma protein (FUS)

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Zi Chao; Chook, Yuh Min [UTSMC

    2012-10-02

    Mutations in the proline/tyrosine–nuclear localization signal (PY-NLS) of the Fused in Sarcoma protein (FUS) cause amyotrophic lateral sclerosis (ALS). Here we report the crystal structure of the FUS PY-NLS bound to its nuclear import receptor Karyopherinβ2 (Kapβ2; also known as Transportin). The FUS PY-NLS occupies the structurally invariant C-terminal arch of Kapβ2, tracing a path similar to that of other characterized PY-NLSs. Unlike other PY-NLSs, which generally bind Kapβ2 in fully extended conformations, the FUS peptide is atypical as its central portion forms a 2.5-turn α-helix. The Kapβ2-binding epitopes of the FUS PY-NLS consist of an N-terminal PGKM hydrophobic motif, a central arginine-rich α-helix, and a C-terminal PY motif. ALS mutations are found almost exclusively within these epitopes. Each ALS mutation site makes multiple contacts with Kapβ2 and mutations of these residues decrease binding affinities for Kapβ2 (KD for wild-type FUS PY-NLS is 9.5 nM) up to ninefold. Thermodynamic analyses of ALS mutations in the FUS PY-NLS show that the weakening of FUS-Kapβ2 binding affinity, the degree of cytoplasmic mislocalization, and ALS disease severity are correlated.

  5. Evolution of structure and local magnetic fields during crystallization of HITPERM glassy alloys studied by in situ diffraction and nuclear forward scattering of synchrotron radiation.

    Science.gov (United States)

    Miglierini, Marcel; Pavlovič, Márius; Procházka, Vít; Hatala, Tomáš; Schumacher, Gerhard; Rüffer, Rudolf

    2015-11-14

    Evolution of structure and local magnetic fields in (Fe(1-x)Co(x))76Mo8Cu1B15 (HITPERM) metallic glass ribbons with various amounts of Co (x = 0, 0.25, 0.5) were studied in situ using diffraction and nuclear forward scattering of synchrotron radiation. It was found that crystallization of all three glasses proceeds in two stages. In the first stage, bcc (Fe,Co) nanocrystals are formed, while in the second stage additional crystalline phases evolve. For all three glasses, the crystallization temperatures at the wheel side were found to be lower than at the air side of the ribbon. The crystallization temperatures were found to decrease with increasing Co content. The lattice parameters of the bcc nanocrystals decrease up to about 550 °C and then increase pointing to squeezing Mo atoms out of the nanograins or to interface effects between the nanocrystals and the glassy matrix. Nuclear forward scattering enabled separate evaluation of the contributions that stem from structurally different regions within the investigated samples including the newly formed nanocrystals and the residual amorphous matrix. Even minor Co content (x = 0.25) has a substantial effect not only upon the magnetic behaviour of the alloy but also upon its structure. Making use of hyperfine magnetic fields, it was possible to unveil structurally diverse positions of Fe atoms that reside in a nanocrystalline lattice with different numbers of Co nearest neighbours.

  6. Local and integral ulrasonic gauges for two-phase flow instrumentation in nuclear reactor and safety technology. Final report

    International Nuclear Information System (INIS)

    The present project was executed in the frame of a co-operation agreement between FZR and the scientific group of Prof. Melnikov of the technical University of Nishny Novgorod (TUNN) in the Russian Federation. It is part of the Federal Government's programme for the provision of advice for Eastern Europe on the building up of democracy and social market economy (TRANSFORM Programme). New methods of two-phase flow instrumentation were developed: Intrusive wave-guide probes can be used for local void fraction measurements. The new ultrasonic mesh sensors allow a fast two-phase flow visualisation with about 250 frames per second. Experiments carried out at the test loop in Rossendorf, but also the tests at the DESIRE facility in Delft have shown that both local wave-guide probes and ultrasonic mesh sensors can be successfully applied under the conditions of high pressure and temperature steam-water mixture, as well as in organic liquids and refrigerants. Furthermore, non-intrusive wave-guide sensors as well as density sensors based on the measurement of the wave propagation velocity in wave-guides immersed into the measuring liquid were developed and tested. In the present stage of the development, the non-intrusive sensors can rather be used for a qualitative gas respectively level detection than for void fraction measurements. The wave-guide density sensor was successfully demonstrated that it is able to measure densities of single-phase liquids. It requires further development of the electronic circuitry. The main innovation was achieved by the development of the ultrasonic mesh sensor, the resoluting capability of which is comparable to methods like electrical wire-mesh sensors and ultra-fast X-ray tomography, while the device itself is robust and low expensive. (orig.)

  7. Macro-localization study of a commercial CAREM nuclear power plant in the province of Formosa : state of progress

    International Nuclear Information System (INIS)

    The framework agreement signed between Formosa Province and the National Atomic Energy Commission (CNEA) in order to establish formal relations of cooperation with regard to projects of common interest, research and development, technology services, human resource training in the areas inherent to CNEA functions, resulted in a specific agreement in order to analyze the potential location of a CAREM commercial nuclear power plant in the Province. Thus, the site survey study which is under development aims at the determination of one or more sites, which meet the criteria set - security related and not related - and is/are eligible for the installation of the plant. The site selection methodology used to develop the study was based on documentation and bibliography of the International Atomic Energy Agency and CNEA. Taking into account these references, the study began divided into 3 levels; currently, Level I is approved and the analysis of Level II is completed. Each of these levels covers different aspects that must be evaluated during the selection process. That one is a complex analysis, which was carried out through the participation and joint efforts of a multidisciplinary team consisting of CNEA’s staff and the Province of Formosa, which in turn had the cooperation of various institutions. Partial results of the Level II document, determine the pre selection of three potential areas in the province. In turn, it is important to remember that the site survey (regional) study is the first stage in the selection process. Then, the site evaluation stage (micro siting study) and the preparation of the Preliminary Safety Report have to be implemented, in order to establish the definitive site for the plant installation. (author)

  8. Transcriptional activity and nuclear localization of Cabut, the Drosophila ortholog of vertebrate TGF-β-inducible early-response gene (TIEG proteins.

    Directory of Open Access Journals (Sweden)

    Yaiza Belacortu

    Full Text Available BACKGROUND: Cabut (Cbt is a C(2H(2-class zinc finger transcription factor involved in embryonic dorsal closure, epithelial regeneration and other developmental processes in Drosophila melanogaster. Cbt orthologs have been identified in other Drosophila species and insects as well as in vertebrates. Indeed, Cbt is the Drosophila ortholog of the group of vertebrate proteins encoded by the TGF-ß-inducible early-response genes (TIEGs, which belong to Sp1-like/Krüppel-like family of transcription factors. Several functional domains involved in transcriptional control and subcellular localization have been identified in the vertebrate TIEGs. However, little is known of whether these domains and functions are also conserved in the Cbt protein. METHODOLOGY/PRINCIPAL FINDINGS: To determine the transcriptional regulatory activity of the Drosophila Cbt protein, we performed Gal4-based luciferase assays in S2 cells and showed that Cbt is a transcriptional repressor and able to regulate its own expression. Truncated forms of Cbt were then generated to identify its functional domains. This analysis revealed a sequence similar to the mSin3A-interacting repressor domain found in vertebrate TIEGs, although located in a different part of the Cbt protein. Using β-Galactosidase and eGFP fusion proteins, we also showed that Cbt contains the bipartite nuclear localization signal (NLS previously identified in TIEG proteins, although it is non-functional in insect cells. Instead, a monopartite NLS, located at the amino terminus of the protein and conserved across insects, is functional in Drosophila S2 and Spodoptera exigua Sec301 cells. Last but not least, genetic interaction and immunohistochemical assays suggested that Cbt nuclear import is mediated by Importin-α2. CONCLUSIONS/SIGNIFICANCE: Our results constitute the first characterization of the molecular mechanisms of Cbt-mediated transcriptional control as well as of Cbt nuclear import, and demonstrate the

  9. Light Water Reactor Sustainability Program: Evaluation of Localized Cable Test Methods for Nuclear Power Plant Cable Aging Management Programs

    Energy Technology Data Exchange (ETDEWEB)

    Glass, Samuel W. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Fifield, Leonard S. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Hartman, Trenton S. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2016-05-30

    This Pacific Northwest National Laboratory (PNNL) milestone report describes progress to date on the investigation of nondestructive test (NDE) methods focusing particularly on local measurements that provide key indicators of cable aging and damage. The work includes a review of relevant literature as well as hands-on experimental verification of inspection capabilities. As NPPs consider applying for second, or subsequent, license renewal (SLR) to extend their operating period from 60 years to 80 years it important to understand how the materials installed in plant systems and components will age during that time and develop aging management programs (AMPs) to assure continued safe operation under normal and design basis events (DBE). Normal component and system tests typically confirm the cables can perform their normal operational function. The focus of the cable test program is directed toward the more demanding challenge of assuring the cable function under accident or DBE. Most utilities already have a program associated with their first life extension from 40 to 60 years. Regrettably there is neither a clear guideline nor a single NDE that can assure cable function and integrity for all cables. Thankfully, however, practical implementation of a broad range of tests allows utilities to develop a practical program that assures cable function to a high degree. The industry has adopted 50% elongation at break (EAB) relative to the un-aged cable condition as the acceptability standard. All tests are benchmarked against the cable EAB test. EAB is a destructive test so the test programs must apply an array of other NDE tests to assure or infer the overall set of cable’s system integrity. These cable NDE programs vary in rigor and methodology. As the industry gains experience with the efficacy of these programs, it is expected that implementation practice will converge to a more common approach. This report addresses the range of local NDE cable tests that are or

  10. Light Water Reactor Sustainability Program: Evaluation of Localized Cable Test Methods for Nuclear Power Plant Cable Aging Management Programs

    Energy Technology Data Exchange (ETDEWEB)

    Glass, Samuel W. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Fifield, Leonard S. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Hartman, Trenton S. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2016-05-30

    This Pacific Northwest National Laboratory (PNNL) milestone report describes progress to date on the investigation of nondestructive test (NDE) methods focusing particularly on local measurements that provide key indicators of cable aging and damage. The work includes a review of relevant literature as well as hands-on experimental verification of inspection capabilities. As NPPs consider applying for second, or subsequent, license renewal (SLR) to extend their operating period from 60 years to 80 years, it important to understand how the materials installed in plant systems and components will age during that time and develop aging management programs (AMPs) to assure continued safe operation under normal and design basis events (DBE). Normal component and system tests typically confirm the cables can perform their normal operational function. The focus of the cable test program is directed toward the more demanding challenge of assuring the cable function under accident or DBE. Most utilities already have a program associated with their first life extension from 40 to 60 years. Regrettably there is neither a clear guideline nor a single NDE that can assure cable function and integrity for all cables. Thankfully, however, practical implementation of a broad range of tests allows utilities to develop a practical program that assures cable function to a high degree. The industry has adopted 50% elongation at break (EAB) relative to the un-aged cable condition as the acceptability standard. All tests are benchmarked against the cable EAB test. EAB is a destructive test so the test programs must apply an array of other NDE tests to assure or infer the overall set of cable’s system integrity. These cable NDE programs vary in rigor and methodology. As the industry gains experience with the efficacy of these programs, it is expected that implementation practice will converge to a more common approach. This report addresses the range of local NDE cable tests that are or

  11. Light Water Reactor Sustainability Program: Evaluation of Localized Cable Test Methods for Nuclear Power Plant Cable Aging Management Programs

    Energy Technology Data Exchange (ETDEWEB)

    Glass, Samuel W. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Fifield, Leonard S. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Hartman, Trenton S. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2016-05-30

    This Pacific Northwest National Laboratory (PNNL) milestone report describes progress to date on the investigation of nondestructive test (NDE) methods focusing particularly on local measurements that provide key indicators of cable aging and damage. The work includes a review of relevant literature as well as hands-on experimental verification of inspection capabilities. As NPPs consider applying for second, or subsequent, license renewal (SLR) to extend their operating period from 60 years to 80 years, it important to understand how the materials installed in plant systems and components will age during that time and develop aging management programs (AMPs) to assure continued safe operation under normal and design basis events (DBE). Normal component and system tests typically confirm the cables can perform their normal operational function. The focus of the cable test program is directed toward the more demanding challenge of assuring the cable function under accident or DBE. Most utilities already have a program associated with their first life extension from 40 to 60 years. Regrettably, there is neither a clear guideline nor a single NDE that can assure cable function and integrity for all cables. Thankfully, however, practical implementation of a broad range of tests allows utilities to develop a practical program that assures cable function to a high degree. The industry has adopted 50% elongation at break (EAB) relative to the un-aged cable condition as the acceptability standard. All tests are benchmarked against the cable EAB test. EAB is a destructive test so the test programs must apply an array of other NDE tests to assure or infer the overall set of cable’s system integrity. These cable NDE programs vary in rigor and methodology. As the industry gains experience with the efficacy of these programs, it is expected that implementation practice will converge to a more common approach. This report addresses the range of local NDE cable tests that are

  12. Local structure and magnetism of L10-type FeNi alloy films with perpendicular magnetic anisotropy studied through 57Fe nuclear probes

    International Nuclear Information System (INIS)

    The local structure and magnetism of FeNi alloy films prepared by alternate deposition of Fe and Ni monatomic layers, where perpendicular magnetic anisotropy has been observed, were investigated through 57Fe nuclear probes using Mössbauer spectroscopy. It was confirmed that the films are composed of L10-type ordered FeNi phase and A1-type disordered FeNi phase. For the films grown at 40–70 °C, which have no perpendicular anisotropy, the A1-disordered phase is dominant, whereas for the films grown at 100–190 °C, which have a stronger perpendicular anisotropy, the relative amount of the L10-ordered phase reaches 40% or more. It was clearly shown that the magnetic anisotropy of these films is strongly correlated with the local environments of Fe in the films. The results imply that if a further increase in the ratio of the L10-ordered phase is successfully achieved, one would obtain films with a stronger magnetic anisotropy applicable to perpendicular magnetic recording. (paper)

  13. Nuclear magnetic resonance solution structure of the peptidoglycan-binding SPOR domain from Escherichia coli DamX: insights into septal localization.

    Science.gov (United States)

    Williams, Kyle B; Yahashiri, Atsushi; Arends, S J Ryan; Popham, David L; Fowler, C Andrew; Weiss, David S

    2013-01-29

    SPOR domains are present in thousands of bacterial proteins and probably bind septal peptidoglycan (PG), but the details of the SPOR-PG interaction have yet to be elucidated. Here we characterize the structure and function of the SPOR domain for an Escherichia coli division protein named DamX. Nuclear magnetic resonance revealed the domain comprises a four-stranded antiparallel β-sheet buttressed on one side by two α-helices. A third helix, designated α3, associates with the other face of the β-sheet, but this helix is relatively mobile. Site-directed mutagenesis revealed the face of the β-sheet that interacts with α3 is important for septal localization and binding to PG sacculi. The position and mobility of α3 suggest it might regulate PG binding, but although α3 deletion mutants still localized to the septal ring, they were too unstable to use in a PG binding assay. Finally, to assess the importance of the SPOR domain in DamX function, we constructed and characterized E. coli mutants that produced DamX proteins with SPOR domain point mutations or SPOR domain deletions. These studies revealed the SPOR domain is important for multiple activities associated with DamX: targeting the protein to the division site, conferring full resistance to the bile salt deoxycholate, improving the efficiency of cell division when DamX is produced at normal levels, and inhibiting cell division when DamX is overproduced.

  14. A nuclear localized protein ZCCHC9 is expressed in cerebral cortex and suppresses the MAPK signal pathway

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The CCHC-type zinc finger motif has numerous biological activities (such as DNA binding and RNA binding) and can also mediate protein-protein interaction. This article gives a primary report about the human ZCCHC9 gene. Protein ZCCHC9 contains four CCHC motifs and is highly conserved in humans, mice, and rats. The whole cDNA sequence of the ZCCHC9 gene has been amplified by PCR and a number of plasmids have been constructed for further study. The results show that ZCCHC9 is localized in the nucleus, and especially concentrated in the nucleolus. It is highly expressed in the brain and testicles of the mouse. This has been confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR). In situ hybridization of the mouse brain indicates that ZCCHC9 is mainly expressed in the cerebral cortex. Reporter gene assay shows that ZCCHC9 suppresses the transcription activities of NF-kappa B and SRE,and may play roles in the Mitogen-Activated Protein Kinase (MAPK) signaling transduction pathway.

  15. THE SENSITIVITY OF CARBON STEELS' SUSCEPTIBILITY TO LOCALIZED CORROSION TO THE PH OF NITRATE BASED NUCLEAR WASTES

    Energy Technology Data Exchange (ETDEWEB)

    BOOMER KD

    2010-01-14

    The Hanford tank reservation contains approximately 50 million gallons of liquid legacy radioactive waste from cold war weapons production, which is stored in 177 underground storage tanks. The tanks will be in use until waste processing operations are completed. The wastes tend to be high pH (over 10) and nitrate based. Under these alkaline conditions carbon steels tend to be passive and undergo relatively slow uniform corrosion. However, the presence of nitrate and other aggressive species, can lead to pitting and stress corrosion cracking. This work is a continuation of previous work that investigated the propensity of steels to suffer pitting and stress corrosion cracking in various waste simulants. The focus of this work is an investigation of the sensitivity of the steels' pitting and stress corrosion cracking susceptibility tosimulant pH. Previous work demonstrated that wastes that are high in aggressive nitrate and low in inhibitory nitrite are susceptible to localized corrosion. However, the previous work involved wastes with pH 12 or higher. The current work involves wastes with lower pH of 10 or 11. It is expected that at these lower pHs that a higher nitrite-to-nitrate ratio will be necessary to ensure tank integrity. This experimental work involved both electrochemical testing, and slow strain rate testing at either the free corrosion potential or under anodic polarization. The results of the current work will be discussed, and compared to work previously presented.

  16. Expression of hemagglutinin protein from the avian influenza virus H5N1 in a baculovirus/insect cell system significantly enhanced by suspension culture

    Directory of Open Access Journals (Sweden)

    Spencer Lynn

    2006-02-01

    Full Text Available Abstract Background Prevention of a possible avian influenza pandemic necessitates the development of rapid diagnostic tests and the eventual production of a vaccine. Results For vaccine production, hemagglutinin (HA1 from avian influenza H5N1 was expressed from a recombinant baculovirus. Recombinant HA1 was expressed in monolayer or suspension culture insect cells by infection with the recombinant baculovirus. The yield of rHA1 from the suspension culture was 68 mg/l, compared to 6 mg/l from the monolayer culture. Immunization of guinea pigs with 50 μg of rHA1 yielded hemagglutinin inhibition and virus neutralization titers of 1:160 after two times vaccination with rHA1 protein. Conclusion Thus, the production of rHA1 using an insect suspension cell system provides a promising basis for economical production of a H5 antigen.

  17. Induction of robust immunity response in mice by dual-expression-system-based recombinant baculovirus expressing the capsid protein of porcine circovirus type 2

    OpenAIRE

    Ye, Yu; Cheng, Xiaoliang; Jie ZHANG; Tong, Tiezhu; Lin, Wenyao; Liao, Ming; Fan, Huiying

    2013-01-01

    Background Porcine circovirus type 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome (PMWS), an emerging swine disease that causes progressive weight loss, dyspnea, tachypnea, anemia, jaundice, and diarrhea in piglets. Although baculovirus is an enveloped virus that infects insects in nature, it has emerged as a vaccine vector, and we used it to develop a novel candidate vaccine for a preventive or therapeutic strategy to control PCV2 infections. Methods Immunoblotting a...

  18. Expression of an Innate Immune Element (Mouse Hepcidin-1) in Baculovirus Expression System and the Comparison of Its Function with Synthetic Human Hepcidin-25

    OpenAIRE

    Yazdani, Yaghoub; Sadeghi, Hamid; Alimohammadian, Mohammad; Andalib, Alireza; Moazen, Fatemeh; Rezaei, Abbas

    2011-01-01

    Hepcidin is an innate immune element which decreases the iron absorption from diet and iron releasing from macrophage cell. In contrast to the chemical iron chelators, there has been limited effort applied to the specific use of hepcidin as a new drug for decreasing the iron overload. Hepcidin is produced in different biological systems. For instance, E-coli is used for human hepcidin expression, however, post-translational modification is impaired. We have used a simple baculovirus expressio...

  19. Mucosal Delivery of ACNPV Baculovirus Driving Expression of the Gal-Lectin LC3 Fragment Confers Protection against Amoebic Liver Abscess in Hamster

    Directory of Open Access Journals (Sweden)

    DM Meneses-Ruiz, JP Laclette, H Aguilar-Díaz, J Hernández-Ruiz, A Luz-Madrigal, A Sampieri, L Vaca, JC Carrero

    2011-01-01

    Full Text Available Mucosal vaccination against amoebiasis using the Gal-lectin of E. histolytica has been proposed as one of the leading strategies for controlling this human disease. However, most mucosal adjuvants used are toxic and the identification of safe delivery systems is necessary. Here, we evaluate the potential of a recombinant Autographa californica baculovirus driving the expression of the LC3 fragment of the Gal-lectin to confer protection against amoebic liver abscess (ALA in hamsters following oral or nasal immunization. Hamsters immunized by oral route showed complete absence (57.9% or partial development (21% of ALA, resulting in some protection in 78.9% of animals when compared with the wild type baculovirus and sham control groups. In contrast, nasal immunization conferred only 21% of protection efficacy. Levels of ALA protection showed lineal correlation with the development of an anti-amoebic cellular immune response evaluated in spleens, but not with the induction of seric IgG anti-amoeba antibodies. These results suggest that baculovirus driving the expression of E. histolytica vaccine candidate antigens is useful for inducing protective cellular and humoral immune responses following oral immunization, and therefore it could be used as a system for mucosal delivery of an anti-amoebic vaccine.

  20. Mucosal delivery of ACNPV baculovirus driving expression of the Gal-lectin LC3 fragment confers protection against amoebic liver abscess in hamster.

    Science.gov (United States)

    Meneses-Ruiz, D M; Laclette, J P; Aguilar-Díaz, H; Hernández-Ruiz, J; Luz-Madrigal, A; Sampieri, A; Vaca, L; Carrero, J C

    2011-01-01

    Mucosal vaccination against amoebiasis using the Gal-lectin of E. histolytica has been proposed as one of the leading strategies for controlling this human disease. However, most mucosal adjuvants used are toxic and the identification of safe delivery systems is necessary. Here, we evaluate the potential of a recombinant Autographa californica baculovirus driving the expression of the LC3 fragment of the Gal-lectin to confer protection against amoebic liver abscess (ALA) in hamsters following oral or nasal immunization. Hamsters immunized by oral route showed complete absence (57.9%) or partial development (21%) of ALA, resulting in some protection in 78.9% of animals when compared with the wild type baculovirus and sham control groups. In contrast, nasal immunization conferred only 21% of protection efficacy. Levels of ALA protection showed lineal correlation with the development of an anti-amoebic cellular immune response evaluated in spleens, but not with the induction of seric IgG anti-amoeba antibodies. These results suggest that baculovirus driving the expression of E. histolytica vaccine candidate antigens is useful for inducing protective cellular and humoral immune responses following oral immunization, and therefore it could be used as a system for mucosal delivery of an anti-amoebic vaccine.

  1. Mucosal Delivery of ACNPV Baculovirus Driving Expression of the Gal-Lectin LC3 Fragment Confers Protection against Amoebic Liver Abscess in Hamster

    Science.gov (United States)

    Meneses-Ruiz, DM; Laclette, JP; Aguilar-Díaz, H; Hernández-Ruiz, J; Luz-Madrigal, A; Sampieri, A; Vaca, L; Carrero, JC

    2011-01-01

    Mucosal vaccination against amoebiasis using the Gal-lectin of E. histolytica has been proposed as one of the leading strategies for controlling this human disease. However, most mucosal adjuvants used are toxic and the identification of safe delivery systems is necessary. Here, we evaluate the potential of a recombinant Autographa californica baculovirus driving the expression of the LC3 fragment of the Gal-lectin to confer protection against amoebic liver abscess (ALA) in hamsters following oral or nasal immunization. Hamsters immunized by oral route showed complete absence (57.9%) or partial development (21%) of ALA, resulting in some protection in 78.9% of animals when compared with the wild type baculovirus and sham control groups. In contrast, nasal immunization conferred only 21% of protection efficacy. Levels of ALA protection showed lineal correlation with the development of an anti-amoebic cellular immune response evaluated in spleens, but not with the induction of seric IgG anti-amoeba antibodies. These results suggest that baculovirus driving the expression of E. histolytica vaccine candidate antigens is useful for inducing protective cellular and humoral immune responses following oral immunization, and therefore it could be used as a system for mucosal delivery of an anti-amoebic vaccine. PMID:22110386

  2. Predictions of local, regional and global radiation doses from iodine-129 for four different disposal methods and an all-nuclear future

    International Nuclear Information System (INIS)

    The radioactive isotope 129I produced by nuclear fission becomes globally distributed if released to the environment. The consequences of several options for management of 129I are discussed. Estimates are presented of radiation doses from the 129I produced in generating 1000 GW(e).a of nuclear electricity, the total production expected in Canada up to about 2040. Individual thyroid dose rates from 129I, and accumulated collective effective dose equivalents to three groups are compared for four 129I management strategies. These groups are: local persons living in the immediate vicinity of a discharge zone from a fuel reprocessing facility or waste disposal vault in the Canadian Shield, regional persons living in the lower Great Lakes basin, and average global persons. The four management strategies studied were: atmospheric discharge from a reprocessing facility, ocean dumping, subseabed disposal, and isolation in a deep vault in plutonic rock in the Canadian Shield. Doses associated with each of these options are compared with each other and with proposed Canadian regulatory limits. Estimates are also presented of doses to the same groups from fission-product 129I, if fission supplied all future world energy needs. The two management options considered were geological disposal in vaults that would each eventually discharge 129I at the rate estimated for a disposal vault in the Canadian Shield, and ocean dumping. Again, doses arising from these options are compared with each other and with proposed regulatory limits. The second comparison allows estimates to be made of the time at which such intensive use of fission could produce unacceptable levels of 129I in the environment

  3. Physiological and receptor-selective retinoids modulate interferon gamma signaling by increasing the expression, nuclear localization, and functional activity of interferon regulatory factor-1.

    Science.gov (United States)

    Luo, Xin M; Ross, A Catharine

    2005-10-28

    Synergistic actions between all-trans-retinoic acid (atRA) and interferon gamma (IFNgamma) on modulation of cellular functions have been reported both in vitro and in vivo. However, the mechanism of atRA-mediated regulation of IFNgamma signaling is poorly understood. In this study, we have used the human lung epithelial cell line A549 to examine the effect of atRA on IFNgamma-induced expression of IFN regulatory factor-1 (IRF-1), an important transcription factor involved in cell growth and apoptosis, differentiation, and antiviral and antibacterial immune responses. At least 4 h of pretreatment with atRA followed by suboptimal concentrations of IFNgamma induced a faster, higher, and more stable expression of IRF-1 than IFNgamma alone. Actinomycin D completely blocked the induction of IRF-1 by the combination, suggesting regulation at the transcriptional level. Further, we found that activation of signal transducer and activator of transcription-1 was induced more dramatically by atRA and IFNgamma than by IFNgamma alone. Expression of IFNgamma receptor-1 on the cell surface was also increased upon atRA pretreatment. Experiments using receptor-selective retinoids revealed that ligands for retinoic acid receptor-alpha (RARalpha), including atRA, 9-cis-retinoic acid, and Am580, sequentially increased the levels of IFNgamma receptor-1, activated signal transducer and activator of transcription-1, and IRF-1 and that an RARalpha antagonist was able to inhibit the effects of atRA and Am580. In addition, atRA pretreatment affected the transcriptional functions of IFNgamma-induced IRF-1, increasing its nuclear localization and DNA binding activity as well as the transcript levels of IRF-1 target genes. These results suggest that atRA, an RARalpha ligand, regulates IFNgamma-induced IRF-1 by affecting multiple components of the IFNgamma signaling pathway, from the plasma membrane to the nuclear transcription factors.

  4. Post-transcriptional regulation of cyclins D1, D3 and G1 and proliferation of human cancer cells depend on IMP-3 nuclear localization.

    Science.gov (United States)

    Rivera Vargas, T; Boudoukha, S; Simon, A; Souidi, M; Cuvellier, S; Pinna, G; Polesskaya, A

    2014-05-29

    RNA-binding proteins of the IMP family (insulin-like growth factor 2 (IGF2) mRNA-binding proteins 1-3) are important post-transcriptional regulators of gene expression. Multiple studies have linked high expression of IMP proteins, and especially of IMP-3, to an unfavorable prognosis in numerous types of cancer. The specific importance of IMP-3 for cancer transformation remains poorly understood. We here show that all three IMPs can directly bind the mRNAs of cyclins D1, D3 and G1 (CCND1, D3 and G1) in vivo and in vitro, and yet only IMP-3 regulates the expression of these cyclins in a significant manner in six human cancer cell lines of different origins. In the absence of IMP-3, the levels of CCND1, D3 and G1 proteins fall dramatically, and the cells accumulate in the G1 phase of the cell cycle, leading to almost complete proliferation arrest. Our results show that, compared with IMP-1 and IMP-2, IMP-3 is enriched in the nucleus, where it binds the transcripts of CCND1, D3 and G1. The nuclear localization of IMP-3 depends on its protein partner HNRNPM and is indispensable for the post-transcriptional regulation of expression of the cyclins. Cytoplasmic retention of IMP-3 and HNRNPM in human cancer cells leads to significant drop in proliferation. In conclusion, a nuclear IMP-3-HNRNPM complex is important for the efficient synthesis of CCND1, D3 and G1 and for the proliferation of human cancer cells.

  5. Emission of 14C by the Almirante Alvaro Alberto Nuclear Power Plant 1 and 2 and their local effects on the environmental levels

    International Nuclear Information System (INIS)

    14C is a is a long-lived beta-emitting nuclide (T1/2 = 5730 years) produced naturally in the upper atmosphere as a result of reactions between neutrons and stable 14N(14N(n,p)14C). Although in a lesser extent, nuclear power plants produce 14C as well during their routine operation. Since it is converted in 14CO2 and mixed throughout the atmosphere, it is incorporated into plant tissues, via photosynthesis process, and hence in food chain. Because of the biological importance of 14C and long half-life, it is of interest to quantify the amounts released by nuclear industry. The Brazilian nuclear central named Nuclear Central Admiral Alvaro Alberto (CNAAA) has two nuclear reactors of PWR type in operation, Angra I (657 MWe) and Angra II (1350 MWe), and one under construction, Angra III (1309 MWe PWR). The aim of this study was to determine the strength of the sources and the 14C content in the environment through analyses of air, vegetation and soils taken within 5 km (the influenced area) of CNAAA. The thesis consists of an extensive review about the subject (part one) and of four papers (part two). The first paper is about the determination of 14C concentrations released by reactors (source strength). For Angra I, a device was developed in order to sample the gaseous effluents and for Angra II, a commercial monitoring system had already been implemented since its initial operation (2001). The 14C can be emitted as hydrocarbons, CO or CO2, depending on the type of reactor. For PWRs, the main chemical form released is hydrocarbons (80 %). The monitoring system of Angra I was planned to determine both CO2 and hydrocarbon fractions but in Angra II, all hydrocarbons are converted to CO2 by using a Pd/Al2O3 catalyst at 450 deg C. The liquid scintillation was the method employed to measure the samples. The second one concerns the atmospheric dispersion of the released radiocarbon through measurements of air samples taken with 3 km from power plants, in five different wind

  6. Application of ultrasonic echo technique to the measurement of 2-dimensional local instantaneous liquid film thickness on a simulated nuclear fuel rod

    International Nuclear Information System (INIS)

    A subchannel analysis for nuclear reactor core thermal hydraulics solves the basic conservation equations for mass, momentum and energy for each sub-channel, taking into account the effect of cross flows between neighboring subchannels. The three fluid model formulation is often considered for the analysis of droplet dispersed annular flow regions where the liquid film flow on the fuel rods is determined by a balance among entrainment and deposition rates of liquid droplets in the vapor flow and evaporation rate on a heated wall. The critical heat flux in a BWR is therefore characterized by film dryout. It is well known that in a typical BWR fuel rod assembly there are mechanical spacers placed at, for example, every 50 centimeters in axial direction to keep the rod clearance adequately which in turn result in flow obstacles consequently, and thus local time dependent film flow structure is changed before and after these spacers. The previous studies revealed a general trend that the liquid film thickness normally reduces in a spacer region due to droplet entrainment rate enhanced by an accelerated vapor flow, whereas in a region downstream of the spacers it recovers again because of highly agitated nature of the flow behind the spacers and increased deposition rate. Thus the accurate prediction of time dependent 2-dimensional film thickness on the fuel rods is extremely indispensable for the evaluation of the critical heat flux for BWR fuel assemblies. However, for the moment, we do not have reliable data-base for non-homogeneous liquid film flow on the fuel rods mainly because of a lack in accurate non-intrusive measuring techniques. The purpose of this work is therefore to present the outline of a new development in high speed ultrasonic echo technique which we are now pursuing at our laboratory with a view to applying it to measurement of time-sequential 2-dimensional thickness of the film flow around a simulated nuclear fuel rod. The scanning time required to

  7. Hydrophobic Interactions Involved in Attachment of a Baculovirus to Hydrophobic Surfaces

    OpenAIRE

    Small, Deirdre A.; Moore, Norman F.; Entwistle, Philip F.

    1986-01-01

    The hydrophobic interactions of Trichoplusia ni nuclear polyhedrosis virus were characterized by hydrophobic interaction chromatography. The determination of the hydrophobic force and some of the factors that influence its size is discussed in relation to the attachment to leaf surfaces of polyhedra during their use as biological control agents against insect pests.

  8. Essential C-Terminal region of the baculovirus minor capsid protein VP80 binds DNA

    NARCIS (Netherlands)

    Marek, M.; Merten, O.W.; Francis-Devaraj, F.; Oers, van M.M.

    2012-01-01

    The essential Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) minor capsid protein VP80 has been recently shown to interact with the virus-triggered, nuclear F-actin cytoskeleton. A role for VP80 in virus morphogenesis has been proposed in the maturation of progeny nucleocapsids and

  9. Kicking Triturus arntzeni when it's down: large-scale nuclear genetic data confirm that newts from the type locality are genetically admixed.

    Science.gov (United States)

    Wielstra, B; Arntzen, J W

    2014-01-01

    We collected nuclear DNA data (52 markers) with next-generation sequencing for nine Triturus newt specimens, including the holotype and two of the paratypes of T. arntzeni, from the type locality at Vrtovać in eastern Serbia. We compare these data to a reference set composed of the four crested newt species distributed in eastern Serbia namely T. cristatus, T. dobrogicus, T. ivanbureschi and T. macedonicus to determine to which of these species the newts from the type locality of T. arntzeni should be attributed. The majority of alleles in individuals from Vrtovać is derived from T. macedonicus, but a considerable number of T. ivanbureschi alleles is also present; alleles typical for T. cristatus and T. dobrogicus are found at low frequency. Accordingly, we interpret Vrtovać as a T. macedonicus - T. ivanbureschi hybrid population, albeit not composed of F1 hybrids but of genetically admixed individuals derived through multiple generations of backcrossing. The data support the notion that the name T. arntzeni should not be applied to a species newly distinguished in T. karelinii sensu lato (to which the name T. ivanbureschi has been given). We conclude that because of the hybrid nature of the individuals from Vrtovać, the name T. arntzeni should be placed not only in the synonymy of T. macedonicus but also in the synonymy of T. ivanbureschi. In this study we demonstrate that next-generation sequencing can provide high quality data for type material with degraded DNA and therefore can play an important role in taxonomy. PMID:24871018

  10. 59Co nuclear magnetic resonance study of the local distribution of atoms in the Heusler compound Co2FeAl0.5Si0.5

    Science.gov (United States)

    Wurmehl, Sabine; Kohlhepp, Jürgen T.; Swagten, Henk J. M.; Koopmans, Bert

    2012-02-01

    In this work, the spin-echo nuclear magnetic resonance (NMR) technique is used to probe the local structure of Co2FeAl0.5Si0.5 bulk samples. The 59Co NMR spectrum of the Heusler compound Co2FeAl0.5Si0.5 consists of four main resonance lines with an underlying sub-structure. The splitting into the main resonance lines is explained by contributions of the B2 type structure. The sub-lines are attributed to a random distribution of Al and Si. By comparing the experimental results with an appropriate multinomial distribution, the fraction of the Al/Si intermixing and the ratio between the contributing structure types is assigned. The main structural contribution of as-cast bulk samples is of B2 type with 38% of L21 contributions. The L21 contribution can be enhanced to 59% by an appropriate annealing process. However, B2 contributions are still present after annealing. Additional foreign phases such as fcc-Co and Co-Al, with relative contributions of less than one percent, are also found in both as-cast and annealed samples. Resonance lines related to slight amounts of the ternary, parental Heusler compounds Co2FeAl and Co2FeSi are also observed.

  11. Geological disposal of radioactive waste: national commitment, local and regional involvement - A Collective Statement of the OECD Nuclear Energy Agency Radioactive Waste Management Committee Adopted March 2012

    International Nuclear Information System (INIS)

    Disposal in engineered facilities built in stable, deep geological formations is the reference solution for permanently isolating long-lived radioactive waste from the human biosphere. This management method is designed to be intrinsically safe and final, meaning that it is not dependent on human presence or intervention in order to fulfil its safety goal. Selecting the site of a waste repository brings up a range of issues involving scientific knowledge, technical capacity, ethical values, territorial planning, community well-being and more. Bringing to fruition the multi-decade task of siting and developing a repository demands a strong national commitment and significant regional and local involvement. This collective statement by the Radioactive Waste Management Committee of the OECD Nuclear Energy Agency recognises the advances made towards greater transparency and dialogue among the diverse stakeholders concerned and identifies the fundamental elements needed to support national commitment and to foster territorial involvement. It concludes that technical and societal partners can develop shared confidence in the safety of geological repositories and jointly carry these projects forward

  12. Human nuclear NAD+ ADP-ribosyltransferase: Localization of the gene on chromosome 1q41-q42 and expression of an active human enzyme in Escherichia coli

    International Nuclear Information System (INIS)

    The gene for human nuclear NAD+ ADP-ribosyltransferase was localized to chromosome 1 at q41-q42 by in situ hybridization with a pADPRT-specific cDNA probe. Expression of a pAD-PRT cDNA under control of the lac promoter in Escherichia coli induces the synthesis of a group of related proteins that were immunoreactive with pADPRT antibody and that had catalytic properties very similar to those of the human enzyme. Purification of this enzymatic activity was performed essentially as described for the human enzyme. The Km, pH optimum, optimal reaction temperature, and inhibition by 3-aminobenzamide and 3-methoxybenzamide were found to be similar for the recombinant and the human enzymes. The purified recombinant enzyme consists of two major proteins of Mr 99,000 and Mr 89,000. Both proteins show pADPRT activity in activity gel analysis with [32P]NAD+ as substrate. Microsequencing of these two proteins isolated by denaturing gel electrophoresis and deletion mutagenesis of the pADPRT expression plasmid shows that the Mr 99,000 and Mr 89,000 proteins derive from initiation of translation at interval translational start signals located within the pADPRT cDNA

  13. Expression Analysis and Nuclear Import Study of Full-length Isoforms Importin α as 6x Histidin-tagged Fusion Protein on the Intracellular Localization of Recombinant HBV Core Protein

    Directory of Open Access Journals (Sweden)

    Aris Haryanto

    2015-10-01

    Full Text Available Isoform importin α molecules play a central role in the classical nuclear import pathway, that occurs throughthe nuclear pore complex (NPC and typically requires a specific nuclear localization signal (NLS. In this study,it was investigated the role of isoforms importin α in the nuclear import of wild type recombinant hepatitis B viruscore protein (WT rHBc, phosphorylated recombinant HBV core (rHBc and recombinant HBV core without NLSby co-immunoprecipitation. Four recombinant full-length isoforms importin α as 6x histidin-tagged fusion proteinwere expressed and analysed from expression plasmid vectors Rch1, pHM 1969, pHM 1967 and pHM 1965. Theresults indicated that importin α-1, importin α-3, importin α-4 and importin α-5 can be expressed and isolatedfrom E. coli transformed recombinant DNA plasmid as protein in size around 58-60 kDa. By the nuclear transportstudy shown that isoforms importin α are involved in the nuclear import of WT rHBc, phosphorylated rHBc andrHBc without NLS. It also indicated that they have an important role for nuclear transport of from cytoplasm intothe nucleus.Keywords: NPC, NLS, importin α, importin β, isoforms importin α as 6x histidin-tagged fusion protein, WTrHBc, SV40 Tag, co-immunoprecipitation, westernblotting.

  14. Virus-like particles of porcine bocavirus generated by recombinant baculoviruses can be applied to sero-epidemic studies.

    Science.gov (United States)

    Zhang, Wenjing; Sano, Natsuha; Kataoka, Michiyo; Ami, Yasushi; Suzaki, Yuriko; Wakita, Takaji; Ikeda, Hidetoshi; Li, Tian-Cheng

    2016-06-01

    Porcine bocaviruses (PBoVs), new members of the Bocavirus genus, have been identified in swine worldwide. However, the antigenicity and epidemiology of PBoVs are still unclear. Here we used a recombinant baculovirus expression system to express the main capsid protein VP2 of Japan strain JY31b in insect Tn5 cells, and successfully produced the virus-like particles of PBoV (PBoV-LPs). The diameter and densities of the PBoV-LPs were estimated to be 30nm and 1.300g/cm(3), respectively, which were similar to the values for the native virion of PBoV. Antigenic analysis demonstrated that the PBoV-LPs were not cross-reactive with porcine circovirus 2, but were cross-reactive with human bocavirus 1, 2, 3 and 4. An ELISA for detection of anti-PBoV IgG antibodies was established using PBoV-LPs as antigen, which proved to be useful for monitoring PBoV infection in both swine and wild boars. The preliminary epidemiology research showed that 90.7% of pigs and 59.5% of wild boars were positive for the anti-PBoV-IgG, suggesting that both species were also widely infected with PBoV. The seven PBoV strains detected in wild boars separated into four subgroups, demonstrating the genetic diversity of PBoV. PMID:26959654

  15. Development of an ELISA based on the baculovirus-expressed capsid protein of porcine circovirus type 2 as antigen.

    Science.gov (United States)

    Liu, Changming; Ihara, Takeshi; Nunoya, Tetsuo; Ueda, Susumu

    2004-03-01

    The genome of porcine circovirus type 2 (PCV2) contains two major open reading frames, which have been shown to encode the virus capsid and replication-associated proteins. The capsid protein is a major structural protein of the virus; it can be a suitable target antigen for detecting PCV2-specific antibodies to monitor PCV2 infection. To produce the antigen, the capsid protein coding sequence was cloned into a baculovirus transfer vector, and a recombinant capsid (rC) protein of PCV2 was expressed as a combined fusion protein in frame with a C-terminal peptide of six histidines. The affinity-purified rC protein was used as coating antigen to develop an ELISA for detecting the virus-specific antibodies in swine sera. The rC protein-based ELISA (rcELISA) was evaluated by examining a panel of 49 PCV2-positive and 49 PCV2-negative swine sera. In comparative experiments of immunoperoxidase monolayer assay (IPMA) using 102 field sera, there was 89.2% coincidence between data obtained by the rcELISA and IPMA. The rcELISA achieved 88.5% specificity and 89.4% sensitivity for detection of PCV2 antibody in the field sera. The assay showed no cross-reactivity with antibodies to PCV type 1, porcine reproductive and respiratory syndrome virus and porcine parvovirus. The results suggest that the rcELISA is suitable for routine serodiagnosis and epidemiological surveys of PCV2-associated diseases. PMID:15107550

  16. Pathogen persistence in the environment and insect-baculovirus interactions: disease-density thresholds, epidemic burnout, and insect outbreaks.

    Science.gov (United States)

    Fuller, Emma; Elderd, Bret D; Dwyer, Greg

    2012-03-01

    Classical epidemic theory focuses on directly transmitted pathogens, but many pathogens are instead transmitted when hosts encounter infectious particles. Theory has shown that for such diseases pathogen persistence time in the environment can strongly affect disease dynamics, but estimates of persistence time, and consequently tests of the theory, are extremely rare. We consider the consequences of persistence time for the dynamics of the gypsy moth baculovirus, a pathogen transmitted when larvae consume foliage contaminated with particles released from infectious cadavers. Using field-transmission experiments, we are able to estimate persistence time under natural conditions, and inserting our estimates into a standard epidemic model suggests that epidemics are often terminated by a combination of pupation and burnout rather than by burnout alone, as predicted by theory. Extending our models to allow for multiple generations, and including environmental transmission over the winter, suggests that the virus may survive over the long term even in the absence of complex persistence mechanisms, such as environmental reservoirs or covert infections. Our work suggests that estimates of persistence times can lead to a deeper understanding of environmentally transmitted pathogens and illustrates the usefulness of experiments that are closely tied to mathematical models. PMID:22322229

  17. Expression and enzyme activity determination of human cyclooxygenase-1 and -2 in a baculovirus-insect cell system

    Institute of Scientific and Technical Information of China (English)

    Wei-yu ZHANG; Xin-ning YANG; Dao-zhong JIN; Xing-zu ZHU

    2004-01-01

    AIM: To develop an in vitro intact cell-based assay for screening selective cyclooxygenase inhibitors. METHODS:Human cyclooxygenase-1 (hCOX-1) and cyclooxygenase-2 (hCOX-2) genes were cloned from human monocyte cell line THP-1 cells and expressed in Spodopterafrugiperda (sf9) insect cell line by Bac-to-Bac baculovirus expression systems. Infected sr9 cells were harvested 24 h post-infection (hpi), and distributed to a 24-well plate,preincubated with various nonsteroidal anti-inflammatory drugs, and challenged with 10 mmol/L arachidonic acid;the cyclooxygenase activity was assessed indirectly by prostaglandin E2-specific radioimmunoassay. RESULTS:Polymerase chain reaction detection demonstrated that hCOX-1 and hCOX-2 were transposed to the bacmid.Western blot analysis showed that infected sf9 cells could express hCOX-1 and hCOX-2 proteins. Radioimmunoassay demonstrated that both recombinant proteins functioned well in sf9 cells. CONCLUSION: Human cyclooxygenase-1 and cyclooxygenase-2 were successfully expressed in sf9 insect cell line. It can be utilized for the identification of potent and selective inhibitors of hCOX- 1 and/or hCOX-2.

  18. Studies on Immunogenicity and Antigenicity of Baculovirus-Expressed Binding Region of Plasmodium falciparum EBA-140 Merozoite Ligand.

    Science.gov (United States)

    Zerka, Agata; Rydzak, Joanna; Lass, Anna; Szostakowska, Beata; Nahorski, Wacław; Wroczyńska, Agnieszka; Myjak, Przemyslaw; Krotkiewski, Hubert; Jaskiewicz, Ewa

    2016-04-01

    The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum erythrocyte binding antigens (EBA) family, which are considered as prospective candidates for malaria vaccine development. EBA proteins were identified as important targets for naturally acquired inhibitory antibodies. Natural antibody response against EBA-140 ligand was found in individuals living in malaria-endemic areas. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They both share homology of domain structure, including the binding region (Region II), which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte receptor interaction during merozoite invasion. It was shown that the erythrocyte receptor for EBA-140 ligand is glycophorin C-a minor human erythrocyte sialoglycoprotein. In studies on the immunogenicity of P. falciparum EBA ligands, the recombinant proteins are of great importance. In this report, we have demonstrated that the recombinant baculovirus-obtained EBA-140 Region II is immunogenic and antigenic. It can raise specific antibodies in rabbits, and it is recognized by natural antibodies present in sera of patients with malaria, and thus, it may be considered for inclusion in multicomponent blood-stage vaccines. PMID:26439848

  19. Functional exchangeability of the nuclear localization signal (NLS of capsid protein between PCV1 and PCV2 in vitro: Implications for the role of NLS in viral replication

    Directory of Open Access Journals (Sweden)

    He Yongqiang

    2011-07-01

    Full Text Available Abstract Background Porcine circovirus type 2 (PCV2 is believed to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS. It is supposed that capsid protein of PCV may contribute to replication control via interaction between Cap and Rep in the nucleoplasm. In this study, we described the construction and in vitro characterization of NLS-exchanged PCV DNA clones based on a PMWS-associated PCV2b isolate from China to determine the role of ORF2 NLS in PCV replication. Results The PCV1, PCV2, PCV2-NLS1 and PCV1-NLS2 DNA clone were generated by ligating a copy of respective genome in tandem with a partial duplication. The PCV2-NLS1 and PCV1-NLS2 DNA clone contained a chimeric genome in which the ORF2 NLS was exchanged. The four DNA clones were all confirmed to be infectious in vitro when transfected into PK-15 cells, as PCV capsid protein were expressed in approximately 10-20% of the transfected cells. The in vitro growth characteristics of the DNA clones were then determined and compared. All the recovered progeny viruses gave rise to increasing infectious titers during passages and were genetically stable by genomic sequencing. The chimeric PCV1-NLS2 and PCV2-NLS1 viruses had the final titers of about 104.2 and 103.8 TCID50/ml, which were significantly lower than that of PCV1 and PCV2 (105.6 and 105.0 TCID50/ml, respectively. When the ORF2 NLS exchanged, the mutant PCV2 (PCV2-NLS1 still replicated less efficiently and showed lower infectious titer than did PCV1 mutant (PCV1-NLS2, which was consistent with the distinction between wild type PCV1 and PCV2. Conclusions Recovery of the chimeiric PCV1-NLS2 and PCV2-NLS1 progeny viruses indicate that the nuclear localization signal sequence of capsid protein are functionally exchangeable between PCV1 and PCV2 with respect to the role of nuclear importing and propagation. The findings also reveal that ORF2 NLS play an accessory role in the replication of PCV. However, we found

  20. Troponin T3 regulates nuclear localization of the calcium channel Ca{sub v}β{sub 1a} subunit in skeletal muscle

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Tan; Taylor, Jackson; Jiang, Yang [Department of Internal Medicine-Gerontology, Wake Forest School of Medicine, Winston-Salem, NC 27157 (United States); Pereyra, Andrea S. [Department of Histology, National University of La Plata, 1900 La Plata (Argentina); Messi, Maria Laura; Wang, Zhong-Min [Department of Internal Medicine-Gerontology, Wake Forest School of Medicine, Winston-Salem, NC 27157 (United States); Hereñú, Claudia [Department of Histology, National University of La Plata, 1900 La Plata (Argentina); Delbono, Osvaldo, E-mail: odelbono@wakehealth.edu [Department of Internal Medicine-Gerontology, Wake Forest School of Medicine, Winston-Salem, NC 27157 (United States); Neuroscience Program, Wake Forest School of Medicine, Winston-Salem, NC 27157 (United States)

    2015-08-15

    The voltage-gated calcium channel (Ca{sub v}) β{sub 1a} subunit (Ca{sub v}β{sub 1a}) plays an important role in excitation–contraction coupling (ECC), a process in the myoplasm that leads to muscle-force generation. Recently, we discovered that the Ca{sub v}β{sub 1a} subunit travels to the nucleus of skeletal muscle cells where it helps to regulate gene transcription. To determine how it travels to the nucleus, we performed a yeast two-hybrid screening of the mouse fast skeletal muscle cDNA library and identified an interaction with troponin T3 (TnT3), which we subsequently confirmed by co-immunoprecipitation and co-localization assays in mouse skeletal muscle in vivo and in cultured C2C12 muscle cells. Interacting domains were mapped to the leucine zipper domain in TnT3 COOH-terminus (160–244 aa) and Ca{sub v}β{sub 1a} NH{sub 2}-terminus (1–99 aa), respectively. The double fluorescence assay in C2C12 cells co-expressing TnT3/DsRed and Ca{sub v}β{sub 1a}/YFP shows that TnT3 facilitates Ca{sub v}β{sub 1a} nuclear recruitment, suggesting that the two proteins play a heretofore unknown role during early muscle differentiation in addition to their classical role in ECC regulation. - Highlights: • Previously, we demonstrated that Ca{sub v}β{sub 1a} is a gene transcription regulator. • Here, we show that TnT3 interacts with Ca{sub v}β{sub 1a}. • We mapped TnT3 and Ca{sub v}β{sub 1a} interaction domain. • TnT3 facilitates Ca{sub v}β{sub 1a} nuclear enrichment. • The two proteins play a heretofore unknown role during early muscle differentiation.

  1. Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector.

    OpenAIRE

    Smith, G.E.; Ju, G; Ericson, B L; Moschera, J; Lahm, H W; Chizzonite, R; Summers, M D

    1985-01-01

    A cDNA coding for human interleukin 2 (IL-2) was inserted into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the polyhedrin promoter. Cells infected with recombinant virus produced high levels of Mr 15,500 IL-2 polypeptide, the majority of which was secreted into the culture medium during infection. The recombinant IL-2 was able to stimulate the growth of an IL-2-dependent cell line. The N-terminal amino acid sequence of the insect-derived IL-2 was identical to t...

  2. Residues R{sup 199}H{sup 200} of prototype foamy virus transactivator Bel1 contribute to its binding with LTR and IP promoters but not its nuclear localization

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Qinglin; Tan, Juan [Key Laboratory of Molecular Microbiology and Biotechnology (Ministry of Education) and Key Laboratory of Microbial Functional Genomics (Tianjin), College of Life Sciences, Nankai University, Tianjin 300071 (China); Cui, Xiaoxu [Key Laboratory of Molecular Microbiology and Biotechnology (Ministry of Education) and Key Laboratory of Microbial Functional Genomics (Tianjin), College of Life Sciences, Nankai University, Tianjin 300071 (China); Centre Laboratory, TianJin 4th Centre Hospital, Tianjin 300140 (China); Luo, Di; Yu, Miao [Key Laboratory of Molecular Microbiology and Biotechnology (Ministry of Education) and Key Laboratory of Microbial Functional Genomics (Tianjin), College of Life Sciences, Nankai University, Tianjin 300071 (China); Liang, Chen [Lady Davis Institute, Jewish General Hospital, Montreal, QC, Canada H3T 1E2 (Canada); Departments of Medicine McGill University, Montreal, QC (Canada); Microbiology and Immunology, McGill University, Montreal, QC (Canada); Qiao, Wentao, E-mail: wentaoqiao@nankai.edu.cn [Key Laboratory of Molecular Microbiology and Biotechnology (Ministry of Education) and Key Laboratory of Microbial Functional Genomics (Tianjin), College of Life Sciences, Nankai University, Tianjin 300071 (China)

    2014-01-20

    Prototype foamy virus encodes a transactivator called Bel1 that enhances viral gene transcription and is essential for PFV replication. Nuclear localization of Bel1 has been reported to rely on two proximal basic motifs R{sup 199}H{sup 200} and R{sup 221}R{sup 222}R{sup 223} that likely function together as a bipartite nuclear localization signal. In this study, we report that mutating R{sup 221}R{sup 222}R{sup 223}, but not R{sup 199}H{sup 200}, relocates Bel1 from the nucleus to the cytoplasm, suggesting an essential role for R{sup 221}R{sup 222}R{sup 223} in the nuclear localization of Bel1. Although not affecting the nuclear localization of Bel1, mutating R{sup 199}H{sup 200} disables Bel1 from transactivating PFV promoters. Results of EMSA reveal that the R{sup 199}H{sup 200} residues are vital for the binding of Bel1 to viral promoter DNA. Moreover, mutating R{sup 199}H{sup 200} in Bel1 impairs PFV replication to a much greater extent than mutating R{sup 221}R{sup 222}R{sup 223}. Collectively, our findings suggest that R{sup 199}H{sup 200} directly participate in Bel1 binding to viral promoter DNA and are indispensible for Bel1 transactivation activity. - Highlights: • The R{sup 221}R{sup 222}R{sup 223} residues are essential for the nuclear localization of Bel1. • Although not affecting the nuclear localization of Bel1, mutating R{sup 199}H{sup 200} disables Bel1 from transactivating PFV promoters. • The R{sup 199}H{sup 200} residues directly participate in Bel1 binding to viral promoter DNA. • Mutating R{sup 199}H{sup 200} in Bel1 impairs PFV replication to a much greater extent than mutating R{sup 221}R{sup 222}R{sup 223}.

  3. Taperin (c9orf75, a mutated gene in nonsyndromic deafness, encodes a vertebrate specific, nuclear localized protein phosphatase one alpha (PP1α docking protein

    Directory of Open Access Journals (Sweden)

    Tony Ferrar

    2012-02-01

    The promiscuous activity of protein phosphatase one (PP1 is controlled in the cell by associated proteins termed regulatory or targeting subunits. Using biochemical and proteomic approaches we demonstrate that the autosomal recessive nonsyndromic hearing loss gene, taperin (C9orf75, encodes a protein that preferentially docks the alpha isoform of PP1. Taperin associates with PP1 through a classic ‘RVxF’ motif and suppresses the general phosphatase activity of the enzyme. The steady-state localization of taperin is predominantly nuclear, however we demonstrate here that the protein can shuttle between the nucleus and cytoplasm and that it is found complexed to PP1 in both of these cellular compartments. Although originally identified as a hearing loss gene, Western blot analyses with taperin-specific antibodies revealed that the protein is widely expressed across mammalian tissues as multiple splice variants. Taperin is a recent proteome addition appearing during the vertebrate lineage with the PP1 binding site embedded within the most conserved region of the protein. Taperin also shares an ancestral relationship with the cytosolic actin binding protein phostensin, another PP1 interacting partner. Quantitative Stable Isotope Labeling by Amino acids in Culture (SILAC-based mass spectrometry was employed to uncover additional taperin binding partners, and revealed an interaction with the DNA damage response proteins Ku70, Ku80, PARP and topoisomerases I and IIα. Consistent with this, we demonstrate the active recruitment of taperin to sites of DNA damage. This makes taperin a new addition to the family of PP1 targeting subunits involved in the DNA damage repair pathway.

  4. A genome-wide integrative genomic study localizes genetic factors influencing antibodies against Epstein-Barr virus nuclear antigen 1 (EBNA-1.

    Directory of Open Access Journals (Sweden)

    Rohina Rubicz

    Full Text Available Infection with Epstein-Barr virus (EBV is highly prevalent worldwide, and it has been associated with infectious mononucleosis and severe diseases including Burkitt lymphoma, Hodgkin lymphoma, nasopharyngeal lymphoma, and lymphoproliferative disorders. Although EBV has been the focus of extensive research, much still remains unknown concerning what makes some individuals more sensitive to infection and to adverse outcomes as a result of infection. Here we use an integrative genomics approach in order to localize genetic factors influencing levels of Epstein Barr virus (EBV nuclear antigen-1 (EBNA-1 IgG antibodies, as a measure of history of infection with this pathogen, in large Mexican American families. Genome-wide evidence of both significant linkage and association was obtained on chromosome 6 in the human leukocyte antigen (HLA region and replicated in an independent Mexican American sample of large families (minimum p-value in combined analysis of both datasets is 1.4×10(-15 for SNPs rs477515 and rs2516049. Conditional association analyses indicate the presence of at least two separate loci within MHC class II, and along with lymphocyte expression data suggest genes HLA-DRB1 and HLA-DQB1 as the best candidates. The association signals are specific to EBV and are not found with IgG antibodies to 12 other pathogens examined, and therefore do not simply reveal a general HLA effect. We investigated whether SNPs significantly associated with diseases in which EBV is known or suspected to play a role (namely nasopharyngeal lymphoma, Hodgkin lymphoma, systemic lupus erythematosus, and multiple sclerosis also show evidence of associated with EBNA-1 antibody levels, finding an overlap only for the HLA locus, but none elsewhere in the genome. The significance of this work is that a major locus related to EBV infection has been identified, which may ultimately reveal the underlying mechanisms by which the immune system regulates infection with this

  5. Effects of long- and short-term passage of insect cells in different culture media on baculovirus replication.

    Science.gov (United States)

    Lynn, D E

    2000-10-01

    Two insect cell lines that had been maintained in both serum-free (SFM) and serum-containing (SCM) media for over 5 years were each tested for their ability to replicate baculovirus. The gypsy moth cell line, IPLB-LdEIta (Ld), produced similar (not statistically different) amounts of gypsy moth nucleopolyhedrovirus (LdMNPV) occlusion bodies (OBs) in the two media (serum-free Ex-Cell 400 and TC-100 with 9% (v/v) fetal bovine serum, SCM(1)) but produced more of the Autographa californica nucleopolyhedrovirus (AcMNPV) OBs in SFM than in SCM(1). When Ld cells normally grown in SCM(1) were switched to SFM, production of OBs from both viruses improved and, after three passages, reached higher levels of AcMNPV production than in cells normally maintained in that medium. Alternatively, cells switched from SFM to SCM(1) initially produced as much (in the case of LdMNPV) or higher (in the case of AcMNPV) levels of virus OBs than cells normally maintained in SCM(1) but productivity dropped off over subsequent passages such that after five passages in SCM(1), cells produced substantially fewer OBs of both viruses. A fall armyworm cell line (IPLB-SF21AE; Sf) showed slightly different effects from long- and short-term passage in SFM (Ex-Cell 400) or SCM(2) (TMN-FH). Cells maintained in SFM produced about 20 times more AcMNPV OBs than cells maintained long-term in SCM. Sf cells switched from SFM to SCM maintained the level of production of that seen in SFM at the first passage, but quickly dropped off OB production levels to that normally seen in SCM. Alternatively, SCM-maintained Sf cells produced higher levels at the first passage in SFM and, within five passages in SFM, reached levels found in cells maintained for long term in this medium. Under the conditions in which these two cell lines were infected, the highest levels of AcMNPV OB production in Ld cells were about five times that of Sf cells. In a separate series of experiments, cells normally grown in SFM were passaged

  6. Duplex real-time PCR for detection and quantification of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) in Penaeus monodon.

    Science.gov (United States)

    Tang, Kathy F J; Lightner, Donald V

    2011-02-22

    We describe a duplex real-time PCR assay using TaqMan probes for the simultaneous detection of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). Both MBV and HPV are shrimp enteric viruses that infect intestinal and hepatopancreatic epithelial cells. Both viruses can cause significant mortalities and depressed growth in infected larval, postlarval, and early juvenile stages of shrimp, and thus present a risk to commercial aquaculture. In this duplex assay, we combined 2 single real-time PCRs, amplifying MBV and HPV, in a one-tube PCR reaction. The 2 viruses were distinguished by specific fluorescent labels at the 5' end of TaqMan probes: the MBV probe was labeled with dichlorodimethoxyfluorescein (JOE), and the HPV probe was labeled with 6-carboxyfluorescein (FAM). The duplex real-time PCR assay was performed in a multi-channel real-time PCR detection system, and MBV and HPV amplification signals were separately detected by the JOE and FAM channels. This duplex assay was validated to be specific to the target viruses and found to have a detection limit of single copies for each virus. The dynamic range was found to be from 1 to 1 x 10(8) copies per reaction. This assay was further applied to quantify MBV and HPV in samples of infected Penaeus monodon collected from Malaysia, Indonesia, and Thailand. The specificity and sensitivity of this duplex real-time PCR assay offer a valuable tool for routine diagnosis and quantification of MBV and HPV from both wild and farmed shrimp stocks.

  7. High-Level Production of a Functional Recombinant Hepatitis B Virus Polymerase in Insect Cells with a Baculovirus Expression System

    Institute of Scientific and Technical Information of China (English)

    WANG Xiaoyan; GAO Linlin; DENG Fei; ZHANG Yanfang; LI Yan; LIN Jusheng

    2007-01-01

    HBV polymerase has intrinsic RNA-dependent reverse transcriptase, DNA-dependent DNA polymerase as well as RNaseH activity. Analysis of HBV polymerase has been hampered for many years due to the inability to express functional enzyme in a recombinant system. To obtain active polymerase at a high level, we have taken advantage of baculovirus expression system. The gene of HBV polymerase was amplified by PCR and cloned into pFastBac Dual to construct the recombinant plasmid pFastbac Dual-pol. The recombinant donor plasmid, pFastbac Dual-pol, was constructed by inserting HBV polymerase gene into EcoRI and PstI sites controlled by polyhedrin promoter. The recombinant donor plasmid was transformed into DH10Bac competent cells for transposition. Recombinant bacmid was constructed by inserting of the mini-Tn7 element from the donor plasmid into the mini-attTn7 attachment site on the bacmid. The recombinant bacmid DNA was isolated and transfected into the Sf9 cells to produce the recombinant virus, and healthy insect Sf9 cells were infected with the recombinant virus containing HBV polymerse gene to express the target protein. HBV polymerse expressed in insect cells was analyzed by SDS-PAGE. PCR results showed recombinant donor plasmid, pFastbac Dual-pol, was constructed successfully. The recombinant hepatitis B virus polymerase was expressed in insect cells at high level. The recombinant hepatitis B virus polymerase should facilitate the analysis of HBV polymerase biological characteristics, allow the investigation for new anti-HBV drugs specifically blocking HBV polymerase.

  8. Highly Efficient and Economical Baculovirus Expression System for Preparing Human Papillomavirus Type16 Virus-like Particle

    Institute of Scientific and Technical Information of China (English)

    Jin ZHENG; Jun MA; Xiao-Feng YANG; Hong-Li LIU; Hong-Wei CHENG; Lu-Sheng SI; Yi-Li WANG

    2004-01-01

    To improve the existing human papillomavirus type16(HPV16)virus-like particle(VLP)preparation,a highly efficient,economical and timesaving system was established.Sf-9 cells were infected with recombinant baculovirus containing the target gene encoding HPV16L1 protein with 6xHis tag,and harvested 72 h postinfection(p.i.)at 27 ℃.The ProBondTM purification system was used for protein purification.The molecular weight of expressed HPV16L1 protein was 58 kD as revealed by SDS-PAGE,and confirmed by Western blot.The purity of denatured and native HPVL 1 proteins that were prepared were 91.9% and 71.5%,respectively,which corresponded to a yield of 2.26 mg denatured protein and 1.84 mg native protein per 2 x 107 cells.The proteins were further analyzed by mouse erythrocyte hemagglutination assay and hemagglutination inhibition assay,and there effects on VLP formation were also visualized by transmission electron microscopy.Results showed that the native protein purified was biologically active as natural HPVL1 protein,inducing the murine erythrocyte agglutination and VLP formation.In addition,the purified recombinant HPV16L1 native protein with 6xHis tag could self-assemble into virions in vitro.Hopefully,the present expression and purification system is promising to be convenient,timesaving and economical for preparation ofHPV16 VLP vaccine.

  9. Nuclear medicine

    International Nuclear Information System (INIS)

    The task of the Expert Committee was to review the technical development and efficacy of nuclear medicine methods and to recommend the best possible means of establishing nuclear medicine services at various levels of medical care in different countries. After reviewing the contributions which nuclear medicine can make, the various types of medical institutions and hospitals in existence, the requirements, organization and funding of nuclear medicine services, and the cost/effectiveness of nuclear medicine, a number of recommendations were made. IAEA and WHO should make information on existing methods of cost/effectiveness analysis widely available; invite governments to include a description of such analysis methods in training programmes of their health officers; assist in the acquisition of the necessary data; and encourage and eventually support actual applications of such analyses to carefully selected nuclear medicine procedures in varying medicosocial environments. They were further recommended to study possible ways of improving reliability and ease of servicing nuclear medicine equipment, and extent of possible local construction; the possibility of making available supplies of matched characterized reagents for radioimmunoassay and related techniques; and to study the advantages of establishing a network of collaborating centres on an international basis

  10. Preparation of ChlL-2 and IBDV VP2 Fusion Protein by Baculovirus Expression System

    Institute of Scientific and Technical Information of China (English)

    Yan Liu; Yongwei Wei; Xiaofeng Wu; Lian Yu

    2005-01-01

    This study aims to produce an effective subunit vaccine against infectious bursal disease virus (IBDV). The genes of chicken interleukin-2 (ChIL-2) and IBDV viral protein 2 (VP2) were amplified and fused by splice overlap extension-polymerase chain reaction (SOE-PCR). The fusion gene was digested by EcoR I/Kpn I and inserted into pBacPAK8 vector, resulting in recombinant transfer plasmid pBacPakVP2-IL2. The recombinant plasmid was transfected into Sf-9 cells accompanied with hybrid nuclear polyhedrosis virus (HyNPV) genome DNA and lipofectin. Plaque-purification indicated that we had got the recombinant Hy-VP2-IL2. Fusion protein VP2-IL2was expressed effectively both in insect cells and bombyx mori. The expression of fusion protein was confirmed by ELISA, SDS-PAGE and Western blotting assay, respectively. This efficient system allows us to meet the need for inexpensive vaccines required by the poultry industry.

  11. A protein tyrosine phosphatase-like protein from baculovirus has RNA 5'-triphosphatase and diphosphatase activities.

    Science.gov (United States)

    Takagi, T; Taylor, G S; Kusakabe, T; Charbonneau, H; Buratowski, S

    1998-08-18

    The superfamily of protein tyrosine phosphatases (PTPs) includes at least one enzyme with an RNA substrate. We recently showed that the RNA triphosphatase domain of the Caenorhabditis elegans mRNA capping enzyme is related to the PTP enzyme family by sequence similarity and mechanism. The PTP most similar in sequence to the capping enzyme triphosphatase is BVP, a dual-specificity PTP encoded by the Autographa californica nuclear polyhedrosis virus. Although BVP previously has been shown to have modest tyrosine and serine/threonine phosphatase activity, we find that it is much more potent as an RNA 5'-phosphatase. BVP sequentially removes gamma and beta phosphates from the 5' end of triphosphate-terminated RNA, leaving a 5'-monophosphate end. The activity was specific for polynucleotides; nucleotide triphosphates were not hydrolyzed. A mutant protein in which the active site cysteine was replaced with serine was inactive. Three other dual-specificity PTPs (VH1, VHR, and Cdc14) did not exhibit detectable RNA phosphatase activity. Therefore, capping enzyme and BVP are members of a distinct PTP-like subfamily that can remove phosphates from RNA. PMID:9707557

  12. Yield and depth Estimation of Selected NTS Nuclear and SPE Chemical Explosions Using Source Equalization by modeling Local and Regional Seismograms (Invited)

    Science.gov (United States)

    Saikia, C. K.; Roman-nieves, J. I.; Woods, M. T.

    2013-12-01

    Source parameters of nuclear and chemical explosions are often estimated by matching either the corner frequency and spectral level of a single event or the spectral ratio when spectra from two events are available with known source parameters for one. In this study, we propose an alternative method in which waveforms from two or more events can be simultaneously equalized by setting the differential of the processed seismograms at one station from any two individual events to zero. The method involves convolving the equivalent Mueller-Murphy displacement source time function (MMDSTF) of one event with the seismogram of the second event and vice-versa, and then computing their difference seismogram. MMDSTF is computed at the elastic radius including both near and far-field terms. For this method to yield accurate source parameters, an inherent assumption is that green's functions for the any paired events from the source to a receiver are same. In the frequency limit of the seismic data, this is a reasonable assumption and is concluded based on the comparison of green's functions computed for flat-earth models at various source depths ranging from 100m to 1Km. Frequency domain analysis of the initial P wave is, however, sensitive to the depth phase interaction, and if tracked meticulously can help estimating the event depth. We applied this method to the local waveforms recorded from the three SPE shots and precisely determined their yields. These high-frequency seismograms exhibit significant lateral path effects in spectrogram analysis and 3D numerical computations, but the source equalization technique is independent of any variation as long as their instrument characteristics are well preserved. We are currently estimating the uncertainty in the derived source parameters assuming the yields of the SPE shots as unknown. We also collected regional waveforms from 95 NTS explosions at regional stations ALQ, ANMO, CMB, COR, JAS LON, PAS, PFO and RSSD. We are

  13. Emission of {sup 14}C by the Almirante Alvaro Alberto Nuclear Power Plant 1 and 2 and their local effects on the environmental levels; Emissao de {sup 14}C pelas unidades 1 e 2 da Central Nuclear Almirante Alvaro Alberto (CNAAA) e seu efeito local nos niveis ambientais

    Energy Technology Data Exchange (ETDEWEB)

    Dias, Cintia Melazo

    2006-07-01

    {sup 14}C is a is a long-lived beta-emitting nuclide (T{sub 1/2} = 5730 years) produced naturally in the upper atmosphere as a result of reactions between neutrons and stable {sup 14}N({sup 14}N(n,p){sup 14}C). Although in a lesser extent, nuclear power plants produce {sup 14}C as well during their routine operation. Since it is converted in {sup 14}CO{sub 2} and mixed throughout the atmosphere, it is incorporated into plant tissues, via photosynthesis process, and hence in food chain. Because of the biological importance of {sup 14}C and long half-life, it is of interest to quantify the amounts released by nuclear industry. The Brazilian nuclear central named Nuclear Central Admiral Alvaro Alberto (CNAAA) has two nuclear reactors of PWR type in operation, Angra I (657 MWe) and Angra II (1350 MWe), and one under construction, Angra III (1309 MWe PWR). The aim of this study was to determine the strength of the sources and the {sup 14}C content in the environment through analyses of air, vegetation and soils taken within 5 km (the influenced area) of CNAAA. The thesis consists of an extensive review about the subject (part one) and of four papers (part two). The first paper is about the determination of {sup 14}C concentrations released by reactors (source strength). For Angra I, a device was developed in order to sample the gaseous effluents and for Angra II, a commercial monitoring system had already been implemented since its initial operation (2001). The {sup 14}C can be emitted as hydrocarbons, CO or CO{sub 2}, depending on the type of reactor. For PWRs, the main chemical form released is hydrocarbons (80 %). The monitoring system of Angra I was planned to determine both CO{sub 2} and hydrocarbon fractions but in Angra II, all hydrocarbons are converted to CO{sub 2} by using a Pd/Al{sub 2}O{sub 3} catalyst at 450 deg C. The liquid scintillation was the method employed to measure the samples. The second one concerns the atmospheric dispersion of the released

  14. Conserved Structural Motifs at the C-Terminus of Baculovirus Protein IE0 are Important for its Functions in Transactivation and Supporting hr5-mediated DNA Replication

    Directory of Open Access Journals (Sweden)

    Neta Luria

    2012-05-01

    Full Text Available IE0 and IE1 are transactivator proteins of the most studied baculovirus, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV. IE0 is a 72.6 kDa protein identical to IE1 with the exception of its 54 N-terminal amino acid residues. To gain some insight about important structural motifs of IE0, we expressed the protein and C‑terminal mutants of it under the control of the Drosophila heat shock promoter and studied the transactivation and replication functions of the transiently expressed proteins. IE0 was able to promote replication of a plasmid bearing the hr5 origin of replication of AcMNPV in transient transfections with a battery of eight plasmids expressing the AcMNPV genes dnapol, helicase, lef-1, lef-2, lef-3, p35, ie-2 and lef-7. IE0 transactivated expression of the baculovirus 39K promoter. Both functions of replication and transactivation were lost after introduction of selected mutations at the basic domain II and helix-loop-helix conserved structural motifs in the C-terminus of the protein. These IE0 mutants were unable to translocate to the cell nucleus. Our results point out the important role of some structural conserved motifs to the proper functioning of IE0.

  15. Enhanced expression of full-length human cytomegalovirus fusion protein in non-swelling baculovirus-infected cells with a minimal fed-batch strategy.

    Directory of Open Access Journals (Sweden)

    Marco Patrone

    Full Text Available Human cytomegalovirus congenital infection represents an unmet medical issue and attempts are ongoing to develop an effective vaccine. The virion fusion players of this enveloped virus are the natural targets to achieve this goal and to develop novel anti-viral therapies. The secreted ectodomain of the viral fusion factor glycoprotein B (gB has been exploited so far as an alternative to the cumbersome expression of the wild type trans-membrane protein. In the soluble form, gB showed encouraging but limited potential as antigen candidate calling for further efforts. Here, the exhaustive evaluation of the Baculovirus/insect cell expression system has been coupled to an orthogonal screening for expression additives to produce full-length gB. In detail, rapamycin was found to prolong gB intracellular accumulation while inhibiting the infection-induced cell swelling. Not obvious to predict, this inhibition did not affect Baculovirus growth, revealing that the virus-induced cell size increase is a dispensable side phenotype. In parallel, a feeding strategy for the limiting nutrient cysteine has been set up which improved gB stability. This multi-modal scheme allowed the production of full-length, mutation-free gB in the milligram scale. The recombinant full-length gB obtained was embedded into a stable mono-dispersed particle substantially larger than the protein trimer itself, according to the reported association of this protein with detergent-resistant lipid domains.

  16. Nuclear Sphingolipid Metabolism

    Science.gov (United States)

    Lucki, Natasha C.; Sewer, Marion B.

    2014-01-01

    Nuclear lipid metabolism is implicated in various processes, including transcription, splicing, and DNA repair. Sphingolipids play roles in numerous cellular functions, and an emerging body of literature has identified roles for these lipid mediators in distinct nuclear processes. Different sphingolipid species are localized in various subnuclear domains, including chromatin, the nuclear matrix, and the nuclear envelope, where sphingolipids exert specific regulatory and structural functions. Sphingomyelin, the most abundant nuclear sphingolipid, plays both structural and regulatory roles in chromatin assembly and dynamics in addition to being an integral component of the nuclear matrix. Sphingosine-1-phosphate modulates histone acetylation, sphingosine is a ligand for steroidogenic factor 1, and nuclear accumulation of ceramide has been implicated in apoptosis. Finally, nuclear membrane–associated ganglioside GM1 plays a pivotal role in Ca2+ homeostasis. This review highlights research on the factors that control nuclear sphingolipid metabolism and summarizes the roles of these lipids in various nuclear processes. PMID:21888508

  17. Argentine nuclear program

    International Nuclear Information System (INIS)

    The paper describes Argentina's nuclear program, detailing its objectives, the schedule of construction of nuclear plants and local production of required equipment. The technologies adopted so far, the local industrial and engineering participation, the preliminary study for the construction of the next power station and Argentina's nonproliferation nuclear policy are analyzed. Argentina's point of view on Canadian nonproliferation policy and CANDU reactor export is discussed

  18. Nitrogen starvation and TorC1 inhibition differentially affect nuclear localization of the Gln3 and Gat1 transcription factors through the rare glutamine tRNACUG in Saccharomyces cerevisiae.

    Science.gov (United States)

    Tate, Jennifer J; Rai, Rajendra; Cooper, Terrance G

    2015-02-01

    A leucine, leucyl-tRNA synthetase-dependent pathway activates TorC1 kinase and its downstream stimulation of protein synthesis, a major nitrogen consumer. We previously demonstrated, however, that control of Gln3, a transcription activator of catabolic genes whose products generate the nitrogenous precursors for protein synthesis, is not subject to leucine-dependent TorC1 activation. This led us to conclude that excess nitrogen-dependent down-regulation of Gln3 occurs via a second mechanism that is independent of leucine-dependent TorC1 activation. A major site of Gln3 and Gat1 (another GATA-binding transcription activator) control occurs at their access to the nucleus. In excess nitrogen, Gln3 and Gat1 are sequestered in the cytoplasm in a Ure2-dependent manner. They become nuclear and activate transcription when nitrogen becomes limiting. Long-term nitrogen starvation and treatment of cells with the glutamine synthetase inhibitor methionine sulfoximine (Msx) also elicit nuclear Gln3 localization. The sensitivity of Gln3 localization to glutamine and inhibition of glutamine synthesis prompted us to investigate the effects of a glutamine tRNA mutation (sup70-65) on nitrogen-responsive control of Gln3 and Gat1. We found that nuclear Gln3 localization elicited by short- and long-term nitrogen starvation; growth in a poor, derepressive medium; Msx or rapamycin treatment; or ure2Δ mutation is abolished in a sup70-65 mutant. However, nuclear Gat1 localization, which also exhibits a glutamine tRNACUG requirement for its response to short-term nitrogen starvation or growth in proline medium or a ure2Δ mutation, does not require tRNACUG for its response to rapamycin. Also, in contrast with Gln3, Gat1 localization does not respond to long-term nitrogen starvation. These observations demonstrate the existence of a specific nitrogen-responsive component participating in the control of Gln3 and Gat1 localization and their downstream production of nitrogenous precursors. This

  19. Nuclear waste

    International Nuclear Information System (INIS)

    This extract from the House of Commons Hansard publication for Wednesday 12th July 1995 considers the current debate on the desirability or otherwise of disposing of low level radioactive waste in landfill sites. It covers wastes generated both by the nuclear industry and by medical processes in local hospitals, and the transport of such waste from source to disposal site. The questions raised lead to a debate about plans to sell off commercially desirable aspects of the nuclear electric generation industry while leaving the costs associated with decommissioning of Magnox reactors as a liability on the public purse. (UK)

  20. Introduction of the anti-apoptotic baculovirus p35 gene in passion fruit induces herbicide tolerance, reduced bacterial lesions, but does not inhibits passion fruit woodiness disease progress induced by cowpea aphid-borne mosaic virus (CABMV

    NARCIS (Netherlands)

    Freitas, D.S.; Coelho, M.C.F.; Souza, M.T.; Marques, A.; Ribeiro, B.M.

    2007-01-01

    The introduction of anti-apoptotic genes into plants leads to resistance to environmental stress and broad-spectrum disease resistance. The anti-apoptotic gene (p35) from a baculovirus was introduced into the genome of passion fruit plants by biobalistics. Eleven regenerated plants showed the presen

  1. The C3H-type zinc finger protein GDS1/C3H42 is a nuclear-speckle-localized protein that is essential for normal growth and development in Arabidopsis.

    Science.gov (United States)

    Kim, Dae Won; Jeon, Su Jeong; Hwang, Sung Min; Hong, Jong Chan; Bahk, Jeong Dong

    2016-09-01

    Eukaryotic C3H-type zinc finger proteins (Znfs) comprise a large family of regulatory proteins involved in many aspects of plant stress response, growth and development. However, compared to mammalian, only a few plant Znfs have been functionally characterized. Here, T-DNA inserted gds1 (growth, development and splicing 1) mutant, displayed abnormal growth throughout the lifecycle owing to the reduction of cell size and number. Inverse PCR analysis revealed that the abnormal growth was caused by the disruption of At3g47120, which encodes a C3H42 protein belonging to the C-X7-C-X5-C-X3-H class of the Znf family. GDS1 was ubiquitously transcribed, but shows high levels of expression in young seedling and unexpanded new leaves. In gds1, the transcripts of many growth- and development-related genes were down-regulated, and the auxin response was dramatically reduced. A fluorescence-based assay revealed that the GDS1 protein was localized to the nucleus, prominently in the speckle compartments. Its arginine/serine dipeptide-rich-like (RS-like) domain was essential for nuclear localization. In addition, the SR1, SRm102 and U1-70K components of the U1 spliceosome interacted with GDS1 in the nuclear speckle compartments. Taken together, these suggest that GDS1, a nuclear-speckle-associated Znf, might play a significant role in splicing during plant growth and development. PMID:27457991

  2. Nuclear techniques used in the localization of fracture zone from granites of Itu-SP; Tecnicas nucleares usadas na localizacao de zonas de fraturas nos granitos de Itu-SP

    Energy Technology Data Exchange (ETDEWEB)

    Hiodo, Francisco Yukio; Porsani, Jorge Luis; Elis, Vagner Roberto, E-mail: francisc@iag.usp.br, E-mail: porsani@iag.usp.br, E-mail: vagnelis@iag.usp.br [Universidade de Sao Paulo (IAG/USP), SP (Brazil). Inst. de Astronomia, Geofisica e Ciencias Atmosfericas. Dept. de Geofisica

    2002-05-15

    Nowadays there are many geophysical techniques of rock fractures mapping as GPR, electromagnetic and dipole-dipole geoelectrical sounding. These methods don't give information about the content of fracture zone, that can be fulfilled of clay. Using nuclear methods as gamma spectrometry and radon emanometry become possible to identify the nature of mineral composition of fracture material and to study the freshwater circulation using the radon transport by water. In this work the integration of nuclear, GPR and geoelectric techniques in Itu granitic intrusive became very interesting because it was possible to find and identify fracture in granitic rock with clay and freshwater content. (author)

  3. Feasibility of a novel positive feedback effect of {sup 131}I-promoted Bac-Egr1-hNIS expression in malignant glioma via baculovirus

    Energy Technology Data Exchange (ETDEWEB)

    Guo Rui [Department of Nuclear Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025 (China); Tian Lipeng [Department of Neurology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025 (China); Han Bing [Department of Endocrine, The 9th Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025 (China); Xu Haoping; Zhang Miao [Department of Nuclear Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025 (China); Li Biao, E-mail: lb10363@rjh.com.c [Department of Nuclear Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025 (China)

    2011-05-15

    Purpose: As intracellular iodine is released rapidly, increased expression of sodium/iodide symporter (NIS) is required for effective radioiodine treatment of tumor. As Egr1 promoter is activated by {sup 131}I and may promote human NIS (hNIS) expression, hNIS also induces {sup 131}I uptake and activates Egr1, so the existence of a positive feedback effect of {sup 131}I-promoted Egr1-hNIS expression is possible. Our purpose was to investigate the possible existence of this positive feedback effect through a series of in vitro pioneer studies. Method: Recombinant baculovirus (Bac-Egr1-hNIS) encoding the hNIS gene under the control of a radiation-inducible Egrl promoter was constructed. To test {sup 131}I-promoted hNIS expression, human malignant glioma U87 cells were transfected with Bac-Egr1-hNIS, stimulated with or without {sup 131}I; the expression of hNIS protein was detected by immunofluorescence and flow cytometry test. In addition, the uptake and efflux of {sup 131}I were determined after the incubation of Bac-Egr1-hNIS-transfected U87 cells with or without {sup 131}I. Results: Immunocytochemical staining and flow cytometry test showed a higher hNIS protein expression in Bac-Egr1-hNIS-transfected U87 cells with {sup 131}I stimulation than in cells without stimulation. Bac-Egr1-hNIS-transfected U87 cells accumulated up to about 4.05 times of {sup 131}I after {sup 131}I stimulation. The amount of {sup 131}I uptake in both groups showed a baculovirus dose-dependent manner. However, rapid efflux of radioactivity was observed in both groups, with 50% lost during the first 2 min after the {sup 131}I-containing medium had been replaced by a nonradioactive medium. Conclusion: Our results indicated that an improved transgene expression of {sup 131}I-stimulated hNIS in U87 cells using a baculovirus vector containing the Egr1 promoter is possible, and the increased expression of hNIS is responsible for a higher {sup 131}I uptake. It might provide a reference for the

  4. Chinese nuclear insurance and Chinese nuclear insurance pool

    International Nuclear Information System (INIS)

    Chinese Nuclear Insurance Started with Daya Bay Nuclear Power Station, PICC issued the insurance policy. Nuclear insurance cooperation between Chinese and international pool's organizations was set up in 1989. In 1996, the Chinese Nuclear Insurance Pool was prepared. The Chinese Nuclear Insurance Pool was approved by The Chinese Insurance Regulatory Committee in May of 1999. The principal aim is to centralize maximum the insurance capacity for nuclear insurance from local individual insurers and to strengthen the reinsurance relations with international insurance pools so as to provide the high quality insurance service for Chinese nuclear industry. The Member Company of Chinese Nuclear Pool and its roles are introduced in this article

  5. Nuclear Medicine Scans for Cancer

    Science.gov (United States)

    ... My ACS » Your Local Offices Close + - Text Size Nuclear Medicine Scans for Cancer Other names for these ... inflammation, or cancer. Use of monoclonal antibodies in nuclear scans A special type of antibody made in ...

  6. Development of a Polymerase Chain Reaction (PCR Assay for the Detection of Philippine Isolates of the Penaeus monodon-type Baculovirus (MBV

    Directory of Open Access Journals (Sweden)

    Christopher Marlowe A. Caipang

    2011-07-01

    Full Text Available Penaeus monodon-type baculovirus (MBV is a DNA virus that infects postlarvae and early juveniles of shrimp, Penaeus monodon. Several variants of this virus occur through nucleotide analysis of its genomic DNA. In the present study, a one-step PCR method was developed for the detection of the Philippine isolates of MBV by designing PCR primers on the least conserved region of the Philippine MBV. Using genomic DNA of MBV-infected shrimp postlarvae, the PCR assay amplified a 193-bp PCR product. Its sensitivity was comparable to the published PCR assays. The strain-specific primers did not cross-react with other DNA viruses including White Spot Syndrome Virus (WSSV, Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV and hepatopancreatic parvovirus (HPV. This PCR assay could be used for regular monitoring and surveillance of MBV in shrimp as well as tracing the movement of the Philippine MBV in shrimp farms in different geographic regions.

  7. Problems concerned with local erosion-corrosion of welded connections in the pipelines of a power unit at a nuclear power station

    Science.gov (United States)

    Tomarov, G. V.; Lovchev, V. N.; Gutsev, D. F.; Shipkov, A. A.; Golubeva, T. N.; Greblov, P. N.

    2012-08-01

    Results from a study aimed at analyzing the damageability and determining the mechanism due to which thinning occurs in the metal of welded connections in the feed water supply control assemblies used in power units of nuclear power stations equipped with RBMK-1000 reactors are presented together with the outcomes from calculation studies aimed at analyzing the effect of hydrodynamic characteristics on the kinetics characterizing the thinning of a welded joint and adjacent elements of a pipeline. Data from a calculation of the erosion-corrosion rate of welded connections in the divergent parts of assemblies controlling the supply of feed water in power units of nuclear power stations equipped with RBMK-1000 reactors are given.

  8. Nuclear Localization of the ERK MAP Kinase Mediated by Drosophila αPS2βPS Integrin and Importin-7

    OpenAIRE

    James, Brian P.; Bunch, Thomas A.; Krishnamoorthy, Srinivasan; Perkins, Lizabeth A.; Brower, Danny L.

    2007-01-01

    The control of gene expression by the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (ERK) requires its translocation into the nucleus. In Drosophila S2 cells nuclear accumulation of diphospho-ERK (dpERK) is greatly reduced by interfering double-stranded RNA against Drosophila importin-7 (DIM-7) or by the expression of integrin mutants, either during active cell spreading or after stimulation by insulin. In both cases, total ERK phosphorylation (on Westerns) is n...

  9. Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose

    Energy Technology Data Exchange (ETDEWEB)

    Ju, Huanyu; Wei, Na; Wang, Qian; Wang, Chunyuan; Jing, Zhiqiang; Guo, Lu; Liu, Dapeng; Gao, Mingchun; Ma, Bo [College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030 (China); Wang, Junwei, E-mail: jwwang@neau.edu.cn [College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030 (China)

    2011-05-27

    Highlights: {yields} All three capsid proteins can be expressed in insect cells in baculovirus expression system. {yields} All three recombinant proteins were spontaneously self-assemble into virus-like particles whose size and appearance were similar to those of native purified GPV virions. {yields} The immunogenicity of GPV-VLPs was better than commercial inactivated vaccine and attenuated vaccine. -- Abstract: Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs.

  10. Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

    Directory of Open Access Journals (Sweden)

    B.M. Ribeiro

    1998-06-01

    Full Text Available The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs. Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV. The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

  11. Easy expression of the C-terminal heavy chain domain of botulinum neurotoxin serotype A as a vaccine candidate using a bi-cistronic baculovirus system.

    Science.gov (United States)

    Villaflores, Oliver B; Hsei, Chein-Ming; Teng, Chao-Yi; Chen, Ying-Ju; Wey, Jiunn-Jye; Tsui, Pei-Yi; Shyu, Rong-Hwa; Tung, Kuo-Lun; Yeh, Jui-Ming; Chiao, Der-Jiang; Wu, Tzong-Yuan

    2013-04-01

    Clostridial botulinum neurotoxin (BoNT) is one of the most toxic proteins causing the food borne disease, botulism. In previous studies, recombinant BoNT production by Escherichia coli and yeast Pichia pastoris has been hampered by high AT content and codon bias in the gene encoding BoNT and required a synthetic gene to resolve this intrinsic bottleneck. This paper reports the simultaneous expression of the C-terminal heavy chain domain of BoNT (rBoNT/A-HC-6h) and enhanced green fluorescent protein (EGFP) using a bi-cistronic baculovirus-insect cell expression system. The expression of EGFP facilitated the monitoring of viral infection, virus titer determination, and isolation of the recombinant virus. Protein fusion with hexa-His-tag and one-step immobilized metal-ion affinity chromatography (IMAC) purification produced a homogenous, stable, and immunologically active 55-kDa rBoNT/A-HC-6h (about 3mg/L) with >90% purity. Furthermore, measured levels of serum titers were 8-folds for mice vaccinated with the purified rBoNT/A-HC-6h (2μg) than for mice administered with botulinum toxoid after initial immunization. Challenge experiment with botulinum A toxin demonstrated the immunoprotective activity of purified rBoNT/A-HC-6h providing the mice full protection against 10(2) LD50 botulinum A toxin with a dose as low as 0.2μg. This study provided supportive evidence for the use of a bi-cistronic baculovirus-Sf21 insect cell expression system in the facile expression of an immunogenically active rBoNT/A-HC. PMID:23313783

  12. Human herpesvirus 6B U19 protein is a PML-regulated transcriptional activator that localizes to nuclear foci in a PML-independent manner

    DEFF Research Database (Denmark)

    Kofod-Olsen, Emil; Ross-Hansen, Katrine; Mikkelsen, Jacob Giehm;

    2008-01-01

    Human herpesvirus 6B (HHV-6B) contains an IE-B domain spanning open reading frames U16/17-U19, based on homology with human cytomegalovirus. Here, the protein product, U19, of the HHV-6B U19 gene is identified as a 47 kDa transcriptional activator. HHV-6B infection or overexpression of U19...... on the RANTES promoter. Thus, U19 is a transcriptional activator that co-localizes with PML and localizes to ND10-like foci independently of PML, yet is regulated negatively by PML or its associated proteins....

  13. The LSD1-Type Zinc Finger Motifs of Pisum sativa LSD1 Are a Novel Nuclear Localization Signal and Interact with Importin Alpha

    OpenAIRE

    Shanping He; Kuowei Huang; Xu Zhang; Xiangchun Yu; Ping Huang; Chengcai An

    2011-01-01

    BACKGROUND: Genetic studies of the Arabidopsis mutant lsd1 highlight the important role of LSD1 in the negative regulation of plant programmed cell death (PCD). Arabidopsis thaliana LSD1 (AtLSD1) contains three LSD1-type zinc finger motifs, which are involved in the protein-protein interaction. METHODOLOGY/PRINCIPAL FINDINGS: To further understand the function of LSD1, we have analyzed cellular localization and functional localization domains of Pisum sativa LSD1 (PsLSD1), which is a homolog ...

  14. 16. national conference of the presidents of the local commissions of information (C.L.I.) around nuclear facilities; 16. conference nationale des presidents de commissions locales d'information (CLI) autour des installations nucleaires

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2005-07-01

    This conference contained two plenary sessions. The first one concerned the epidemiology with the following subjects: the epidemiology, its contributions and its limits; the epidemiological surveillance of cancers in France: role of registers within the device; the aggregates of leukaemia near the nuclear installations; perception and management of the risk; the point of view of the scientific committee of the A.N.C.L.I.; Followed a work in a workshop on the subjects relative to the functioning of the C.L.I. (Organization, relations, information) then the second plenary session with as subjects: organization and functioning of C.L.I., relations and links with A.N.C.L.I., information received and diffused by the C.L.I. (N.C.)

  15. Nuclear Medicine

    Science.gov (United States)

    ... Parents/Teachers Resource Links for Students Glossary Nuclear Medicine What is nuclear medicine? What are radioactive tracers? ... funded researchers advancing nuclear medicine? What is nuclear medicine? Nuclear medicine is a medical specialty that uses ...

  16. Polish Nuclear Physics Network

    International Nuclear Information System (INIS)

    In June 2002 the representatives of thirteen Polish nuclear physics units decided to create Polish Nuclear Physics Network (PNPN) and to contact Czech Republic, Hungary and Slovakia with a suggestion to establish a larger network of nuclear physics laboratories in these countries and in Poland. In spring 2003 North-East European Network (NEEN) was established. Its planned networking activities, their objectives and expected outcomes were submitted to EURONS Coordinator. During the same period the nuclear physics laboratories from Bulgaria, Croatia, Greece, Romania, Serbia and Turkey formed South-East European Network (SEEN) and also applied for EURONS support. Eventually, following the EURONS advice, the merge of NEEN and SEEN was decided by representatives of both networks and, in 2004, a common network EWON (East - West Outreach) was included in the EURONS initiative. The indicated EU financial contribution to EWON includes only the support of NEEN, whereas SEEN, for practical reasons, is financed separately. The nuclear physics activity in Poland can be conveniently divided into a few subgroups: - experimental nuclear physics using local facilities; - experimental nuclear physics using external facilities; - theoretical nuclear physics; - applications of nuclear physics to other domains of science; - medical applications. Most of these activities are presented in this report, at least partly, in the form of review articles and short communications. The special place in the Polish nuclear physics landscape occupies the theoretical physics. Not limited by severe financial restrictions which affects local experimental facilities, the flourishing of this domain is especially evident in the nuclear structure theory

  17. Influence of Local Natural Circulation on Passive Residual Heat Removal in Nuclear Reactor%反应堆内局部自然循环对非能动余热排出的影响

    Institute of Scientific and Technical Information of China (English)

    于雷; 鄢炳火; 刘守相

    2012-01-01

    为研究反应堆堆内局部自然循环对非能动余热排出的影响,利用改进的RELAP5/MOD3.2程序对核动力装置及非能动余热排出系统进行数学建模与理论研究,并利用试验数据进行了校核.研究表明:在核动力装置自然循环运行条件下,由于反应堆上封头旁流及反应堆入口漏流通道的存在,在反应堆活性区、上封头、环腔及下腔室之间构成了局部自然循环流动现象;在主回路自然循环能力较弱时,堆内产生的局部自然循环流动占优,反应堆衰变热无法顺利带出.%The nuclear power plant and passive residual heat removal system were modeled and investigated with the advanced code developed from RELAP5/MOD3. 2 code, for analyzing the influence of local natural circulation on the passive residual heat removal in nuclear reactor. The simulation results were validated with experimental data. The results indicate that the local natural circulation occurs in the connected region consists of reactor radiation zone, upper head, annular channel and lower plenum in case of natural circulation operation, due to the upper head by-passing flow and the leak flow at the core inlet. As the natural circulation capability is weak, the local natural circulation in the core is so significant that the decay heat can not be removed successfully.

  18. Model changing and equipment management improvement for local transmitter of nuclear power plant%核电站就地变送器换型及设备管理改进

    Institute of Scientific and Technical Information of China (English)

    张鹏

    2011-01-01

    目前核电站的装机容量越来越大,为了使高性能的主体设备充分发挥其可用率,提高电站功率因子,对保护、控制监视核电站的信号的要求也越来越高。可靠、稳定、准确是对这些信号最基本的要求,而这些信号主要来自于就地测量仪表。对秦山核电有限公司变送器换型前后发生的事例,从测量原理、历史状态、以及事件发生可能的原因,结合现场的实际检修过程进行分析,介绍就地仪控设备的管理以及改进。%With technology development,the current installed capacity of nuclear power plants keeps growing.In order to give full play to the availability of main high-performance equipment and improve the plant power factor,the signals for protection,monitor and control are strictly required on reliability,stability,and accuracy.These features are the basic requirements of signals which are mainly form the local measurement instruments.In this paper,according to the local transmitter maintenance experience of Qinshan Nuclear Power Co.,Ltd.,the paper is talking about the management and improvement of local instrument devices,and focus on analysis of measurement principles,history status,and possible causes of the incident.

  19. Localización extra nuclear de receptores esteroides y activación de mecanismos no genómicos Extra nuclear localization of steroid receptors and non genomic activation mechanisms

    Directory of Open Access Journals (Sweden)

    María Cecilia Bottino

    2010-04-01

    Full Text Available Los receptores de hormonas esteroides han sido considerados históricamente como factores de transcripción nucleares. Sin embargo, en los últimos años surgieron evidencias que indican que su activación desencadena eventos rápidos, independientes de la transcripción y que involucran a diferentes segundos mensajeros; muchos de estos receptores han sido localizados en la membrana celular. Por otra parte, se han caracterizado varios receptores de hormonas esteroides noveles, de estructura molecular diferente al receptor clásico, localizados principalmente en la membrana celular. Esta revisión enfoca los diferentes efectos iniciados por los glucocorticoides, mineralocorticoides, andrógenos, estrógenos y progesterona, y los posibles receptores involucrados en los mismos.Steroid hormone receptors have been historically considered as nuclear transcription factors. Nevertheless, in the last years, many of them have been detected in the cellular membrane. It has been postulated that their activation can induce transcription independent rapid events involving different second messengers. In addition, several novel steroid hormone receptors, showing a different molecular structure than the classical ones, have also been characterized and most of them are also located in the plasmatic membrane. This review focuses on the variety of effects initiated by glucocorticoids, mineralocorticoids, androgens, estrogens and progesterone, and the possible receptors involved mediating these effects.