Fusion reactor design studies

International Nuclear Information System (INIS)

Emmert, G.A.; Kulcinski, G.L.; Santarius, J.F.

1990-01-01

This report discusses the following topics on the ARIES tokamak: systems; plasma power balance; impurity control and fusion ash removal; fusion product ripple loss; energy conversion; reactor fueling; first wall design; shield design; reactor safety; and fuel cost and resources

Localization of a Region in the Fusion Protein of Avian Metapneumovirus That Modulates Cell-Cell Fusion

Science.gov (United States)

Wei, Yongwei; Feng, Kurtis; Yao, Xiangjie; Cai, Hui; Li, Junan; Mirza, Anne M.; Iorio, Ronald M.

2012-01-01

The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented. PMID:22915815

Localization of a region in the fusion protein of avian metapneumovirus that modulates cell-cell fusion.

Science.gov (United States)

Wei, Yongwei; Feng, Kurtis; Yao, Xiangjie; Cai, Hui; Li, Junan; Mirza, Anne M; Iorio, Ronald M; Li, Jianrong

2012-11-01

The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented.

Genetically engineered endostatin-lidamycin fusion proteins effectively inhibit tumor growth and metastasis

International Nuclear Information System (INIS)

Jiang, Wen-guo; Zhen, Yong-su; Lu, Xin-an; Shang, Bo-yang; Fu, Yan; Zhang, Sheng-hua; Zhou, Daifu; Li, Liang; Li, Yi; Luo, Yongzhang

2013-01-01

Endostatin (ES) inhibits endothelial cell proliferation, migration, invasion, and tube formation. It also shows antiangiogenesis and antitumor activities in several animal models. Endostatin specifically targets tumor vasculature to block tumor growth. Lidamycin (LDM), which consists of an active enediyne chromophore (AE) and a non-covalently bound apo-protein (LDP), is a member of chromoprotein family of antitumor antibiotics with extremely potent cytotoxicity to cancer cells. Therefore, we reasoned that endostatin-lidamycin (ES-LDM) fusion proteins upon energizing with enediyne chromophore may obtain the combined capability targeting tumor vasculature and tumor cell by respective ES and LDM moiety. In this study, we designed and obtained two new endostatin-based fusion proteins, endostatin-LDP (ES-LDP) and LDP-endostatin (LDP-ES). In vitro, the antiangiogenic effect of fusion proteins was determined by the wound healing assay and tube formation assay and the cytotoxicity of their enediyne-energized analogs was evaluated by CCK-8 assay. Tissue microarray was used to analyze the binding affinity of LDP, ES or ES-LDP with specimens of human lung tissue and lung tumor. The in vivo efficacy of the fusion proteins was evaluated with human lung carcinoma PG-BE1 xenograft and the experimental metastasis model of 4T1-luc breast cancer. ES-LDP and LDP-ES disrupted the formation of endothelial tube structures and inhibited endothelial cell migration. Evidently, ES-LDP accumulated in the tumor and suppressed tumor growth and metastasis. ES-LDP and ES show higher binding capability than LDP to lung carcinoma; in addition, ES-LDP and ES share similar binding capability. Furthermore, the enediyne-energized fusion protein ES-LDP-AE demonstrated significant efficacy against lung carcinoma xenograft in athymic mice. The ES-based fusion protein therapy provides some fundamental information for further drug development. Targeting both tumor vasculature and tumor cells by endostatin

Structural characterization of Mumps virus fusion protein core

International Nuclear Information System (INIS)

Liu Yueyong; Xu Yanhui; Lou Zhiyong; Zhu Jieqing; Hu Xuebo; Gao, George F.; Qiu Bingsheng; Rao Zihe; Tien, Po

2006-01-01

The fusion proteins of enveloped viruses mediating the fusion between the viral and cellular membranes comprise two discontinuous heptad repeat (HR) domains located at the ectodomain of the enveloped glycoproteins. The crystal structure of the fusion protein core of Mumps virus (MuV) was determined at 2.2 A resolution. The complex is a six-helix bundle in which three HR1 peptides form a central highly hydrophobic coiled-coil and three HR2 peptides pack against the hydrophobic grooves on the surface of central coiled-coil in an oblique antiparallel manner. Fusion core of MuV, like those of simian virus 5 and human respiratory syncytium virus, forms typical 3-4-4-4-3 spacing. The similar charecterization in HR1 regions, as well as the existence of O-X-O motif in extended regions of HR2 helix, suggests a basic rule for the formation of the fusion core of viral fusion proteins

Cellulose binding domain fusion proteins

Science.gov (United States)

Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

1998-01-01

A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

A fluorescent cassette-based strategy for engineering multiple domain fusion proteins

Directory of Open Access Journals (Sweden)

Khorchid Ahmad

2003-07-01

Full Text Available Abstract Background The engineering of fusion proteins has become increasingly important and most recently has formed the basis of many biosensors, protein purification systems, and classes of new drugs. Currently, most fusion proteins consist of three or fewer domains, however, more sophisticated designs could easily involve three or more domains. Using traditional subcloning strategies, this requires micromanagement of restriction enzymes sites that results in complex workaround solutions, if any at all. Results Therefore, to aid in the efficient construction of fusion proteins involving multiple domains, we have created a new expression vector that allows us to rapidly generate a library of cassettes. Cassettes have a standard vector structure based on four specific restriction endonuclease sites and using a subtle property of blunt or compatible cohesive end restriction enzymes, they can be fused in any order and number of times. Furthermore, the insertion of PCR products into our expression vector or the recombination of cassettes can be dramatically simplified by screening for the presence or absence of fluorescence. Conclusions Finally, the utility of this new strategy was demonstrated by the creation of basic cassettes for protein targeting to subcellular organelles and for protein purification using multiple affinity tags.

Structural Transition and Antibody Binding of EBOV GP and ZIKV E Proteins from Pre-Fusion to Fusion-Initiation State

Directory of Open Access Journals (Sweden)

Anna Lappala

2018-05-01

Full Text Available Membrane fusion proteins are responsible for viral entry into host cells—a crucial first step in viral infection. These proteins undergo large conformational changes from pre-fusion to fusion-initiation structures, and, despite differences in viral genomes and disease etiology, many fusion proteins are arranged as trimers. Structural information for both pre-fusion and fusion-initiation states is critical for understanding virus neutralization by the host immune system. In the case of Ebola virus glycoprotein (EBOV GP and Zika virus envelope protein (ZIKV E, pre-fusion state structures have been identified experimentally, but only partial structures of fusion-initiation states have been described. While the fusion-initiation structure is in an energetically unfavorable state that is difficult to solve experimentally, the existing structural information combined with computational approaches enabled the modeling of fusion-initiation state structures of both proteins. These structural models provide an improved understanding of four different neutralizing antibodies in the prevention of viral host entry.

Fluorescent sensors based on bacterial fusion proteins

International Nuclear Information System (INIS)

Mateu, Batirtze Prats; Pum, Dietmar; Sleytr, Uwe B; Toca-Herrera, José L; Kainz, Birgit

2014-01-01

Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins. (paper)

Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

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A.T. Da Poian

2005-06-01

Full Text Available Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.

Computational and biological characterization of fusion proteins of two insecticidal proteins for control of insect pests.

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Javaid, Shaista; Naz, Sehrish; Amin, Imran; Jander, Georg; Ul-Haq, Zaheer; Mansoor, Shahid

2018-03-19

Sucking pests pose a serious agricultural challenge, as available transgenic technologies such as Bacillus thuringiensis crystal toxins (Bt) are not effective against them. One approach is to produce fusion protein toxins for the control of these pests. Two protein toxins, Hvt (ω-atracotoxin from Hadronyche versuta) and onion leaf lectin, were translationally fused to evaluate the negative effects of fusion proteins on Phenacoccus solenopsis (mealybug), a phloem-feeding insect pest. Hvt was cloned both N-terminally (HL) and then C-terminally (LH) in the fusion protein constructs, which were expressed transiently in Nicotiana tabacum using a Potato Virus X (PVX) vector. The HL fusion protein was found to be more effective against P. solenopsis, with an 83% mortality rate, as compared to the LH protein, which caused 65% mortality. Hvt and lectin alone caused 42% and 45%, respectively, under the same conditions. Computational studies of both fusion proteins showed that the HL protein is more stable than the LH protein. Together, these results demonstrate that translational fusion of two insecticidal proteins improved the insecticidal activity relative to each protein individually and could be expressed in transgenic plants for effective control of sucking pests.

Design activities of a fusion experimental breeder

International Nuclear Information System (INIS)

Huang, J.; Feng, K.; Sheng, G.

1999-01-01

The fusion reactor design studies in China are under the support of a fusion-fission hybrid reactor research Program. The purpose of this program is to explore the potential near-term application of fusion energy to support the long-term fusion energy on the one hand and the fission energy development on the other. During 1992-1996 a detailed consistent and integral conceptual design of a Fusion Experimental Breeder, FEB was completed. Beginning from 1996, a further design study towards an Engineering Outline Design of the FEB, FEB-E, has started. The design activities are briefly given. (author)

Design activities of a fusion experimental breeder

International Nuclear Information System (INIS)

Huang, J.; Feng, K.; Sheng, G.

2001-01-01

The fusion reactor design studies in China are under the support of a fusion-fission hybrid reactor research Program. The purpose of this program is to explore the potential near-term application of fusion energy to support the long-term fusion energy on the one hand and the fission energy development on the other. During 1992-1996 a detailed consistent and integral conceptual design of a Fusion Experimental Breeder, FEB was completed. Beginning from 1996, a further design study towards an Engineering Outline Design of the FEB, FEB-E, has started. The design activities are briefly given. (author)

Protein functional links in Trypanosoma brucei, identified by gene fusion analysis

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Trimpalis Philip

2011-07-01

Full Text Available Abstract Background Domain or gene fusion analysis is a bioinformatics method for detecting gene fusions in one organism by comparing its genome to that of other organisms. The occurrence of gene fusions suggests that the two original genes that participated in the fusion are functionally linked, i.e. their gene products interact either as part of a multi-subunit protein complex, or in a metabolic pathway. Gene fusion analysis has been used to identify protein functional links in prokaryotes as well as in eukaryotic model organisms, such as yeast and Drosophila. Results In this study we have extended this approach to include a number of recently sequenced protists, four of which are pathogenic, to identify fusion linked proteins in Trypanosoma brucei, the causative agent of African sleeping sickness. We have also examined the evolution of the gene fusion events identified, to determine whether they can be attributed to fusion or fission, by looking at the conservation of the fused genes and of the individual component genes across the major eukaryotic and prokaryotic lineages. We find relatively limited occurrence of gene fusions/fissions within the protist lineages examined. Our results point to two trypanosome-specific gene fissions, which have recently been experimentally confirmed, one fusion involving proteins involved in the same metabolic pathway, as well as two novel putative functional links between fusion-linked protein pairs. Conclusions This is the first study of protein functional links in T. brucei identified by gene fusion analysis. We have used strict thresholds and only discuss results which are highly likely to be genuine and which either have already been or can be experimentally verified. We discuss the possible impact of the identification of these novel putative protein-protein interactions, to the development of new trypanosome therapeutic drugs.

Premature activation of the paramyxovirus fusion protein before target cell attachment with corruption of the viral fusion machinery.

Science.gov (United States)

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

2011-11-04

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

Impact of fluorescent protein fusions on the bacterial flagellar motor.

Science.gov (United States)

Heo, M; Nord, A L; Chamousset, D; van Rijn, E; Beaumont, H J E; Pedaci, F

2017-10-03

Fluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side effects, but such insight is still lacking for many applications. This is particularly relevant in the study of the internal dynamics of motor proteins, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to the stator of the Bacterial Flagellar Motor (BFM) have previously been used to unveil the motor subunit dynamics. Here we report the effects on single motors of three fluorescent proteins fused to the stators, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry remained unaffected. MotB fusions decreased the switching frequency and induced a novel bias-dependent asymmetry in the speed in the two directions. These effects could be mitigated by inserting a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions to the stator on BFM dynamics and their alleviation- new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.

Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies

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Hideki Kusunoki

2017-03-01

Full Text Available Hepatitis B virus X protein (HBx is a multifunctional protein that interacts directly with many host proteins. For example, HBx interacts with anti-apoptotic proteins, Bcl-2 and Bcl-xL, through its BH3-like motif, which leads to elevated cytosolic calcium levels, efficient viral DNA replication and the induction of apoptosis. To facilitate sample preparation and perform detailed structural characterization of the complex between HBx and Bcl-xL, we designed and purified a recombinant HBx BH3-like motif-linker-Bcl-xL fusion protein produced in E. coli. The fusion protein was characterized by size exclusion chromatography, circular dichroism and nuclear magnetic resonance experiments. Our results show that the fusion protein is a monomer in aqueous solution, forms a stable intramolecular complex, and likely retains the native conformation of the complex between Bcl-xL and the HBx BH3-like motif. Furthermore, the HBx BH3-like motif of the intramolecular complex forms an α-helix. These observations indicate that the fusion protein should facilitate structural studies aimed at understanding the interaction between HBx and Bcl-xL at the atomic level.

Design of Fusion Proteins for Efficient and Soluble Production of Immunogenic Ebola Virus Glycoprotein in Escherichia coli.

Science.gov (United States)

Ji, Yang; Lu, Yuan; Yan, Yishu; Liu, Xinxin; Su, Nan; Zhang, Chong; Bi, Shengli; Xing, Xin-Hui

2018-03-03

The Ebola hemorrhagic fever caused by Ebola virus is an extremely dangerous disease, and effective therapeutic agents are still lacking. Platforms for the efficient production of vaccines are crucial to ensure quick response against an Ebola virus outbreak. Ebola virus glycoprotein (EbolaGP) on the virion surface is responsible for membrane binding and virus entry, thus becoming the key target for vaccine development. However, heterologous expression of this protein still faces engineering challenges such as low production levels and insoluble aggregation. Here, the authors design and compare various fusion strategies, attaching great importance to the solubility-enhancing effect, and tag removal process. It is found that a C-terminal intein-based tag greatly enhances the solubility of EbolaGP and allows one-step chromatographic purification of the untagged EbolaGP through thiol-catalyzed self-cleavage. The purified untagged EbolaGP alone or with Freund's adjuvant are highly immunogenic, as confirmed in a mouse model. Consequently, the present study puts forward a new strategy for the efficient and soluble expression of untagged immunogenic EbolaGP. The intein-based protein fusion approach may be of importance for the large-scale production of Ebola virus subunit vaccine. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Impact of fluorescent protein fusions on the bacterial flagellar motor

NARCIS (Netherlands)

Heo, M.; Nord, A. L.; Chamousset, D.; van Rijn, E.; Beaumont, H.J.E.; Pedaci, F.

2017-01-01

Fluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side

The Multifaceted Role of SNARE Proteins in Membrane Fusion.

Science.gov (United States)

Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A

2017-01-01

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined.

Design study of laser fusion rocket

International Nuclear Information System (INIS)

Nakashima, Hideki; Shoyama, Hidetoshi; Kanda, Yukinori

1991-01-01

A design study was made on a rocket powered by laser fusion. Dependence of its flight performance on target gain, driver repetition rate and fuel composition was analyzed to obtain optimal design parameters of the laser fusion rocket. The results indicate that the laser fusion rocket fueled with DT or D 3 He has the potential advantages over other propulsion systems such as fission rocket for interplanetary travel. (author)

Comparative immunoblot analysis with 10 different, partially overlapping recombinant fusion proteins derived from 5 different cytomegalovirus proteins

NARCIS (Netherlands)

van Zanten, J.; LAZZAROTTO, T; CAMPISI, B; VORNHAGEN, R; JAHN, G; LANDINI, MP; The, T. Hauw

Ten fusion proteins derived from five various CMV encoded proteins were used for the detection of specific antibody response by immunoblot technique in sera from renal transplant recipients. The fusion proteins were derived from the following CMV specific proteins: the assembly protein ppUL80a with

Chaperone activity of human small heat shock protein-GST fusion proteins.

Science.gov (United States)

Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A

2017-07-01

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST

Engineering a pharmacologically superior form of granulocyte-colony-stimulating factor by fusion with gelatin-like-protein polymer.

Science.gov (United States)

Huang, Yan-Shan; Wen, Xiao-Fang; Wu, Yi-Liang; Wang, Ye-Fei; Fan, Min; Yang, Zhi-Yu; Liu, Wei; Zhou, Lin-Fu

2010-03-01

The plasma half-life of therapeutic proteins is a critical factor in many clinical applications. Therefore, new strategies to prolong plasma half-life of long-acting peptides and protein drugs are in high demand. Here, we designed an artificial gelatin-like protein (GLK) and fused this hydrophilic GLK polymer to granulocyte-colony-stimulating factor (G-CSF) to generate a chimeric GLK/G-CSF fusion protein. The genetically engineered recombinant GLK/G-CSF (rGLK/G-CSF) fusion protein was purified from Pichia pastoris. In vitro studies demonstrated that rGLK/G-CSF possessed an enlarged hydrodynamic radius, improved thermal stability and retained full bioactivity compared to unfused G-CSF. Following a single subcutaneous administration to rats, the rGLK/G-CSF fusion protein displayed a slower plasma clearance rate and stimulated greater and longer lasting increases in circulating white blood cells than G-CSF. Our findings indicate that fusion with this artificial, hydrophilic, GLK polymer provides many advantages in the construction of a potent hematopoietic factor with extended plasma half-life. This approach could be easily applied to other therapeutic proteins and have important clinical applications. (c) 2009 Elsevier B.V. All rights reserved.

Protein-induced fusion can be modulated by target membrane lipids through a structural switch at the level of the fusion peptide

NARCIS (Netherlands)

Pecheur, EI; Martin, [No Value; Bienvenue, A; Ruysschaert, JM; Hoekstra, D

2000-01-01

Regulatory features of protein-induced membrane fusion are largely unclear, particularly at the level of the fusion peptide. Fusion peptides being part of larger protein complexes, such investigations are met with technical limitations. Here, we show that the fusion activity of influenza virus or

Advances in laser solenoid fusion reactor design

International Nuclear Information System (INIS)

Steinhauer, L.C.; Quimby, D.C.

1978-01-01

The laser solenoid is an alternate fusion concept based on a laser-heated magnetically-confined plasma column. The reactor concept has evolved in several systems studies over the last five years. We describe recent advances in the plasma physics and technology of laser-plasma coupling. The technology advances include progress on first walls, inner magnet design, confinement module design, and reactor maintenance. We also describe a new generation of laser solenoid fusion and fusion-fission reactor designs

Health physics in fusion reactor design

International Nuclear Information System (INIS)

Wong, K.Y.; Dinner, P.J.

1984-06-01

Experience in the control of tritium exposures to workers and the public gained through the design and operation of Ontario Hydro's nuclear stations has been applied to fusion projects and to design studies on emerging fusion reactor concepts. Ontario Hydro performance in occupational tritium exposure control and environmental impact is reviewed. Application of tritium control technologies and dose management methodology during facility design is highlighted

Fusion target design

International Nuclear Information System (INIS)

Bangerter, R.O.

1978-01-01

Most detailed fusion target design is done by numerical simulation using large computers. Although numerical simulation is briefly discussed, this lecture deals primarily with the way in which basic physical arguments, driver technology considerations and economical power production requirements are used to guide and augment the simulations. Physics topics discussed include target energetics, preheat, stability and symmetry. A specific design example is discussed

Elastin-like-polypeptide based fusion proteins for osteogenic factor delivery in bone healing.

Science.gov (United States)

McCarthy, Bryce; Yuan, Yuan; Koria, Piyush

2016-07-08

Modern treatments of bone injuries and diseases are becoming increasingly dependent on the usage of growth factors to stimulate bone growth. Bone morphogenetic protein-2 (BMP-2), a potent osteogenic inductive protein, exhibits promising results in treatment models, but recently has had its practical efficacy questioned due to the lack of local retention, ectopic bone formation, and potentially lethal inflammation. Where a new delivery technique of the BMP-2 is necessary, here we demonstrate the viability of an elastin-like peptide (ELP) fusion protein containing BMP-2 for delivery of the BMP-2. This fusion protein retains the performance characteristics of both the BMP-2 and ELP. The fusion protein was found to induce osteogenic differentiation of mesenchymal stem cells as evidenced by the production of alkaline phosphatase and extracellular calcium deposits in response to treatment by the fusion protein. Retention of the ELPs inverse phase transition property has allowed for expression of the fusion protein within a bacterial host (such as Escherichia coli) and easy and rapid purification using inverse transition cycling. The fusion protein formed self-aggregating nanoparticles at human-body temperature. The data collected suggests the viability of these fusion protein nanoparticles as a dosage-efficient and location-precise noncytotoxic delivery vehicle for BMP-2 in bone treatment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1029-1037, 2016. © 2016 American Institute of Chemical Engineers.

Current fusion power plant design concepts

International Nuclear Information System (INIS)

Gore, B.F.; Murphy, E.S.

1976-09-01

Nine current U.S. designs for fusion power plants are described in this document. Summary tabulations include a tenth concept, for which the design document was unavailable during preparation of the descriptions. The information contained in the descriptions was used to define an envelope of fusion power plant characteristics which formed the basis for definition of reference first commercial fusion power plant design. A brief prose summary of primary plant features introduces each of the descriptions contained in the body of this document. In addition, summary tables are presented. These tables summarize in side-by-side fashion, plant parameters, processes, combinations of materials used, requirements for construction materials, requirements for replacement materials during operation, and production of wastes

Fusion peptides from oncogenic chimeric proteins as putative specific biomarkers of cancer.

Science.gov (United States)

Conlon, Kevin P; Basrur, Venkatesha; Rolland, Delphine; Wolfe, Thomas; Nesvizhskii, Alexey I; MacCoss, Michael J; Lim, Megan S; Elenitoba-Johnson, Kojo S J

2013-10-01

Chromosomal translocations encoding chimeric fusion proteins constitute one of the most common mechanisms underlying oncogenic transformation in human cancer. Fusion peptides resulting from such oncogenic chimeric fusions, though unique to specific cancer subtypes, are unexplored as cancer biomarkers. Here we show, using an approach termed fusion peptide multiple reaction monitoring mass spectrometry, the direct identification of different cancer-specific fusion peptides arising from protein chimeras that are generated from the juxtaposition of heterologous genes fused by recurrent chromosomal translocations. Using fusion peptide multiple reaction monitoring mass spectrometry in a clinically relevant scenario, we demonstrate the specific, sensitive, and unambiguous detection of a specific diagnostic fusion peptide in clinical samples of anaplastic large cell lymphoma, but not in a diverse array of benign lymph nodes or other forms of primary malignant lymphomas and cancer-derived cell lines. Our studies highlight the utility of fusion peptides as cancer biomarkers and carry broad implications for the use of protein biomarkers in cancer detection and monitoring.

Biodistribution and tumor imaging of an anti-CEA single-chain antibody-albumin fusion protein

International Nuclear Information System (INIS)

Yazaki, Paul J.; Kassa, Thewodros; Cheung, Chia-wei; Crow, Desiree M.; Sherman, Mark A.; Bading, James R.; Anderson, Anne-Line J.; Colcher, David; Raubitschek, Andrew

2008-01-01

Albumin fusion proteins have demonstrated the ability to prolong the in vivo half-life of small therapeutic proteins/peptides in the circulation and thereby potentially increase their therapeutic efficacy. To evaluate if this format can be employed for antibody-based imaging, an anticarcinoembryonic antigen (CEA) single-chain antibody(scFv)-albumin fusion protein was designed, expressed and radiolabeled for biodistribution and imaging studies in athymic mice bearing human colorectal carcinoma LS-174T xenografts. The [ 125 I]-T84.66 fusion protein demonstrated rapid tumor uptake of 12.3% injected dose per gram (ID/g) at 4 h that reached a plateau of 22.7% ID/g by 18 h. This was a dramatic increase in tumor uptake compared to 4.9% ID/g for the scFv alone. The radiometal [ 111 In]-labeled version resulted in higher tumor uptake, 37.2% ID/g at 18 h, which persisted at the tumor site with tumor: blood ratios reaching 18:1 and with normal tissues showing limited uptake. Based on these favorable imaging properties, a pilot [ 64 Cu]-positron emission tomography imaging study was performed with promising results. The anti-CEA T84.66 scFv-albumin fusion protein demonstrates highly specific tumor uptake that is comparable to cognate recombinant antibody fragments. The radiometal-labeled version, which shows lower normal tissue accumulation than these recombinant antibodies, provides a promising and novel platform for antibody-based imaging agents

Cloning, Expression and Purification of the Recombinant HIV-1 Tat-Nef Fusion Protein in Prokaryotic Expression System

Directory of Open Access Journals (Sweden)

Somayeh Kadkhodayan

2016-07-01

Full Text Available Abstract Background: Nef is one of the HIV-1 critical proteins, because it is essential for viral replication and AIDS disease progression and induction of immune response against it can partially inhibit viral infection. Moreover, a domain of the HIV-1 Trans-Activator of Transcription (Tat, 48-60 aa could act as a cell penetrating peptide (CPP. In current study, cloning and expression of Tat-Nef fusion protein was performed in E. coli for the first time. The protein expression was confirmed by western blot analysis and was purified using reverse staining method. Materials and Methods: In this experimental study, primarily, cloning of Tat-Nef fusion gene was done in pGEX6p2 expression vector. Then, the expression of Tat-Nef recombinat protein in E.coli BL21 (DE3 strain was performed by using IPTG inducer. The protein expression was confirmed by SDS-PAGE and western blotting using anti-Nef monoclonal antibody. Then, the recombinant fusion protein was purified from gel using reverse staining method. Results: The results of PCR analysis and enzyme digestion showed a clear band of ~ 726 bp in agarose gel indicating the correct Tat-Nef fusion cloning in pGEX6p2 prokaryotic expression vector. In addition, a 54 kDa band of Tat-Nef on SDS-PAGE revealed Tat-Nef protein expression that western blot analysis using anti-Nef monoclonal antibody confirmed it. Conclusion: The purified Tat-Nef recombinant fusion protein will be used as an antigen for protein vaccine design against HIV infection.

Menin-MLL inhibitors reverse oncogenic activity of MLL fusion proteins in leukemia.

Science.gov (United States)

Grembecka, Jolanta; He, Shihan; Shi, Aibin; Purohit, Trupta; Muntean, Andrew G; Sorenson, Roderick J; Showalter, Hollis D; Murai, Marcelo J; Belcher, Amalia M; Hartley, Thomas; Hess, Jay L; Cierpicki, Tomasz

2012-01-29

Translocations involving the mixed lineage leukemia (MLL) gene result in human acute leukemias with very poor prognosis. The leukemogenic activity of MLL fusion proteins is critically dependent on their direct interaction with menin, a product of the multiple endocrine neoplasia (MEN1) gene. Here we present what are to our knowledge the first small-molecule inhibitors of the menin-MLL fusion protein interaction that specifically bind menin with nanomolar affinities. These compounds effectively reverse MLL fusion protein-mediated leukemic transformation by downregulating the expression of target genes required for MLL fusion protein oncogenic activity. They also selectively block proliferation and induce both apoptosis and differentiation of leukemia cells harboring MLL translocations. Identification of these compounds provides a new tool for better understanding MLL-mediated leukemogenesis and represents a new approach for studying the role of menin as an oncogenic cofactor of MLL fusion proteins. Our findings also highlight a new therapeutic strategy for aggressive leukemias with MLL rearrangements.

Introduction to magnetic fusion reactor design

International Nuclear Information System (INIS)

Watanabe, Kenji

1988-01-01

Trend of the tokamak reactor design works so far carried out is reviewed, and method of conceptual design for commercial fusion reactor is critically considered concerning the black-box conpepts. System-framework of the engineering of magnetic fusion (commercial) reactor design is proposed as four steps. Based on it the next design studies are recommended in parallel approaches for making real-overcome of reactor material problem, from the view point of technological realization and not from the economical one. Real trials are involved. (author)

Expression of the Acyl-Coenzyme A: Cholesterol Acyltransferase GFP Fusion Protein in Sf21 Insect Cells

Science.gov (United States)

Mahtani, H. K.; Richmond, R. C.; Chang, T. Y.; Chang, C. C. Y.; Rose, M. Franklin (Technical Monitor)

2001-01-01

The enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an important contributor to the pathological expression of plaque leading to artherosclerosis n a major health problem. Adequate knowledge of the structure of this protein will enable pharmaceutical companies to design drugs specific to the enzyme. ACAT is a membrane protein located in the endoplasmic reticulum.t The protein has never been purified to homogeneity.T.Y. Chang's laboratory at Dartmouth College provided a 4-kb cDNA clone (K1) coding for a structural gene of the protein. We have modified the gene sequence and inserted the cDNA into the BioGreen His Baculovirus transfer vector. This was successfully expressed in Sf2l insect cells as a GFP-labeled ACAT protein. The advantage to this ACAT-GFP fusion protein (abbreviated GCAT) is that one can easily monitor its expression as a function of GFP excitation at 395 nm and emission at 509 nm. Moreover, the fusion protein GCAT can be detected on Western blots with the use of commercially available GFP antibodies. Antibodies against ACAT are not readily available. The presence of the 6xHis tag in the transfer vector facilitates purification of the recombinant protein since 6xHis fusion proteins bind with high affinity to Ni-NTA agarose. Obtaining highly pure protein in large quantities is essential for subsequent crystallization. The purified GCAT fusion protein can readily be cleaved into distinct GFP and ACAT proteins in the presence of thrombin. Thrombin digests the 6xHis tag linking the two protein sequences. Preliminary experiments have indicated that both GCAT and ACAT are expressed as functional proteins. The ultimate aim is to obtain large quantities of the ACAT protein in pure and functional form appropriate for protein crystal growth. Determining protein structure is the key to the design and development of effective drugs. X-ray analysis requires large homogeneous crystals that are difficult to obtain in the gravity environment of earth

Conceptual design of inertial confinement fusion power plant

International Nuclear Information System (INIS)

Mima, Kunioki; Yamanaka, Tatsuhiko; Nakai, Sadao

1994-01-01

Presented is the status of the conceptual design studies of inertial confinement fusion reactors. The recent achievements of the laser fusion research enable us to refine the conceptual design of the power plant. In the paper, main features of several new conceptual designs of ICF reactor; KOYO, SIRIUS-P, HYLIFE-II and so on are summarized. In particular, the target design and the reactor chamber design are described. Finally, the overview of the laser fusion reactor and the irradiation system is also described. (author)

Protein design on computers. Five new proteins: Shpilka, Grendel, Fingerclasp, Leather, and Aida.

Science.gov (United States)

Sander, C; Vriend, G; Bazan, F; Horovitz, A; Nakamura, H; Ribas, L; Finkelstein, A V; Lockhart, A; Merkl, R; Perry, L J

1992-02-01

What is the current state of the art in protein design? This question was approached in a recent two-week protein design workshop sponsored by EMBO and held at the EMBL in Heidelberg. The goals were to test available design tools and to explore new design strategies. Five novel proteins were designed: Shpilka, a sandwich of two four-stranded beta-sheets, a scaffold on which to explore variations in loop topology; Grendel, a four-helical membrane anchor, ready for fusion to water-soluble functional domains; Finger-clasp, a dimer of interdigitating beta-beta-alpha units, the simplest variant of the "handshake" structural class; Aida, an antibody binding surface intended to be specific for flavodoxin; Leather--a minimal NAD binding domain, extracted from a larger protein. Each design is available as a set of three-dimensional coordinates, the corresponding amino acid sequence and a set of analytical results. The designs are placed in the public domain for scrutiny, improvement, and possible experimental verification.

The coronavirus spike protein : mechanisms of membrane fusion and virion incorporation

NARCIS (Netherlands)

Bosch, B.J.

2004-01-01

The coronavirus spike protein is a membrane-anchored glycoprotein responsible for virus-cell attachment and membrane fusion, prerequisites for a successful virus infection. In this thesis, two aspects are described regarding the molecular biology of the coronavirus spike protein: its membrane fusion

Expression and Activation of Horseradish Peroxidase-Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes.

Science.gov (United States)

Xxxx, Patmawati; Minamihata, Kosuke; Tatsuke, Tsuneyuki; Lee, Jae Man; Kusakabe, Takahiro; Kamiya, Noriho

2018-06-01

Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Distinct roles for key karyogamy proteins during yeast nuclear fusion.

Science.gov (United States)

Melloy, Patricia; Shen, Shu; White, Erin; Rose, Mark D

2009-09-01

During yeast mating, cell fusion is followed by the congression and fusion of the two nuclei. Proteins required for nuclear fusion are found at the surface (Prm3p) and within the lumen (Kar2p, Kar5p, and Kar8p) of the nuclear envelope (NE). Electron tomography (ET) of zygotes revealed that mutations in these proteins block nuclear fusion with different morphologies, suggesting that they act in different steps of fusion. Specifically, prm3 zygotes were blocked before formation of membrane bridges, whereas kar2, kar5, and kar8 zygotes frequently contained them. Membrane bridges were significantly larger and occurred more frequently in kar2 and kar8, than in kar5 mutant zygotes. The kinetics of NE fusion in prm3, kar5, and kar8 mutants, measured by live-cell fluorescence microscopy, were well correlated with the size and frequency of bridges observed by ET. However the kar2 mutant was defective for transfer of NE lumenal GFP, but not diffusion within the lumen, suggesting that transfer was blocked at the NE fusion junction. These observations suggest that Prm3p acts before initiation of outer NE fusion, Kar5p may help dilation of the initial fusion pore, and Kar2p and Kar8p act after outer NE fusion, during inner NE fusion.

Ceramic sphere-pac breeder design for fusion blankets

International Nuclear Information System (INIS)

Gierszewski, P.J.; Sullivan, J.D.

1991-01-01

Randomly packed beds of ceramic spheres are a practical approach to surrounding fusion plasmas with tritium-breeding material. This paper examines the general properties of sphere-pac beds for application in fusion breeder blankets. The design considerations and models are reviewed for packing, tritium breeding and recovery, thermal conductivity, purge-gas pressure drop, mechanical behavior and fabrication. The design correlations are compared against available fusion ceramic data. Specific conclusions are that ternary (three-size) beds are not attractive for fusion blankets, and that the fusion spheres should be as large as possible subject primarily to packing constraints. (orig.)

Myristylation of gag-onc fusion proteins in mammalian transforming retroviruses

International Nuclear Information System (INIS)

Schultz, A.; Oroszlan, S.

1984-01-01

Four cell lines producing transforming proteins encoded by three mammalian oncogenes (fes, abl, and ras) were investigated for incorporation of [ 3 H]myristate into gag-onc fusion proteins. Using 5-min pulse-labelings, fusion proteins of Abelson murine leukemia virus, Gardner-Arnstein strain of feline sarcoma virus (FeSV), and Snyder-Theilen strain of FeSV were shown to be myristylated. In a 4-hr pulse, p29gag-ras of rat sarcoma virus (RaSV) was also shown to incorporate radiolabel. The fatty acid was recovered from this labeled protein by acid hydrolysis, and identified by reverse-phase thin-layer chromatography to be [ 3 H]myristic acid. The results indicate that substitution of viral gag sequences by cellular oncogene sequences does not abolish their ability to become post-translationally modified by this long chain fatty acid. It is assumed that in the fusion proteins the myristyl moiety is linked through an amide linkage to the amino-terminal glycine as previously found for several retroviral gag precursor polyproteins. The possible role of myristylation of transforming proteins is discussed

Myristylation of gag-onc fusion proteins in mammalian transforming retroviruses

Energy Technology Data Exchange (ETDEWEB)

Schultz, A.; Oroszlan, S.

1984-03-01

Four cell lines producing transforming proteins encoded by three mammalian oncogenes (fes, abl, and ras) were investigated for incorporation of (/sup 3/H)myristate into gag-onc fusion proteins. Using 5-min pulse-labelings, fusion proteins of Abelson murine leukemia virus, Gardner-Arnstein strain of feline sarcoma virus (FeSV), and Snyder-Theilen strain of FeSV were shown to be myristylated. In a 4-hr pulse, p29gag-ras of rat sarcoma virus (RaSV) was also shown to incorporate radiolabel. The fatty acid was recovered from this labeled protein by acid hydrolysis, and identified by reverse-phase thin-layer chromatography to be (/sup 3/H)myristic acid. The results indicate that substitution of viral gag sequences by cellular oncogene sequences does not abolish their ability to become post-translationally modified by this long chain fatty acid. It is assumed that in the fusion proteins the myristyl moiety is linked through an amide linkage to the amino-terminal glycine as previously found for several retroviral gag precursor polyproteins. The possible role of myristylation of transforming proteins is discussed.

Fusion reactor design studies: standard accounts for cost estimates

International Nuclear Information System (INIS)

Schulte, S.C.; Willke, T.L.; Young, J.R.

1978-05-01

The fusion reactor design studies--standard accounts for cost estimates provides a common format from which to assess the economic character of magnetically confined fusion reactor design concepts. The format will aid designers in the preparation of design concept costs estimates and also provide policymakers with a tool to assist in appraising which design concept may be economically promising. The format sets forth a categorization and accounting procedure to be used when estimating fusion reactor busbar energy cost that can be easily and consistently applied. Reasons for developing the procedure, explanations of the procedure, justifications for assumptions made in the procedure, and the applicability of the procedure are described in this document. Adherence to the format when evaluating prospective fusion reactor design concepts will result in the identification of the more promising design concepts thus enabling the fusion power alternatives with better economic potential to be quickly and efficiently developed

The dengue virus type 2 envelope protein fusion peptide is essential for membrane fusion

International Nuclear Information System (INIS)

Huang, Claire Y.-H.; Butrapet, Siritorn; Moss, Kelly J.; Childers, Thomas; Erb, Steven M.; Calvert, Amanda E.; Silengo, Shawn J.; Kinney, Richard M.; Blair, Carol D.; Roehrig, John T.

2010-01-01

The flaviviral envelope (E) protein directs virus-mediated membrane fusion. To investigate membrane fusion as a requirement for virus growth, we introduced 27 unique mutations into the fusion peptide of an infectious cDNA clone of dengue 2 virus and recovered seven stable mutant viruses. The fusion efficiency of the mutants was impaired, demonstrating for the first time the requirement for specific FP AAs in optimal fusion. Mutant viruses exhibited different growth kinetics and/or genetic stabilities in different cell types and adult mosquitoes. Virus particles could be recovered following RNA transfection of cells with four lethal mutants; however, recovered viruses could not re-infect cells. These viruses could enter cells, but internalized virus appeared to be retained in endosomal compartments of infected cells, thus suggesting a fusion blockade. Mutations of the FP also resulted in reduced virus reactivity with flavivirus group-reactive antibodies, confirming earlier reports using virus-like particles.

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

Science.gov (United States)

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

2012-06-01

The production bioreactor step of an Fc-Fusion protein manufacturing cell culture process was characterized following Quality by Design principles. Using scientific knowledge derived from the literature and process knowledge gathered during development studies and manufacturing to support clinical trials, potential critical and key process parameters with a possible impact on product quality and process performance, respectively, were determined during a risk assessment exercise. The identified process parameters were evaluated using a design of experiment approach. The regression models generated from the data allowed characterizing the impact of the identified process parameters on quality attributes. The main parameters having an impact on product titer were pH and dissolved oxygen, while those having the highest impact on process- and product-related impurities and variants were pH and culture duration. The models derived from characterization studies were used to define the cell culture process design space. The design space limits were set in such a way as to ensure that the drug substance material would consistently have the desired quality. Copyright © 2012 Elsevier B.V. All rights reserved.

Osteoinductive recombinant silk fusion proteins for bone regeneration.

Science.gov (United States)

Dinjaski, Nina; Plowright, Robyn; Zhou, Shun; Belton, David J; Perry, Carole C; Kaplan, David L

2017-02-01

Protein polymers provide a unique opportunity for tunable designs of material systems due to the genetic basis of sequence control. To address the challenge of biomineralization interfaces with protein based materials, we genetically engineered spider silks to design organic-inorganic hybrid systems. The spider silk inspired domain (SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQGT) 15 served as an organic scaffold to control material stability and to allow multiple modes of processing, whereas the hydroxyapatite binding domain VTKHLNQISQSY (VTK), provided control over osteogenesis. The VTK domain was fused either to the N-, C- or both terminals of the spider silk domain to understand the effect of position on material properties and mineralization. The addition of the VTK domain to silk did not affect the physical properties of the silk recombinant constructs, but it had a critical role in the induction of biomineralization. When the VTK domain was placed on both the C- and N-termini the formation of crystalline hydroxyapatite was significantly increased. In addition, all of the recombinant proteins in film format supported the growth and proliferation of human mesenchymal stem cells (hMSCs). Importantly, the presence of the VTK domain enhanced osteoinductive properties up to 3-fold compared to the control (silk alone without VTK). Therefore, silk-VTK fusion proteins have been shown suitable for mineralization and functionalization for specific biomedical applications. Organic-inorganic interfaces are integral to biomaterial functions in many areas of repair and regeneration. Several protein polymers have been investigated for this purpose. Despite their success the limited options to fine-tune their material properties, degradation patterns and functionalize them for each specific biomedical application limits their application. Various studies have shown that the biological performance of such proteins can be improved by genetic engineering. The present study provides data

Inhibitory effect of PTD-OD-HA fusion protein on Bcr-Abl in K562 cells

Directory of Open Access Journals (Sweden)

Miao GAO

2012-10-01

Full Text Available Objective　To study the transduction dynamics, location of PTD-OD-HA fusion protein and its interaction with Bcr-Abl oncoprotein in K562 cell lines, and explore the influence of PTD-OD-HA fusion protein on oligomerization and tyrosine kinase activity of Bcr-Abl. Methods　PTD-OD-HA fusion protein was labeled with FITC and co-cultured with K562 cells. The transduction efficiency of labeled PTD-OD-HA at different doses and time intervals was observed under fluorescence microscope. The location of labeled PTD-OD-HA fusion protein in K562 cells was detected by confocal microscopy. The interaction of PTD-OD-HA fusion protein with Bcr-Abl oncoprotein was confirmed by coimmunoprecipitation. The phosphorylation of Bcr-Abl oncoprotein was detected by Western blotting. Results　PTD-OD-HA fusion protein labeled with FITC was transduced into K562 cells in a dose- and time-dependent manner. PTD-OD-HA fusion protein was located in the cytoplasm of K562 cells and was consistent with the location of Bcr-Abl oncoprotein. The interaction of PTD-OD-HA fusion protein with Bcr-Abl oncoprotein was proved in K562 cells. This interaction could interrupt the homologous oligomerization of Bcr-Abl oncoprotein and reduce the phosphorylation of Bcr-Abl oncoprotein. Conclusion　PTD-OD-HA fusion protein could be transduced into K562 cells efficiently, inhibit the oligomerization and reduce the phosphorylation of Bcr-Abl oncoprotein.

Production of recombinant proteins in Escherichia coli tagged with the fusion protein CusF3H.

Science.gov (United States)

Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

2017-04-01

Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

Production of N-acetyl-D-neuraminic acid using two sequential enzymes overexpressed as double-tagged fusion proteins

Directory of Open Access Journals (Sweden)

Cheng Chung-Hsien

2009-07-01

Full Text Available Abstract Background Two sequential enzymes in the production of sialic acids, N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase, were overexpressed as double-tagged gene fusions. Both were tagged with glutathione S-transferase (GST at the N-terminus, but at the C-terminus, one was tagged with five contiguous aspartate residues (5D, and the other with five contiguous arginine residues (5R. Results Both fusion proteins were overexpressed in Escherichia coli and retained enzymatic activity. The fusions were designed so their surfaces were charged under enzyme reaction conditions, which allowed isolation and immobilization in a single step, through a simple capture with either an anionic or a cationic exchanger (Sepharose Q or Sepharose SP that electrostatically bound the 5D or 5R tag. The introduction of double tags only marginally altered the affinity of the enzymes for their substrates, and the double-tagged proteins were enzymatically active in both soluble and immobilized forms. Combined use of the fusion proteins led to the production of N-acetyl-D-neuraminic acid (Neu5Ac from N-acetyl-D-glucosamine (GlcNAc. Conclusion Double-tagged gene fusions were overexpressed to yield two enzymes that perform sequential steps in sialic acid synthesis. The proteins were easily immobilized via ionic tags onto ionic exchange resins and could thus be purified by direct capture from crude protein extracts. The immobilized, double-tagged proteins were effective for one-pot enzymatic production of sialic acid.

Rapamycin-induced oligomer formation system of FRB-FKBP fusion proteins.

Science.gov (United States)

Inobe, Tomonao; Nukina, Nobuyuki

2016-07-01

Most proteins form larger protein complexes and perform multiple functions in the cell. Thus, artificial regulation of protein complex formation controls the cellular functions that involve protein complexes. Although several artificial dimerization systems have already been used for numerous applications in biomedical research, cellular protein complexes form not only simple dimers but also larger oligomers. In this study, we showed that fusion proteins comprising the induced heterodimer formation proteins FRB and FKBP formed various oligomers upon addition of rapamycin. By adjusting the configuration of fusion proteins, we succeeded in generating an inducible tetramer formation system. Proteins of interest also formed tetramers by fusing to the inducible tetramer formation system, which exhibits its utility in a broad range of biological applications. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Functional NifD-K fusion protein in Azotobacter vinelandii is a homodimeric complex equivalent to the native heterotetrameric MoFe protein

International Nuclear Information System (INIS)

Lahiri, Surobhi; Pulakat, Lakshmi; Gavini, Nara

2005-01-01

The MoFe protein of the complex metalloenzyme nitrogenase folds as a heterotetramer containing two copies each of the homologous α and β subunits, encoded by the nifD and the nifK genes respectively. Recently, the functional expression of a fusion NifD-K protein of nitrogenase was demonstrated in Azotobacter vinelandii, strongly implying that the MoFe protein is flexible as it could accommodate major structural changes, yet remain functional [M.H. Suh, L. Pulakat, N. Gavini, J. Biol. Chem. 278 (2003) 5353-5360]. This finding led us to further explore the type of interaction between the fused MoFe protein units. We aimed to determine whether an interaction exists between the two fusion MoFe proteins to form a homodimer that is equivalent to native heterotetrameric MoFe protein. Using the Bacteriomatch Two-Hybrid System, translationally fused constructs of NifD-K (fusion) with the full-length λCI of the pBT bait vector and also NifD-K (fusion) with the N-terminal α-RNAP of the pTRG target vector were made. To compare the extent of interaction between the fused NifD-K proteins to that of the β-β interactions in the native MoFe protein, we proceeded to generate translationally fused constructs of NifK with the α-RNAP of the pTRG vector and λCI protein of the pBT vector. The strength of the interaction between the proteins in study was determined by measuring the β-galactosidase activity and extent of ampicillin resistance of the colonies expressing these proteins. This analysis demonstrated that direct protein-protein interaction exists between NifD-K fusion proteins, suggesting that they exist as homodimers. As the interaction takes place at the β-interfaces of the NifD-K fusion proteins, we propose that these homodimers of NifD-K fusion protein may function in a similar manner as that of the heterotetrameric native MoFe protein. The observation that the extent of protein-protein interaction between the β-subunits of the native MoFe protein in Bacterio

SOLASE: a conceptual laser fusion reactor design

International Nuclear Information System (INIS)

Conn, R.W.; Abdel-Khalik, S.I.; Moses, G.A.

1977-12-01

The SOLASE conceptual laser fusion reactor has been designed to elucidate the technological problems posed by inertial confinement fusion reactors. This report contains a detailed description of all aspects of the study including the physics of pellet implosion and burn, optics and target illumination, last mirror design, laser system analysis, cavity design, pellet fabrication and delivery, vacuum system requirements, blanket design, thermal hydraulics, tritium analysis, neutronics calculations, radiation effects, stress analysis, shield design, reactor and plant building layout, maintenance procedures, and power cycle design. The reactor is designed as a 1000 MW/sub e/ unit for central station electric power generation

SOLASE: a conceptual laser fusion reactor design

International Nuclear Information System (INIS)

Conn, R.W.; Abdel-Khalik, S.I.; Moses, G.A.

1977-12-01

The SOLASE conceptual laser fusion reactor has been designed to elucidate the technological problems posed by inertial confinement fusion ractors. This report contains a detailed description of all aspects of the study including the physics of pellet implosion and burn, optics and target illumination, last mirror design, laser system analysis, cavity design, pellet fabrication and delivery, vacuum system requirements, blanket design, thermal hydraulics, tritium analysis, neutronics calculations, radiation effects, stress analysis, shield design, reactor and plant building layout, maintenance procedures, and power cycle design. The reactor is designed as a 1000 MW/sub e/ unit for central station electric power generation

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

Science.gov (United States)

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

2016-07-08

Avian metapneumovirus (aMPV) fusion (F) protein mediates virus-cell membrane fusion to initiate viral infection, which requires F protein binding to its receptor(s) on the host cell surface. However, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV (aMPV/B) F proteins contain a conserved Arg-Asp-Asp (RDD) motif, suggesting that the aMPV/B F protein may mediate membrane fusion via the binding of RDD to integrin. When blocked with integrin-specific peptides, aMPV/B F protein fusogenicity and viral replication were significantly reduced. Specifically we identified integrin αv and/or β1-mediated F protein fusogenicity and viral replication using antibody blocking, small interfering RNAs (siRNAs) knockdown, and overexpression. Additionally, overexpression of integrin αv and β1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. When RDD was altered to RAE (Arg-Ala-Glu), aMPV/B F protein binding and fusogenic activity were profoundly impaired. These results suggest that integrin αvβ1 is a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which will provide new insights on the fusogenic mechanism and pathogenesis of aMPV. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Science.gov (United States)

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

2016-01-01

Avian metapneumovirus (aMPV) fusion (F) protein mediates virus-cell membrane fusion to initiate viral infection, which requires F protein binding to its receptor(s) on the host cell surface. However, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV (aMPV/B) F proteins contain a conserved Arg-Asp-Asp (RDD) motif, suggesting that the aMPV/B F protein may mediate membrane fusion via the binding of RDD to integrin. When blocked with integrin-specific peptides, aMPV/B F protein fusogenicity and viral replication were significantly reduced. Specifically we identified integrin αv and/or β1-mediated F protein fusogenicity and viral replication using antibody blocking, small interfering RNAs (siRNAs) knockdown, and overexpression. Additionally, overexpression of integrin αv and β1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. When RDD was altered to RAE (Arg-Ala-Glu), aMPV/B F protein binding and fusogenic activity were profoundly impaired. These results suggest that integrin αvβ1 is a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which will provide new insights on the fusogenic mechanism and pathogenesis of aMPV. PMID:27226547

Radiological design criteria for fusion power test facilities

International Nuclear Information System (INIS)

Singh, M.S.; Campbell, G.W.

1982-01-01

The quest for fusion power and understanding of plasma physics has resulted in planning, design, and construction of several major fusion power test facilities, based largely on magnetic and inertial confinement concepts. We have considered radiological design aspects of the Joint European Torus (JET), Livermore Mirror and Inertial Fusion projects, and Princeton Tokamak. Our analyses on radiological design criteria cover acceptable exposure levels at the site boundary, man-rem doses for plant personnel and population at large, based upon experience gained for the fission reactors, and on considerations of cost-benefit analyses

Fusion reactor design: On the road to commercialization

International Nuclear Information System (INIS)

Kulcinski, G.L.

1984-01-01

The worldwide effort in fusion is now approximately 2 billion dollars per year and over 12 billion dollars has been invested since 1951 in developing this energy source for the 21st century. A vital component of the past efforts in fusion research has been the conceptual design activities performed by scientists and engineers around the world. Almost 80 such major designs of Tokamak, Mirror, Laser and Ion Beam Reactors have been published and this article discusses how recent conceptual designs have afftected our perception of future fusion reactor performance. (orig.) [de

Conservation of a proteinase cleavage site between an insect retrovirus (gypsy) Env protein and a baculovirus envelope fusion protein

International Nuclear Information System (INIS)

Pearson, Margot N.; Rohrmann, George F.

2004-01-01

The predicted Env protein of insect retroviruses (errantiviruses) is related to the envelope fusion protein of a major division of the Baculoviridae. The highest degree of homology is found in a region that contains a furin cleavage site in the baculovirus proteins and an adjacent sequence that has the properties of a fusion peptide. In this investigation, the homologous region in the Env protein of the gypsy retrovirus of Drosophila melanogaster (DmegypV) was investigated. Alteration of the predicted DmegypV Env proteinase cleavage site from RIAR to AIAR significantly reduced cleavage of Env in both Spodoptera frugiperda (Sf-9) and D. melanogaster (S2) cell lines. When the predicted DmegypV Env cleavage site RIAR was substituted for the cleavage sequence RRKR in the Lymantria dispar nucleopolyhedrovirus fusion protein (LD130) sequence, cleavage of the hybrid LD130 molecules still occurred, although at a reduced level. The conserved 21-amino acid sequence just downstream of the cleavage site, which is thought to be the fusion peptide in LD130, was also characterized. When this sequence from DmegypV Env was substituted for the homologous sequence in LD130, cleavage still occurred, but no fusion was observed in either cell type. In addition, although a DmegypV-Env-green fluorescent protein construct localized to cell membranes, no cell fusion was observed

Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli.

Science.gov (United States)

Khademi, Farzad; Yousefi-Avarvand, Arshid; Derakhshan, Mohammad; Meshkat, Zahra; Tafaghodi, Mohsen; Ghazvini, Kiarash; Aryan, Ehsan; Sankian, Mojtaba

2017-10-01

The purpose of this study was to clone, express, and purify a novel multidomain fusion protein of Micobacterium tuberculosis (Mtb) in a prokaryotic system. An hspX/esxS gene construct was synthesized and ligated into a pGH plasmid, E. coli TOP10 cells were transformed, and the vector was purified. The vector containing the construct and pET-21b (+) plasmid were digested with the same enzymes and the construct was ligated into pET-21b (+). The accuracy of cloning was confirmed by colony PCR and sequencing. E. coli BL21 cells were transformed with the pET-21b (+)/hspX/esxS expression vector and protein expression was evaluated. Finally, the expressed fusion protein was purified on a Ni-IDA column and verified by SDS-PAGE and western blotting. The hspX/esxS gene construct was inserted into pET-21b (+) and recombinant protein expression was induced with IPTG in E. coli BL21 cells. Various concentrations of IPTG were tested to determine the optimum concentration for expression induction. The recombinant protein was expressed in insoluble inclusion bodies. Three molar guanidine HCl was used to solubilize the insoluble protein. An HspX/EsxS Mtb fusion protein was expressed in E. coli and the recombinant protein was purified. After immunological analysis, the HspX/EsxS fusion protein might be an anti-tuberculosis vaccine candidate in future clinical trial studies.

Fusion Engineering Device. Volume II. Design description

International Nuclear Information System (INIS)

1981-10-01

This volume summarizes the design of the FED. It includes a description of the major systems and subsystems, the supporting plasma design analysis, a projected device cost and associated construction schedule, and a description of the facilities to house and support the device. This effort represents the culmination of the FY81 studies conducted at the Fusion Engineering Design Center (FEDC). Unique in these design activities has been the collaborative involvement of the Design Center personnel and numerous resource physicists from the fusion community who have made significant contributions in the physics design analysis as well as the physics support of the engineering design of the major FED systems and components

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

International Nuclear Information System (INIS)

Mukai, Atsushi; Hashimoto, Naohiro

2008-01-01

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

Novel fusion protein approach for efficient high-throughput screening of small molecule-mediating protein-protein interactions in cells and living animals.

Science.gov (United States)

Paulmurugan, Ramasamy; Gambhir, Sanjiv S

2005-08-15

Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule-mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction-mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGSFACGSLSCGSF. A 9 +/- 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation.

Regulation of Exocytotic Fusion Pores by SNARE Protein Transmembrane Domains

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Zhenyong Wu

2017-10-01

Full Text Available Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs. Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble proteins called Complexins (Cpx and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs of SNARE proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores

Conceptual design study for a laser fusion hybrid

International Nuclear Information System (INIS)

Maniscalco, J.A.

1976-01-01

Lawrence Livermore Laboratory and Bechtel Corporation have been involved in a joint effort to conceptually design a laser fusion hybrid reactor. The design which has evolved is a depleted-uranium fueled fast-fission blanket which produces fissile plutonium and electricity. A major objective of the design study was to evaluate the feasibility of producing fissile fuel with laser fusion. This feasibility evaluation was carried out by analyzing the integrated engineering performance of the complete conceptual design and by identifying the required laser/pellet performance. The performance of the laser fusion hybrid has also been compared to a typical fast breeder reactor. The results show that the laser fusion hybrid produces enough fissile material to fuel more than six light water reactors (LWRs) of equivalent thermal power while operating in a regime which requires an order of magnitude less laser and pellet performance than pure laser fusion. In comparison to a fast breeder reactor the hybrid produces 10 times more fissile fuel. An economic analysis of the design shows that the cost of electricity in a combined hybrid-LWR scenario increases by only 20 to 40 percent when the capital cost of the hybrid ranges from 2 to 3 times more than an LWR

Conceptual design study for a laser fusion hybrid

International Nuclear Information System (INIS)

Maniscalco, J.A.

1976-09-01

Lawrence Livermore Laboratory and Bechtel Corporation have been involved in a joint effort to conceptually design a laser fusion hybrid reactor. The design which has evolved is a depleted-uranium fueled fast-fission blanket which produces fissile plutonium and electricity. A major objective of the design study was to evaluate the feasibility of producing fissile fuel with laser fusion. This feasibility evaluation was carried out by analyzing the integrated engineering performance of the complete conceptual design and by identifying the required laser/pellet performance. The performance of the laser fusion hybrid has also been compared to a typical fast breeder reactor. The results show that the laser fusion hybrid produces enough fissile material to fuel more than six light water reactors (LWR's) of equivalent thermal power while operating in a regime which requires an order of magnitude less laser and pellet performance than pure laser fusion. In comparison to a fast breeder reactor the hybrid produces 10 times more fissile fuel. An economic analysis of the design shows that the cost of electricity in a combined hybrid-LWR scenario is insensitive to the capital cost of the hybrid, increasing by only 20 to 40 percent when the capital cost of the hybrid ranges from 2 to 3 times more than an LWR

Fusion proteins useful for producing pinene

Energy Technology Data Exchange (ETDEWEB)

Peralta-Yahya, Pamela P.; Keasling, Jay D

2016-06-28

The present invention provides for a modified host cell comprising a heterologous pinene synthase (PS), or enzymatically active fragment or variant thereof, and optionally a geranyl pyrophosphate synthase (GPPS), or enzymatically active fragment or variant thereof, or a fusion protein comprising: (a) a PS and (b) a GPPS linked by a linker.

Neutronics issues in fusion-fission hybrid reactor design

International Nuclear Information System (INIS)

Liu Chengan

1995-01-01

The coupled neutron and γ-ray transport equations and nuclear number density equations, and its computer program systems concerned in fusion-fission hybrid reactor design are briefly described. The current status and focal point for coming work of nuclear data used in fusion reactor design are explained

Characterization of the fusion core in zebrafish endogenous retroviral envelope protein

Energy Technology Data Exchange (ETDEWEB)

Shi, Jian [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072 (China); State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China); Zhang, Huaidong [CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China); Gong, Rui, E-mail: gongr@wh.iov.cn [CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China); Xiao, Gengfu, E-mail: xiaogf@wh.iov.cn [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072 (China); State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China)

2015-05-08

Zebrafish endogenous retrovirus (ZFERV) is the unique endogenous retrovirus in zebrafish, as yet, containing intact open reading frames of its envelope protein gene in zebrafish genome. Similarly, several envelope proteins of endogenous retroviruses in human and other mammalian animal genomes (such as syncytin-1 and 2 in human, syncytin-A and B in mouse) were identified and shown to be functional in induction of cell–cell fusion involved in placental development. ZFERV envelope protein (Env) gene appears to be also functional in vivo because it is expressible. After sequence alignment, we found ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR) which were crucial for membrane fusion. We expressed the regions of N + C protein in the ZFERV Env (residues 459–567, including predicted NHR and CHR) to characterize the fusion core structure. We found N + C protein could form a stable coiled-coil trimer that consists of three helical NHR regions forming a central trimeric core, and three helical CHR regions packing into the grooves on the surface of the central core. The structural characterization of the fusion core revealed the possible mechanism of fusion mediated by ZFERV Env. These results gave comprehensive explanation of how the ancient virus infects the zebrafish and integrates into the genome million years ago, and showed a rational clue for discovery of physiological significance (e.g., medicate cell–cell fusion). - Highlights: • ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes. • The fusion core of ZFERV Env forms stable coiled-coil trimer including three NHRs and three CHRs. • The structural mechanism of viral entry mediated by ZFERV Env is disclosed. • The results are helpful for further discovery of physiological function of ZFERV Env in zebrafish.

Present status of inertial confinement fusion reactor design

International Nuclear Information System (INIS)

Mima, Kunioki; Ido, Shunji; Nakai, Sadao.

1986-01-01

Since inertial nuclear fusion reactors do not require high vacuum and high magnetic field, the structure of the reactor cavity becomes markedly simple as compared with tokamak type fusion reactors. In particular, since high vacuum is not necessary, liquid metals such as lithium and lead can be used for the first wall, and the damage of reactor structures by neutrons can be prevented. As for the core, the energy efficiency of lasers is not very high, accordingly it must be designed so that the pellet gain due to nuclear fusion becomes sufficiently high, and typically, the gain coefficient from 100 to 200 is necessary. In this paper, the perspective of pellet gain, the plan from the present status to the practical reactors, and the conceptual design of the practical reactors are discussed. The plan of fuel ignition, energy break-even and high gain by the implosion mode, of which the uncertain factor due to uneven irradiation and instability was limited to the minimum, was clarified. The scenario of the development of laser nuclear fusion reactors is presented, and the concept of the reactor system is shown. The various types of nuclear fusion-fission hybrid reactors are explained. As for the design of inertial fusion power reactors, the engineering characteristics of the core, the conceptual design, water fall type reactors and DD fuel reactors are discussed. (Kako, I.)

A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

Science.gov (United States)

Kajino, Tsutomu; Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Udaka, Shigezo; Yamada, Yukio; Takahashi, Haruo

2000-01-01

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system. PMID:10653729

A design of steady state fusion burner

International Nuclear Information System (INIS)

Hasegawa, Akira; Hatori, Tadatsugu; Itoh, Kimitaka; Ikuta, Takashi; Kodama, Yuji.

1975-01-01

We present a brief design of a steady state fusion burner in which a continuous burning of nuclear fuel may be achieved with output power of a gigawatt. The laser fusion is proposed to ignite the fuel. (auth.)

Expression, purification, crystallization and preliminary X-ray crystallographic studies of hepatitis B virus core fusion protein corresponding to octahedral particles

International Nuclear Information System (INIS)

Kikuchi, Masaki; Iwabuchi, Shinichiro; Kikkou, Tatsuhiko; Noguchi, Keiichi; Odaka, Masafumi; Yohda, Masafumi; Kawata, Masaaki; Sato, Chikara; Matsumoto, Osamu

2013-01-01

Novel hepatitis B virus-like particles of recombinant dimeric core–GFP fusion protein were expressed, purified and crystallized. The crystals diffracted to 2.15 Å resolution and belonged to space group F432, with unit-cell parameters a = b = c = 219.7 Å. Recombinant hepatitis B virus core proteins dimerize to form building blocks that are capable of self-assembly into a capsid. A core capsid protein dimer (CPD) linked to a green fluorescent protein variant, EGFP, at the C-terminus has been designed. The recombinant fusion CPD was expressed in Escherichia coli, assembled into virus-like particles (VLPs), purified and crystallized. The single crystal diffracted to 2.15 Å resolution and belonged to the cubic space group F432, with unit-cell parameters a = b = c = 219.7 Å. The fusion proteins assembled into icosahedral VLPs in aqueous solution, but were rearranged into octahedral symmetry through the crystal-packing process under the crystallization conditions

The yeast cell fusion protein Prm1p requires covalent dimerization to promote membrane fusion.

Directory of Open Access Journals (Sweden)

Alex Engel

2010-05-01

Full Text Available Prm1p is a multipass membrane protein that promotes plasma membrane fusion during yeast mating. The mechanism by which Prm1p and other putative regulators of developmentally controlled cell-cell fusion events facilitate membrane fusion has remained largely elusive. Here, we report that Prm1p forms covalently linked homodimers. Covalent Prm1p dimer formation occurs via intermolecular disulfide bonds of two cysteines, Cys-120 and Cys-545. PRM1 mutants in which these cysteines have been substituted are fusion defective. These PRM1 mutants are normally expressed, retain homotypic interaction and can traffic to the fusion zone. Because prm1-C120S and prm1-C545S mutants can form covalent dimers when coexpressed with wild-type PRM1, an intermolecular C120-C545 disulfide linkage is inferred. Cys-120 is adjacent to a highly conserved hydrophobic domain. Mutation of a charged residue within this hydrophobic domain abrogates formation of covalent dimers, trafficking to the fusion zone, and fusion-promoting activity. The importance of intermolecular disulfide bonding informs models regarding the mechanism of Prm1-mediated cell-cell fusion.

Antibody-cytokine fusion proteins for improving efficacy and safety of cancer therapy.

Science.gov (United States)

Valedkarimi, Zahra; Nasiri, Hadi; Aghebati-Maleki, Leili; Majidi, Jafar

2017-11-01

Cytokines are key players in the regulation of immune responses both in physiological and pathological states. A number of cytokines have been evaluated in clinical trials and shown promising results in the treatment of different malignancies. Despite this, the clinical application of these molecules may be plagued by undesirable side effects The development of recombinant antibody-cytokine fusion proteins, which offer a means for target delivery of cytokines toward the tumor site, has significantly improved the therapeutic index of these immunomodulatory molecules. Selective tumor localization is provided by the monoclonal antibody component of the fusion protein that binds to the molecules present on the surface of tumor cells or accumulated preferentially in the diseased site. In this manner, the cytokine element is specifically located at the tumor site and can stimulate immune cells with appropriate cytokine receptors. Over the recent years, several antibody-cytokine fusion proteins have been developed with the capacity to target a wide variety of cancers whose application, in some cases, has led to complete rejection of the tumor. These findings support the notion that antibody-cytokine fusion proteins represent huge potential for cancer therapy. This review presents an overview of the advances made in the field of targeted cytokine delivery, which is made possible by genetically engineering antibody-cytokine fusion proteins. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Overview of Fusion-Fission Hybrid Reactor Design Study in China

International Nuclear Information System (INIS)

Huang Jinhua; Feng Kaiming; Deng Baiquan; Deng, P.Zh.; Zhang Guoshu; Hu Gang; He Kaihui; Wu Yican; Qiu Lijian; Huang Qunying; Xiao Bingjia; Liu Xiaoping; Chen Yixue; Kong, M.H.

2002-01-01

The motivation for developing fusion-fission hybrid reactors is discussed in the context of electricity power requirements by 2050 in China. A detailed conceptual design of the Fusion Experimental Breeder (FEB) was developed from 1986-1995. The FEB has a subignited tokamak fusion core with a major radius of 4.0 m, a fusion power of 145 MW, and a fusion energy gain Q of 3. Based on this, an engineering outline design study of the FEB, FEB-E, has been performed. This design study is a transition from conceptual to engineering design in this research. The main results beyond that given in the detailed conceptual design are included in this paper, namely, the design studies of the blanket, divertor, test blanket, and tritium and environment issues. In-depth analyses have been performed to support the design. Studies of related advanced concepts such as the waste transmutation blanket concept and the spherical tokamak core concept are also presented

Challenges of designing fusion reactors for remote maintainability

International Nuclear Information System (INIS)

Masson, L.S.

1981-01-01

One of the major problems faced by the fusion community is the development of the high level of reliability required to assure that fusion will be a viable commercial power source. Much of the responsibility for solving this problem falls directly on the designer in developing concepts that have a high level of maintainability for the next generation engineering oriented reactors; and long range, in developing full maintainability for the more complicated commercial concepts with their required high level of on-line time. The near-term challenge will include development of unique design concepts to perform inspection, maintenance, replacement, and testing under the stringent conditions imposed by the next generation engineering oriented machines. The long range challenge will focus on basic design concepts that will enable the full maintainability required by commercial fusion. In addition to the purely technical challenges, the fusion community is also faced with the problem of developing programmatic means to assure that reactor maintenance issues are given proper and timely emphasis as the nuclear phase of fusion is approached

Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization

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Sanjukta Chakrabarti

2016-06-01

Full Text Available Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1–D3-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities.

Applying design principles to fusion reactor configurations for propulsion in space

International Nuclear Information System (INIS)

Carpenter, S.A.; Deveny, M.E.; Schulze, N.R.

1993-01-01

The application of fusion power to space propulsion requires rethinking the engineering-design solution to controlled-fusion energy. Whereas the unit cost of electricity (COE) drives the engineering-design solution for utility-based fusion reactor configurations; initial mass to low earth orbit (IMLEO), specific jet power (kW(thrust)/kg(engine)), and reusability drive the engineering-design solution for successful application of fusion power to space propulsion. Three design principles (DP's) were applied to adapt and optimize three candidate-terrestrial-fusion-reactor configurations for propulsion in space. The three design principles are: provide maximum direct access to space for waste radiation, operate components as passive radiators to minimize cooling-system mass, and optimize the plasma fuel, fuel mix, and temperature for best specific jet power. The three candidate terrestrial fusion reactor configurations are: the thermal barrier tandem mirror (TBTM), field reversed mirror (FRM), and levitated dipole field (LDF). The resulting three candidate space fusion propulsion systems have their IMLEO minimized and their specific jet power and reusability maximized. A preliminary rating of these configurations was performed, and it was concluded that the leading engineering-design solution to space fusion propulsion is a modified TBTM that we call the Mirror Fusion Propulsion System (MFPS)

Advances in fusion reactor design

International Nuclear Information System (INIS)

Baker, C.C.

1987-01-01

The author addresses the tokamak as a power reactor. Contrary to popular opinion, there are still a few people that think a tokamak might make a good fusion power reactor. In thinking about advances in fusion reactor design, in the U.S., at least, that generally means advances relevant to the Starfire design. He reviews some of the features of Starfire. Starfire is the last major study done of the tokamak as a reactor in this country. It is now over eight years old in the sense that eight years ago was really the time in which major decisions were made as to its features. Starfire was a tokamak with a major radius of seven meters, about twice the linear dimensions of a machine like TIBER

Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins.

Science.gov (United States)

Mousa, Jarrod J; Binshtein, Elad; Human, Stacey; Fong, Rachel H; Alvarado, Gabriela; Doranz, Benjamin J; Moore, Martin L; Ohi, Melanie D; Crowe, James E

2018-02-01

Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.

Materials design data for fusion reactors

International Nuclear Information System (INIS)

Tavassoli, A.A.F.

1998-01-01

Design data needed for fusion reactors are characterized by the diversity of materials and the complexity of loading situations found in these reactors. In addition, advanced fabrication techniques, such as hot isostatic pressing, envisaged for fabrication of single and multilayered in-vessel components, could significantly change the original materials properties for which the current design rules are written. As a result, additional materials properties have had to be generated for fusion reactors and new structural design rules formulated. This paper recalls some of the materials properties data generated for ITER and DEMO, and gives examples of how these are converted into design criteria. In particular, it gives specific examples for the properties of 316LN-IG and modified 9Cr-1Mo steels, and CuCrZr alloy. These include, determination of tension, creep, isochronous, fatigue, and creep-fatigue curves and their analysis and conversion into design limits. (orig.)

Materials design data for fusion reactors

Energy Technology Data Exchange (ETDEWEB)

Tavassoli, A.A.F. [CEA Commissariat a l`Energie Atomique, Gif sur Yvette (France). CEREM

1998-10-01

Design data needed for fusion reactors are characterized by the diversity of materials and the complexity of loading situations found in these reactors. In addition, advanced fabrication techniques, such as hot isostatic pressing, envisaged for fabrication of single and multilayered in-vessel components, could significantly change the original materials properties for which the current design rules are written. As a result, additional materials properties have had to be generated for fusion reactors and new structural design rules formulated. This paper recalls some of the materials properties data generated for ITER and DEMO, and gives examples of how these are converted into design criteria. In particular, it gives specific examples for the properties of 316LN-IG and modified 9Cr-1Mo steels, and CuCrZr alloy. These include, determination of tension, creep, isochronous, fatigue, and creep-fatigue curves and their analysis and conversion into design limits. (orig.) 19 refs.

Initial trade and design studies for the fusion engineering device

International Nuclear Information System (INIS)

Flanagan, C.A.; Steiner, D.; Smith, G.E.

1981-06-01

The Magnetic Fusion Energy Engineering Act of 1980 calls for the operation of a Fusion Engineering Device (FED) by 1990. It is the intent of the Act that the FED, in combination with other testing facilities, will establish the engineering feasibility of magnetic fusion energy. The Fusion Engineering Design Center (FEDC), under the guidance of a Technical Management Board (TMB), initiated a program of trade and design studies in October 1980 to support the selection of the FED concept. This document presents the results of these initial trade and design studies. Based on these results, a baseline configuration has been identified and the Design Center effort for the remainder of the fiscal year will be devoted to the development of a self-consistent FED design description

Blood-brain barrier drug delivery of IgG fusion proteins with a transferrin receptor monoclonal antibody.

Science.gov (United States)

Pardridge, William M

2015-02-01

Biologic drugs are large molecules that do not cross the blood- brain barrier (BBB). Brain penetration is possible following the re-engineering of the biologic drug as an IgG fusion protein. The IgG domain is a MAb against an endogenous BBB receptor such as the transferrin receptor (TfR). The TfRMAb acts as a molecular Trojan horse to ferry the fused biologic drug into the brain via receptor-mediated transport on the endogenous BBB TfR. This review discusses TfR isoforms, models of BBB transport of transferrin and TfRMAbs, and the genetic engineering of TfRMAb fusion proteins, including BBB penetrating IgG-neurotrophins, IgG-decoy receptors, IgG-lysosomal enzyme therapeutics and IgG-avidin fusion proteins, as well as BBB transport of bispecific antibodies formed by fusion of a therapeutic antibody to a TfRMAb targeting antibody. Also discussed are quantitative aspects of the plasma pharmacokinetics and brain uptake of TfRMAb fusion proteins, as compared to the brain uptake of small molecules, and therapeutic applications of TfRMAb fusion proteins in mouse models of neural disease, including Parkinson's disease, stroke, Alzheimer's disease and lysosomal storage disorders. The review covers the engineering of TfRMAb-avidin fusion proteins for BBB targeted delivery of biotinylated peptide radiopharmaceuticals, low-affinity TfRMAb Trojan horses and the safety pharmacology of chronic administration of TfRMAb fusion proteins. The BBB delivery of biologic drugs is possible following re-engineering as a fusion protein with a molecular Trojan horse such as a TfRMAb. The efficacy of this technology will be determined by the outcome of future clinical trials.

Induction of protein body formation in plant leaves by elastin-like polypeptide fusions

Directory of Open Access Journals (Sweden)

Joensuu Jussi J

2009-08-01

Full Text Available Abstract Background Elastin-like polypeptides are synthetic biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the simple non-chromatographic purification of recombinant proteins. In addition, elastin-like polypeptide fusions have been shown to enhance the accumulation of a range of different recombinant proteins in plants, thus addressing the major limitation of plant-based expression systems, which is a low production yield. This study's main objectives were to determine the general utility of elastin-like polypeptide protein fusions in various intracellular compartments and to elucidate elastin-like polypeptide's mechanism of action for increasing recombinant protein accumulation in the endoplasmic reticulum of plants. Results The effect of elastin-like polypeptide fusions on the accumulation of green fluorescent protein targeted to the cytoplasm, chloroplasts, apoplast, and endoplasmic reticulum was evaluated. The endoplasmic reticulum was the only intracellular compartment in which an elastin-like polypeptide tag was shown to significantly enhance recombinant protein accumulation. Interestingly, endoplasmic reticulum-targeted elastin-like polypeptide fusions induced the formation of a novel type of protein body, which may be responsible for elastin-like polypeptide's positive effect on recombinant protein accumulation by excluding the heterologous protein from normal physiological turnover. Although expressed in the leaves of plants, these novel protein bodies appeared similar in size and morphology to the prolamin-based protein bodies naturally found in plant seeds. The elastin-like polypeptide-induced protein bodies were highly mobile organelles, exhibiting various dynamic patterns of movement throughout the cells, which were dependent on intact actin microfilaments and a functional actomyosin motility system. Conclusion An endoplasmic reticulum-targeted elastin-like polypeptide fusion approach

A fungal biofilm reactor based on metal structured packing improves the quality of a Gla::GFP fusion protein produced by Aspergillus oryzae.

Science.gov (United States)

Zune, Q; Delepierre, A; Gofflot, S; Bauwens, J; Twizere, J C; Punt, P J; Francis, F; Toye, D; Bawin, T; Delvigne, F

2015-08-01

Fungal biofilm is known to promote the excretion of secondary metabolites in accordance with solid-state-related physiological mechanisms. This work is based on the comparative analysis of classical submerged fermentation with a fungal biofilm reactor for the production of a Gla::green fluorescent protein (GFP) fusion protein by Aspergillus oryzae. The biofilm reactor comprises a metal structured packing allowing the attachment of the fungal biomass. Since the production of the target protein is under the control of the promoter glaB, specifically induced in solid-state fermentation, the biofilm mode of culture is expected to enhance the global productivity. Although production of the target protein was enhanced by using the biofilm mode of culture, we also found that fusion protein production is also significant when the submerged mode of culture is used. This result is related to high shear stress leading to biomass autolysis and leakage of intracellular fusion protein into the extracellular medium. Moreover, 2-D gel electrophoresis highlights the preservation of fusion protein integrity produced in biofilm conditions. Two fungal biofilm reactor designs were then investigated further, i.e. with full immersion of the packing or with medium recirculation on the packing, and the scale-up potentialities were evaluated. In this context, it has been shown that full immersion of the metal packing in the liquid medium during cultivation allows for a uniform colonization of the packing by the fungal biomass and leads to a better quality of the fusion protein.

Fusion protein-based biofilm fabrication composed of recombinant azurin–myoglobin for dual-level biomemory application

Energy Technology Data Exchange (ETDEWEB)

Lee, Taek [Research Institute for Basic Science, Sogang University, Heukseok-dong, Dongjak-gu, Seoul 156-756 (Korea, Republic of); Chung, Yong-Ho; Yoon, Jinho [Department of Chemical and Biomolecular Engineering, Sogang University, Heukseok-dong, Dongjak-gu, 35 Baekbeom-ro (Sinsu-dong), Mapo-gu, Seoul 121-742 (Korea, Republic of); Min, Junhong [School of Integrative Engineering, Chung-Ang University, Heukseok-dong, Dongjak-gu, Seoul 156-756 (Korea, Republic of); Choi, Jeong-Woo, E-mail: jwchoi@sogang.ac.kr [Department of Chemical and Biomolecular Engineering, Sogang University, Heukseok-dong, Dongjak-gu, 35 Baekbeom-ro (Sinsu-dong), Mapo-gu, Seoul 121-742 (Korea, Republic of)

2014-11-30

Graphical abstract: - Highlights: • We developed the fusion protein-based biofilm on the inorganic surface. • For making the fusion protein, the recombinant azurin and the myoglobin was conjugated by the native chemical ligation method. • The developed fusion protein shows unique electrochemical property. • The proposed fusion protein biofilm appears to be a good method for dual-level biomemory device. - Abstract: In the present study, a fusion protein-based biofilm composed of a recombinant azurin–myoglobin (Azu-Myo) has been developed and confirmed its original electrochemical property for dual-level biomemory device application. For this purpose, the azurin was modified with cysteine residues for direct immobilization and conjugation. Then, the recombinant azurin was conjugated with the myoglobin via a sulfo-SMCC bifunctional linker using the chemical ligation method (CLM). The SDS-PAGE and UV–vis spectroscopy were performed to examine the fusion protein conjugates. The prepared Azu-Myo fusion protein was self-assembled onto Au substrate for the biofilm fabrication. Then, the atomic force microscopy (AFM) was used to confirm the immobilization and the surface-enhanced Raman spectroscopy (SERS) was carried out to the surface analysis. Also, the cyclic voltammetry (CV) was carried out to observe an electrochemical property of fabricated biofilm. As a result, the two pair of redox potential values was obtained for dual-level biomemory device application. Then, the dual-level biomemory function was verified by the multi-potential chronoamperometry (MPCA). The results indicate a new fabrication method and material combination for advances in bioelectronic device development.

Fusion protein-based biofilm fabrication composed of recombinant azurin–myoglobin for dual-level biomemory application

International Nuclear Information System (INIS)

Lee, Taek; Chung, Yong-Ho; Yoon, Jinho; Min, Junhong; Choi, Jeong-Woo

2014-01-01

Graphical abstract: - Highlights: • We developed the fusion protein-based biofilm on the inorganic surface. • For making the fusion protein, the recombinant azurin and the myoglobin was conjugated by the native chemical ligation method. • The developed fusion protein shows unique electrochemical property. • The proposed fusion protein biofilm appears to be a good method for dual-level biomemory device. - Abstract: In the present study, a fusion protein-based biofilm composed of a recombinant azurin–myoglobin (Azu-Myo) has been developed and confirmed its original electrochemical property for dual-level biomemory device application. For this purpose, the azurin was modified with cysteine residues for direct immobilization and conjugation. Then, the recombinant azurin was conjugated with the myoglobin via a sulfo-SMCC bifunctional linker using the chemical ligation method (CLM). The SDS-PAGE and UV–vis spectroscopy were performed to examine the fusion protein conjugates. The prepared Azu-Myo fusion protein was self-assembled onto Au substrate for the biofilm fabrication. Then, the atomic force microscopy (AFM) was used to confirm the immobilization and the surface-enhanced Raman spectroscopy (SERS) was carried out to the surface analysis. Also, the cyclic voltammetry (CV) was carried out to observe an electrochemical property of fabricated biofilm. As a result, the two pair of redox potential values was obtained for dual-level biomemory device application. Then, the dual-level biomemory function was verified by the multi-potential chronoamperometry (MPCA). The results indicate a new fabrication method and material combination for advances in bioelectronic device development

Integrated Chamber Design for the Laser Inertial Fusion Energy (LIFE) Engine

International Nuclear Information System (INIS)

Latkowski, J.F.; Kramer, K.J.; Abbott, R.P.; Morris, K.R.; DeMuth, J.; Divol, L.; El-Dasher, B.; Lafuente, A.; Loosmore, G.; Reyes, S.; Moses, G.A.; Fratoni, M.; Flowers, D.; Aceves, S.; Rhodes, M.; Kane, J.; Scott, H.; Kramer, R.; Pantano, C.; Scullard, C.; Sawicki, R.; Wilks, S.; Mehl, M.

2010-01-01

The Laser Inertial Fusion Energy (LIFE) concept is being designed to operate as either a pure fusion or hybrid fusion-fission system. A key component of a LIFE engine is the fusion chamber subsystem. The present work details the chamber design for the pure fusion option. The fusion chamber consists of the first wall and blanket. This integrated system must absorb the fusion energy, produce fusion fuel to replace that burned in previous targets, and enable both target and laser beam transport to the ignition point. The chamber system also must mitigate target emissions, including ions, x-rays and neutrons and reset itself to enable operation at 10-15 Hz. Finally, the chamber must offer a high level of availability, which implies both a reasonable lifetime and the ability to rapidly replace damaged components. An integrated LIFE design that meets all of these requirements is described herein.

Integrated Chamber Design for the Laser Inertial Fusion Energy (LIFE) Engine

Energy Technology Data Exchange (ETDEWEB)

Latkowski, J F; Kramer, K J; Abbott, R P; Morris, K R; DeMuth, J; Divol, L; El-Dasher, B; Lafuente, A; Loosmore, G; Reyes, S; Moses, G A; Fratoni, M; Flowers, D; Aceves, S; Rhodes, M; Kane, J; Scott, H; Kramer, R; Pantano, C; Scullard, C; Sawicki, R; Wilks, S; Mehl, M

2010-12-07

The Laser Inertial Fusion Energy (LIFE) concept is being designed to operate as either a pure fusion or hybrid fusion-fission system. A key component of a LIFE engine is the fusion chamber subsystem. The present work details the chamber design for the pure fusion option. The fusion chamber consists of the first wall and blanket. This integrated system must absorb the fusion energy, produce fusion fuel to replace that burned in previous targets, and enable both target and laser beam transport to the ignition point. The chamber system also must mitigate target emissions, including ions, x-rays and neutrons and reset itself to enable operation at 10-15 Hz. Finally, the chamber must offer a high level of availability, which implies both a reasonable lifetime and the ability to rapidly replace damaged components. An integrated LIFE design that meets all of these requirements is described herein.

In vivo immobilization of fusion proteins on bioplastics by the novel tag BioF.

Science.gov (United States)

Moldes, Cristina; García, Pedro; García, José L; Prieto, María A

2004-06-01

A new protein immobilization and purification system has been developed based on the use of polyhydroxyalkanoates (PHAs, or bioplastics), which are biodegradable polymers accumulated as reserve granules in the cytoplasm of certain bacteria. The N-terminal domain of the PhaF phasin (a PHA-granule-associated protein) from Pseudomonas putida GPo1 was used as a polypeptide tag (BioF) to anchor fusion proteins to PHAs. This tag provides a novel way to immobilize proteins in vivo by using bioplastics as supports. The granules carrying the BioF fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble BioF fusion protein can be obtained by a mild detergent treatment of the granule. The efficiency of this system has been demonstrated by constructing two BioF fusion products, including a functional BioF-beta-galactosidase. This is the first example of an active bioplastic consisting of a biodegradable matrix carrying an active enzyme.

Spatiotemporal dynamics of membrane remodeling and fusion proteins during endocytic transport.

Science.gov (United States)

Arlt, Henning; Auffarth, Kathrin; Kurre, Rainer; Lisse, Dominik; Piehler, Jacob; Ungermann, Christian

2015-04-01

Organelles of the endolysosomal system undergo multiple fission and fusion events to combine sorting of selected proteins to the vacuole with endosomal recycling. This sorting requires a consecutive remodeling of the organelle surface in the course of endosomal maturation. Here we dissect the remodeling and fusion machinery on endosomes during the process of endocytosis. We traced selected GFP-tagged endosomal proteins relative to exogenously added fluorescently labeled α-factor on its way from the plasma membrane to the vacuole. Our data reveal that the machinery of endosomal fusion and ESCRT proteins has similar temporal localization on endosomes, whereas they precede the retromer cargo recognition complex. Neither deletion of retromer nor the fusion machinery with the vacuole affects this maturation process, although the kinetics seems to be delayed due to ESCRT deletion. Of importance, in strains lacking the active Rab7-like Ypt7 or the vacuolar SNARE fusion machinery, α-factor still proceeds to late endosomes with the same kinetics. This indicates that endosomal maturation is mainly controlled by the early endosomal fusion and remodeling machinery but not the downstream Rab Ypt7 or the SNARE machinery. Our data thus provide important further understanding of endosomal biogenesis in the context of cargo sorting. © 2015 Arlt et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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

International Nuclear Information System (INIS)

Nordlund, Henri R.; Laitinen, Olli H.; Uotila, Sanna T.H.; Kulmala, Minna; Kalkkinen, Nisse; Kulomaa, Markku S.

2005-01-01

The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches

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

Science.gov (United States)

Nordlund, Henri R; Laitinen, Olli H; Uotila, Sanna T H; Kulmala, Minna; Kalkkinen, Nisse; Kulomaa, Markku S

2005-10-14

The presented green fluorescent protein and streptavidin core-based tripartite fusion system provides a simple and efficient way for the production of proteins fused to it in insect cells. This fusion protein forms a unique tag, which serves as a multipurpose device enabling easy optimization of production, one-step purification via streptavidin-biotin interaction, and visualization of the fusion protein during downstream processing and in applications. In the present study, we demonstrate the successful production, purification, and detection of a natural rubber latex allergen Hev b5 with this system. We also describe the production of another NRL allergen with the system, Hev b1, which formed large aggregates and gave small yields in purification. The aggregates were detected at early steps by microscopical inspection of the infected insect cells producing this protein. Therefore, this fusion system can also be utilized as a fast indicator of the solubility of the expressed fusion proteins and may therefore be extremely useful in high-throughput expression approaches.

Characterization of HCoV-229E fusion core: Implications for structure basis of coronavirus membrane fusion

International Nuclear Information System (INIS)

Liu Cheng; Feng Youjun; Gao Feng; Zhang Qiangmin; Wang Ming

2006-01-01

Human coronavirus 229E (HCoV-229E), a member of group I coronaviruses, has been identified as one of the major viral agents causing respiratory tract diseases in humans for nearly 40 years. However, the detailed molecular mechanism of the membrane fusion mediated by the spike (S) protein of HCoV-229E remains elusive. Here, we report, for the first time, a rationally designed fusion core of HCoV-229E (HR1-SGGRGG-HR2), which was in vitro produced in GST prokaryotic expression system. Multiple lines of experimental data including gel-filtration, chemical cross-linking, and circular diagram (CD) demonstrated that the HCoV-229E fusion core possesses the typical properties of the trimer of coiled-coil heterodimer (six α-helix bundle). 3D structure modeling presents its most-likely structure, similar to those of coronaviruses that have been well-documented. Collectively, HCoV-229E S protein belongs to the type I fusion protein, which is characterized by the existence of two heptad-repeat regions (HR1 and HR2), furthermore, the available knowledge concerning HCoV-229E fusion core may make it possible to design small molecule or polypeptide drugs targeting the membrane fusion, a crucial step of HCoV-229E infection

Conceptual design of light ion beam inertia nuclear fusion reactors

International Nuclear Information System (INIS)

1983-07-01

Light ion beam, inertia nuclear fusion system drew attention recently as one of the nuclear fusion systems for power reactors in the history of the research on nuclear fusion. Its beginning seemed to be the judgement that the implosion of fusion fuel pellets with light ions can be realized with the light ions which can be obtained in view of accelerator techniques. Of course, in order to generate practically usable nuclear fusion reaction by this system and maintain it, many technical difficulties must be overcome. This research was carried out for the purpose of discovering such technical problems and searching for their solution. At the time of doing the works, the following policy was adopted. Though their is the difference of fine and rough, the design of a whole reactor system is performed conformably. In order to make comparison with other reactor types and nuclear fusion systems, the design is carried out as the power plant of about one million kWe output. As the extent of the design, the works at conceptual design stage are performed to present the concept of design which satisfies the required function. Basically, the design is made from conservative standpoint. This research of design was started in 1981, and in fiscal 1982, the mutual adjustment among the design of respective parts was performed on the basis of the results in 1981, and the possible revision and new proposal were investigated. (Kako, I.)

Fusion protein is the main determinant of metapneumovirus host tropism.

Science.gov (United States)

de Graaf, Miranda; Schrauwen, Eefje J A; Herfst, Sander; van Amerongen, Geert; Osterhaus, Albert D M E; Fouchier, Ron A M

2009-06-01

Human metapneumovirus (HMPV) and avian metapneumovirus subgroup C (AMPV-C) infect humans and birds, respectively. This study confirmed the difference in host range in turkey poults, and analysed the contribution of the individual metapneumovirus genes to host range in an in vitro cell-culture model. Mammalian Vero-118 cells supported replication of both HMPV and AMPV-C in contrast to avian quail fibroblast (QT6) cells in which only AMPV-C replicated to high titres. Inoculation of Vero-118 and QT6 cells with recombinant HMPV in which genes were exchanged with those of AMPV-C revealed that the metapneumovirus fusion (F) protein is the main determinant for host tropism. Chimeric viruses in which polymerase complex proteins were exchanged between HMPV and AMPV-C replicated less efficiently compared with HMPV in QT6 cells. Using mini-genome systems, it was shown that exchanging these polymerase proteins resulted in reduced replication and transcription efficiency in QT6 cells. Examination of infected Vero-118 and QT6 cells revealed that viruses containing the F protein of AMPV-C yielded larger syncytia compared with viruses containing the HMPV F protein. Cell-content mixing assays revealed that the F protein of AMPV-C was more fusogenic compared with the F protein of HMPV, and that the F2 region is responsible for the difference observed between AMPV-C and HMPV F-promoted fusion in QT6 and Vero-118 cells. This study provides insight into the determinants of host tropism and membrane fusion of metapneumoviruses.

Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

International Nuclear Information System (INIS)

Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga; Luque, Daniel; Terrón, María C.; Calder, Lesley J.; Melero, José A.

2014-01-01

Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV F occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV F , we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV F at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy

Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

Energy Technology Data Exchange (ETDEWEB)

Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Luque, Daniel; Terrón, María C. [Unidad de Microscopía Electrónica y Confocal, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Calder, Lesley J. [National Institute for Medical Research, MRC, Mill Hill, London NW7 1AA (United Kingdom); Melero, José A., E-mail: jmelero@isciii.es [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain)

2014-07-15

Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV{sub F} occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV{sub F}, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV{sub F} at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy.

Design of a hyperstable 60-subunit protein icosahedron

Science.gov (United States)

Hsia, Yang; Bale, Jacob B.; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K.; Nattermann, Una; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N.; Gonen, Tamir; King, Neil P.; Baker, David

2016-07-01

The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

Design of a fusion engineering test facility

International Nuclear Information System (INIS)

Sager, P.H.

1980-01-01

The fusion Engineering Test Facility (ETF) is being designed to provide for engineering testing capability in a program leading to the demonstration of fusion as a viable energy option. It will combine power-reactor-type components and subsystems into an integrated tokamak system and provide a test bed to test blanket modules in a fusion environment. Because of the uncertainties in impurity control two basic designs are being developed: a design with a bundle divertor (Design 1) and one with a poloidal divertor (Design 2). The two designs are similar where possible, the latter having somewhat larger toroidal field (TF) coils to accommodate removal of the larger torus sectors required for the single-null poloidal divertor. Both designs have a major radius of 5.4 m, a minor radius of 1.3 m, and a D-shaped plasma with an elongation of 1.6. Ten TF coils are incorporated in both designs, producing a toroidal field of 5.5 T on-axis. The ohmic heating and equilibrium field (EF) coils supply sufficient volt-seconds to produce a flat-top burn of 100 s and a duty cycle of 135 s, including a start of 12 s, a burn termination of 10 s, and a pumpdown of 13 s. The total fusion power during burn is 750 MW, giving a neutron wall loading of 1.5 MW/m 2 . In Design 1 of the poloidal field (PF) coils except the fast-response EF coils are located outside the FT coils and are superconducting. The fast-response coils are located inside the TF coil bore near the torus and are normal conducting so that they can be easily replaced.In Design 2 all of the PF coils are located outside the TF coils and are superconducting. Ignition is achieved with 60 MW of neutral beam injection at 150 keV. Five megawatts of radio frequency heating (electron cyclotron resonance heating) is used to assist in the startup and limit the breakdown requirement to 25 V

Conceptual design study of Fusion Experimental Reactor (FY87FER)

International Nuclear Information System (INIS)

1988-05-01

The design study of Fusion Experimental Reactor(FER) which has been proposed to be the next step fusion device has been conducted by JAERI Reactor System Laboratory since 1982 and by FER design team since 1984. This is the final report of the FER design team program and describes the results obtained in FY1987 (partially in FY1986) activities. The contents of this report consist of the reference design which is based on the guideline in FY1986 by the Subcomitees set up in Nuclear Fusion Council of Atomic Energy Commission of Japan, the Low-Physics-Risk reactor design for achieving physics mission more reliably and the system study of FER design candidates including above two designs. (author)

Incorporation of albumin fusion proteins into fibrin clots in vitro and in vivo: comparison of different fusion motifs recognized by factor XIIIa

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Sheffield William P

2011-12-01

Full Text Available Abstract Background The transglutaminase activated factor XIII (FXIIIa acts to strengthen pathological fibrin clots and to slow their dissolution, in part by crosslinking active α2-antiplasmin (α2AP to fibrin. We previously reported that a yeast-derived recombinant fusion protein comprising α2AP residues 13-42 linked to human serum albumin (HSA weakened in vitro clots but failed to become specifically incorporated into in vivo clots. In this study, our aims were to improve both the stability and clot localization of the HSA fusion protein by replacing α2AP residues 13-42 with shorter sequences recognized more effectively by FXIIIa. Results Expression plasmids were prepared encoding recombinant HSA with the following N-terminal 23 residue extensions: H6NQEQVSPLTLLAG4Y (designated XL1; H6DQMMLPWAVTLG4Y (XL2; H6WQHKIDLPYNGAG4Y (XL3; and their 17 residue non-His-tagged equivalents (XL4, XL5, and XL6. The HSA moiety of XL4- to XL6-HSA proteins was C-terminally His-tagged. All chimerae were efficiently secreted from transformed Pichia pastoris yeast except XL3-HSA, and following nickel chelate affinity purification were found to be intact by amino acid sequencing, as was an N-terminally His-tagged version of α2AP(13-42-HSA. Of the proteins tested, XL5-HSA was cross-linked to biotin pentylamine (BPA most rapidly by FXIIIa, and was the most effective competitor of α2AP crosslinking not only to BPA but also to plasma fibrin clots. In the mouse ferric chloride vena cava thrombosis model, radiolabeled XL5-HSA was retained in the clot to a greater extent than recombinant HSA. In the rabbit jugular vein stasis thrombosis model, XL5-HSA was also retained in the clot, in a urea-insensitive manner indicative of crosslinking to fibrin, to a greater extent than recombinant HSA. Conclusions Fusion protein XL5-HSA (DQMMLPWAVTLG4Y-HSAH6 was found to be more active as a substrate for FXIIIa-mediated transamidation than seven other candidate fusion proteins in

Measles Virus Fusion Protein: Structure, Function and Inhibition

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Philippe Plattet

2016-04-01

Full Text Available Measles virus (MeV, a highly contagious member of the Paramyxoviridae family, causes measles in humans. The Paramyxoviridae family of negative single-stranded enveloped viruses includes several important human and animal pathogens, with MeV causing approximately 120,000 deaths annually. MeV and canine distemper virus (CDV-mediated diseases can be prevented by vaccination. However, sub-optimal vaccine delivery continues to foster MeV outbreaks. Post-exposure prophylaxis with antivirals has been proposed as a novel strategy to complement vaccination programs by filling herd immunity gaps. Recent research has shown that membrane fusion induced by the morbillivirus glycoproteins is the first critical step for viral entry and infection, and determines cell pathology and disease outcome. Our molecular understanding of morbillivirus-associated membrane fusion has greatly progressed towards the feasibility to control this process by treating the fusion glycoprotein with inhibitory molecules. Current approaches to develop anti-membrane fusion drugs and our knowledge on drug resistance mechanisms strongly suggest that combined therapies will be a prerequisite. Thus, discovery of additional anti-fusion and/or anti-attachment protein small-molecule compounds may eventually translate into realistic therapeutic options.

[Research progress in hirudin fusion protein--review].

Science.gov (United States)

Zhang, Chuan-Ling; Yu, Ai-Ping; Jin, Ji-De; Wu, Chu-Tse

2007-02-01

Natural hirudin extracted from the secretion of medical leech salivary gland is a single-chain peptide containing 65 aminoacid residues with molecular weight of 7000 D, and exists in three isomers of HV1, HV2 and HV3. Hirudin possesses three disulfide bridges forming the structure of core cyclic peptides, which binds to the catalytic site of thrombin so as to inhibit the catalysis of thrombin. Its c-terminus rich in acidic aminoacid residues possesses hydrophilicity, and is free on the molecular surface, and can bind with fibrin recognition site of hirudin. The minimal segment of 12 - 16 C-terminal acidic residues keeps the minimal activity of anti-thrombosis. Thus, hirudin, as a potent and specific inhibitor of thrombin, can be used to protect from and to treat clinically thrombosis. As it has some disadvantages such as short half-life, bleeding side-effect and mono-function, and so on, hirudin has been fused with some other functional proteins in recent years. The obtained fusion proteins can prolong the half life of hirudin, or relieve it bleeding side effect, or bring new functions, such as thrombolysis, inhibiting the platelet aggregation, targeting specifically. The research progress in hirudin fusion protein was summarized in this review.

The MARVEL domain protein, Singles Bar, is required for progression past the pre-fusion complex stage of myoblast fusion.

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Estrada, Beatriz; Maeland, Anne D; Gisselbrecht, Stephen S; Bloor, James W; Brown, Nicholas H; Michelson, Alan M

2007-07-15

Multinucleated myotubes develop by the sequential fusion of individual myoblasts. Using a convergence of genomic and classical genetic approaches, we have discovered a novel gene, singles bar (sing), that is essential for myoblast fusion. sing encodes a small multipass transmembrane protein containing a MARVEL domain, which is found in vertebrate proteins involved in processes such as tight junction formation and vesicle trafficking where--as in myoblast fusion--membrane apposition occurs. sing is expressed in both founder cells and fusion competent myoblasts preceding and during myoblast fusion. Examination of embryos injected with double-stranded sing RNA or embryos homozygous for ethane methyl sulfonate-induced sing alleles revealed an identical phenotype: replacement of multinucleated myofibers by groups of single, myosin-expressing myoblasts at a stage when formation of the mature muscle pattern is complete in wild-type embryos. Unfused sing mutant myoblasts form clusters, suggesting that early recognition and adhesion of these cells are unimpaired. To further investigate this phenotype, we undertook electron microscopic ultrastructural studies of fusing myoblasts in both sing and wild-type embryos. These experiments revealed that more sing mutant myoblasts than wild-type contain pre-fusion complexes, which are characterized by electron-dense vesicles paired on either side of the fusing plasma membranes. In contrast, embryos mutant for another muscle fusion gene, blown fuse (blow), have a normal number of such complexes. Together, these results lead to the hypothesis that sing acts at a step distinct from that of blow, and that sing is required on both founder cell and fusion-competent myoblast membranes to allow progression past the pre-fusion complex stage of myoblast fusion, possibly by mediating fusion of the electron-dense vesicles to the plasma membrane.

Design, construction, and characterization of high-performance membrane fusion devices with target-selectivity.

Science.gov (United States)

Kashiwada, Ayumi; Yamane, Iori; Tsuboi, Mana; Ando, Shun; Matsuda, Kiyomi

2012-01-31

Membrane fusion proteins such as the hemagglutinin glycoprotein have target recognition and fusion accelerative domains, where some synergistically working elements are essential for target-selective and highly effective native membrane fusion systems. In this work, novel membrane fusion devices bearing such domains were designed and constructed. We selected a phenylboronic acid derivative as a recognition domain for a sugar-like target and a transmembrane-peptide (Leu-Ala sequence) domain interacting with the target membrane, forming a stable hydrophobic α-helix and accelerating the fusion process. Artificial membrane fusion behavior between the synthetic devices in which pilot and target liposomes were incorporated was characterized by lipid-mixing and inner-leaflet lipid-mixing assays. Consequently, the devices bearing both the recognition and transmembrane domains brought about a remarkable increase in the initial rate for the membrane fusion compared with the devices containing the recognition domain alone. In addition, a weakly acidic pH-responsive device was also constructed by replacing three Leu residues in the transmembrane-peptide domain by Glu residues. The presence of Glu residues made the acidic pH-dependent hydrophobic α-helix formation possible as expected. The target-selective liposome-liposome fusion was accelerated in a weakly acidic pH range when the Glu-substituted device was incorporated in pilot liposomes. The use of this pH-responsive device seems to be a potential strategy for novel applications in a liposome-based delivery system. © 2011 American Chemical Society

High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag.

Science.gov (United States)

Han, Yingqian; Guo, Wanying; Su, Bingqian; Guo, Yujie; Wang, Jiang; Chu, Beibei; Yang, Guoyu

2018-02-01

Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni 2+ . Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of a human protein-derived HIV-1 fusion inhibitor targeting the gp41 fusion core structure.

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Lijun Chao

Full Text Available The HIV-1 envelope glycoprotein (Env gp41 plays a crucial role in the viral fusion process. The peptides derived from the C-terminal heptad repeat (CHR of gp41 are potent HIV fusion inhibitors. However, the activity of these anti-HIV-1 peptides in vivo may be attenuated by their induction of anti-gp41 antibodies. Thus, it is essential to identify antiviral peptides or proteins with low, or no, immunogenicity to humans. Here, we found that the C-terminal fragment (aa 462-521 of the human POB1 (the partner of RalBP1, designated C60, is an HIV-1 fusion inhibitor. It bound to N36, the peptide derived from the N-terminal heptad repeat (NHR of gp41, and to the six-helix bundle (6-HB formed by N36 and C34, a CHR-peptide, but it did not bind to C34. Unlike the CHR-peptides, C60 did not block gp41 6-HB formation. Rather, results suggest that C60 inhibits HIV-1 fusion by binding to the 6-HB, in particular, the residues in the gp41 NHR domain that are exposed on the surface of 6-HB. Since 6-HB plays a crucial role in the late stage of fusion between the viral envelope and endosomal membrane during the endocytic process of HIV-1, C60 may serve as a host restriction factor to suppress HIV-1 entry into CD4+ T lymphocytes. Taken together, it can be concluded from these results that C60 can be used as a lead for the development of anti-HIV-1 therapeutics or microbicides for the treatment and prevention of HIV-1 infection, as well as a molecular probe to study the fusogenic mechanism of HIV-1.

Challenges of designing fusion reactors for remote maintainability

International Nuclear Information System (INIS)

Mason, L.S.

1981-01-01

One of the major problems faced by the fusion community is the development of the high level of reliability required to assure that fusion will be a viable commercial power source. Much of the responsibility for solving this problem falls directly on the designer in developing concepts that have a high level of maintainability. The problems are both near-term, in developing maintainability for next generation engineering oriented reactors; and long range, in developing full maintainability for the more commercial concepts with their required high level of on-line time. The near-time challenge will include development of unqiue design concepts to perform inspection, maintenance, replacement, and testing under the stringent conditions imposed by the next generation engineering oriented machines. The long range challenge will focus on basic design concepts that will enable the full mainatability required by commerical fusion

Resolution of Disulfide Heterogeneity in Nogo Receptor 1 Fusion Proteins by Molecular Engineering

Energy Technology Data Exchange (ETDEWEB)

P Weinreb; D Wen; F Qian; C Wildes; E Garber; L Walus; M Jung; J Wang; J Relton; et al.

2011-12-31

NgRI (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgRI have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgRI are flanked by N- and C-terminal disulfide-containing 'cap' domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgRI(310)-Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgRI truncation site or the spacing between the NgRI and Fc domains, or replacing cysteines within the NgRI or IgG hinge regions. One variant, which incorporates replacements of Cys{sup 266} and Cys{sup 309} with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgRI-Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins.

Design windows of laser fusion power plants and conceptual design of laser-diode pumped slab laser

International Nuclear Information System (INIS)

Kozaki, Y.; Eguchi, T.; Izawa, Y.

1999-01-01

An analysis of the design space available to laser fusion power plants has been carried out, in terms of design key parameters such as target gain, laser energy and laser repetition rate, the number of fusion react ion chambers, and plant size. The design windows of economically attractive laser fusion plants is identified with the constraints of key design parameters and the cost conditions. Especially, for achieving high repetition rate lasers, we have proposed and designed a diode-pumped solid-state laser driver which consists of water-cooled zig-zag path slab amplifiers. (author)

Conceptual design of the SlimCS fusion DEMO reactor

International Nuclear Information System (INIS)

Tobita, Kenji; Nishio, Satoshi; Enoeda, Mikio; Nakamura, Hirofumi; Hayashi, Takumi; Asakura, Nobuyuki; Utoh, Hiroyasu; Tanigawa, Hiroyasu; Nishitani, Takeo; Isono, Takaaki; Sakurai, Shinji; Kurita, Genichi; Hayashi, Takao; Oyama, Naoyuki; Liu Changle; Hamamatsu, Kiyotaka; Inoue, Takashi; Ozeki, Takahisa; Sato, Masayasu; Suzuki, Satoshi; Kawashima, Hisato; Ezato, Koichiro; Tsuru, Daigo; Koizumi, Norikiyo; Sakamoto, Keiji; Ando, Masami; Sakamoto, Yoshiteru; Shibama, Yusuke; Suzuki, Takahiro; Takechi, Manabu; Takahashi, Koji; Hirose, Takanori; Sato, Satoru; Nozawa, Takashi; Tanigawa, Hisashi; Kakudate, Satoshi; Kawamura, Yoshinori; Yamanishi, Toshihiko; Hoshino, Tsuyoshi; Ochiai, Kentaro; Ide, Shunsuke; Aiba, Nobuyuki; Shimizu, Katsuhiro; Honda, Mitsuru; Nakamichi, Masaru; Nishi, Hiroshi; Seki, Yoji; Nakamura, Yukiharu; Tsuchiya, Kunihiko; Yoshida, Tohru; Song Yuntao

2010-08-01

This report describes the results of the conceptual design study of the SlimCS fusion DEMO reactor aiming at demonstrating fusion power production in a plant scale and allowing to assess the economic prospects of a fusion power plant. The design study has focused on a compact and low aspect ratio tokamak reactor concept with a reduced-sized central solenoid, which is novel compared with previous tokamak reactor concept such as SSTR (Steady State Tokamak Reactor). Owing to low aspect ratio, the reactor will be capable of having comparatively high beta limit and high elongation (which can elevate the Greenwald density limit), having potential for high power density. The reactor has the main parameters of a major radius of 5.5 m, aspect ratio of 2.6, elongation of 2.0, normalized beta of 4.3, fusion out put of 2.95 GW and average neutron wall load of 3 MW/m 2 . This report covers various aspects of design study including systematic design, physics design, torus configuration, blanket, superconducting magnet, maintenance and building, which were carried out increase the engineering feasibility of the concept. (author)

The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane

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Juan Fontana

2017-08-01

Full Text Available All enveloped viruses, including herpesviruses, must fuse their envelope with the host membrane to deliver their genomes into target cells, making this essential step subject to interference by antibodies and drugs. Viral fusion is mediated by a viral surface protein that transits from an initial prefusion conformation to a final postfusion conformation. Strikingly, the prefusion conformation of the herpesvirus fusion protein, gB, is poorly understood. Herpes simplex virus (HSV, a model system for herpesviruses, causes diseases ranging from mild skin lesions to serious encephalitis and neonatal infections. Using cryo-electron tomography and subtomogram averaging, we have characterized the structure of the prefusion conformation and fusion intermediates of HSV-1 gB. To this end, we have set up a system that generates microvesicles displaying full-length gB on their envelope. We confirmed proper folding of gB by nondenaturing electrophoresis-Western blotting with a panel of monoclonal antibodies (MAbs covering all gB domains. To elucidate the arrangement of gB domains, we labeled them by using (i mutagenesis to insert fluorescent proteins at specific positions, (ii coexpression of gB with Fabs for a neutralizing MAb with known binding sites, and (iii incubation of gB with an antibody directed against the fusion loops. Our results show that gB starts in a compact prefusion conformation with the fusion loops pointing toward the viral membrane and suggest, for the first time, a model for gB’s conformational rearrangements during fusion. These experiments further illustrate how neutralizing antibodies can interfere with the essential gB structural transitions that mediate viral entry and therefore infectivity.

Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

Science.gov (United States)

Salehi, Nasrin; Peng, Ching-An

2016-07-08

CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. © 2016 American Institute of Chemical Engineers.

Fusion reactor design and technology 1986. V. 1

International Nuclear Information System (INIS)

1987-01-01

The first volume of the Proceedings of the Fourth Technical Committee Meeting and Workshop on Fusion Reactor Design and Technology organized by the IAEA (Yalta, 26 May - 6 June 1986) includes 36 papers devoted to the following topics: fusion programmes (3 papers), tokamaks (15 papers), non-tokamak reactors and open systems (9 papers), inertial confinement concepts (5 papers), fission-fusion hybrids (4 papers). Each of these papers has a separate abstract. Refs, figs and tabs

Conceptual design of Fusion Experimental Reactor (FER)

International Nuclear Information System (INIS)

Tone, T.; Fujisawa, N.

1983-01-01

Conceptual design studies of the Fusion Experimental Reactor (FER) have been performed. The FER has an objective of achieving selfignition and demonstrating engineering feasibility as a next generation tokamak to JT-60. Various concepts of the FER have been considered. The reference design is based on a double-null divertor. Optional design studies with some attractive features based on advanced concepts such as pumped limiter and RF current drive have been carried out. Key design parameters are; fusion power of 440 MW, average neutron wall loading of 1MW/m 2 , major radius of 5.5m, plasma minor radius of 1.1m, plasma elongation of 1.5, plasma current of 5.3MA, toroidal beta of 4%, toroidal field on plasma axis of 5.7T and tritium breeding ratio of above unity

Role of protein disulfide isomerase and other thiol-reactive proteins in HIV-1 envelope protein-mediated fusion

International Nuclear Information System (INIS)

Ou Wu; Silver, Jonathan

2006-01-01

Cell-surface protein disulfide isomerase (PDI) has been proposed to promote disulfide bond rearrangements in HIV-1 envelope protein (Env) that accompany Env-mediated fusion. We evaluated the role of PDI in ways that have not been previously tested by downregulating PDI with siRNA and by overexpressing wild-type or variant forms of PDI in transiently and stably transfected cells. These manipulations, as well as treatment with anti-PDI antibodies, had only small effects on infection or cell fusion mediated by NL4-3 or AD8 strains of HIV-1. However, the cell-surface thiol-reactive reagent 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) had a much stronger inhibitory effect in our system, suggesting that cell-surface thiol-containing molecules other than PDI, acting alone or in concert, have a greater effect than PDI on HIV-1 Env-mediated fusion. We evaluated one such candidate, thioredoxin, a PDI family member reported to reduce a labile disulfide bond in CD4. We found that the ability of thioredoxin to reduce the disulfide bond in CD4 is enhanced in the presence of HIV-1 Env gp120 and that thioredoxin also reduces disulfide bonds in gp120 directly in the absence of CD4. We discuss the implications of these observations for identification of molecules involved in disulfide rearrangements in Env during fusion

Protein fold recognition using geometric kernel data fusion.

Science.gov (United States)

Zakeri, Pooya; Jeuris, Ben; Vandebril, Raf; Moreau, Yves

2014-07-01

Various approaches based on features extracted from protein sequences and often machine learning methods have been used in the prediction of protein folds. Finding an efficient technique for integrating these different protein features has received increasing attention. In particular, kernel methods are an interesting class of techniques for integrating heterogeneous data. Various methods have been proposed to fuse multiple kernels. Most techniques for multiple kernel learning focus on learning a convex linear combination of base kernels. In addition to the limitation of linear combinations, working with such approaches could cause a loss of potentially useful information. We design several techniques to combine kernel matrices by taking more involved, geometry inspired means of these matrices instead of convex linear combinations. We consider various sequence-based protein features including information extracted directly from position-specific scoring matrices and local sequence alignment. We evaluate our methods for classification on the SCOP PDB-40D benchmark dataset for protein fold recognition. The best overall accuracy on the protein fold recognition test set obtained by our methods is ∼ 86.7%. This is an improvement over the results of the best existing approach. Moreover, our computational model has been developed by incorporating the functional domain composition of proteins through a hybridization model. It is observed that by using our proposed hybridization model, the protein fold recognition accuracy is further improved to 89.30%. Furthermore, we investigate the performance of our approach on the protein remote homology detection problem by fusing multiple string kernels. The MATLAB code used for our proposed geometric kernel fusion frameworks are publicly available at http://people.cs.kuleuven.be/∼raf.vandebril/homepage/software/geomean.php?menu=5/. © The Author 2014. Published by Oxford University Press.

General software design for multisensor data fusion

Science.gov (United States)

Zhang, Junliang; Zhao, Yuming

1999-03-01

In this paper a general method of software design for multisensor data fusion is discussed in detail, which adopts object-oriented technology under UNIX operation system. The software for multisensor data fusion is divided into six functional modules: data collection, database management, GIS, target display and alarming data simulation etc. Furthermore, the primary function, the components and some realization methods of each modular is given. The interfaces among these functional modular relations are discussed. The data exchange among each functional modular is performed by interprocess communication IPC, including message queue, semaphore and shared memory. Thus, each functional modular is executed independently, which reduces the dependence among functional modules and helps software programing and testing. This software for multisensor data fusion is designed as hierarchical structure by the inheritance character of classes. Each functional modular is abstracted and encapsulated through class structure, which avoids software redundancy and enhances readability.

Conceptual design of the fusion-driven subcritical system FDS-I

International Nuclear Information System (INIS)

Wu, Y.; Zheng, S.; Zhu, X.; Wang, W.; Wang, H.; Liu, S.; Bai, Y.; Chen, H.; Hu, L.; Chen, M.; Huang, Q.; Huang, D.; Zhang, S.; Li, J.; Chu, D.; Jiang, J.; Song, Y.

2006-01-01

The fusion-driven subcritical system (named FDS-I) was previously proposed as an intermediate step toward the final application of fusion energy. A conceptual design of the FDS-I is presented, which consists of the fusion neutron driver with relatively easy-achieved plasma parameters, and the He-gas/liquid lithium-lead Dual-cooled subcritical Waste Transmutation (DWT) blanket used to transmute long-lived radioactive wastes and to generate energy on the basis of self-sustainable fission and fusion fuel cycle. An overview of the FDS-I is given and the specifications of the design analysis are summarized

Trends in radiation protection: possible effects on fusion power plant design

International Nuclear Information System (INIS)

Eurajoki, Tapani; Frias, Manuel Pascual; Orlandi, Sergio

2003-01-01

Since the design of fusion power plants involves long-term issues, ranging over several decades, it is useful to try to foresee under what kind of regulations the first fusion plants are to be operated. Application of present radiological regulations and practice to a fusion power plant concept is considered. The current design phase of fusion power plants motivates the top-down dose assessment, but it is crucial to aim at bottom-up assessments to ensure radiation doses as low as reasonably achievable. Since several issues, relating both to our knowledge on radiation as well as to the practice of radiation protection, may change in the future, it is necessary to continuously follow the development in the further design of fusion power plants

Nuclear design of a very-low-activation fusion reactor

International Nuclear Information System (INIS)

Cheng, E.T.; Hopkins, G.R.

1983-06-01

An investigation was conducted to study the nuclear design aspects of using very-low-activation materials, such as SiC, MgO, and aluminum for fusion-reactor first wall, blanket, and shield applications. In addition to the advantage of very-low radioactive inventory, it was found that the very-low-activation fusion reactor can also offer an adequate tritium-breeding ratio and substantial amount of blanket nuclear heating as a conventional-material-structured reactor does. The most-stringent design constraint found in a very-low-activation fusion reactor is the limited space available in the inboard region of a tokamak concept for shielding to protect the superconducting toroidal field coil. A reference design was developed which mitigates the constraint by adopting a removable tungsten shield design that retains the inboard dimensions and gives the same shield performance as the reference STARFIRE tokamak reactor design

Neutronic Parametric Study on a Conceptual Design for a Transmutation Fusion Blanket

International Nuclear Information System (INIS)

Tariq Siddique, M.; Kim, Myung Hyun

2011-01-01

Fusion energy may be the one of options of future energy. In all over the world, researchers are putting their efforts for its commercial and economical availability. Fusion-fission hybrid reactors have been studied for various applications in China. First milestone of fusion energy is expected to be the fusion fission hybrid reactors. In fusion-fission hybrid reactor the blanket design is of second prime importance after fusion source. In this study conceptual design of a fusion blanket is initiated for calculation of tritium production, transmutation of minor actinides (MA) and fission products (FP) and energy multiplication calculations

Optimizing HIV-1 protease production in Escherichia coli as fusion protein

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Piubelli Luciano

2011-06-01

Full Text Available Abstract Background Human immunodeficiency virus (HIV is the etiological agent in AIDS and related diseases. The aspartyl protease encoded by the 5' portion of the pol gene is responsible for proteolytic processing of the gag-pol polyprotein precursor to yield the mature capsid protein and the reverse transcriptase and integrase enzymes. The HIV protease (HIV-1Pr is considered an attractive target for designing inhibitors which could be used to tackle AIDS and therefore it is still the object of a number of investigations. Results A recombinant human immunodeficiency virus type 1 protease (HIV-1Pr was overexpressed in Escherichia coli cells as a fusion protein with bacterial periplasmic protein dithiol oxidase (DsbA or glutathione S-transferase (GST, also containing a six-histidine tag sequence. Protein expression was optimized by designing a suitable HIV-1Pr cDNA (for E. coli expression and to avoid autoproteolysis and by screening six different E. coli strains and five growth media. The best expression yields were achieved in E. coli BL21-Codon Plus(DE3-RIL host and in TB or M9 medium to which 1% (w/v glucose was added to minimize basal expression. Among the different parameters assayed, the presence of a buffer system (based on phosphate salts and a growth temperature of 37°C after adding IPTG played the main role in enhancing protease expression (up to 10 mg of chimeric DsbA:HIV-1Pr/L fermentation broth. GST:HIVPr was in part (50% produced as soluble protein while the overexpressed DsbA:HIV-1Pr chimeric protein largely accumulated in inclusion bodies as unprocessed fusion protein. A simple refolding procedure was developed on HiTrap Chelating column that yielded a refolded DsbA:HIV-1Pr with a > 80% recovery. Finally, enterokinase digestion of resolubilized DsbA:HIV-1Pr gave more than 2 mg of HIV-1Pr per liter of fermentation broth with a purity ≤ 80%, while PreScission protease cleavage of soluble GST:HIVPr yielded ~ 0.15 mg of pure HIV-1

Conceptual design of fusion experimental reactor (FER)

International Nuclear Information System (INIS)

1984-03-01

A conceptual design study (option C) has been carried out for the fusion experimental reactor (FER). In addition to design of the tokamak reactor and associated systems based on the reference design specifications, feasibility of a water-shield reactor concept was examined as a topical study. The design study for the reference tokamak reactor has produced a reactor concept for the FER, along with major R D items for the concept, based on close examinations on thermal design, electromagnetics, neutronics and remote maintenance. Particular efforts have been directed to the area of electromagnetics. Detailed analyses with close simulation models have been performed on PF coil arrangements and configurations, shell effects of the blanket for plasma position unstability, feedback control, and eddy currents during disruptions. The major design specifications are as follows; Peak fusion power 437 MW Major radius 5.5 m Minor radius 1.1 m Plasma elongation 1.5 Plasma current 5.3 MA Toroidal beta 4 % Field on axis 5.7 T (author)

Economic comparison of fusion power plant designs

International Nuclear Information System (INIS)

O'Neill, J.E.

1986-01-01

Over the past 10 yr, a number of studies have been developed for fusion power plants of various types (tokamaks, mirrors, etc.) complete with figures of merit such as cost estimates and estimates of the cost of generating electricity (COE). Each of these designs involves unresolved physics and engineering problems which, it is assumed, will eventually be worked out. Because of such uncertainties the figures of merit associated with such designs are not to be compared as absolute measures of worth but as relative indicators of progress within a given concept type. As part of Grumman's involvement in fusion energy development, an effort has been undertaken to compare economic indicators from the referenced studies in order to determine the cost trend in recent reactor design activities

Safety considerations in next step fusion design and beyond

International Nuclear Information System (INIS)

Holland, D.F.

1990-01-01

Recent U.S. and international design studies provide insights into the potential safety and environmental advantages of fusion as well as the development needed to realize this potential. We in the Fusion Safety Program at EG ampersand G Idaho have analyzed the Compact Ignition Tokamak (CIT), the International Thermonuclear Engineering Reactor (ITER), and the Advanced Reactor Innovative Engineering Study (ARIES). I have reviewed these three designs to determine issues related to meeting the safety and the environmental goals that guide fusion development in the U.S. The paper lists safety and environmental issues that are generic to fusion and approaches to favorably resolve each issue. The technical developments that have the highest potential of contributing to improving the safety and environmental attractiveness of fusion are identified and discussed. These developments are in the areas of low-activation materials, plasma- facing components, and plasma physics relating to off-normal plasma events and tritium burn-up. 8 refs., 7 tabs

Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

Science.gov (United States)

Li, Xiao-Jing; Liu, Jin-Ling; Gao, Dong-Sheng; Wan, Wen-Yan; Yang, Xia; Li, Yong-Tao; Chang, Hong-Tao; Chen, Lu; Wang, Chuan-Qing; Zhao, Jun

2016-03-01

Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

Expression and Purification of Neurotrophin-Elastin-Like Peptide Fusion Proteins for Neural Regeneration.

Science.gov (United States)

Johnson, Tamina; Koria, Piyush

2016-04-01

Neural injuries such as spinal cord injuries, traumatic brain injuries, or nerve transection injuries pose a major health problem. Neurotrophins such as nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) have been shown to improve the outcome of neural injuries in several pre-clinical models, but their use in clinics is limited by the lack of a robust delivery system that enhances their bioavailability and half-life. We describe two fusion proteins comprising NGF or BDNF fused with elastin-like peptides (ELPs). The aim of this study was to investigate the biological activity of neurotrophin-ELP (N-ELP) fusion proteins via in vitro culture models. NGF and BDNF were cloned in front of an elastin-like polypeptide sequence V40C2. These proteins were expressed in bacteria as inclusion bodies. These fusion proteins underwent solubilization via 8 M urea and purification via inverse transition cycling (ITC). We measured the particle size and the effect of temperature on precipitated particles using dynamic light scattering (DLS). We used western blot analysis to confirm the specificity of NGF-ELP to tropomyosin receptor kinase A (TrkA) antibody and to confirm the specificity of BDNF-ELP to TrkB antibody. PC12 cells were used to perform a neurite outgrowth assay to determine the biological activity of NGF-ELP. Bioactivity of BDNF-ELP was ascertained via transfecting human epithelial kidney (HEK 293-T) cells to express the TrkB receptor. The proteins were successfully purified to high homogeneity by exploiting the phase transition property of ELPs and urea, which solubilize inclusion bodies. Using PC12 neurite outgrowth assay, we further demonstrated that the biological activity of NGF was retained in the fusion. Similarly, BDNF-ELP phosphorylated the TrkB receptor, suggesting the biological activity of BDNF was also retained in the fusion. We further show that owing to the phase transition property of ELPs in the fusion, these proteins self-assembled into

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

Science.gov (United States)

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

2009-05-01

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

Electrostatic Architecture of the Infectious Salmon Anemia Virus (ISAV) Core Fusion Protein Illustrates a Carboxyl-Carboxylate pH Sensor.

Science.gov (United States)

Cook, Jonathan D; Soto-Montoya, Hazel; Korpela, Markus K; Lee, Jeffrey E

2015-07-24

Segment 5, ORF 1 of the infectious salmon anemia virus (ISAV) genome, encodes for the ISAV F protein, which is responsible for viral-host endosomal membrane fusion during a productive ISAV infection. The entry machinery of ISAV is composed of a complex of the ISAV F and ISAV hemagglutinin esterase (HE) proteins in an unknown stoichiometry prior to receptor engagement by ISAV HE. Following binding of the receptor to ISAV HE, dissociation of the ISAV F protein from HE, and subsequent endocytosis, the ISAV F protein resolves into a fusion-competent oligomeric state. Here, we present a 2.1 Å crystal structure of the fusion core of the ISAV F protein determined at low pH. This structure has allowed us to unambiguously demonstrate that the ISAV entry machinery exhibits typical class I viral fusion protein architecture. Furthermore, we have determined stabilizing factors that accommodate the pH-dependent mode of ISAV transmission, and our structure has allowed the identification of a central coil that is conserved across numerous and varied post-fusion viral glycoprotein structures. We then discuss a mechanistic model of ISAV fusion that parallels the paramyxoviral class I fusion strategy wherein attachment and fusion are relegated to separate proteins in a similar fashion to ISAV fusion. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype.

Science.gov (United States)

Fahrenkrog, Birthe; Martinelli, Valérie; Nilles, Nadine; Fruhmann, Gernot; Chatel, Guillaume; Juge, Sabine; Sauder, Ursula; Di Giacomo, Danika; Mecucci, Cristina; Schwaller, Jürg

2016-01-01

Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

Enhanced SUMOylation of proteins containing a SUMO-interacting motif by SUMO-Ubc9 fusion

International Nuclear Information System (INIS)

Kim, Eui Tae; Kim, Kyeong Kyu; Matunis, Mike J.; Ahn, Jin-Hyun

2009-01-01

Identifying new targets for SUMO and understanding the function of protein SUMOylation are largely limited by low level of SUMOylation. It was found recently that Ubc9, the SUMO E2 conjugating enzyme, is covalently modified by SUMO at a lysine 14 in the N-terminal alpha helix, and that SUMO-modified Ubc9 has enhanced conjugation activity for certain target proteins containing a SUMO-interacting motif (SIM). Here, we show that, compared to intact Ubc9, the SUMO-Ubc9 fusion protein has higher conjugating activity for SIM-containing targets such as Sp100 and human cytomegalovirus IE2. Assays using an IE2 SIM mutant revealed the requirement of SIM for the enhanced IE2 SUMOylation by SUMO-Ubc9. In pull-down assays with cell extracts, the SUMO-Ubc9 fusion protein bound to more diverse cellular proteins and interacted with some SIM-containing proteins with higher affinities than Ubc9. Therefore, the devised SUMO-Ubc9 fusion will be useful for identifying SIM-containing SUMO targets and producing SUMO-modified proteins.

The TIP30 protein complex, arachidonic acid and coenzyme A are required for vesicle membrane fusion.

Directory of Open Access Journals (Sweden)

Chengliang Zhang

Full Text Available Efficient membrane fusion has been successfully mimicked in vitro using artificial membranes and a number of cellular proteins that are currently known to participate in membrane fusion. However, these proteins are not sufficient to promote efficient fusion between biological membranes, indicating that critical fusogenic factors remain unidentified. We have recently identified a TIP30 protein complex containing TIP30, acyl-CoA synthetase long-chain family member 4 (ACSL4 and Endophilin B1 (Endo B1 that promotes the fusion of endocytic vesicles with Rab5a vesicles, which transport endosomal acidification enzymes vacuolar (H⁺-ATPases (V-ATPases to the early endosomes in vivo. Here, we demonstrate that the TIP30 protein complex facilitates the fusion of endocytic vesicles with Rab5a vesicles in vitro. Fusion of the two vesicles also depends on arachidonic acid, coenzyme A and the synthesis of arachidonyl-CoA by ACSL4. Moreover, the TIP30 complex is able to transfer arachidonyl groups onto phosphatidic acid (PA, producing a new lipid species that is capable of inducing close contact between membranes. Together, our data suggest that the TIP30 complex facilitates biological membrane fusion through modification of PA on membranes.

Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein.

Science.gov (United States)

Welch, Brett D; Liu, Yuanyuan; Kors, Christopher A; Leser, George P; Jardetzky, Theodore S; Lamb, Robert A

2012-10-09

The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein.

Eradication of Human Hepatic and Pulmonary Melanoma Metastases in SCID Mice by Antibody--Interleukin 2 Fusion Proteins

Science.gov (United States)

Becker, Jurgen C.; Pancook, James D.; Gillies, Stephen D.; Mendelsohn, John; Reisfeld, Ralph A.

1996-04-01

Antibody--cytokine fusion proteins combine the unique targeting ability of antibodies with the multifunctional activity of cytokines. Here, we demonstrate the therapeutic efficacy of such constructs for the treatment of hepatic and pulmonary metastases of different melanoma cell lines. Two antibody--interleukin 2 (IL-2) fusion proteins, ch225-IL2 and ch14.18-IL2, constructed by fusion of a synthetic sequence coding for human IL-2 to the carboxyl end of the Cγ 1 gene of the corresponding antibodies, were tested for their therapeutic efficacy against xenografted human melanoma in vivo. Tumorspecific fusion proteins completely inhibited the growth of hepatic and pulmonary metastases in C.B-17 scid/scid mice previously reconstituted with human lymphokine-activated killer cells, whereas treatment with combinations of the corresponding antibodies plus recombinant IL-2 only reduced the tumor load. Even when treatment with fusion proteins was delayed up to 8 days after inoculation of tumor cells, it still resulted in complete eradication of micrometastases that were established at that time point. Selection of tumor cell lines expressing or lacking the targeted antigen of the administered fusion protein proved the specificity of the observed antitumor effect. Biodistribution analysis demonstrated that the tumorspecific fusion protein accumulated not only in subcutaneous tumors but also in lungs and livers affected with micrometastases. Survival times of animals treated with the fusion protein were more than doubled as compared to those treated with the combination of the corresponding antibody plus IL-2. Our data demonstrate that an immunotherapeutic approach using cytokines targeted by antibodies to tumor sites has potent effects against disseminated human melanoma.

US-DOE Fusion-Breeder Program: blanket design and system performance

International Nuclear Information System (INIS)

Lee, J.D.

1983-01-01

Conceptual design studies are being used to assess the technical and economic feasibility of fusion's potential to produce fissile fuel. A reference design of a fission-suppressed blanket using conventional materials is under development. Theoretically, a fusion breeder that incorporates this fusion-suppressed blanket surrounding a 3000-MW tandem mirror fusion core produces its own tritium plus 5600 kg of 233 U per year. The 233 U could then provide fissile makeup for 21 GWe of light-water reactor (LWR) power using a denatured thorium fuel cycle with full recycle. This is 16 times the net electric power produced by the fusion breeder (1.3 GWe). The cost of electricity from this fusion-fission system is estimated to be only 23% higher than the cost from LWRs that have makeup from U 3 O 8 at present costs (55 $/kg). Nuclear performance, magnetohydrodynamics (MHD), radiation effects, and other issues concerning the fission-suppressed blanket are summarized, as are some of the present and future objectives of the fusion breeder program

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

International Nuclear Information System (INIS)

Kim, Kyoungmi; Kim, Hwain; Lee, Daekee

2009-01-01

Site-specific recombination (SSR) by Cre recombinase and its target sequence, loxP, is a valuable tool in genetic analysis of gene function. Recently, several studies reported successful application of Cre fusion protein containing protein transduction peptide for inducing gene modification in various mammalian cells including ES cell as well as in the whole animal. In this study, we show that a short incubation of preimplantation mouse embryos with purified cell-permeable Cre fusion protein results in efficient SSR. X-Gal staining of preimplantation embryos, heterozygous for Gtrosa26 tm1Sor , revealed that treatment of 1-cell or 2-cell embryos with 3 μM of Cre fusion protein for 2 h leads to Cre-mediated excision in 70-85% of embryos. We have examined the effect of the concentration of the Cre fusion protein and the duration of the treatment on embryonic development, established a condition for full term development and survival to adulthood, and demonstrated the germ line transmission of excised Gtrosa26 allele. Potential applications and advantages of the highly efficient technique described here are discussed.

Laser solenoid fusion--fission design

International Nuclear Information System (INIS)

Steinhauer, L.C.; Taussig, R.T.

1976-01-01

The dependence of breeding performance on system engineering parameters is examined for laser solenoid fusion-fission reactors. Reactor performance is found to be relatively insensitive to most of the engineering parameters, and compact designs can be built based on reasonable technologies. Point designs are described for the prototype series of reactors (mid-term technologies) and for second generation systems (advanced technologies). It is concluded that the laser solenoid has a good probability of timely application to fuel breeding needs

Magnet design considerations for Tokamak fusion reactors

International Nuclear Information System (INIS)

Purcell, J.R.; Chen, W.; Thomas, R.

1976-01-01

Design problems for superconducting ohmic heating and toroidal field coils for large Tokamak fusion reactors are discussed. The necessity for making these coils superconducting is explained, together with the functions of these coils in a Tokamak reactor. Major problem areas include materials related aspects and mechanical design and cryogenic considerations. Projections and comparisons are made based on existing superconducting magnet technology. The mechanical design of large-scale coils, which can contain the severe electromagnetic loading and stress generated in the winding, are emphasized. Additional major tasks include the development of high current conductors for pulsed applications to be used in fabricating the ohmic heating coils. It is important to note, however, that no insurmountable technical barriers are expected in the course of developing superconducting coils for Tokamak fusion reactors. (Auth.)

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

Directory of Open Access Journals (Sweden)

Waka Omata

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

Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase

Energy Technology Data Exchange (ETDEWEB)

Wang, Yechun; Yi, Hankuil; Wang, Melissa; Yu, Oliver; Jez, Joseph M. (WU); (Danforth)

2012-10-24

To increase the biochemical efficiency of biosynthetic systems, metabolic engineers have explored different approaches for organizing enzymes, including the generation of unnatural fusion proteins. Previous work aimed at improving the biosynthesis of resveratrol, a stilbene associated a range of health-promoting activities, in yeast used an unnatural engineered fusion protein of Arabidopsis thaliana (thale cress) 4-coumaroyl-CoA ligase (At4CL1) and Vitis vinifera (grape) stilbene synthase (VvSTS) to increase resveratrol levels 15-fold relative to yeast expressing the individual enzymes. Here we present the crystallographic and biochemical analysis of the 4CL::STS fusion protein. Determination of the X-ray crystal structure of 4CL::STS provides the first molecular view of an artificial didomain adenylation/ketosynthase fusion protein. Comparison of the steady-state kinetic properties of At4CL1, VvSTS, and 4CL::STS demonstrates that the fusion protein improves catalytic efficiency of either reaction less than 3-fold. Structural and kinetic analysis suggests that colocalization of the two enzyme active sites within 70 {angstrom} of each other provides the basis for enhanced in vivo synthesis of resveratrol.

Site-directed antibody immobilization using a protein A-gold binding domain fusion protein for enhanced SPR immunosensing.

Science.gov (United States)

de Juan-Franco, Elena; Caruz, Antonio; Pedrajas, J R; Lechuga, Laura M

2013-04-07

We have implemented a novel strategy for the oriented immobilization of antibodies onto a gold surface based on the use of a fusion protein, the protein A-gold binding domain (PAG). PAG consists of a gold binding peptide (GBP) coupled to the immunoglobulin-binding domains of staphylococcal protein A. This fusion protein provides an easy and fast oriented immobilization of antibodies preserving its native structure, while leaving the antigen binding sites (Fab) freely exposed. Using this immobilization strategy, we have demonstrated the performance of the immunosensing of the human Growth Hormone by SPR. A limit of detection of 90 ng mL(-1) was obtained with an inter-chip variability lower than 7%. The comparison of this method with other strategies for the direct immobilization of antibodies over gold surfaces has showed the enhanced sensitivity provided by the PAG approach.

Membrane fusion and exocytosis.

Science.gov (United States)

Jahn, R; Südhof, T C

1999-01-01

Membrane fusion involves the merger of two phospholipid bilayers in an aqueous environment. In artificial lipid bilayers, fusion proceeds by means of defined transition states, including hourglass-shaped intermediates in which the proximal leaflets of the fusing membranes are merged whereas the distal leaflets are separate (fusion stalk), followed by the reversible opening of small aqueous fusion pores. Fusion of biological membranes requires the action of specific fusion proteins. Best understood are the viral fusion proteins that are responsible for merging the viral with the host cell membrane during infection. These proteins undergo spontaneous and dramatic conformational changes upon activation. In the case of the paradigmatic fusion proteins of the influenza virus and of the human immunodeficiency virus, an amphiphilic fusion peptide is inserted into the target membrane. The protein then reorients itself, thus forcing the fusing membranes together and inducing lipid mixing. Fusion of intracellular membranes in eukaryotic cells involves several protein families including SNAREs, Rab proteins, and Sec1/Munc-18 related proteins (SM-proteins). SNAREs form a novel superfamily of small and mostly membrane-anchored proteins that share a common motif of about 60 amino acids (SNARE motif). SNAREs reversibly assemble into tightly packed helical bundles, the core complexes. Assembly is thought to pull the fusing membranes closely together, thus inducing fusion. SM-proteins comprise a family of soluble proteins that bind to certain types of SNAREs and prevent the formation of core complexes. Rab proteins are GTPases that undergo highly regulated GTP-GDP cycles. In their GTP form, they interact with specific proteins, the effector proteins. Recent evidence suggests that Rab proteins function in the initial membrane contact connecting the fusing membranes but are not involved in the fusion reaction itself.

Systems-design and energy-balance considerations for impact fusion

International Nuclear Information System (INIS)

Krakowski, R.A.; Miller, R.L.

1979-01-01

Areas of concern and potential problems for impact fusion are qualitatively considered within an overall systems context. A parametric and qualitative description of the general energy balance and systems considerations for an Impact Fusion Reactor (IFR) design is discussed. Reactor systems design considerations for an IFR are presented. An attempt to assess the IFR viability is made based on highly simplified but limiting projectile-target energy balances and thermonuclear burn models

Fusion breeder sphere - PAC blanket design

International Nuclear Information System (INIS)

Sullivan, J.D.; Palmer, B.J.F.

1987-11-01

There is a considerable world-wide effort directed toward the production of materials for fusion reactors. Many ceramic fabrication groups are working on making lithium ceramics in a variety of forms, to be incorporated into the tritium breeding blanket which will surround the fusion reactor. Current blanket designs include ceramic in either monolithic or packed sphere bed (sphere-pac) forms. The major thrust at AECL is the production of lithium aluminate spheres to be incorporated in a sphere-pac bed. Contemporary studies on breeder blanket design offer little insight into the requirements on the sizes of the spheres. This study examined the parameters which determine the properties of pressure drop and coolant requirements. It was determined that an optimised sphere-pac bed would be composed of two diameters of spheres: 75 weight % at 3 mm and 25 weight % at 0.3 mm

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

Science.gov (United States)

Suh, Jin Sook; Lee, Jue Yeon; Choi, Yoon Jung; You, Hyung Keun; Hong, Seong-Doo; Chung, Chong Pyoung; Park, Yoon Jeong

2014-01-01

Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP), and a transcriptional coactivator with a PDZ-binding motif (TAZ) protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC) differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in alginate gel for the purpose of localization and controlled release. The LMWP-TAZ fusion protein-loaded alginate gel matrix significantly increased bone formation in rabbit calvarial defects compared with alginate gel matrix mixed with free TAZ protein. The protein transduction of TAZ fused with cell-penetrating LMWP peptide was able selectively to stimulate osteogenesis in vitro and in vivo. Taken together, this fusion protein-transduction technology for osteogenic protein can thus be applied in combination with biomaterials for tissue regeneration and controlled release for tissue

Viral membrane fusion

International Nuclear Information System (INIS)

Harrison, Stephen C.

2015-01-01

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

Viral membrane fusion

Energy Technology Data Exchange (ETDEWEB)

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

2015-05-15

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

Low Resolution Structure of RAR1-GST-Tag Fusion Protein in Solution

International Nuclear Information System (INIS)

Taube, M.; Kozak, M.; Jarmolowski, A.

2010-01-01

RAR1 is a protein required for resistance mediated by many R genes and function upstream of signaling pathways leading to H 2 O 2 accumulation. The structure and conformation of RAR1-GST-Tag fusion protein from barley (Hordeum vulgare) in solution was studied by the small angle scattering of synchrotron radiation. It was found that the dimer of RAR1-GST-Tag protein is characterized in solution by radius of gyration R G = 6.19 nm and maximal intramolecular vector D max = 23 nm. On the basis of the small angle scattering of synchrotron radiation SAXS data two bead models obtained by ab initio modeling are proposed. Both models show elongated conformations. We also concluded that molecules of fusion protein form: dimers in solution via interaction of GST domains. (authors)

Engineering of a parainfluenza virus type 5 fusion protein (PIV-5 F): development of an autonomous and hyperfusogenic protein by a combinational mutagenesis approach.

Science.gov (United States)

Terrier, O; Durupt, F; Cartet, G; Thomas, L; Lina, B; Rosa-Calatrava, M

2009-12-01

The entry of enveloped viruses into host cells is accomplished by fusion of the viral envelope with the target cell membrane. For the paramyxovirus parainfluenza virus type 5 (PIV-5), this fusion involves an attachment protein (HN) and a class I viral fusion protein (F). We investigated the effect of 20 different combinations of 12 amino-acid substitutions within functional domains of the PIV-5 F glycoprotein, by performing cell surface expression measurements, quantitative fusion and syncytia assays. We found that combinations of mutations conferring an autonomous phenotype with mutations leading to an increased fusion activity were compatible and generated functional PIV-5 F proteins. The addition of mutations in the heptad-repeat domains led to both autonomous and hyperfusogenic phenotypes, despite the low cell surface expression of the corresponding mutants. Such engineering approach may prove useful not only for deciphering the fundamental mechanism behind viral-mediated membrane fusion but also in the development of potential therapeutic applications.

Single chain Fc-dimer-human growth hormone fusion protein for improved drug delivery.

Science.gov (United States)

Zhou, Li; Wang, Hsuan-Yao; Tong, Shanshan; Okamoto, Curtis T; Shen, Wei-Chiang; Zaro, Jennica L

2017-02-01

Fc fusion protein technology has been successfully used to generate long-acting forms of several protein therapeutics. In this study, a novel Fc-based drug carrier, single chain Fc-dimer (sc(Fc) 2 ), was designed to contain two Fc domains recombinantly linked via a flexible linker. Since the Fc dimeric structure is maintained through the flexible linker, the hinge region was omitted to further stabilize it against proteolysis and reduce FcγR-related effector functions. The resultant sc(Fc) 2 candidate preserved the neonatal Fc receptor (FcRn) binding. sc(Fc) 2 -mediated delivery was then evaluated using a therapeutic protein with a short plasma half-life, human growth hormone (hGH), as the protein drug cargo. This novel carrier protein showed a prolonged in vivo half-life and increased hGH-mediated bioactivity compared to the traditional Fc-based drug carrier. sc(Fc) 2 technology has the potential to greatly advance and expand the use of Fc-technology for improving the pharmacokinetics and bioactivity of protein therapeutics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.

Science.gov (United States)

Pandey, Naresh; Nobles, Christopher L; Zechiedrich, Lynn; Maresso, Anthony W; Silberg, Jonathan J

2015-05-15

Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein-protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein-protein interactions within whole animals.

Anti-Diabetic Effects of CTB-APSL Fusion Protein in Type 2 Diabetic Mice

Directory of Open Access Journals (Sweden)

Yunlong Liu

2014-03-01

Full Text Available To determine whether cholera toxin B subunit and active peptide from shark liver (CTB-APSL fusion protein plays a role in treatment of type 2 diabetic mice, the CTB-APSL gene was cloned and expressed in silkworm (Bombyx mori baculovirus expression vector system (BEVS, then the fusion protein was orally administrated at a dose of 100 mg/kg for five weeks in diabetic mice. The results demonstrated that the oral administration of CTB-APSL fusion protein can effectively reduce the levels of both fasting blood glucose (FBG and glycosylated hemoglobin (GHb, promote insulin secretion and improve insulin resistance, significantly improve lipid metabolism, reduce triglycerides (TG, total cholesterol (TC and low density lipoprotein (LDL levels and increase high density lipoprotein (HDL levels, as well as effectively improve the inflammatory response of type 2 diabetic mice through the reduction of the levels of inflammatory cytokines tumor necrosis factor-α (TNF-α and interleukin-6 (IL-6. Histopathology shows that the fusion protein can significantly repair damaged pancreatic tissue in type 2 diabetic mice, significantly improve hepatic steatosis and hepatic cell cloudy swelling, reduce the content of lipid droplets in type 2 diabetic mice, effectively inhibit renal interstitial inflammatory cells invasion and improve renal tubular epithelial cell nucleus pyknosis, thus providing an experimental basis for the development of a new type of oral therapy for type 2 diabetes.

Purification, crystallization and X-ray diffraction analysis of human dynamin-related protein 1 GTPase-GED fusion protein

International Nuclear Information System (INIS)

Klinglmayr, Eva; Wenger, Julia; Mayr, Sandra; Bossy-Wetzel, Ella; Puehringer, Sandra

2012-01-01

The crystallization and initial diffraction analysis of human Drp1 GTPase-GED fusion protein are reported. The mechano-enzyme dynamin-related protein 1 plays an important role in mitochondrial fission and is implicated in cell physiology. Dysregulation of Drp1 is associated with abnormal mitochondrial dynamics and neuronal damage. Drp1 shares structural and functional similarities with dynamin 1 with respect to domain organization, ability to self-assemble into spiral-like oligomers and GTP-cycle-dependent membrane scission. Structural studies of human dynamin-1 have greatly improved the understanding of this prototypical member of the dynamin superfamily. However, high-resolution structural information for full-length human Drp1 covering the GTPase domain, the middle domain and the GTPase effector domain (GED) is still lacking. In order to obtain mechanistic insights into the catalytic activity, a nucleotide-free GTPase-GED fusion protein of human Drp1 was expressed, purified and crystallized. Initial X-ray diffraction experiments yielded data to 2.67 Å resolution. The hexagonal-shaped crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 53.59, b = 151.65, c = 43.53 Å, one molecule per asymmetric unit and a solvent content of 42%. Expression of selenomethionine-labelled protein is currently in progress. Here, the expression, purification, crystallization and X-ray diffraction analysis of the Drp1 GTPase-GED fusion protein are presented, which form a basis for more detailed structural and biophysical analysis

OSIRIS and SOMBRERO Inertial Fusion Power Plant Designs, Volume 2: Designs, Assessments, and Comparisons

Energy Technology Data Exchange (ETDEWEB)

Meier, W. R.; Bieri, R. L.; Monsler, M. J.; Hendricks, C. D.; Laybourne, P.; Shillito, K. R.

1992-03-01

This is a comprehensive design study of two Inertial Fusion Energy (IFE) electric power plants. Conceptual designs are presented for a fusion reactor (called Osiris) using an induction-linac heavy-ion beam driver, and another (called SOMBRERO) using a KrF laser driver. The designs covered all aspects of IFE power plants, including the chambers, heat transport and power conversion systems, balance-of-plant facilities, target fabrication, target injection and tracking, as well as the heavy-ion and KrF drivers. The point designs were assessed and compared in terms of their environmental & safety aspects, reliability and availability, economics, and technology development needs.

[Construction of cTnC-linker-TnI (P) Genes, Expression of Fusion Protein and Preparation of Lyophilized Protein].

Science.gov (United States)

Song, Xiaoli; Liu, Xiaoyun; Cai, Lei; Wu, Jianwei; Wang, Jihua

2015-12-01

In order to construct and express human cardiac troponin C-linker-troponin I(P) [ cTnC-linker-TnI(P)] fusion protein, detect its activity and prepare lyophilized protein, we searched the CDs of human cTnC and cTnI from GenBank, synthesized cTnC and cTnI(30-110aa) into cloning vector by a short DNA sequence coding for 15 neutral amino acid residues. pCold I-cTnC-linker-TnI(P) was constructed and transformed into E. coli BL21(DE3). Then, cTnC-linker-TnI(P) fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Soluable expression of cTnC-linker-TnI(P) in prokaryotic system was successfully obtained. The fusion protein was purified by Ni²⁺ Sepharose 6 Fast Flow affinity chromatography with over 95% purity and prepared into lyophilized protein. The activity of purified cTnC-linker-TnI(P) and its lyophilized protein were detected by Wondfo Finecare™ cTnI Test. Lyophilized protein of cTnC-linker-TnI(P) was stable for 10 or more days at 37 °C and 4 or more months at 25 °C and 4 °C. The expression system established in this research is feasible and efficient. Lyophilized protein is stable enough to be provided as biological raw materials for further research.

ELMO Bumpy Torus fusion-reactor design study

International Nuclear Information System (INIS)

Bathke, C.G.; Krakowski, R.A.

1981-01-01

A complete power plant design of a 1200-MWe ELMO Bumpy Torus Reactor (EBTR) is described that emphasizes those features that are unique to the EBT confinement concept, with subsystems and balance-of-plant items that are generic to magnetic fusion being adopted from past, more extensive tokamak reactor designs

Coating Nanoparticles with Plant-Produced Transferrin-Hydrophobin Fusion Protein Enhances Their Uptake in Cancer Cells

DEFF Research Database (Denmark)

Reuter, Lauri J.; Shahbazi, Mohammad-Ali; Makila, Ermei M.

2017-01-01

can be expressed in Nicotiana benthamiana plants as a fusion with Trichoderma reesei hydrophobins HFBI, HFBII, or HFBIV. Transferrin-HFBIV was further expressed in tobacco BY-2 suspension cells. Both partners of the fusion protein retained their functionality; the hydrophobin moiety enabled migration...... to a surfactant phase in an aqueous two-phase system, and the transferrin moiety was able to reversibly bind iron. Coating porous silicon nanoparticles with the fusion protein resulted in uptake of the nanoparticles in human cancer cells. This study provides a proof-of concept for the functionalization...

Status of fusion reactor blanket design

International Nuclear Information System (INIS)

Smith, D.L.; Sze, D.K.

1986-02-01

The recent Blanket Comparison and Selection Study (BCSS), which was a comprehensive evaluation of fusion reactor blanket design and the status of blanket technology, serves as an excellent basis for further development of blanket technology. This study provided an evaluation of over 130 blanket concepts for the reference case of electric power producing, DT fueled reactors in both Tokamak and Tandem Mirror (TMR) configurations. Based on a specific set of reactor operating parameters, the current understanding of materials and blanket technology, and a uniform evaluation methodology developed as part of the study, a limited number of concepts were identified that offer the greatest potential for making fusion an attractive energy source

Development of a novel fluorescent imaging probe for tumor hypoxia by use of a fusion protein with oxygen-dependent degradation domain of HIF-1α

Science.gov (United States)

Tanaka, Shotaro; Kizaka-Kondoh, Shinae; Harada, Hiroshi; Hiraoka, Masahiro

2007-02-01

More malignant tumors contain more hypoxic regions. In hypoxic tumor cells, expression of a series of hypoxiaresponsive genes related to malignant phenotype such as angiogenesis and metastasis are induced. Hypoxia-inducible factor-1 (HIF-1) is a master transcriptional activator of such genes, and thus imaging of hypoxic tumor cells where HIF-1 is active, is important in cancer therapy. We have been developing PTD-ODD fusion proteins, which contain protein transduction domain (PTD) and the VHL-mediated protein destruction motif in oxygen-dependent degradation (ODD) domain of HIF-1 alpha subunit (HIF-1α). Thus PTD-ODD fusion proteins can be delivered to any tissue in vivo through PTD function and specifically stabilized in hypoxic cells through ODD function. To investigate if PTD-ODD fusion protein can be applied to construct hypoxia-specific imaging probes, we first constructed a fluorescent probe because optical imaging enable us to evaluate a probe easily, quickly and economically in a small animal. We first construct a model fusion porein PTD-ODD-EGFP-Cy5.5 named POEC, which is PTD-ODD protein fused with EGFP for in vitro imaging and stabilization of fusion protein, and conjugated with a near-infrared dye Cy5.5. This probe is designed to be degraded in normoxic cells through the function of ODD domain and followed by quick clearance of free fluorescent dye. On the other hand, this prove is stabilized in hypoxic tumor cells and thus the dye is stayed in the cells. Between normoxic and hypoxic conditions, the difference in the clearance rate of the dye will reveals suited contrast for tumor-hypoxia imaging. The optical imaging probe has not been optimized yet but the results presented here exhibit a potential of PTD-ODD fusion protein as a hypoxia-specific imaging probe.

TALE-PvuII fusion proteins--novel tools for gene targeting.

Science.gov (United States)

Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

2013-01-01

Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.

Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay

International Nuclear Information System (INIS)

Morton, Craig J.; Cameron, Rachel; Lawrence, Lynne J.; Lin Bo; Lowe, Melinda; Luttick, Angela; Mason, Anthony; McKimm-Breschkin, Jenny; Parker, Michael W.; Ryan, Jane; Smout, Michael; Sullivan, Jayne; Tucker, Simon P.; Young, Paul R.

2003-01-01

Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors

Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological, biochemical and therapeutic properties.

Science.gov (United States)

Soleimanpour, Saman; Hassannia, Tahereh; Motiee, Mahdieh; Amini, Abbas Ali; Rezaee, S A R

2017-05-01

Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor-ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies.

Research and Application of Autodesk Fusion360 in Industrial Design

Science.gov (United States)

Song, P. P.; Qi, Y. M.; Cai, D. C.

2018-05-01

In 2016, Fusion 360, a productintroduced byAutodesk and integrating industrial design, structural design, mechanical simulation, and CAM, turns out a design platform supportingcollaboration and sharing both cross-platform and via the cloud. In previous products, design and manufacturing use to be isolated. In the course of design, research and development, the communication between designers and engineers used to go on through different software products, tool commands, and even industry terms. Moreover, difficulty also lies with the communication between design thoughts and machining strategies. Naturally, a difficult product design and R & D process would trigger a noticeable gap between the design model and the actual product. A complete product development process tends to cover several major areas, such as industrial design, mechanical design, rendering and animation, computer aided emulation (CAE), and computer aided manufacturing (CAM). Fusion 360, a perfect design solving the technical problems of cross-platform data exchange, realizes the effective control of cross-regional collaboration and presents an overview of collaboration and breaks the barriers between art and manufacturing, andblocks between design and processing. The “Eco-development of Fusion360 Industrial Chain” is both a significant means to and an inevitable trend forthe manufacturers and industrial designers to carry out innovation in China.

Study on Fusion Protein and Its gene in Baculovirus Specificity

International Nuclear Information System (INIS)

Nemr, W.A.H.

2012-01-01

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

Mechanistic insight provided by glutaredoxin within a fusion to redox-sensitive yellow fluorescent protein

DEFF Research Database (Denmark)

Björnberg, Olof; Østergaard, Henrik; Winther, Jakob R

2006-01-01

Redox-sensitive yellow fluorescent protein (rxYFP) contains a dithiol disulfide pair that is thermodynamically suitable for monitoring intracellular glutathione redox potential. Glutaredoxin 1 (Grx1p) from yeast is known to catalyze the redox equilibrium between rxYFP and glutathione, and here, we...... have generated a fusion of the two proteins, rxYFP-Grx1p. In comparison to isolated subunits, intramolecular transfer of reducing equivalents made the fusion protein kinetically superior in reactions with glutathione. The rate of GSSG oxidation was thus improved by a factor of 3300. The reaction...... separately and in the fusion. This could not be ascribed to the lack of an unproductive side reaction to glutaredoxin disulfide. Instead, slower alkylation kinetics with iodoacetamide indicates a better leaving-group capability of the remaining cysteine residue, which can explain the increased activity....

Domain fusion analysis by applying relational algebra to protein sequence and domain databases.

Science.gov (United States)

Truong, Kevin; Ikura, Mitsuhiko

2003-05-06

Domain fusion analysis is a useful method to predict functionally linked proteins that may be involved in direct protein-protein interactions or in the same metabolic or signaling pathway. As separate domain databases like BLOCKS, PROSITE, Pfam, SMART, PRINTS-S, ProDom, TIGRFAMs, and amalgamated domain databases like InterPro continue to grow in size and quality, a computational method to perform domain fusion analysis that leverages on these efforts will become increasingly powerful. This paper proposes a computational method employing relational algebra to find domain fusions in protein sequence databases. The feasibility of this method was illustrated on the SWISS-PROT+TrEMBL sequence database using domain predictions from the Pfam HMM (hidden Markov model) database. We identified 235 and 189 putative functionally linked protein partners in H. sapiens and S. cerevisiae, respectively. From scientific literature, we were able to confirm many of these functional linkages, while the remainder offer testable experimental hypothesis. Results can be viewed at http://calcium.uhnres.utoronto.ca/pi. As the analysis can be computed quickly on any relational database that supports standard SQL (structured query language), it can be dynamically updated along with the sequence and domain databases, thereby improving the quality of predictions over time.

Standard mirror fusion reactor design study

International Nuclear Information System (INIS)

Moir, R.W.

1978-01-01

This report covers the work of the Magnetic Fusion Energy Division's reactor study group during FY 1976 on the standard mirror reactor. The ''standard'' mirror reactor is characterized as a steady state, neutral beam sustained, D-T fusioning plasma confined by a Yin-Yang magnetic mirror field. The physics parameters are obtained from the same physics model that explains the 2XIIB experiment. The model assumes that the drift cyclotron loss cone mode occurs on the boundary of the plasma, and that it is stabilized by warm plasma with negligible energy investment. The result of the study was a workable mirror fusion power plant, steady-state blanket removal made relatively simple by open-ended geometry, and no impurity problem due to the positive plasma potential. The Q (fusion power/injected beam power) turns out to be only 1.1 because of loss out the ends from Coulomb collisions, i.e., classical losses. This low Q resulted in 77% of the gross electrical power being used to power the injectors, thereby causing the net power cost to be high. The low Q stimulated an intensive search for Q-enhancement concepts, resulting in the LLL reactor design effort turning to the field reversal mirror and the tandem mirror, each having Q of order 5

Macrophage fusion is controlled by the cytoplasmic protein tyrosine phosphatase PTP-PEST/PTPN12.

Science.gov (United States)

Rhee, Inmoo; Davidson, Dominique; Souza, Cleiton Martins; Vacher, Jean; Veillette, André

2013-06-01

Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as β-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading.

Optical design considerations for laser fusion reactors

International Nuclear Information System (INIS)

Monsler, M.J.; Maniscalco, J.A.

1977-09-01

The plan for the development of commercial inertial confinement fusion (ICF) power plants is discussed, emphasizing the utilization of the unique features of laser fusion to arrive at conceptual designs for reactors and optical systems which minimize the need for advanced materials and techniques requiring expensive test facilities. A conceptual design for a liquid lithium fall reactor is described which successfully deals with the hostile x-ray and neutron environment and promises to last the 30 year plant lifetime. Schemes for protecting the final focusing optics are described which are both compatible with this reactor system and show promise of surviving a full year in order to minimize costly downtime. Damage mechanisms and protection techniques are discussed, and a recommendation is made for a high f-number metal mirror final focusing system

C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage

International Nuclear Information System (INIS)

Tzeng, W.-P.; Frey, Teryl K.

2006-01-01

Rubella virus (RUB) replicons are derivatives of the RUB infectious cDNA clone that retain the nonstructural open reading frame (NS-ORF) that encodes the replicase proteins but not the structural protein ORF (SP-ORF) that encodes the virion proteins. RUB defective interfering (DI) RNAs contain deletions within the SP-ORF and thus resemble replicons. DI RNAs often retain the 5' end of the capsid protein (C) gene that has been shown to modulate virus-specific RNA synthesis. However, when replicons either with or without the C gene were passaged serially in the presence of wt RUB as a source of the virion proteins, it was found that neither replicon was maintained and DI RNAs were generated. The majority DI RNA species contained in-frame deletions in the SP-ORF leading to a fusion between the 5' end of the C gene and the 3' end of the E1 glycoprotein gene. DI infectious cDNA clones were constructed and transcripts from these DI infectious cDNA clones were maintained during serial passage with wt RUB. The C-E1 fusion protein encoded by the DI RNAs was synthesized and was required for maintenance of the DI RNA during serial passage. This is the first report of a functional novel gene product resulting from deletion during DI RNA generation. Thus far, the role of the C-E1 fusion protein in maintenance of DI RNAs during serial passage remained elusive as it was found that the fusion protein diminished rather than enhanced DI RNA synthesis and was not incorporated into virus particles

Optimization of the Expression of DT386-BR2 Fusion Protein in Escherichia coli using Response Surface Methodology.

Science.gov (United States)

Shafiee, Fatemeh; Rabbani, Mohammad; Jahanian-Najafabadi, Ali

2017-01-01

The aim of this study was to determine the best condition for the production of DT386-BR2 fusion protein, an immunotoxin consisting of catalytic and translocation domains of diphtheria toxin fused to BR2, a cancer specific cell penetrating peptide, for targeted eradication of cancer cells, in terms of the host, cultivation condition, and culture medium. Recombinant pET28a vector containing the codons optimized for the expression of the DT386-BR2 gene was transformed to different strains of Escherichia coli ( E. coli BL21 DE3, E. coli Rosetta DE3 and E. coli Rosetta-gami 2 DE3), followed by the induction of expression using 1 mM IPTG. Then, the strain with the highest ability to produce recombinant protein was selected and used to determine the best expression condition using response surface methodology (RSM). Finally, the best culture medium was selected. Densitometry analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the expressed fusion protein showed that E. coli Rosetta DE3 produced the highest amounts of the recombinant fusion protein when quantified by 1 mg/ml bovine serum albumin (178.07 μg/ml). Results of RSM also showed the best condition for the production of the recombinant fusion protein was induction with 1 mM IPTG for 2 h at 37°C. Finally, it was established that terrific broth could produce higher amounts of the fusion protein when compared to other culture media. In this study, we expressed the recombinant DT386-BR2 fusion protein in large amounts by optimizing the expression host, cultivation condition, and culture medium. This fusion protein will be subjected to purification and evaluation of its cytotoxic effects in future studies.

Optimization of the Expression of DT386-BR2 Fusion Protein in Escherichia coli using Response Surface Methodology

Directory of Open Access Journals (Sweden)

Fatemeh Shafiee

2017-01-01

Full Text Available Background: The aim of this study was to determine the best condition for the production of DT386-BR2 fusion protein, an immunotoxin consisting of catalytic and translocation domains of diphtheria toxin fused to BR2, a cancer specific cell penetrating peptide, for targeted eradication of cancer cells, in terms of the host, cultivation condition, and culture medium. Materials and Methods: Recombinant pET28a vector containing the codons optimized for the expression of the DT386-BR2 gene was transformed to different strains of Escherichia coli (E. coli BL21 DE3, E. coli Rosetta DE3 and E. coli Rosetta-gami 2 DE3, followed by the induction of expression using 1 mM IPTG. Then, the strain with the highest ability to produce recombinant protein was selected and used to determine the best expression condition using response surface methodology (RSM. Finally, the best culture medium was selected. Results: Densitometry analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the expressed fusion protein showed that E. coli Rosetta DE3 produced the highest amounts of the recombinant fusion protein when quantified by 1 mg/ml bovine serum albumin (178.07 μg/ml. Results of RSM also showed the best condition for the production of the recombinant fusion protein was induction with 1 mM IPTG for 2 h at 37°C. Finally, it was established that terrific broth could produce higher amounts of the fusion protein when compared to other culture media. Conclusion: In this study, we expressed the recombinant DT386-BR2 fusion protein in large amounts by optimizing the expression host, cultivation condition, and culture medium. This fusion protein will be subjected to purification and evaluation of its cytotoxic effects in future studies.

Purification method for recombinant proteins based on a fusion between the target protein and the C-terminus of calmodulin

Science.gov (United States)

Schauer-Vukasinovic, Vesna; Deo, Sapna K.; Daunert, Sylvia

2002-01-01

Calmodulin (CaM) was used as an affinity tail to facilitate the purification of the green fluorescent protein (GFP), which was used as a model target protein. The protein GFP was fused to the C-terminus of CaM, and a factor Xa cleavage site was introduced between the two proteins. A CaM-GFP fusion protein was expressed in E. coli and purified on a phenothiazine-derivatized silica column. CaM binds to the phenothiazine on the column in a Ca(2+)-dependent fashion and it was, therefore, used as an affinity tail for the purification of GFP. The fusion protein bound to the affinity column was then subjected to a proteolytic digestion with factor Xa. Pure GFP was eluted with a Ca(2+)-containing buffer, while CaM was eluted later with a buffer containing the Ca(2+)-chelating agent EGTA. The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.

Interactions involved in pH protection of the alphavirus fusion protein

Energy Technology Data Exchange (ETDEWEB)

Fields, Whitney; Kielian, Margaret, E-mail: margaret.kielian@einstein.yu.edu

2015-12-15

The alphavirus membrane protein E1 mediates low pH-triggered fusion of the viral and endosome membranes during virus entry. During virus biogenesis E1 associates as a heterodimer with the transmembrane protein p62. Late in the secretory pathway, cellular furin cleaves p62 to the mature E2 protein and a peripheral protein E3. E3 remains bound to E2 at low pH, stabilizing the heterodimer and thus protecting E1 from the acidic pH of the secretory pathway. Release of E3 at neutral pH then primes the virus for fusion during entry. Here we used site-directed mutagenesis and revertant analysis to define residues important for the interactions at the E3–E2 interface. Our data identified a key residue, E2 W235, which was required for E1 pH protection and alphavirus production. Our data also suggest additional residues on E3 and E2 that affect their interacting surfaces and thus influence the pH protection of E1 during alphavirus exit.

Designing the Cascade inertial confinement fusion reactor

International Nuclear Information System (INIS)

Pitts, J.H.

1987-01-01

The primary goal in designing inertial confinement fusion (ICF) reactors is to produce electrical power as inexpensively as possible, with minimum activation and without compromising safety. This paper discusses a method for designing the Cascade rotating ceramic-granule-blanket reactor (Pitts, 1985) and its associated power plant (Pitts and Maya, 1985). Although focus is on the cascade reactor, the design method and issues presented are applicable to most other ICF reactors

Inertial fusion reactor designs

International Nuclear Information System (INIS)

Meier, W.

1987-01-01

In this paper, a variety of reactor concepts are proposed. One of the prime concerns is dealing with the x-rays and debris that are emitted by the target. Internal neutron shielding can reduce radiation damage and activation, leading to longer life systems, reduced activation and fewer safety concerns. There is really no consensus on what the best reactor concept is at this point. There has been virtually no chamber technology development to date. This is the flip side of the coin of the separability of the target physics and the reactor design. Since reactor technology has not been required to do target experiments, it's not being developed. Economic analysis of conceptual designs indicates that ICF can be economically competitive with magnetic fusion, fission and fossil plants

Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes.

Science.gov (United States)

Debaize, Lydie; Jakobczyk, Hélène; Rio, Anne-Gaëlle; Gandemer, Virginie; Troadec, Marie-Bérengère

2017-01-01

Genetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy. We describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm. We propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells. The optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients' cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. Therefore, the optimized PLA protocol provides a new tool that can be adopted in a wide range of applications in the biological field.

Influence of rhTPO/GM-CSF fusion protein on hemopoiesis in mice irradiated with 60Co γ-rays

International Nuclear Information System (INIS)

Cao Hua; Ge Zhongliang; Zhang Qunwei; Liu Xiuzhen

1999-01-01

Objective: To find a new biological therapy for secondary hematopoietic failure including anemia, infection and hemorrhage after administration of chemotherapeutic drugs etc. Methods: hGM-CSF gene was ligated with hTPO gene isolated from human fetal liver mRNA and a new fusion protein rh TPO/GM-CSF obtained. Results: The new fusion protein could promote recovery of peripheral WBC and PLT of 5.0 Gy irradiated mice. BFU-E, CFU-Meg and CFU-GM in bone marrow of mice after irradiation recovered significantly by treatment with rhTPO/GM-CSF fusion protein for 10 days. Conclusion: These results suggest that the new fusion protein has the biological activity of both hTPO and hGM-CSF simultaneously and can stimulate the proliferation of megakaryocytes and granulocyte progenitors

First preliminary design of an experimental fusion reactor

International Nuclear Information System (INIS)

1977-09-01

A preliminary design of a tokamak experimental fusion reactor to be built in the near future is under way. The goals of the reactor are to achieve reactor-level plasma conditions for a sufficiently long operation period and to obtain design, construction and operational experience for the main components of full-scale power reactors. This design covers overall reactor system including plasma characteristics, reactor structure, blanket neutronics, shielding, superconducting magnets, neutral beam injector, electric power supply system, fuel circulating system, reactor cooling system, tritium recovery system and maintenance scheme. The main design parameters are as follows: the reactor fusion power 100 MW, torus radius 6.75 m, plasma radius 1.5 m, first wall radius 1.75 m, toroidal magnet field on axis 6 T, blanket fertile material Li 2 O, coolant He, structural material 316SS and tritium breeding ratio 0.9. (auth.)

The conserved glycine residues in the transmembrane domain of the Semliki Forest virus fusion protein are not required for assembly and fusion

International Nuclear Information System (INIS)

Liao Maofu; Kielian, Margaret

2005-01-01

The alphavirus Semliki Forest virus (SFV) infects cells via a low pH-triggered fusion reaction mediated by the viral E1 protein. Both the E1 fusion peptide and transmembrane (TM) domain are essential for membrane fusion, but the functional requirements for the TM domain are poorly understood. Here we explored the role of the five TM domain glycine residues, including the highly conserved glycine pair at E1 residues 415/416. SFV mutants with alanine substitutions for individual or all five glycine residues (5G/A) showed growth kinetics and fusion pH dependence similar to those of wild-type SFV. Mutants with increasing substitution of glycine residues showed an increasingly more stringent requirement for cholesterol during fusion. The 5G/A mutant showed decreased fusion kinetics and extent in fluorescent lipid mixing assays. TM domain glycine residues thus are not required for efficient SFV fusion or assembly but can cause subtle effects on the properties of membrane fusion

Design aspects of low activation fusion ignition experiments

International Nuclear Information System (INIS)

Cheng, E.T.; Creedon, R.L.; Hopkins, G.R.; Trester, P.W.; Wong, C.P.C.; Schultz, K.R.

1986-01-01

Preliminary design studies have been done exploring (1) materials selection, (2) shutdown biological dose rates, (3) mechanical design and (4) thermal design of a fusion ignition experiment made of low activation materials. From the results of these preliminary design studies it appears that an ignition experiment could be built of low activation materials, and that this design would allow hands-on access for maintenance

The B isozyme creatine kinase is active as a fusion protein in Escherichia coli

International Nuclear Information System (INIS)

Koretsky, A.P.; Traxler, B.A.

1989-01-01

A cDNA encoding the B isozyme of creatine kinase CK B has been expressed in Escherichia coli from a fusion with lacZ carried by λgtll. Western blots indicate that a stable polypeptide with the appropriate mobility for the Β-galactosidase-creatine kinase Β-gal-CK B ) fusion protein cross-reacts with both Β-gal and CK B antiserum. No significant CK activity is detected in control E. coli; however, extracts from cells containing the λgtll-CK B construct have a CK activity of 1.54j0.07 μmol/min per mg protein. The fusion protein appears to provide this activity bacause immunoprecipitation of protein with Β-gal antiserum leads to a loss of CK activity from extracts. That the enzyme is active in vivo was demonstrated by detection of a phosphocreatine (PCr) peak in the 31 P NMR spectrum from E. coli grown on medium supplemented with creatine. As in mammalian brain and muscle, the PCr peak detected was sensitive to the energy status of the E. coli. (author). 17 refs.; 3 figs.; 1 tab

Pharmacokinetics and safety in rhesus monkeys of a monoclonal antibody-GDNF fusion protein for targeted blood-brain barrier delivery.

Science.gov (United States)

Pardridge, William M; Boado, Ruben J

2009-10-01

Glial-derived neurotrophic factor (GDNF) is a potential therapy for stroke, Parkinson's disease, or drug addiction. However, GDNF does not cross the blood-brain barrier (BBB). GDNF is re-engineered as a fusion protein with a chimeric monoclonal antibody (MAb) to the human insulin receptor (HIR), which acts as a molecular Trojan horse to deliver the GDNF across the BBB. The pharmacokinetics (PK), toxicology, and safety pharmacology of the HIRMAb-GDNF fusion protein were investigated in Rhesus monkeys. The fusion protein was administered as an intravenous injection at doses up to 50 mg/kg over a 60 h period to 56 Rhesus monkeys. The plasma concentration of the HIRMAb-GDNF fusion protein was measured with a 2-site sandwich ELISA. No adverse events were observed in a 2-week terminal toxicology study, and no neuropathologic changes were observed. The PK analysis showed a linear relationship between plasma AUC and dose, a large systemic volume of distribution, as well as high clearance rates of 8-10 mL/kg/min. A no-observable-adverse-effect level is established in the Rhesus monkey for the acute administration of the HIRMAb-GDNF fusion protein. The fusion protein targeting the insulin receptor has a PK profile similar to a classical small molecule.

Conceptual design study of a scyllac fusion test reactor

International Nuclear Information System (INIS)

Thomassen, K.I.

1975-07-01

The report describes a conceptual design study of a fusion test reactor based on the Scyllac toroidal theta-pinch approach to fusion. It is not the first attempt to describe the physics and technology required for demonstrating scientific feasibility of the approach, but it is the most complete design in the sense that the physics necessary to achieve the device goals is extrapolated from experimentally tested MHD theories of toroidal systems,and it uses technological systems whose engineering performance has been carefully calculated to ensure that they meet the machine requirements

Overproduction, purification and characterization of human interferon alpha2a-human serum albumin fusion protein produced in methilotropic yeast Pichia pastoris

Science.gov (United States)

Ningrum, R. A.; Santoso, A.; Herawati, N.

2017-05-01

Human interferon alpha2a (hIFNα2a) is a therapeutic protein that used in cancer and hepatitis B/C therapy. The main problem of using hIFNα-2a is its short elimination half life due to its low molecular weight. Development of higher molecular weight protein by albumin fusion technology is a rational strategy to solve the problem. In our previous research we constructed an open reading frame (ORF) encoding hIFNα2a-human serum albumin (HSA) fusion protein that expressed in Pichia pastoris (P. pastoris) protease deficient strain SMD1168. This research was performed to overproduce, purify and characterize the fusion protein. To overproduce the protein, cultivation was performed in buffered complex medium containing glyserol (BMGY) for 24 h and protein overproduction was applied in buffered complex medium containing methanol (BMMY) for 48 hours at 30°C. The fusion protein was purified by blue sepharose affinity chromatography. Molecular weight characterization by SDS PAGE corresponds with its theoretical size, 85 kDa. Western blot analysis demonstrated that the fusion protein was recognized by anti hIFNα2 and anti HSA monoclonal antibody as well. Amino acid sequence of the fusion protein was determined by LC MS/MS2 mass spectrometry with trypsin as proteolitic enzyme. There were three fragments that identified as hIFNα2a and seven fragments that identified as HSA. Total identified amino acids were 150 residues with 20% coverage from total residues. To conclude, hIFNα2a-HSA fusion protein was overproduced, purified and characterized. Characterization based on molecular weight, antibody recognition and amino acid sequence confirmed that the fusion protein has correct identity as theoretically thought.

Overproduction, purification and characterization of human interferon alpha2a-human serum albumin fusion protein produced in methilotropic yeast Pichia pastoris

International Nuclear Information System (INIS)

Ningrum, R A; Santoso, A; Herawati, N

2017-01-01

Human interferon alpha2a (hIFNα2a) is a therapeutic protein that used in cancer and hepatitis B/C therapy. The main problem of using hIFNα-2a is its short elimination half life due to its low molecular weight. Development of higher molecular weight protein by albumin fusion technology is a rational strategy to solve the problem. In our previous research we constructed an open reading frame (ORF) encoding hIFNα2a-human serum albumin (HSA) fusion protein that expressed in Pichia pastoris (P. pastoris) protease deficient strain SMD1168. This research was performed to overproduce, purify and characterize the fusion protein. To overproduce the protein, cultivation was performed in buffered complex medium containing glyserol (BMGY) for 24 h and protein overproduction was applied in buffered complex medium containing methanol (BMMY) for 48 hours at 30°C. The fusion protein was purified by blue sepharose affinity chromatography. Molecular weight characterization by SDS PAGE corresponds with its theoretical size, 85 kDa. Western blot analysis demonstrated that the fusion protein was recognized by anti hIFNα2 and anti HSA monoclonal antibody as well. Amino acid sequence of the fusion protein was determined by LC MS/MS2 mass spectrometry with trypsin as proteolitic enzyme. There were three fragments that identified as hIFNα2a and seven fragments that identified as HSA. Total identified amino acids were 150 residues with 20% coverage from total residues. To conclude, hIFNα2a-HSA fusion protein was overproduced, purified and characterized. Characterization based on molecular weight, antibody recognition and amino acid sequence confirmed that the fusion protein has correct identity as theoretically thought. (paper)

Extrap conceptual fusion reactor design study

International Nuclear Information System (INIS)

Eninger, J.E; Lehnert, B.

1987-12-01

A study has recently been initiated to asses the fusion reactor potential of the Extrap concept. A reactor model is defined that fulfills certain economic and environmental criteria. This model is applied to Extrap and a reference reactor is outlined. The design is optimized by varying parameters subject to both physics and engineering constraints. Several design options are examined and key engineering issues are identified and addressed. Some preliminary results and conclusions of this work are summarized. (authors)

Updated reference design of a liquid metal cooled tandem mirror fusion breeder

International Nuclear Information System (INIS)

Berwald, D.H.; Whitley, R.H.; Garner, J.K.

1985-09-01

Detailed studies of key techinical issues for liquid metal cooled fusion breeder (fusion-fission hybrid blankets) have been performed during the period 1983-4. Based upon the results of these studies, the 1982 reference liquid metal cooled tandem mirror fusion breeder blanket design was updated and is described. The updated reference blankets provides increased breeding and lower technological risk in comparison with the original reference blanket. In addition to the blanket design revisions, a plant concept, cost, and fuel cycle economics assessment is provided. The fusion breeder continues to promise an economical source of fissile fuel for the indefinite future

Updated reference design of a liquid metal cooled tandem mirror fusion breeder

Energy Technology Data Exchange (ETDEWEB)

Berwald, D.H.; Whitley, R.H.; Garner, J.K.; Gromada, R.J.; McCarville, T.J.; Moir, R.W.; Lee, J.D.; Bandini, B.R.; Fulton, F.J.; Wong, C.P.C.; Maya, I.; Hoot, C.G.; Schultz, K.R.; Miller, L.G.; Beeston, J.M.; Harris, B.L.; Westman, R.A.; Ghoniem, N.M.; Orient, G.; Wolfer, M.; DeVan, J.H.; Torterelli, P.

1985-09-01

Detailed studies of key techinical issues for liquid metal cooled fusion breeder (fusion-fission hybrid blankets) have been performed during the period 1983-4. Based upon the results of these studies, the 1982 reference liquid metal cooled tandem mirror fusion breeder blanket design was updated and is described. The updated reference blankets provides increased breeding and lower technological risk in comparison with the original reference blanket. In addition to the blanket design revisions, a plant concept, cost, and fuel cycle economics assessment is provided. The fusion breeder continues to promise an economical source of fissile fuel for the indefinite future.

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

Science.gov (United States)

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

2015-09-01

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

Osiris and SOMBRERO inertial fusion power plant designs - summary, conclusions, and recommendations

International Nuclear Information System (INIS)

Meier, Wayne R.

1994-01-01

An 18 month study to evaluate the potential of inertial fusion energy (IFE) for electric power production has been completed. The primary objective of the study was to provide the US Department of Energy with an evaluation of the potential of inertial fusion for electric power production. The study included the conceptual design of two inertial fusion power plants. Osiris uses an induction linac heavy ion beam driver, and SOMBRERO uses a krypton fluoride laser driver. Conceptual designs were completed for the reactors, power conversion and plant facilities, and drivers. Environmental and safety aspects, technical issues, technology development needs, and economics of the final point designs were assessed and compared. This paper summarizes the results and conclusions of the conceptual designs and results of the assessment studies. We conclude that IFE has the potential of producing technically credible designs with environmental, safety, and economics characteristics that are just as attractive as magnetic fusion. Realizing this potential will require additional research and development on target physics, chamber design, target production and injection systems, and drivers. ((orig.))

A novel fusion protein of IP10-scFv retains antibody specificity and chemokine function

International Nuclear Information System (INIS)

Guo Junqing; Chen Liu; Ai Hongwu; Jing Jiannian; Zhou Jiyong; Zhang Chuyu; You Shangyou

2004-01-01

We combined the specificity of tumor-specific antibody with the chemokine function of interferon-γ inducible protein 10 (IP-10) to recruit immune effector cells in the vicinity of tumor cells. A novel fusion protein of IP10-scFv was constructed by fusing mouse IP-10 to V H region of single-chain Fv fragment (scFv) against acidic isoferritin (AIF), and expressed in NS0 murine myeloma cells. The IP10-scFv fusion protein was shown to maintain the specificity of the antiAIF scFv with similar affinity constant, and bind to the human hepatocarcinoma SMMC 7721 cells secreting AIF as well as the activated mouse T lymphocytes expressing CXCR3 receptor. Furthermore, the IP10-scFv protein either in solution or bound on the surface of SMMC 7721 cells induced significant chemotaxis of mouse T cells in vitro. The results indicate that the IP10-scFv fusion protein possesses both bioactivities of the tumor-specific antibody and IP-10 chemokine, suggesting its possibility to induce an enhanced immune response against the residual tumor cells in vivo

Trypsin- and low pH-mediated fusogenicity of avian metapneumovirus fusion proteins is determined by residues at positions 100, 101 and 294.

Science.gov (United States)

Yun, Bingling; Guan, Xiaolu; Liu, Yongzhen; Gao, Yanni; Wang, Yongqiang; Qi, Xiaole; Cui, Hongyu; Liu, Changjun; Zhang, Yanping; Gao, Li; Li, Kai; Gao, Honglei; Gao, Yulong; Wang, Xiaomei

2015-10-26

Avian metapneumovirus (aMPV) and human metapneumovirus (hMPV) are members of the genus Metapneumovirus in the subfamily Pneumovirinae. Metapneumovirus fusion (F) protein mediates the fusion of host cells with the virus membrane for infection. Trypsin- and/or low pH-induced membrane fusion is a strain-dependent phenomenon for hMPV. Here, we demonstrated that three subtypes of aMPV (aMPV/A, aMPV/B, and aMPV/C) F proteins promoted cell-cell fusion in the absence of trypsin. Indeed, in the presence of trypsin, only aMPV/C F protein fusogenicity was enhanced. Mutagenesis of the amino acids at position 100 and/or 101, located at a putative cleavage region in aMPV F proteins, revealed that the trypsin-mediated fusogenicity of aMPV F proteins is regulated by the residues at positions 100 and 101. Moreover, we demonstrated that aMPV/A and aMPV/B F proteins mediated cell-cell fusion independent of low pH, whereas the aMPV/C F protein did not. Mutagenesis of the residue at position 294 in the aMPV/A, aMPV/B, and aMPV/C F proteins showed that 294G played a critical role in F protein-mediated fusion under low pH conditions. These findings on aMPV F protein-induced cell-cell fusion provide new insights into the molecular mechanisms underlying membrane fusion and pathogenesis of aMPV.

Production and characterization of active recombinant interleukin-12/eGFP fusion protein in stably-transfected DF1 chicken cells.

Science.gov (United States)

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

2015-01-01

The adjuvant activity of chicken interleukin-12 (chIL-12) protein has been described as similar to that of mammalian IL-12. Recombinant chIL-12 can be produced using several methods, but chIL-12 production in eukaryotic cells is lower than that in prokaryotic cells. Stimulating compounds, such as dimethyl sulfoxide (DMSO), can be added to animal cell cultures to overcome this drawback. In this study, we constructed a cell line, DF1/chIL-12 which stably expressed a fusion protein, chIL-12 and enhanced green fluorescent protein (eGFP) connected by a (G4 S)3 linker sequence. Fusion protein production was increased when cells were cultured in the presence of DMSO. When 1 × 10(6) DF1/chIL-12 cells were inoculated in a T-175 flask containing 30 mL of media, incubated for 15 h, and further cultivated in the presence of 4% DMSO for 48 h, the production of total fusion protein was mostly enhanced compared with the production of total fusion protein by using cell lysates induced with DMSO at other concentrations. The concentrations of the unpurified and purified total fusion proteins in cell lysates were 2,781 ± 2.72 ng mL(-1) and 2,207 ± 3.28 ng mL(-1) , respectively. The recovery rate was 79%. The fusion protein stimulated chicken splenocytes to produce IFN-γ, which was measured using an enzyme-linked immunosorbent assay, in the culture supernatant, indicating that treating DF1/chIL-12 cells with DMSO or producing chIL-12 in a fusion protein form does not have adverse effects on the bioactivity of chIL-12. © 2015 American Institute of Chemical Engineers.

A compact, multifunctional fusion module directs cholesterol-dependent homomultimerization and syncytiogenic efficiency of reovirus p10 FAST proteins.

Directory of Open Access Journals (Sweden)

Tim Key

2014-03-01

Full Text Available The homologous p10 fusion-associated small transmembrane (FAST proteins of the avian (ARV and Nelson Bay (NBV reoviruses are the smallest known viral membrane fusion proteins, and are virulence determinants of the fusogenic reoviruses. The small size of FAST proteins is incompatible with the paradigmatic membrane fusion pathway proposed for enveloped viral fusion proteins. Understanding how these diminutive viral fusogens mediate the complex process of membrane fusion is therefore of considerable interest, from both the pathogenesis and mechanism-of-action perspectives. Using chimeric ARV/NBV p10 constructs, the 36-40-residue ectodomain was identified as the major determinant of the differing fusion efficiencies of these homologous p10 proteins. Extensive mutagenic analysis determined the ectodomain comprises two distinct, essential functional motifs. Syncytiogenesis assays, thiol-specific surface biotinylation, and liposome lipid mixing assays identified an ∼25-residue, N-terminal motif that dictates formation of a cystine loop fusion peptide in both ARV and NBV p10. Surface immunofluorescence staining, FRET analysis and cholesterol depletion/repletion studies determined the cystine loop motif is connected through a two-residue linker to a 13-residue membrane-proximal ectodomain region (MPER. The MPER constitutes a second, independent motif governing reversible, cholesterol-dependent assembly of p10 multimers in the plasma membrane. Results further indicate that: (1 ARV and NBV homomultimers segregate to distinct, cholesterol-dependent microdomains in the plasma membrane; (2 p10 homomultimerization and cholesterol-dependent microdomain localization are co-dependent; and (3 the four juxtamembrane MPER residues present in the multimerization motif dictate species-specific microdomain association and homomultimerization. The p10 ectodomain therefore constitutes a remarkably compact, multifunctional fusion module that directs syncytiogenic

Junction region of EWS-FLI1 fusion protein has a dominant negative effect in Ewing's sarcoma in vitro.

Science.gov (United States)

Jully, Babu; Vijayalakshmi, Ramshankar; Gopal, Gopisetty; Sabitha, Kesavan; Rajkumar, Thangarajan

2012-11-12

Ewing's sarcoma is a malignancy characterized by a specific 11:22 chromosomal translocation which generates a novel EWS-FLI1 fusion protein functioning as an aberrant transcription factor. In the present study, we have further characterized the junction region of the EWS-FLI1 fusion protein. In-silico model of EWS-FLI1 fusion protein was analysed for ligand binding sites, and a putative region (amino acid (aa) 251-343 of the type 1 fusion protein) in the vicinity of the fusion junction was cloned and expressed using bacterial expression. The recombinant protein was characterized by Circular Dichroism (CD). We then expressed aa 251-280 ectopically in Ewing's sarcoma cell-line and its effect on cell proliferation, tumorigenicity and expression of EWS-FLI1 target genes were analysed. Our modelling analysis indicated that Junction region (aa 251-343) encompasses potential ligand biding sites in the EWS-FLI1 protein and when expressed in bacteria was present as soluble form. Ectopically expressing this region in Ewing's sarcoma cells inhibited tumorigenicity, and EWS-FLI1 target genes indicating a dominant negative biological effect. Junction region can be exploited further as target for drug development in future to specifically target EWS-FLI1 in Ewing's Sarcoma.

[Construction and prokaryotic expression of recombinant gene EGFRvIII HBcAg and immunogenicity analysis of the fusion protein].

Science.gov (United States)

Duan, Xiao-yi; Wang, Jian-sheng; Guo, You-min; Han, Jun-li; Wang, Quan-ying; Yang, Guang-xiao

2007-01-01

To construct recombinant prokaryotic expression plasmid pET28a(+)/c-PEP-3-c and evaluate the immunogenicity of the fusion protein. cDNA fragment encoding PEP-3 was obtained from pGEM-T Easy/PEP-3 and inserted into recombinant plasmid pGEMEX/HBcAg. Then it was subcloned in prokaryotic expression vector and transformed into E.coli BL21(DE3). The fusion protein was expressed by inducing IPTG and purified by Ni(2+)-NTA affinity chromatography. BALB/c mice were immunized with fusion protein and the antibody titre was determined by indirect ELISA. The recombinant gene was confirmed to be correct by restriction enzyme digestion and DNA sequencing. After prokaryotic expression, fusion protein existed in sediment and accounted for 56% of all bacterial lysate. The purified product accounted for 92% of all protein and its concentration was 8 g/L. The antibody titre in blood serum reached 1:16 000 after the fourth immunization and reached 1:2.56x10(5) after the sixth immunization. The titre of anti-PEP-3 antibody reached 1:1.28x10(5) and the titre of anti-HBcAg antibody was less than 1:4x10(3). Fusion gene PEP-3-HBcAg is highly expressed in E.coli BL21. The expressed fusion protein can induce neutralizing antibody with high titer and specificity, which lays a foundation for the study of genetically engineering vaccine for malignant tumors with the high expression of EGFRvIII.

Egg CD9 protein tides correlated with sperm oscillations tune the gamete fusion ability in mammal.

Science.gov (United States)

Ravaux, Benjamin; Favier, Sophie; Perez, Eric; Gourier, Christine

2018-01-23

Mammalian fertilization involves membrane events -adhesion, fusion, sperm engulfment, membrane block to polyspermy- whose causes remain largely unknown. Recently, specific oscillations of the sperm in contact with the egg were shown to be necessary for fusion. Using a microfluidic chip to impose the venue for the encounter of two gametes allowed real-time observation of the membrane remodelling occurring at the sperm/egg interface. The spatiotemporal mapping of egg CD9 revealed that this protein concentrates at the egg/sperm interface as a result of sperm oscillations, until a CD9-rich platform is nucleated on which fusion immediately takes place. Within 2 to 5 minutes after fusion, most of the CD9 leaves the egg for the external aqueous medium. Then an egg membrane wave engulfs the sperm head in approximately 25 minutes. These results show that sperm oscillations initiate the CD9 recruitment that causes gamete fusion after which CD9 and associated proteins leave the membrane in a process likely to contribute to block polyspermy. They highlight that the gamete fusion story in mammals is an unexpected interplay between mechanical constraints and proteins. © The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

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

Directory of Open Access Journals (Sweden)

Luisa Cironi

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

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector

Science.gov (United States)

2013-01-01

Background Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. Results We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit’s component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. Conclusions We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome

Present status of design, research and development of nuclear fusion reactors and problems

International Nuclear Information System (INIS)

1983-04-01

Seven years have elapsed since the publication of ''Progress of nuclear fusion research and perspective toward the development of power reactors'' by the Atomic Energy Society of Japan in August, 1976. During this period, the research and development of nuclear fusion have changed from plasma physics to reactor technology, being conscious of the realization of fusion reactors. There are the R project in the Institute of Plasma Physics, Nagoya University, and the design and construction of JT-60 in Japan Atomic Energy Research Institute, to put it concretely. Now the research and development taking the economical efficiency into account are adopted. However, the type of fusion reactors is not reduced to tokamak type, accordingly the research and development to meet the diverse possibilities are forwarded. The progress of tokamak reactor research, core plasma design, nuclear design and shielding design, thermal structure design, the design of superconducting magnets, disassembling and repair, safety, economical efficiency, the conceptual design of other types than tokamak and others are reported. (Kako, I.)

Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions.

Science.gov (United States)

Tsachaki, Maria; Birk, Julia; Egert, Aurélie; Odermatt, Alex

2015-07-01

Membrane proteins of the endoplasmic reticulum (ER) are involved in a wide array of essential cellular functions. Identification of the topology of membrane proteins can provide significant insight into their mechanisms of action and biological roles. This is particularly important for membrane enzymes, since their topology determines the subcellular site where a biochemical reaction takes place and the dependence on luminal or cytosolic co-factor pools and substrates. The methods currently available for the determination of topology of proteins are rather laborious and require post-lysis or post-fixation manipulation of cells. In this work, we have developed a simple method for defining intracellular localization and topology of ER membrane proteins in living cells, based on the fusion of the respective protein with redox-sensitive green-fluorescent protein (roGFP). We validated the method and demonstrated that roGFP fusion proteins constitute a reliable tool for the study of ER membrane protein topology, using as control microsomal 11β-hydroxysteroid dehydrogenase (11β-HSD) proteins whose topology has been resolved, and comparing with an independent approach. We then implemented this method to determine the membrane topology of six microsomal members of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The results revealed a luminal orientation of the catalytic site for three enzymes, i.e. 17β-HSD6, 7 and 12. Knowledge of the intracellular location of the catalytic site of these enzymes will enable future studies on their biological functions and on the role of the luminal co-factor pool. Copyright © 2015 Elsevier B.V. All rights reserved.

Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein.

Directory of Open Access Journals (Sweden)

Benjamin Dälken

Full Text Available BACKGROUND: The apoptosis-inducing serine protease granzyme B (GrB is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. METHODS AND FINDINGS: We investigated the influence of bacterial maltose-binding protein (MBP fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal α-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. CONCLUSIONS: Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins.

Maltose-Binding Protein Enhances Secretion of Recombinant Human Granzyme B Accompanied by In Vivo Processing of a Precursor MBP Fusion Protein

Science.gov (United States)

Dälken, Benjamin; Jabulowsky, Robert A.; Oberoi, Pranav; Benhar, Itai; Wels, Winfried S.

2010-01-01

Background The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. Methods and Findings We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal α-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. Conclusions Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins. PMID:21203542

Design and evaluation of a laser fusion energy station for industrial applications

International Nuclear Information System (INIS)

Kok, K.D.; Bates, F.J.; Denning, R.S.; Triplett, M.B.; Waddell, J.D.

1978-01-01

The identification and development of long-term energy options is important in the continued growth of industry in the United States. Fusion and particularly laser fusion is one of the possible options. This paper applies the criteria used by industry in the selection of an energy source to the first of a series of conceptual designs for a laser fusion energy station. Several conclusions are presented including the constraints placed on the design by the criteria

An Approach to Automated Fusion System Design and Adaptation

Directory of Open Access Journals (Sweden)

Alexander Fritze

2017-03-01

Full Text Available Industrial applications are in transition towards modular and flexible architectures that are capable of self-configuration and -optimisation. This is due to the demand of mass customisation and the increasing complexity of industrial systems. The conversion to modular systems is related to challenges in all disciplines. Consequently, diverse tasks such as information processing, extensive networking, or system monitoring using sensor and information fusion systems need to be reconsidered. The focus of this contribution is on distributed sensor and information fusion systems for system monitoring, which must reflect the increasing flexibility of fusion systems. This contribution thus proposes an approach, which relies on a network of self-descriptive intelligent sensor nodes, for the automatic design and update of sensor and information fusion systems. This article encompasses the fusion system configuration and adaptation as well as communication aspects. Manual interaction with the flexibly changing system is reduced to a minimum.

An Approach to Automated Fusion System Design and Adaptation.

Science.gov (United States)

Fritze, Alexander; Mönks, Uwe; Holst, Christoph-Alexander; Lohweg, Volker

2017-03-16

Industrial applications are in transition towards modular and flexible architectures that are capable of self-configuration and -optimisation. This is due to the demand of mass customisation and the increasing complexity of industrial systems. The conversion to modular systems is related to challenges in all disciplines. Consequently, diverse tasks such as information processing, extensive networking, or system monitoring using sensor and information fusion systems need to be reconsidered. The focus of this contribution is on distributed sensor and information fusion systems for system monitoring, which must reflect the increasing flexibility of fusion systems. This contribution thus proposes an approach, which relies on a network of self-descriptive intelligent sensor nodes, for the automatic design and update of sensor and information fusion systems. This article encompasses the fusion system configuration and adaptation as well as communication aspects. Manual interaction with the flexibly changing system is reduced to a minimum.

An FPGA-based heterogeneous image fusion system design method

Science.gov (United States)

Song, Le; Lin, Yu-chi; Chen, Yan-hua; Zhao, Mei-rong

2011-08-01

Taking the advantages of FPGA's low cost and compact structure, an FPGA-based heterogeneous image fusion platform is established in this study. Altera's Cyclone IV series FPGA is adopted as the core processor of the platform, and the visible light CCD camera and infrared thermal imager are used as the image-capturing device in order to obtain dualchannel heterogeneous video images. Tailor-made image fusion algorithms such as gray-scale weighted averaging, maximum selection and minimum selection methods are analyzed and compared. VHDL language and the synchronous design method are utilized to perform a reliable RTL-level description. Altera's Quartus II 9.0 software is applied to simulate and implement the algorithm modules. The contrast experiments of various fusion algorithms show that, preferably image quality of the heterogeneous image fusion can be obtained on top of the proposed system. The applied range of the different fusion algorithms is also discussed.

Characterization of a novel fusion protein from IpaB and IpaD of Shigella spp. and its potential as a pan-Shigella vaccine.

Science.gov (United States)

Martinez-Becerra, Francisco J; Chen, Xiaotong; Dickenson, Nicholas E; Choudhari, Shyamal P; Harrison, Kelly; Clements, John D; Picking, William D; Van De Verg, Lillian L; Walker, Richard I; Picking, Wendy L

2013-12-01

Shigellosis is an important disease in the developing world, where about 90 million people become infected with Shigella spp. each year. We previously demonstrated that the type three secretion apparatus (T3SA) proteins IpaB and IpaD are protective antigens in the mouse lethal pulmonary model. In order to simplify vaccine formulation and process development, we have evaluated a vaccine design that incorporates both of these previously tested Shigella antigens into a single polypeptide chain. To determine if this fusion protein (DB fusion) retains the antigenic and protective capacities of IpaB and IpaD, we immunized mice with the DB fusion and compared the immune response to that elicited by the IpaB/IpaD combination vaccine. Purification of the DB fusion required coexpression with IpgC, the IpaB chaperone, and after purification it maintained the highly α-helical characteristics of IpaB and IpaD. The DB fusion also induced comparable immune responses and retained the ability to protect mice against Shigella flexneri and S. sonnei in the lethal pulmonary challenge. It also offered limited protection against S. dysenteriae challenge. Our results show the feasibility of generating a protective Shigella vaccine comprised of the DB fusion.

Characterization of a Novel Fusion Protein from IpaB and IpaD of Shigella spp. and Its Potential as a Pan-Shigella Vaccine

Science.gov (United States)

Martinez-Becerra, Francisco J.; Chen, Xiaotong; Dickenson, Nicholas E.; Choudhari, Shyamal P.; Harrison, Kelly; Clements, John D.; Picking, William D.; Van De Verg, Lillian L.; Walker, Richard I.

2013-01-01

Shigellosis is an important disease in the developing world, where about 90 million people become infected with Shigella spp. each year. We previously demonstrated that the type three secretion apparatus (T3SA) proteins IpaB and IpaD are protective antigens in the mouse lethal pulmonary model. In order to simplify vaccine formulation and process development, we have evaluated a vaccine design that incorporates both of these previously tested Shigella antigens into a single polypeptide chain. To determine if this fusion protein (DB fusion) retains the antigenic and protective capacities of IpaB and IpaD, we immunized mice with the DB fusion and compared the immune response to that elicited by the IpaB/IpaD combination vaccine. Purification of the DB fusion required coexpression with IpgC, the IpaB chaperone, and after purification it maintained the highly α-helical characteristics of IpaB and IpaD. The DB fusion also induced comparable immune responses and retained the ability to protect mice against Shigella flexneri and S. sonnei in the lethal pulmonary challenge. It also offered limited protection against S. dysenteriae challenge. Our results show the feasibility of generating a protective Shigella vaccine comprised of the DB fusion. PMID:24060976

Design system for in-vessel mainipulator of fusion reactor 'DESIM'

International Nuclear Information System (INIS)

Adachi, Junihci; Kobayashi, Takeshi; Ise, Hideo; Sato, Keisuke; Matsuda, Hirotsugu

1989-01-01

A computer aided design system 'DESIM' for the in-vessel manipulators of nuclear fusion reactors has been developed to design the manipulators efficiently. The DESIM consists of the following subsystems: (1) the design system for arm mechanisms to realize optimum manipulation performance in the specified workspace; (2) the robot simulator to study manipulator movement, postures and interference problems; (3) the CAD system which is used to define the structure object data for robots, and the interface system for the data conversion from the CAD system to the robot simulator. The DESIM has been used to design the in-vessel manipulator for the Fusion Experimental Reactor (FER) to confirm the effectiveness. (author)

Analysis of the selective advantage conferred by a C-E1 fusion protein synthesized by rubella virus DI RNAs

International Nuclear Information System (INIS)

Claus, Claudia; Tzeng, W.-P.; Liebert, Uwe Gerd; Frey, Teryl K.

2007-01-01

During serial passaging of rubella virus (RUB) in cell culture, the dominant species of defective-interfering RNA (DI) generated contains an in-frame deletion between the capsid protein (C) gene and E1 glycoprotein gene resulting in production of a C-E1 fusion protein that is necessary for the maintenance of the DI [Tzeng, W.P., Frey, T.K. (2006). C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage. Virology 356 198-207.]. A BHK cell line stably expressing the RUB structural proteins was established which was used to package DIs into virus particles following transfection with in vitro transcripts from DI infectious cDNA constructs. Packaging of a DI encoding an in-frame C-GFP-E1 reporter fusion protein corresponding to the C-E1 fusion protein expressed in a native DI was only marginally more efficient than packaging of a DI encoding GFP, indicating that the C-E1 fusion protein did not function by enhancing packaging. However, infection with the DI encoding the C-GFP-E1 fusion protein (in the absence of wt RUB helper virus) resulted in formation of clusters of GFP-positive cells and the percentage of GFP-positive cells in the culture following infection remained relatively constant. In contrast, a DI encoding GFP did not form GFP-positive clusters and the percentage of GFP-positive cells declined by roughly half from 2 to 4 days post-infection. Cluster formation and sustaining the percentage of infected (GFP-positive) cells required the C part of the fusion protein, including the downstream but not the upstream of two arginine clusters (both of which are associated with RNA binding and association with mitochondrial p32 protein) and the E1 part through the transmembrane sequence, but not the C-terminal cytoplasmic tail. Among a collection of mutant DI constructs, cluster formation and sustaining infected cell percentage correlated with maintenance during serial passage with wt RUB. We hypothesize that cluster formation and

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

Science.gov (United States)

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

2016-05-01

In filamentous fungi, communication is essential for the formation of an interconnected, multinucleate, syncytial network, which is constructed via hyphal fusion or fusion of germinated asexual spores (germlings). Anastomosis in filamentous fungi is comparable to other somatic cell fusion events resulting in syncytia, including myoblast fusion during muscle differentiation, macrophage fusion, and fusion of trophoblasts during placental development. In Neurospora crassa, fusion of genetically identical germlings is a highly dynamic and regulated process that requires components of a MAP kinase signal transduction pathway. The kinase pathway components (NRC-1, MEK-2 and MAK-2) and the scaffold protein HAM-5 are recruited to hyphae and germling tips undergoing chemotropic interactions. The MAK-2/HAM-5 protein complex shows dynamic oscillation to hyphae/germling tips during chemotropic interactions, and which is out-of-phase to the dynamic localization of SOFT, which is a scaffold protein for components of the cell wall integrity MAP kinase pathway. In this study, we functionally characterize HAM-5 by generating ham-5 truncation constructs and show that the N-terminal half of HAM-5 was essential for function. This region is required for MAK-2 and MEK-2 interaction and for correct cellular localization of HAM-5 to "fusion puncta." The localization of HAM-5 to puncta was not perturbed in 21 different fusion mutants, nor did these puncta colocalize with components of the secretory pathway. We also identified HAM-14 as a novel member of the HAM-5/MAK-2 pathway by mining MAK-2 phosphoproteomics data. HAM-14 was essential for germling fusion, but not for hyphal fusion. Colocalization and coimmunoprecipitation data indicate that HAM-14 interacts with MAK-2 and MEK-2 and may be involved in recruiting MAK-2 (and MEK-2) to complexes containing HAM-5. Copyright © 2016 by the Genetics Society of America.

Fusion protein based on Grb2-SH2 domain for cancer therapy

International Nuclear Information System (INIS)

Saito, Yuriko; Furukawa, Takako; Arano, Yasushi; Fujibayashi, Yasuhisa; Saga, Tsuneo

2010-01-01

Research highlights: → Grb2 mediates EGFR signaling through binding to phosphorylate EGFR with SH2 domain. → We generated fusion proteins containing 1 or 2 SH2 domains of Grb2 added with TAT. → The one with 2 SH2 domains (TSSF) interfered ERK phosphorylation. → TSSF significantly delayed the growth of EGFR overexpressing tumor in a mouse model. -- Abstract: Epidermal growth factor receptor (EGFR) is one of the very attractive targets for cancer therapy. In this study, we generated fusion proteins containing one or two Src-homology 2 (SH2) domains of growth factor receptor bound protein 2 (Grb2), which bind to phosphorylated EGFR, added with HIV-1 transactivating transcription for cell membrane penetration (termed TSF and TSSF, respectively). We examined if they can interfere Grb2-mediated signaling pathway and suppress tumor growth as expected from the lack of SH3 domain, which is necessary to intermediate EGFR-Grb2 cell signaling, in the fusion proteins. The transduction efficiency of TSSF was similar to that of TSF, but the binding activity of TSSF to EGFR was higher than that of TSF. Treatment of EGFR-overexpressing cells showed that TSSF decreased p42-ERK phosphorylation, while TSF did not. Both the proteins delayed cell growth but did not induce cell death in culture. TSSF also significantly suppressed tumor growth in vivo under consecutive administration. In conclusion, TSSF showed an ability to inhibit EGFR-Grb2 signaling and could have a potential to treat EGFR-activated cancer.

International Fusion Materials Irradiation Facility conceptual design activity. Present status and perspective

International Nuclear Information System (INIS)

Kondo, Tatsuo; Noda, Kenji; Oyama, Yukio

1998-01-01

For developing the materials for nuclear fusion reactors, it is indispensable to study on the neutron irradiation behavior under fusion reactor conditions, but there is not any high energy neutron irradiation facility that can simulate fusion reactor conditions at present. Therefore, the investigation of the IFMIF was begun jointly by Japan, USA, Europe and Russia following the initiative of IEA. The conceptual design activities were completed in 1997. As to the background and the course, the present status of the research on heavy irradiation and the testing means for fusion materials, the requirement and the technical basis of high energy neutron irradiation, and the international joint design activities are reported. The materials for fusion reactors are exposed to the neutron irradiation with the energy spectra up to 14 MeV. The requirements from the users that the IFMIF should satisfy, the demand of the tests for the materials of prototype and demonstration fusion reactors and the evaluation of the neutron field characteristics of the IFMIF are discussed. As to the conceptual design of the IFMIF, the whole constitution, the operational mode, accelerator system and target system are described. (K.I.)

Designing the Sniper: Improving Targeted Human Cytolytic Fusion Proteins for Anti-Cancer Therapy via Molecular Simulation

Directory of Open Access Journals (Sweden)

Anna Bochicchio

2017-02-01

Full Text Available Targeted human cytolytic fusion proteins (hCFPs are humanized immunotoxins for selective treatment of different diseases including cancer. They are composed of a ligand specifically binding to target cells genetically linked to a human apoptosis-inducing enzyme. hCFPs target cancer cells via an antibody or derivative (scFv specifically binding to e.g., tumor associated antigens (TAAs. After internalization and translocation of the enzyme from endocytosed endosomes, the human enzymes introduced into the cytosol are efficiently inducing apoptosis. Under in vivo conditions such enzymes are subject to tight regulation by native inhibitors in order to prevent inappropriate induction of cell death in healthy cells. Tumor cells are known to upregulate these inhibitors as a survival mechanism resulting in escape of malignant cells from elimination by immune effector cells. Cytosolic inhibitors of Granzyme B and Angiogenin (Serpin P9 and RNH1, respectively, reduce the efficacy of hCFPs with these enzymes as effector domains, requiring detrimentally high doses in order to saturate inhibitor binding and rescue cytolytic activity. Variants of Granzyme B and Angiogenin might feature reduced affinity for their respective inhibitors, while retaining or even enhancing their catalytic activity. A powerful tool to design hCFPs mutants with improved potency is given by in silico methods. These include molecular dynamics (MD simulations and enhanced sampling methods (ESM. MD and ESM allow predicting the enzyme-protein inhibitor binding stability and the associated conformational changes, provided that structural information is available. Such “high-resolution” detailed description enables the elucidation of interaction domains and the identification of sites where particular point mutations may modify those interactions. This review discusses recent advances in the use of MD and ESM for hCFP development from the viewpoints of scientists involved in both fields.

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

Energy Technology Data Exchange (ETDEWEB)

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

2009-04-24

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

[Rapid expression and preparation of the recombinant fusion protein sTNFRII-gAD by adenovirus vector system].

Science.gov (United States)

Lu, Yue; Liu, Dan; Zhang, Xiaoren; Liu, Xuerong; Shen, Wei; Zheng, Gang; Liu, Yunfan; Dong, Xiaoyan; Wu, Xiaobing; Gao, Jimin

2011-08-01

We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFalpha activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFalpha's cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.

Conceptual design study of fusion experimental reactor (FER)

International Nuclear Information System (INIS)

1986-11-01

Since 1980 the design study has been conducted at JAERI for the Fusion Experimental Reactor (FER) which has been proposed to be the next machine to JT-60 in the Japanese long term program of fusion reactor development. During two years from 1984 to 1985 FER concept was reviewed and redesigned. This report is the summary of the results obtained in the review and redesign activities in 1984 and 85. In the first year FER concept was discussed again and its frame work was reestablished. According to the new frame work the major reactor components of FER were designed. In the second year the whole plant system design including plant layout plan was conducted as well as the more detailed design analysis of the reactor conponents. The newly established frame for FER design is as follows: 1) Plasma : Self-ignition. 2) Operation scenario : Quasi-steady state operation with long burn pulse. 3) Neutron fluence on the first wall : 0.3 MWY/M 2 . 4) Blanket : Non-tritium breeding blanket with test modules for breeding blanket development. 5) Magnets : Superconducting Magnets. (author)

New concepts for controlled fusion reactor blanket design

International Nuclear Information System (INIS)

Conn, R.W.; Kulcinski, G.L.; Avci, H.; El-Maghrabi, M.

1975-01-01

Several new concepts for fusion reactor blanket design based on the idea of shifting, or tailoring, the neutron spectrum incident on the first structural wall are presented. The spectral shifter is a nonstructural element which can be made of graphite, silicon carbide, or three dimensionally woven carbon fibers (and containing other materials as appropriate) placed between the neutron source and the first structural wall. The softened neutron spectrum incident on the structural components leads to lower gas production and atom displacement rates than in more standard fusion blanket designs. In turn, this results in longer anticipated lifetimes for the structural materials and can significantly reduce radioactivity and afterheat levels. In addition, the neutron spectrum in the first structural wall can be made to approach the flux shape in fast breeder reactors. Such spectral softening means that existing radiation facilities may be more profitably used to provide relevant materials radiation damage data for the structural materials in these fusion blanket designs. This general class of blanket concepts are referred to as internal spectral shifter and energy converter, or ISSEC concepts. These specific design concepts fall into three main categories: ISSEC/EB concepts based on utilizing existing designs which breed tritium behind the first structural wall; ISSEC/IB concepts based on breeding tritium inside the first vacuum wall; and ISSEC/Bu concepts based on using boron, carbon, and perhaps, beryllium to obtain an energy multiplier and converter design that does not attempt to breed tritium or utilize lithium. The detailed analyses relate specifically to the nuclear performance of ISSEC systems and to a discussion of materials radiation damage problems in the structural material.(U.S.)

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

Science.gov (United States)

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

2015-01-01

To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins. © 2015 S. Karger AG, Basel.

The pharmacological efficacy of the anti-IL17 scFv and sTNFR1 bispecific fusion protein in inflammation mouse stimulated by LPS.

Science.gov (United States)

Yang, Yongbi; Zhang, Teng; Cao, Hongxue; Yu, Dan; Zhang, Tong; Zhao, Shaojuan; Jing, Xiaohui; Song, Liying; Liu, Yunye; Che, Ruixiang; Liu, Xin; Li, Deshan; Ren, Guiping

2017-08-01

Acute lung injury (ALI) is still a leading cause of morbidity and mortality in critically ill patients. Recently, our study found that a bispecific fusion protein treatment can ameliorate the lung injury induced by LPS. However, the molecular mechanisms which bispecific fusion protein ameliorates acute lung injury remain unclear. In this study, we found that the bispecific fusion protein treatment inhibited the nuclear transcription of NF-κB in confocal laser scanning fluorescence microscopy, the bispecific fusion protein exert protective effects in the cell model of ALI induced by lipopolysaccharide (LPS) via inhibiting the nuclear factor κB (NF-κB) signaling pathway and mediate inflammation. Moreover, the treatment of the bispecific fusion protein show its efficacy in animal models stimulated by LPS, the results of real-time PCR and ELISA demonstrate that bispecific fusion protein treatment effectively inhibited the over-expression of inflammatory cytokines(tumor necrosis factor α, interleukin 1β and interleukin 17). In addition, LPS-challenged mice exhibited significant lung injury characterized by the deterioration of histopathology, which was meliorated by bispecific fusion protein treatment. Collectively, these results demonstrate that bispecific fusion protein treatment ameliorates LPS-induced ALI through reducing inflammatory cytokines and lung inflammation, which may be associated with the decreased the nuclear transcription of NF-κB. The bispecific fusion protein may be useful as a novel therapy to treat ALI. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

TALE-PvuII Fusion Proteins – Novel Tools for Gene Targeting

Science.gov (United States)

Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

2013-01-01

Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity. PMID:24349308

Fusion reactor design and technology program in China

International Nuclear Information System (INIS)

Huang, J.H.

1994-01-01

A fusion-fission hybrid reactor program was launched in 1987. The purpose of development of the hybrid reactor is twofold: to solve the problem of nuclear fuel supply for an expected large-scale development of fission reactor plants, and to maintain the momentum of fusion research. The program is described and the activities and progress of the program are presented. Two conceptual designs of an engineering test reactor with tokamak configuration were developed at the Southwestern Institute of Physics and the Institute of Plasma Physics. The results are a tokamak engineering test breeder (TETB) series design and a fusion-fission hybrid reactor design (SSEHR), characterized by a liquid-Li self-cooled blanket and an He-cooled solid tritium breeder blanket respectively. In parallel with the design studies, relevant technological experiments on a small or medium scale have been supported by this program. These include LHCD, ICRH and pellet injection in the area of plasma engineering; neutronics integral experiments with U, Pu, Fe and Be; various irradiation tests of austenitic and ferritic steels, magnetohydrodynamic (MHD) pressure drop experiments using a liquid metal loop; research into permeation barriers for tritium and hydrogen isotopes; solid tritium breeder tests using an in-situ loop in a fission reactor. All these experiments have proceeded successfully. The second step of this program is now starting. It seems reasonable that most of the research carried out in the first step will continue. ((orig.))

A recombined fusion protein PTD-Grb2-SH2 inhibits the proliferation of breast cancer cells in vitro.

Science.gov (United States)

Yin, Jikai; Cai, Zhongliang; Zhang, Li; Zhang, Jian; He, Xianli; Du, Xilin; Wang, Qing; Lu, Jianguo

2013-03-01

The growth factor receptor bound protein 2 (Grb2) is one of the affirmative targets for cancer therapy, especially for breast cancer. In this study, we hypothesized the Src-homology 2 (SH2) domain in Grb2 may serve as a competitive protein-binding agent to interfere with the proliferation of breast cancer cells in vitro. We designed, constructed, expressed and purified a novel fusion protein containing the protein transduction domain (PTD) and Grb2-SH2 domain (we named it after PTD-Grb2-SH2). An immunofluorescence assay was used to investigate the location of PTD-Grb2-SH2 in cells. MTT assay and EdU experiments were applied to detect the proliferation of breast cancer cells. The ultra-structure was observed using transmission electron microscopy. Flow cytometry was used to determine the cytotoxicity of PTD-Grb2-SH2 on cell proliferation. We successfully obtained the PTD-Grb2-SH2 fusion protein in soluble form using a prokaryotic expression system. The new fusion protein successfully passed through both the cellular and nuclear membranes of breast cancer cells. The MTT assay showed that PTD-Grb2-SH2 exhibited significant toxicity to breast cancer cells in a dose- and time-dependent manner in vitro. EdU identified the decreased proliferation rates in treated MDA-MB-231 and SK-BR-3 cells. Observation by transmission electron microscopy and flow cytometry further confirmed the cytotoxicity as apoptosis. Our results show that the HIV1-TAT domain is a useful tool for transporting a low molecular weight protein across the cell membrane in vitro. The PTD-Grb2-SH2 may be a novel agent for breast cancer therapy.

Different sets of ER-resident J-proteins regulate distinct polar nuclear-membrane fusion events in Arabidopsis thaliana.

Science.gov (United States)

Maruyama, Daisuke; Yamamoto, Masaya; Endo, Toshiya; Nishikawa, Shuh-ichi

2014-11-01

Angiosperm female gametophytes contain a central cell with two polar nuclei. In many species, including Arabidopsis thaliana, the polar nuclei fuse during female gametogenesis. We previously showed that BiP, an Hsp70 in the endoplasmic reticulum (ER), was essential for membrane fusion during female gametogenesis. Hsp70 function requires partner proteins for full activity. J-domain containing proteins (J-proteins) are the major Hsp70 functional partners. A. thaliana ER contains three soluble J-proteins, AtERdj3A, AtERdj3B, and AtP58(IPK). Here, we analyzed mutants of these proteins and determined that double-mutant ovules lacking AtP58(IPK) and AtERdj3A or AtERdj3B were defective in polar nuclear fusion. Electron microscopy analysis identified that polar nuclei were in close contact, but no membrane fusion occurred in mutant ovules lacking AtP58(IPK) and AtERdj3A. The polar nuclear outer membrane appeared to be connected via the ER remaining at the inner unfused membrane in mutant ovules lacking AtP58(IPK) and AtERdj3B. These results indicate that ER-resident J-proteins, AtP58(IPK)/AtERdj3A and AtP58(IPK)/AtERdj3B, function at distinct steps of polar nuclear-membrane fusion. Similar to the bip1 bip2 double mutant female gametophytes, the aterdj3a atp58(ipk) double mutant female gametophytes defective in fusion of the outer polar nuclear membrane displayed aberrant endosperm proliferation after fertilization with wild-type pollen. However, endosperm proliferated normally after fertilization of the aterdj3b atp58(ipk) double mutant female gametophytes defective in fusion of the inner membrane. Our results indicate that the polar nuclear fusion defect itself does not cause an endosperm proliferation defect. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

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Suh JS

2014-03-01

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

Identification of two Th1 cell epitopes on the Babesia bovis-encoded 77-kilodalton merozoite protein (Bb-1) by use of truncated recombinant fusion proteins.

Science.gov (United States)

Brown, W C; Zhao, S; Woods, V M; Tripp, C A; Tetzlaff, C L; Heussler, V T; Dobbelaere, D A; Rice-Ficht, A C

1993-01-01

Previous studies have demonstrated the serologic and T-cell immunogenicity for cattle of a recombinant form of the apical complex-associated 77-kDa merozite protein of Babesia bovis, designated Bb-1. The present study characterizes the immunogenic epitopes of the Bb-1 protein. A series of recombinant truncated fusion proteins spanning the majority of the Bb-1 protein were expressed in Escherichia coli, and their reactivities with bovine peripheral blood mononuclear cells and T-cell clones derived from B. bovis-immune cattle and with rabbit antibodies were determined. Lymphocytes from two immune cattle were preferentially stimulated by the N-terminal half of the Bb-1 protein (amino acids 23 to 266, termed Bb-1A), localizing the T-cell epitopes to the Bb-1A portion of the molecule. CD4+ T-cell clones derived by stimulation with the intact Bb-1 fusion protein were used to identify two T-cell epitopes in the Bb-1A protein, consisting of amino acids SVVLLSAFSGN VWANEAEVSQVVK and FSDVDKTKSTEKT (residues 23 to 46 and 82 to 94). In contrast, rabbit antiserum raised against the intact fusion protein reacted only with the C-terminal half of the protein (amino acids 267 to 499, termed Bb-1B), which contained 28 tandem repeats of the tetrapeptide PAEK or PAET. Biological assays and Northern (RNA) blot analyses for cytokines revealed that following activation with concanavalin A, T-cell clones reactive against the two Bb-1A epitopes produced interleukin-2, gamma interferon, and tumor necrosis factors beta and alpha, but not interleukin-4, suggesting that the Bb-1 antigen preferentially stimulates the Th1 subset of CD4+ T cells in cattle. The studies described here report for the first time the characterization, by cytokine production, of the Th1 subset of bovine T cells and show that, as in mice, protozoal antigens can induce Th1 cells in ruminants. This first demonstration of B. bovis-encoded Th1 cell epitopes provides a rationale for incorporation of all or part of the Bb-1

Conceptual design of a laser fusion power plant

International Nuclear Information System (INIS)

Maniscalco, J.A.; Meier, W.R.; Monsler, M.J.

1977-01-01

A conceptual design of a laser fusion power plant is extensively discussed. Recent advances in high gain targets are exploited in the design. A smaller blanket structure is made possible by use of a thick falling region of liquid lithium for a first wall. Major design features of the plant, reactor, and laser systems are described. A parametric analysis of performance and cost vs. design parameters is presented to show feasible design points. A more definitive follow-on conceptual design study is planned

A novel fusion protein of IP10-scFv retains antibody specificity and chemokine function

Energy Technology Data Exchange (ETDEWEB)

Junqing, Guo; Liu, Chen; Hongwu, Ai; Jiannian, Jing; Jiyong, Zhou; Chuyu, Zhang; Shangyou, You

2004-07-23

We combined the specificity of tumor-specific antibody with the chemokine function of interferon-{gamma} inducible protein 10 (IP-10) to recruit immune effector cells in the vicinity of tumor cells. A novel fusion protein of IP10-scFv was constructed by fusing mouse IP-10 to V{sub H} region of single-chain Fv fragment (scFv) against acidic isoferritin (AIF), and expressed in NS0 murine myeloma cells. The IP10-scFv fusion protein was shown to maintain the specificity of the antiAIF scFv with similar affinity constant, and bind to the human hepatocarcinoma SMMC 7721 cells secreting AIF as well as the activated mouse T lymphocytes expressing CXCR3 receptor. Furthermore, the IP10-scFv protein either in solution or bound on the surface of SMMC 7721 cells induced significant chemotaxis of mouse T cells in vitro. The results indicate that the IP10-scFv fusion protein possesses both bioactivities of the tumor-specific antibody and IP-10 chemokine, suggesting its possibility to induce an enhanced immune response against the residual tumor cells in vivo.

Junction region of EWS-FLI1 fusion protein has a dominant negative effect in Ewing’s Sarcoma in vitro

International Nuclear Information System (INIS)

Jully, Babu; Vijayalakshmi, Ramshankar; Gopal, Gopisetty; Sabitha, Kesavan; Rajkumar, Thangarajan

2012-01-01

Ewing’s sarcoma is a malignancy characterized by a specific 11:22 chromosomal translocation which generates a novel EWS-FLI1 fusion protein functioning as an aberrant transcription factor. In the present study, we have further characterized the junction region of the EWS-FLI1 fusion protein. In-silico model of EWS-FLI1 fusion protein was analysed for ligand binding sites, and a putative region (amino acid (aa) 251–343 of the type 1 fusion protein) in the vicinity of the fusion junction was cloned and expressed using bacterial expression. The recombinant protein was characterized by Circular Dichroism (CD). We then expressed aa 251–280 ectopically in Ewing’s sarcoma cell-line and its effect on cell proliferation, tumorigenicity and expression of EWS-FLI1 target genes were analysed. Our modelling analysis indicated that Junction region (aa 251–343) encompasses potential ligand biding sites in the EWS-FLI1 protein and when expressed in bacteria was present as soluble form. Ectopically expressing this region in Ewing’s sarcoma cells inhibited tumorigenicity, and EWS-FLI1 target genes indicating a dominant negative biological effect. Junction region can be exploited further as target for drug development in future to specifically target EWS-FLI1 in Ewing’s Sarcoma

The novel fusion proteins, GnRH-p53 and GnRHIII-p53, expression and their anti-tumor effect.

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Peiyuan Jia

Full Text Available p53, one of the most well studied tumor suppressor factor, is responsible to a variety of damage owing to the induction of apoptosis and cell cycle arrest in the tumor cells. More than 50% of human tumors contain mutation or deletion of p53. Gonadotrophin-releasing hormone (GnRH, as the ligand of Gonadotrophin-releasing hormone receptor (GnRH-R, was used to deliver p53 into tumor cells. The p53 fusion proteins GnRH-p53 and GnRH iii-p53 were expressed and their targeted anti-tumor effects were determined. GnRH mediates its fusion proteins transformation into cancer cells. The intracellular delivery of p53 fusion proteins exerted the inhibition of the growth of H1299 cells in vitro and the reduction of tumor volume in vivo. Their anti-tumor effect was functioned by the apoptosis and cell cycle arrest induced by p53. Hence, the fusion protein could be a novel protein drug for anti-tumor therapy.

Design study of an accelerator for heavy ion fusion

International Nuclear Information System (INIS)

Katayama, T.; Noda, A.; Tokuda, N.; Hirao, Y.

1980-01-01

Design of a demonstration accelerator for heavy ion fusion based on a synchrotron system is briefly described. The proposed complex system of injector linac, rapid cycling synchrotron and five accumulation rings can produce a peak current 1.6 kA, peak power 32 TW and total energy 0.3 MJ. Investigations of the intrabeam scattering give a lifetime of the beam longer than the fusion cycle time of 1 sec

Cell-to-Cell Measles Virus Spread between Human Neurons Is Dependent on Hemagglutinin and Hyperfusogenic Fusion Protein.

Science.gov (United States)

Sato, Yuma; Watanabe, Shumpei; Fukuda, Yoshinari; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji

2018-03-15

Measles virus (MV) usually causes acute infection but in rare cases persists in the brain, resulting in subacute sclerosing panencephalitis (SSPE). Since human neurons, an important target affected in the disease, do not express the known MV receptors (signaling lymphocyte activation molecule [SLAM] and nectin 4), how MV infects neurons and spreads between them is unknown. Recent studies have shown that many virus strains isolated from SSPE patients possess substitutions in the extracellular domain of the fusion (F) protein which confer enhanced fusion activity. Hyperfusogenic viruses with such mutations, unlike the wild-type MV, can induce cell-cell fusion even in SLAM- and nectin 4-negative cells and spread efficiently in human primary neurons and the brains of animal models. We show here that a hyperfusogenic mutant MV, IC323-F(T461I)-EGFP (IC323 with a fusion-enhancing T461I substitution in the F protein and expressing enhanced green fluorescent protein), but not the wild-type MV, spreads in differentiated NT2 cells, a widely used human neuron model. Confocal time-lapse imaging revealed the cell-to-cell spread of IC323-F(T461I)-EGFP between NT2 neurons without syncytium formation. The production of virus particles was strongly suppressed in NT2 neurons, also supporting cell-to-cell viral transmission. The spread of IC323-F(T461I)-EGFP was inhibited by a fusion inhibitor peptide as well as by some but not all of the anti-hemagglutinin antibodies which neutralize SLAM- or nectin-4-dependent MV infection, suggesting the presence of a distinct neuronal receptor. Our results indicate that MV spreads in a cell-to-cell manner between human neurons without causing syncytium formation and that the spread is dependent on the hyperfusogenic F protein, the hemagglutinin, and the putative neuronal receptor for MV. IMPORTANCE Measles virus (MV), in rare cases, persists in the human central nervous system (CNS) and causes subacute sclerosing panencephalitis (SSPE) several

Conceptual design and neutronics analyses of a fusion reactor blanket simulation facility

International Nuclear Information System (INIS)

Beller, D.E.; Ott, K.O.; Terry, W.K.

1987-01-01

A new conceptual design of a fusion reactor blanket simulation facility has been developed. This design follows the principles that have been successfully employed in the Purdue Fast Breeder Blanket Facility (FBBF), where experiments have resulted in the discovery of substantial deficiencies in neutronics predictions. With this design, discrepancies between calculation and experimental data can be nearly fully attributed to calculation methods because design deficiencies that could affect results are insignificant. The conceptual design of this FBBF analog, the Fusion Reactor Blanket Facility, is presented

Magnet Design Considerations for Fusion Nuclear Science Facility

Energy Technology Data Exchange (ETDEWEB)

Zhai, Y. [Princeton Plasma Physics Lab. (PPPL), Princeton, NJ (United States); Kessel, C. [Princeton Plasma Physics Lab. (PPPL), Princeton, NJ (United States); El-Guebaly, L. [Univ. of Wisconsin, Madison, WI (United States) Fusion Technology Institute; Titus, P. [Princeton Plasma Physics Lab. (PPPL), Princeton, NJ (United States)

2016-06-01

The Fusion Nuclear Science Facility (FNSF) is a nuclear confinement facility that provides a fusion environment with components of the reactor integrated together to bridge the technical gaps of burning plasma and nuclear science between the International Thermonuclear Experimental Reactor (ITER) and the demonstration power plant (DEMO). Compared with ITER, the FNSF is smaller in size but generates much higher magnetic field, i.e., 30 times higher neutron fluence with three orders of magnitude longer plasma operation at higher operating temperatures for structures surrounding the plasma. Input parameters to the magnet design from system code analysis include magnetic field of 7.5 T at the plasma center with a plasma major radius of 4.8 m and a minor radius of 1.2 m and a peak field of 15.5 T on the toroidal field (TF) coils for the FNSF. Both low-temperature superconductors (LTS) and high-temperature superconductors (HTS) are considered for the FNSF magnet design based on the state-of-the-art fusion magnet technology. The higher magnetic field can be achieved by using the high-performance ternary restacked-rod process Nb3Sn strands for TF magnets. The circular cable-in-conduit conductor (CICC) design similar to ITER magnets and a high-aspect-ratio rectangular CICC design are evaluated for FNSF magnets, but low-activation-jacket materials may need to be selected. The conductor design concept and TF coil winding pack composition and dimension based on the horizontal maintenance schemes are discussed. Neutron radiation limits for the LTS and HTS superconductors and electrical insulation materials are also reviewed based on the available materials previously tested. The material radiation limits for FNSF magnets are defined as part of the conceptual design studies for FNSF magnets.

Studies with GFP-Vpr fusion proteins: induction of apoptosis but ablation of cell-cycle arrest despite nuclear membrane or nuclear localization

International Nuclear Information System (INIS)

Waldhuber, Megan G.; Bateson, Michael; Tan, Judith; Greenway, Alison L.; McPhee, Dale A.

2003-01-01

The human immunodeficiency virus type 1 (HIV-1) Vpr protein is known to arrest the cell cycle in G 2 /M and induce apoptosis following arrest. The functions of Vpr relative to its location in the cell remain unresolved. We now demonstrate that the location and function of Vpr are dependent on the makeup of fusion proteins and that the functions of G 2 /M arrest and apoptosis are separable. Using green fluorescence protein mutants (EGFP or EYFP), we found that fusion at either the N- or C-terminus compromised the ability of Vpr to arrest cell cycling, relative to that of His-Vpr or wild-type protein. Additionally, utilizing the ability to specifically identify cells expressing the fusion proteins, we confirm that Vpr can induce apoptosis, but appears to be independent of cell-cycle arrest in G 2 /M. Both N- and C-terminal Vpr/EYFP fusion proteins induced apoptosis but caused minimal G 2 /M arrest. These studies with Vpr fusion proteins indicate that the functions of Vpr leading to G 2 /M arrest and apoptosis are separable and that fusion of Vpr to EGFP or EYFP affected the localization of the protein. Our findings suggest that nuclear membrane localization and nuclear import and export are strongly governed by modification of the N-terminus of Vpr

Structural characterization of the fusion core in syncytin, envelope protein of human endogenous retrovirus family W

International Nuclear Information System (INIS)

Gong Rui; Peng Xiaoxue; Kang Shuli; Feng Huixing; Huang Jianying; Zhang Wentao; Lin Donghai; Tien Po; Xiao Gengfu

2005-01-01

Syncytin is a captive retroviral envelope protein, possibly involved in the formation of the placental syncytiotrophoblast layer generated by trophoblast cell fusion at the maternal-fetal interface. We found that syncytin and type I viral envelope proteins shared similar structural profiling, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR). We expressed the predicted regions of NHR (41 aa) and CHR (34 aa) in syncytin as a native single chain (named 2-helix protein) to characterize it. 2-helix protein exists as a trimer and is highly α-helix, thermo-stable, and denatured by low pH. NHR and CHR could form a protease-resistant complex. The complex structure built by the molecular docking demonstrated that NHR and CHR associated in an antiparallel manner. Overall, the 2-helix protein could form a thermo-stable coiled coil trimer. The fusion core structure of syncytin was first demonstrated in endogenous retrovirus. These results support the explanation how syncytin mediates cytotrophoblast cell fusion involved in placental morphogenesis

Conceptual design study for a mirror fusion breeder

International Nuclear Information System (INIS)

Huang Jinhua; Deng Boquan; Li Guiqing

1986-01-01

A mirror fusion breeder, CHD, has been designed for providing plenty of nuclear fuel for light water reactors to meet the needs for rapid development of nuclear power in the first half of next century. The breeder is able to support the nuclear fuel needs for more than 10 LWRs of equal scale in power with fuel enriched directly in CHD without reprocessing. Measures are taken to flatten the power density distribution in the blanket so that fission is suppressed in the region close to the plasma, and by this way fuel production is enhanced for this direct enriched fusion breeder. In order to reduce the MHD pressure drop, LiPb flows in the blanket axially. Though the tritium inventory in the reactor is very low, special material and design have to be developed to reduce the permeation of tritium through the coolant pipes. The cost of electricity from the system, consisting of 11 LWR plants and one fusion breeder is predicted to be 1.05 times of that from a traditional LWR plant. This figure is insensitive both to the cost of CHD and its support ratio

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

International Nuclear Information System (INIS)

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

2010-01-01

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

Peptide-Based Membrane Fusion Inhibitors Targeting HCoV-229E Spike Protein HR1 and HR2 Domains

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Shuai Xia

2018-02-01

Full Text Available Human coronavirus 229E (HCoV-229E infection in infants, elderly people, and immunocompromised patients can cause severe disease, thus calling for the development of effective and safe therapeutics to treat it. Here we reported the design, synthesis and characterization of two peptide-based membrane fusion inhibitors targeting HCoV-229E spike protein heptad repeat 1 (HR1 and heptad repeat 2 (HR2 domains, 229E-HR1P and 229E-HR2P, respectively. We found that 229E-HR1P and 229E-HR2P could interact to form a stable six-helix bundle and inhibit HCoV-229E spike protein-mediated cell-cell fusion with IC50 of 5.7 and 0.3 µM, respectively. 229E-HR2P effectively inhibited pseudotyped and live HCoV-229E infection with IC50 of 0.5 and 1.7 µM, respectively. In a mouse model, 229E-HR2P administered intranasally could widely distribute in the upper and lower respiratory tracts and maintain its fusion-inhibitory activity. Therefore, 229E-HR2P is a promising candidate for further development as an antiviral agent for the treatment and prevention of HCoV-229E infection.

Self-Assembly of Spider Silk-Fusion Proteins Comprising Enzymatic and Fluorescence Activity.

Science.gov (United States)

Humenik, Martin; Mohrand, Madeleine; Scheibel, Thomas

2018-04-18

The recombinant spider silk protein eADF4(C16) was genetically fused either with esterase 2 (EST2) or green fluorescent protein (GFP). The fusions EST-eADF4(C16) and GFP-eADF4(C16) were spectroscopically investigated and showed native structures of EST and GFP. The structural integrity was confirmed by the enzymatic activity of EST and the fluorescence of GFP. The spider silk moiety retained its intrinsically unstructured conformation in solution and the self-assembly into either nanofibrils or nanoparticles could be controlled by the concentration of phosphate. Particles, however, showed significantly lower activity of the EST and GFP domains likely caused by a steric hindrance. However, upon self-assembly of EST-eADF4(C16) and GFP-eADF4(C16) into fibrils the protein activities were retained. In general, the fusion of globular enzymes with the spider silk domain allows the generation of fibrous biomaterials with catalytic or light emitting properties.

Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation.

Science.gov (United States)

Alvarez, M Lucrecia; Topal, Emel; Martin, Federico; Cardineau, Guy A

2010-01-01

Improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems since product yields have a major influence on process economics. Cereal grain evolved to store large amounts of proteins in tightly organized aggregates. In maize, gamma-Zein is the major storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in specialized organelles called protein bodies (PB). Zera (gamma-Zein ER-accumulating domain) is the N-terminal proline-rich domain of gamma-zein that is sufficient to induce the assembly of PB formation. Fusion of the Zera domain to proteins of interest results in assembly of dense PB-like, ER-derived organelles, containing high concentration of recombinant protein. Our main goal was to increase recombinant protein accumulation in plants in order to enhance the efficiency of orally-delivered plant-made vaccines. It is well known that oral vaccination requires substantially higher doses than parental formulations. As a part of a project to develop a plant-made plague vaccine, we expressed our model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without a fused Zera domain. We demonstrated that Zera-F1-V protein accumulation was at least 3x higher than F1-V alone when expressed in three different host plant systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells. We confirmed the feasibility of using Zera technology to induce protein body formation in non-seed tissues. Zera expression and accumulation did not affect plant development and growth. These results confirmed the potential exploitation of Zera technology to substantially increase the accumulation of value-added proteins in plants.

Once for All: A Novel Robust System for Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in Plants

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Guitao Zhong

2017-06-01

Full Text Available Chimeric fluorescent fusion proteins have been employed as a powerful tool to reveal the subcellular localizations and dynamics of proteins in living cells. Co-expression of a fluorescent fusion protein with well-known organelle markers in the same cell is especially useful in revealing its spatial and temporal functions of the protein in question. However, the conventional methods for co-expressing multiple fluorescent tagged proteins in plants have the drawbacks of low expression efficiency, variations in the expression level and time-consuming genetic crossing. Here, we have developed a novel robust system that allows for high-efficient co-expression of multiple chimeric fluorescent fusion proteins in plants in a time-saving fashion. This system takes advantage of employing a single expression vector which consists of multiple semi-independent expressing cassettes for the protein co-expression thereby overcoming the limitations of using multiple independent expressing plasmids. In addition, it is a highly manipulable DNA assembly system, in which modification and recombination of DNA molecules are easily achieved through an optimized one-step assembly reaction. By employing this effective system, we demonstrated that co-expression of two chimeric fluorescent fusion reporter proteins of vacuolar sorting receptor and secretory carrier membrane protein gave rise to their perspective subcellular localizations in plants via both transient expression and stable transformation. Thus, we believed that this technical advance represents a promising approach for multi-color-protein co-expression in plant cells.

Conceptual design of a fast-ignition laser fusion reactor FALCON-D

International Nuclear Information System (INIS)

Goto, T.; Ogawa, Y.; Okano, K.; Hiwatari, R.; Asaoka, Y.; Someya, Y.; Sunahara, A.; Johzaki, T.

2008-10-01

A new conceptual design of the laser fusion power plant FALCON-D (Fast ignition Advanced Laser fusion reactor CONcept with a Dry wall chamber) has been proposed. The fast ignition method can achieve the sufficient fusion gain for a commercial operation (∼100) with about 10 times smaller fusion yield than the conventional central ignition method. FALCON-D makes full use of this property and aims at designing with a compact dry wall chamber (5 - 6 m radius). 1-D/2-D hydrodynamic simulations showed the possibility of the sufficient gain achievement with a 40 MJ target yield. The design feasibility of the compact dry wall chamber and solid breeder blanket system was shown through the thermomechanical analysis of the dry wall and neutronics analysis of the blanket system. A moderate electric output (∼400 MWe) can be achieved with a high repetition (30 Hz) laser. This dry wall concept not only reduces some difficulties accompanied with a liquid wall but also enables a simple cask maintenance method for the replacement of the blanket system, which can shorten the maintenance time. The basic idea of the maintenance method for the final optics system has also been proposed. Some critical R and D issues required for this design are also discussed. (author)

Mission and design of the Fusion Ignition Research Experiment (FIRE)

International Nuclear Information System (INIS)

Meade, D.M.; Jardin, S.C.; Schmidt, J.

2001-01-01

Experiments are needed to test and extend present understanding of confinement, macroscopic stability, alpha-driven instabilities, and particle/power exhaust in plasmas dominated by alpha heating. A key issue is to what extent pressure profile evolution driven by strong alpha heating will act to self-organize advanced configurations with large bootstrap current fractions and internal transport barriers. A design study of a Fusion Ignition Research Experiment (FIRE) is underway to assess near term opportunities for advancing the scientific understanding of self-heated fusion plasmas. The emphasis is on understanding the behavior of fusion plasmas dominated by alpha heating (Q≥5) that are sustained for durations comparable to the characteristic plasma time scales (≥20 τ E and ∼τ skin , where τ skin is the time for the plasma current profile to redistribute at fixed current). The programmatic mission of FIRE is to attain, explore, understand and optimize alpha-dominated plasmas to provide knowledge for the design of attractive magnetic fusion energy systems. The programmatic strategy is to access the alpha-heating-dominated regime with confidence using the present advanced tokamak data base (e.g., Elmy-H-mode, ≤0.75 Greenwald density) while maintaining the flexibility for accessing and exploring other advanced tokamak modes (e. g., reversed shear, pellet enhanced performance) at lower magnetic fields and fusion power for longer durations in later stages of the experimental program. A major goal is to develop a design concept that could meet these physics objectives with a construction cost in the range of $1B. (author)

Preconceptual design of hyfire. A fusion driven high temperature electrolysis plant

International Nuclear Information System (INIS)

Varljen, T.C.; Chi, J.W.H.; Karbowski, J.S.

1983-01-01

Brookhaven National Laboratory has been engaged in a scoping study to investigate the potential merits of coupling a fusion reactor with a high temperature blanket to a high temperature electrolysis (HTE) process to produce hydrogen and oxygen. Westinghouse is assisting this study in the areas of systems design integration, plasma engineering, balance of plant design and electrolyzer technology. The aim of the work done in the past year has been to focus on a reference design point for the plant, which has been designated HYFIRE. In prior work, the STARFIRE commercial tokamak fusion reactor was directly used as the fusion driver. This report describes a new design obtained by scaling the basic STARFIRE design to permit the achievement of a blanket power of 6000 MWt. The high temperature blanket design employs a thermally insulated refractory oxide region which provides high temperature (>1000 deg. C) steam at moderate pressures to high temperature electrolysis units. The electrolysis process selected is based on the high temperature, solid electrolyte fuel cell technology developed by Westinghouse. An initial process design and plant layout has been completed; component cost and plant economics studies are now underway to develop estimates of hydrogen production costs and to determine the sensitivity of this cost to changes in major design parameters. (author)

Conceptual design report for a Fusion Engineering Device sector-handling machine and movable manipulator system

International Nuclear Information System (INIS)

Watts, K.D.; Masson, L.S.; McPherson, R.S.

1982-10-01

Design requirements, trade studies, design descriptions, conceptual designs, and cost estimates have been completed for the Fusion Engineering Device sector handling machine, movable manipulator system, subcomponent handling machine, and limiter blade handling machine. This information will be used by the Fusion Engineering Design Center to begin to determine the cost and magnitude of the effort required to perform remote maintenance on the Fusion Engineering Device. The designs presented are by no means optimum, and the costs estimates are rough-order-of-magnitude

Cleaved thioredoxin fusion protein enables the crystallization of poorly soluble ERα in complex with synthetic ligands

International Nuclear Information System (INIS)

Cura, Vincent; Gangloff, Monique; Eiler, Sylvia; Moras, Dino; Ruff, Marc

2007-01-01

A new crystallization strategy: the presence of cleaved thioredoxin fusion is critical for crystallization of the estrogen nuclear receptor ligand binding domain in complex with synthetic ligands. This novel technique should be regarded as an interesting alternative for crystallization of difficult proteins. The ligand-binding domain (LBD) of human oestrogen receptor α was produced in Escherichia coli as a cleavable thioredoxin (Trx) fusion in order to improve solubility. Crystallization trials with either cleaved and purified LBD or with the purified fusion protein both failed to produce crystals. In another attempt, Trx was not removed from the LBD after endoproteolytic cleavage and its presence promoted nucleation and subsequent crystal growth, which allowed the structure determination of two different LBD–ligand–coactivator peptide complexes at 2.3 Å resolution. This technique is likely to be applicable to other low-solubility proteins

Solid-State Nuclear Magnetic Resonance Investigation of the Structural Topology and Lipid Interactions of a Viral Fusion Protein Chimera Containing the Fusion Peptide and Transmembrane Domain.

Science.gov (United States)

Yao, Hongwei; Lee, Myungwoon; Liao, Shu-Yu; Hong, Mei

2016-12-13

The fusion peptide (FP) and transmembrane domain (TMD) of viral fusion proteins play important roles during virus-cell membrane fusion, by inducing membrane curvature and transient dehydration. The structure of the water-soluble ectodomain of viral fusion proteins has been extensively studied crystallographically, but the structures of the FP and TMD bound to phospholipid membranes are not well understood. We recently investigated the conformations and lipid interactions of the separate FP and TMD peptides of parainfluenza virus 5 (PIV5) fusion protein F using solid-state nuclear magnetic resonance. These studies provide structural information about the two domains when they are spatially well separated in the fusion process. To investigate how these two domains are structured relative to each other in the postfusion state, when the ectodomain forms a six-helix bundle that is thought to force the FP and TMD together in the membrane, we have now expressed and purified a chimera of the FP and TMD, connected by a Gly-Lys linker, and measured the chemical shifts and interdomain contacts of the protein in several lipid membranes. The FP-TMD chimera exhibits α-helical chemical shifts in all the membranes examined and does not cause strong curvature of lamellar membranes or membranes with negative spontaneous curvature. These properties differ qualitatively from those of the separate peptides, indicating that the FP and TMD interact with each other in the lipid membrane. However, no 13 C- 13 C cross peaks are observed in two-dimensional correlation spectra, suggesting that the two helices are not tightly associated. These results suggest that the ectodomain six-helix bundle does not propagate into the membrane to the two hydrophobic termini. However, the loosely associated FP and TMD helices are found to generate significant negative Gaussian curvature to membranes that possess spontaneous positive curvature, consistent with the notion that the FP-TMD assembly may

Revealing Surface Waters on an Antifreeze Protein by Fusion Protein Crystallography Combined with Molecular Dynamic Simulations.

Science.gov (United States)

Sun, Tianjun; Gauthier, Sherry Y; Campbell, Robert L; Davies, Peter L

2015-10-08

Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters.

A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L

International Nuclear Information System (INIS)

Pager, Cara Theresia; Craft, Willie Warren; Patch, Jared; Dutch, Rebecca Ellis

2006-01-01

The Nipah virus fusion (F) protein is proteolytically processed to F 1 + F 2 subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsins can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form

Generation of a selectively cytotoxic fusion protein against p53 mutated cancers

International Nuclear Information System (INIS)

Kousparou, Christina A; Yiacoumi, Efthymia; Deonarain, Mahendra P; Epenetos, Agamemnon A

2012-01-01

A significant number of cancers are caused by defects in p21 causing functional defects in p21 or p53 tumour-suppressor proteins. This has led to many therapeutic approaches including restoration by gene therapy with wild-type p53 or p21 using viral or liposomal vectors, which have toxicity or side-effect limitations. We set out to develop a safer, novel fusion protein which has the ability to reconstitute cancer cell lines with active p21 by protein transduction. The fusion protein was produced from the cell-translocating peptide Antennapedia (Antp) and wild-type, full-length p21 (Antp-p21). This was expressed and refolded from E. coli and tested on a variety of cell lines and tumours (in a BALB/c nude xenograft model) with differing p21 or p53 status. Antp-p21 penetrated and killed cancer cells that do not express wild type p53 or p21. This included cells that were matched to cogenic parental cell lines. Antp-p21 killed cancer cells selectively that were malignant as a result of mutations or nuclear exclusion of the p53 and p21 genes and over-expression of MDM2. Non-specific toxicity was excluded by showing that Antp-p21 penetrated but did not kill p53- or p21- wild-type cells. Antp-p21 was not immunogenic in normal New Zealand White rabbits. Recombinant Antp peptide alone was not cytotoxic, showing that killing was due to the transduction of the p21 component of Antp-p21. Antp-p21 was shown to penetrate cancer cells engrafted in vivo and resulted in tumour eradication when administered with conventionally-used chemotherapeutic agents, which alone were unable to produce such an effect. Antp-p21 may represent a new and promising targeted therapy for patients with p53-associated cancers supporting the concept that rational design of therapies directed against specific cancer mutations will play a part in the future of medical oncology

Generation of a selectively cytotoxic fusion protein against p53 mutated cancers

Directory of Open Access Journals (Sweden)

Kousparou Christina A

2012-08-01

Full Text Available Abstract Background A significant number of cancers are caused by defects in p21 causing functional defects in p21 or p53 tumour-suppressor proteins. This has led to many therapeutic approaches including restoration by gene therapy with wild-type p53 or p21 using viral or liposomal vectors, which have toxicity or side-effect limitations. We set out to develop a safer, novel fusion protein which has the ability to reconstitute cancer cell lines with active p21 by protein transduction. Methods The fusion protein was produced from the cell-translocating peptide Antennapedia (Antp and wild-type, full-length p21 (Antp-p21. This was expressed and refolded from E. coli and tested on a variety of cell lines and tumours (in a BALB/c nude xenograft model with differing p21 or p53 status. Results Antp-p21 penetrated and killed cancer cells that do not express wild type p53 or p21. This included cells that were matched to cogenic parental cell lines. Antp-p21 killed cancer cells selectively that were malignant as a result of mutations or nuclear exclusion of the p53 and p21 genes and over-expression of MDM2. Non-specific toxicity was excluded by showing that Antp-p21 penetrated but did not kill p53- or p21- wild-type cells. Antp-p21 was not immunogenic in normal New Zealand White rabbits. Recombinant Antp peptide alone was not cytotoxic, showing that killing was due to the transduction of the p21 component of Antp-p21. Antp-p21 was shown to penetrate cancer cells engrafted in vivo and resulted in tumour eradication when administered with conventionally-used chemotherapeutic agents, which alone were unable to produce such an effect. Conclusions Antp-p21 may represent a new and promising targeted therapy for patients with p53-associated cancers supporting the concept that rational design of therapies directed against specific cancer mutations will play a part in the future of medical oncology.

Public acceptance of fusion energy and scientific feasibility of a fusion reactor. Design of inductively driven long pulse tokamak reactors: IDLT

International Nuclear Information System (INIS)

Ogawa, Yuichi

1998-01-01

Based on scientific data based adopted for designing ITER plasmas and on the advancement of fusion nuclear technology from the recent R and D program, the scientific feasibility of inductively-driven tokamak fusion reactors is studied. A low wall-loading DEMO fusion reactor is designed, which utilizes an austenitic stainless steel in conjunction with significant data bases and operating experiences, since we have given high priority to the early and reliable realization of a tokamak fusion plasma over the cost performance. Since the DEMO reactor with the relatively large volume (i.e., major radius of 10 m) is employed, plasma ignition is achievable with a low fusion power of 0.8 GW, and an operation period of 4 - 5 hours is available only with inductive current drive. Disadvantages of pulsed operation in commercial fusion reactors include fatigue in structural materials and the necessity of an energy storage system to compensate the electric power during the dwell time. To overcome these disadvantages, a pulse length is prolonged up to about 10 hours, resulting in the remarkable reduction of the total cycle number to 10 4 during the life of the fusion plant. (author)

Definition and conceptual design of a small fusion reactor

International Nuclear Information System (INIS)

1979-04-01

The objective of this project is to evaluate various mirror fusion reactor concepts that might result in small systems for the effective production of electrical power or stored energy (e.g., nuclear and chemical fuels). The basic two-year program goal is to select a particular concept and develop the conceptual design of a pilot plant that could provide a useful output from fusion. The pilot plant would be built and operated in the late 1980s

Nuclear-thermal-coupled optimization code for the fusion breeding blanket conceptual design

International Nuclear Information System (INIS)

Li, Jia; Jiang, Kecheng; Zhang, Xiaokang; Nie, Xingchen; Zhu, Qinjun; Liu, Songlin

2016-01-01

Highlights: • A nuclear-thermal-coupled predesign code has been developed for optimizing the radial build arrangement of fusion breeding blanket. • Coupling module aims at speeding up the efficiency of design progress by coupling the neutronics calculation code with the thermal-hydraulic analysis code. • Radial build optimization algorithm aims at optimal arrangement of breeding blanket considering one or multiple specified objectives subject to the design criteria such as material temperature limit and available TBR. - Abstract: Fusion breeding blanket as one of the key in-vessel components performs the functions of breeding the tritium, removing the nuclear heat and heat flux from plasma chamber as well as acting as part of shielding system. The radial build design which determines the arrangement of function zones and material properties on the radial direction is the basis of the detailed design of fusion breeding blanket. For facilitating the radial build design, this study aims for developing a pre-design code to optimize the radial build of blanket with considering the performance of nuclear and thermal-hydraulic simultaneously. Two main features of this code are: (1) Coupling of the neutronics analysis with the thermal-hydraulic analysis to speed up the analysis progress; (2) preliminary optimization algorithm using one or multiple specified objectives subject to the design criteria in the form of constrains imposed on design variables and performance parameters within the possible engineering ranges. This pre-design code has been applied to the conceptual design of water-cooled ceramic breeding blanket in project of China fusion engineering testing reactor (CFETR).

Nuclear-thermal-coupled optimization code for the fusion breeding blanket conceptual design

Energy Technology Data Exchange (ETDEWEB)

Li, Jia, E-mail: lijia@ustc.edu.cn [School of Nuclear Science and Technology, University of Science and Technology of China, Hefei 230027, Anhui (China); Jiang, Kecheng; Zhang, Xiaokang [Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031, Anhui (China); Nie, Xingchen [School of Nuclear Science and Technology, University of Science and Technology of China, Hefei 230027, Anhui (China); Zhu, Qinjun; Liu, Songlin [Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031, Anhui (China)

2016-12-15

Highlights: • A nuclear-thermal-coupled predesign code has been developed for optimizing the radial build arrangement of fusion breeding blanket. • Coupling module aims at speeding up the efficiency of design progress by coupling the neutronics calculation code with the thermal-hydraulic analysis code. • Radial build optimization algorithm aims at optimal arrangement of breeding blanket considering one or multiple specified objectives subject to the design criteria such as material temperature limit and available TBR. - Abstract: Fusion breeding blanket as one of the key in-vessel components performs the functions of breeding the tritium, removing the nuclear heat and heat flux from plasma chamber as well as acting as part of shielding system. The radial build design which determines the arrangement of function zones and material properties on the radial direction is the basis of the detailed design of fusion breeding blanket. For facilitating the radial build design, this study aims for developing a pre-design code to optimize the radial build of blanket with considering the performance of nuclear and thermal-hydraulic simultaneously. Two main features of this code are: (1) Coupling of the neutronics analysis with the thermal-hydraulic analysis to speed up the analysis progress; (2) preliminary optimization algorithm using one or multiple specified objectives subject to the design criteria in the form of constrains imposed on design variables and performance parameters within the possible engineering ranges. This pre-design code has been applied to the conceptual design of water-cooled ceramic breeding blanket in project of China fusion engineering testing reactor (CFETR).

Osiris and SOMBRERO inertial confinement fusion power plant designs

International Nuclear Information System (INIS)

Meier, W.R.; Bieri, R.L.; Monsler, M.J.

1992-03-01

The primary objective of the of the IFE Reactor Design Studies was to provide the Office of Fusion Energy with an evaluation of the potential of inertial fusion for electric power production. The term reactor studies is somewhat of a misnomer since these studies included the conceptual design and analysis of all aspects of the IFE power plants: the chambers, heat transport and power conversion systems, other balance of plant facilities, target systems (including the target production, injection, and tracking systems), and the two drivers. The scope of the IFE Reactor Design Studies was quite ambitious. The majority of our effort was spent on the conceptual design of two IFE electric power plants, one using an induction linac heavy ion beam (HIB) driver and the other using a Krypton Fluoride (KrF) laser driver. After the two point designs were developed, they were assessed in terms of their (1) environmental and safety aspects; (2) reliability, availability, and maintainability; (3) technical issues and technology development requirements; and (4) economics. Finally, we compared the design features and the results of the assessments for the two designs

Conceptual design of the JAERI demonstration fusion reactor

International Nuclear Information System (INIS)

Sako, K.; Tone, T.; Seki, Y.

1976-01-01

Conceptual design of a tokamak demonstration fusion reactor is carried out. This design is an extended and improved version of the previous design which was presented at the 5th IAEA Conference. The main design parameters are as follows: the reactor thermal power 2000 MW, torus radius 10.5 m, plasma radius 2.7 m, first wall radius 3.0 m, toroidal magnetic field on axis 6T, blanket fertile material Li 2 O, coolant He, structural material Mo-alloy and tritium breeding ratio 1.2

Comparison of immune responses against foot-and-mouth disease virus induced by fusion proteins using the swine IgG heavy chain constant region or β-galactosidase as a carrier of immunogenic epitopes

International Nuclear Information System (INIS)

Li Guangjin; Chen Weizao; Yan Weiyao; Zhao Kai; Liu Mingqiu; Zhang Jun; Fei Liang; Xu Quanxing; Sheng Zutian; Lu Yonggan; Zheng Zhaoxin

2004-01-01

Previously, we demonstrated that a fusion protein (Gal-FMDV) consisting of β-galactosidase and an immunogenic peptide, amino acids (141-160)-(21-40)-(141-160), of foot-and-mouth disease virus (FMDV) VP1 protein induced protective immune responses in guinea pigs and swine. We now designed a new potential recombinant protein vaccine against FMDV in swine. The immunogenic peptide, amino acids (141-160)-(21-40)-(141-160) from the VP1 protein of serotype O FMDV, was fused to the carboxy terminus of a swine immunoglobulin G single heavy chain constant region and expressed in Escherichia coli. The expressed fusion protein (IgG-FMDV) was purified and emulsified with oil adjuvant. Vaccination twice at an interval of 3 weeks with the emulsified IgG-FMDV fusion protein induced an FMDV-specific spleen proliferative T-cell response in guinea pigs and elicited high levels of neutralizing antibody in guinea pigs and swine. All of the immunized animals were efficiently protected against FMDV challenge. There was no significant difference between IgG-FMDV and Gal-FMDV in eliciting immunity after vaccination twice in swine. However, when evaluating the efficacy of a single inoculation of the fusion proteins, we found that IgG-FMDV could elicit a protective immune response in swine, while Gal-FMDV only elicited a weak neutralizing activity and could not protect the swine against FMDV challenge. Our results suggest that the IgG-FMDV fusion protein is a promising vaccine candidate for FMD in swine

Recent designs for advanced fusion reactor blankets

International Nuclear Information System (INIS)

Sze, D.K.

1994-06-01

A series of reactor design studies based on the Tokamak configuration have been carried out under the direction of Professor Robert Conn of UCLA. They are called ARIES-1 through 4 and PULSAR 1 and 2. The key mission of these studies is to evaluate the attractiveness of fusion assuming different degrees of advancement in either physics or engineering development. Also, the requirements of engineering and physics systems for a pulsed reactor were evaluated by the PULSAR design studies. This paper discusses the directions and conclusions of the blanket and related engineering systems for those design studies

The reversed-field-pinch (RFP) fusion neutron source: A conceptual design

International Nuclear Information System (INIS)

Bathke, C.G.; Krakowski, R.A.; Miller, R.L.; Werley, K.A.

1989-01-01

The conceptual design of an ohmically heated, reversed-field pinch (RFP) operating at ∼5-MW/m 2 steady-state DT fusion neutron wall loading and ∼124-MW total fusion power is presented. These results are useful in projecting the development of a cost effective, low input power (∼206 MW) source of DT neutrons for large-volume (∼10 m 3 ), high-fluence (3.4 MW yr/m 2 ) fusion nuclear materials and technology testing. 19 refs., 15 figs., 9 tabs

IFMIF : International Fusion Materials Irradiation Facility Conceptual Design Activity: Final report

International Nuclear Information System (INIS)

Martone, M.

1997-01-01

This report documents the results of the Conceptual Design Activity (CDA) on the International Fusion Materials Irradiation Facility (IFMIF), conducted during 1995 and 1996. The activity is under the auspices of the International Energy Agency (IEA) Implementing Agreement for a Programme of Research and Development on Fusion Materials. An IEA Fusion Materials Executive Subcommittee was charged with overseeing the IFMIF-CDA work. Participants in the CDA are the European Union, Japan, and the United States, with the Russian Federation as an associate member

IFMIF : International Fusion Materials Irradiation Facility Conceptual Design Activity: Final report

Energy Technology Data Exchange (ETDEWEB)

Martone, M [ENEA, Centro Ricerche Frascati, Rome (Italy)

1997-01-01

This report documents the results of the Conceptual Design Activity (CDA) on the International Fusion Materials Irradiation Facility (IFMIF), conducted during 1995 and 1996. The activity is under the auspices of the International Energy Agency (IEA) Implementing Agreement for a Programme of Research and Development on Fusion Materials. An IEA Fusion Materials Executive Subcommittee was charged with overseeing the IFMIF-CDA work. Participants in the CDA are the European Union, Japan, and the United States, with the Russian Federation as an associate member.

In-silico determination of insecticidal potential of Vip3Aa-Cry1Ac fusion protein against Lepidopteran targets using molecular docking

Directory of Open Access Journals (Sweden)

Aftab eAhmad

2015-12-01

Full Text Available Study and research of Bt (Bacillus thuringiensis transgenic plants have opened new ways to combat insect pests. Over the decades, however, insect pests, especially the Lepidopteran, have developed tolerance against Bt delta-endotoxins. Such issues can be addressed through the development of novel toxins with greater toxicity and affinity against a broad range of insect receptors. In this computational study, functional domains of Bacillus thuringiensis crystal delta-endotoxin (Cry1Ac insecticidal protein and vegetative insecticidal protein (Vip3Aa have been fused to develop a broad-range Vip3Aa-Cry1Ac fusion protein. Cry1Ac and Vip3Aa are non-homologous insecticidal proteins possessing receptors against different targets within the midgut of insects. The insecticidal proteins were fused to broaden the insecticidal activity. Molecular docking analysis of the fusion protein against aminopeptidase-N (APN and cadherin receptors of five Lepidopteran insects (Agrotis ipsilon, Helicoverpa armigera, Pectinophora gossypiella, Spodoptera exigua and Spodoptera litura revealed that the Ser290, Ser293, Leu337, Thr340 and Arg437 residues of the fusion protein are involved in the interaction with insect receptors. The Helicoverpa armigera cadherin receptor, however, showed no interaction, which might be due to either loss or burial of interactive residues inside the fusion protein. These findings revealed that the Vip3Aa-Cry1Ac fusion protein has a strong affinity against Lepidopteran insect receptors and hence has a potential to be an efficient broad-range insecticidal protein.

The small G-proteins Rac1 and Cdc42 are essential for myoblast fusion in the mouse

DEFF Research Database (Denmark)

Vasyutina, Elena; Martarelli, Benedetta; Brakebusch, Cord

2009-01-01

Rac1 and Cdc42 are small G-proteins that regulate actin dynamics and affect plasma membrane protrusion and vesicle traffic. We used conditional mutagenesis in mice to demonstrate that Rac1 and Cdc42 are essential for myoblast fusion in vivo and in vitro. The deficit in fusion of Rac1 or Cdc42 mut...... genetic analysis demonstrates thus that the function of Rac in myoblast fusion is evolutionarily conserved from insects to mammals and that Cdc42, a molecule hitherto not implicated in myoblast fusion, is essential for the fusion of murine myoblasts....

Conceptual design of tritium production fusion reactor based on spherical torus

International Nuclear Information System (INIS)

He Kaihui; Huang Jinhua

2003-01-01

Conceptual design of an advanced tritium production fusion reactor based on spherical torus, which is intermediate application of fusion energy, was presented in this paper. Differing from the traditional tokamak tritium production reactor design, advanced plasma physics performance and compact structural characteristics of ST were used to minimize tritium leakage and maximize tritium breeding ratio with arrangement of tritium production blankets within vacuum vessel as possible in order to produce 1 kg excess tritium except need of self-sufficient plasma core with 40% or more corresponding plant availability. Based on 2D neutronics calculation, preliminary conceptual design of ST-TPR was presented, providing the backgrounds and reference for next detailed conceptual design

Safety issues relating to the design of fusion power facilities

International Nuclear Information System (INIS)

Stasko, R.R.; Wong, K.Y.; Russell, S.B.

1986-06-01

In order to make fusion power a viable future source of energy, it will be necessary to ensure that the cost of power for fusion electric generation is competitive with advanced fission concepts. In addition, fusion power will have to live up to its original promise of being a more radiologically benign technology than fission, and be able to demonstrate excellent operational safety performance. These two requirements are interrelated, since the selection of an appropriate safety philosophy early in the design phase could greatly reduce or eliminate the capital costs of elaborate safety related and protective sytems. This paper will briefly overview a few of the key safety issues presently recognized as critical to the ultimate achievement of licensable, environmentally safe and socially acceptable fusion power facilities. 12 refs

Reduction in Brain Heparan Sulfate with Systemic Administration of an IgG Trojan Horse-Sulfamidase Fusion Protein in the Mucopolysaccharidosis Type IIIA Mouse.

Science.gov (United States)

Boado, Ruben J; Lu, Jeff Zhiqiang; Hui, Eric Ka-Wai; Pardridge, William M

2018-02-05

Mucopolysaccharidosis Type IIIA (MPSIIIA), also known as Sanfilippo A syndrome, is an inherited neurodegenerative disease caused by mutations in the lysosomal enzyme, N-sulfoglucosamine sulfohydrolase (SGSH), also known as sulfamidase. Mutations in the SGSH enzyme, the only mammalian heparan N-sulfatase, cause accumulation of lysosomal inclusion bodies in brain cells comprising heparan sulfate (HS) glycosaminoglycans (GAGs). Treatment of MPSIIIA with intravenous recombinant SGSH is not possible because this large molecule does not cross the blood-brain barrier (BBB). BBB penetration by SGSH was enabled in the present study by re-engineering this enzyme as an IgG-SGSH fusion protein, where the IgG domain is a chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR), designated the cTfRMAb. The IgG domain of the fusion protein acts as a molecular Trojan horse to deliver the enzyme into brain via transport on the endogenous BBB TfR. The cTfRMAb-SGSH fusion protein bound to the mouse TfR with high affinity, ED 50 = 0.74 ± 0.07 nM, and retained high SGSH enzyme activity, 10 043 ± 1003 units/mg protein, which is comparable to recombinant human SGSH. Male and female MPSIIIA mice, null for the SGSH enzyme, were treated for 6 weeks with thrice-weekly intraperitoneal injections of vehicle, 5 mg/kg of the cTfRMAb alone, or 5 mg/kg of the cTfRMAb-SGSH fusion protein, starting at the age of 2 weeks, and were euthanized 1 week after the last injection. Brain and liver HS, as determined by liquid chromatography-mass spectrometry, were elevated 30-fold and 36-fold, respectively, in the MPSIIIA mouse. Treatment of the mice with the cTfRMAb-SGSH fusion protein caused a 70% and 85% reduction in brain and liver HS, respectively. The reduction in brain HS was associated with a 28% increase in latency on the rotarod test of motor activity in male mice. The mice exhibited no injection related reactions, and only a low titer end of study antidrug antibody

A Maltose-Binding Protein Fusion Construct Yields a Robust Crystallography Platform for MCL1.

Directory of Open Access Journals (Sweden)

Matthew C Clifton

Full Text Available Crystallization of a maltose-binding protein MCL1 fusion has yielded a robust crystallography platform that generated the first apo MCL1 crystal structure, as well as five ligand-bound structures. The ability to obtain fragment-bound structures advances structure-based drug design efforts that, despite considerable effort, had previously been intractable by crystallography. In the ligand-independent crystal form we identify inhibitor binding modes not observed in earlier crystallographic systems. This MBP-MCL1 construct dramatically improves the structural understanding of well-validated MCL1 ligands, and will likely catalyze the structure-based optimization of high affinity MCL1 inhibitors.

Computational Protein Design

DEFF Research Database (Denmark)

Johansson, Kristoffer Enøe

Proteins are the major functional group of molecules in biology. The impact of protein science on medicine and chemical productions is rapidly increasing. However, the greatest potential remains to be realized. The fi eld of protein design has advanced computational modeling from a tool of support...... to a central method that enables new developments. For example, novel enzymes with functions not found in natural proteins have been de novo designed to give enough activity for experimental optimization. This thesis presents the current state-of-the-art within computational design methods together...... with a novel method based on probability theory. With the aim of assembling a complete pipeline for protein design, this work touches upon several aspects of protein design. The presented work is the computational half of a design project where the other half is dedicated to the experimental part...

Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP-fusion proteins and multimode FRET microscopy

NARCIS (Netherlands)

Vermeer, J.E.M.; van Munster, E.B.; Vischer, N.O.; Gadella, T.

2004-01-01

Multimode fluorescence resonance energy transfer (FRET) microscopy was applied to study the plasma membrane organization using different lipidated green fluorescent protein (GFP)-fusion proteins co-expressed in cowpea protoplasts. Cyan fluorescent protein (CFP) was fused to the hyper variable region

Tritium system design studies of fusion experimental breeder

International Nuclear Information System (INIS)

Deng Baiquan; Huang Jinhua

2003-01-01

A summary of the tritium system design studies for the engineering outline design of a fusion experimental breeder (FEB-E) is presented. This paper is divided into three sections. In first section, the geometry, loading features and tritium concentrations in liquid lithium of tritium breeding zones of blanket are described. The tritium flow chart corresponding to the tritium fuel cycle system has been constructed, and the inventories in ten subsystems are calculated using SWITRIM code in section 2. Results show that the necessary initial tritium storage to start up FEB-E with fusion power of 143 MW is about 319 g. In final section, the tritium leakage issues under different operation circumstances have been analyzed. It was found that the potential danger of tritium leakage could be resulted from the exhausted gas of the diverter system. It is important to elevate the tritium burnup fraction and reduce the tritium throughput. (authors)

Pellet design for a laser fusion reactor

International Nuclear Information System (INIS)

Thiessen, A.R.; Nuckolls, J.

1974-01-01

The requirements for laser fusion pellet design are discussed. Computer calculations are presented of a capsule consisting of a spherical solid drop of DT surrounded by a concentric shell of DT. Gains greater than 40 fold are achieved with laser energies of approximately 0.5 MJ, and peak powers of about 10 16 W. (U.S.)

F-18 Labeled Diabody-Luciferase Fusion Proteins for Optical-ImmunoPET

Energy Technology Data Exchange (ETDEWEB)

Wu, Anna M. [Univ. of California, Los Angeles, CA (United States)

2013-01-18

The goal of the proposed work is to develop novel dual-labeled molecular imaging probes for multimodality imaging. Based on small, engineered antibodies called diabodies, these probes will be radioactively tagged with Fluorine-18 for PET imaging, and fused to luciferases for optical (bioluminescence) detection. Performance will be evaluated and validated using a prototype integrated optical-PET imaging system, OPET. Multimodality probes for optical-PET imaging will be based on diabodies that are dually labeled with 18F for PET detection and fused to luciferases for optical imaging. 1) Two sets of fusion proteins will be built, targeting the cell surface markers CEA or HER2. Coelenterazine-based luciferases and variant forms will be evaluated in combination with native substrate and analogs, in order to obtain two distinct probes recognizing different targets with different spectral signatures. 2) Diabody-luciferase fusion proteins will be labeled with 18F using amine reactive [18F]-SFB produced using a novel microwave-assisted, one-pot method. 3) Sitespecific, chemoselective radiolabeling methods will be devised, to reduce the chance that radiolabeling will inactivate either the target-binding properties or the bioluminescence properties of the diabody-luciferase fusion proteins. 4) Combined optical and PET imaging of these dual modality probes will be evaluated and validated in vitro and in vivo using a prototype integrated optical-PET imaging system, OPET. Each imaging modality has its strengths and weaknesses. Development and use of dual modality probes allows optical imaging to benefit from the localization and quantitation offered by the PET mode, and enhances the PET imaging by enabling simultaneous detection of more than one probe.

Effect of Bcl-xL overexpression on sialylation of Fc-fusion protein in recombinant Chinese hamster ovary cell cultures.

Science.gov (United States)

Lee, Jong Hyun; Kim, Yeon-Gu; Lee, Gyun Min

2015-01-01

The sialic acid of glycoproteins secreted by recombinant Chinese hamster ovary (rCHO) cells can be impaired by sialidase under culture conditions which promote the extracellular accumulation of this enzyme. To investigate the effect of Bcl-xL overexpression on the sialylation of glycoproteins produced in rCHO cell culture, two rCHO cell lines producing the same Fc-fusion protein, which were derived from DUKX-B11 and DG44, respectively, were engineered to have regulated Bcl-xL overexpression using the Tet-off system. For both cell lines, Bcl-xL overexpression improved cell viability and extended culture longevity in batch cultures. As a result, a maximum Fc-fusion protein titer increased by Bcl-xL overexpression though the extent of titer enhancement differed between the two cell lines. With Bcl-xL overexpression, the sialylation of Fc-fusion protein, which was assessed by isoelectric focusing gel and sialic acid content analyses, decreased more slowly toward the end of batch cultures. This was because Bcl-xL overexpression delayed the extracellular accumulation of sialidase activity by reducing cell lysis during batch cultures. Taken together, Bcl-xL overexpression in rCHO cell culture increased Fc-fusion protein production and also reduced the impairment of sialylation of Fc-fusion protein by maintaining high viability during batch cultures. © 2015 American Institute of Chemical Engineers.

New sensitive and specific assay for human immunodeficiency virus antibodies using labeled recombinant fusion protein and time-resolved fluoroimmunoassay.

OpenAIRE

Siitari, H; Turunen, P; Schrimsher, J; Nunn, M

1990-01-01

A new, rapid method for the detection of human immunodeficiency virus type 1 (HIV-1) antibody by time-resolved fluoroimmunoassay (TR-FIA) was developed. In this assay format, microtitration strips were coated with a recombinant fusion protein, and the same protein was labeled with europium and added into the wells simultaneously with the test specimens. The recombinant fusion protein contained the HIV-1 p24 gag protein sequence that carried an insertion, near the carboxyl terminus, of a 23-am...

The fusion protein of wild-type canine distemper virus is a major determinant of persistent infection

International Nuclear Information System (INIS)

Plattet, Philippe; Rivals, Jean-Paul; Zuber, BenoIt; Brunner, Jean-Marc; Zurbriggen, Andreas; Wittek, Riccardo

2005-01-01

The wild-type A75/17 canine distemper virus (CDV) strain induces a persistent infection in the central nervous system but infects cell lines very inefficiently. In contrast, the genetically more distant Onderstepoort CDV vaccine strain (OP-CDV) induces extensive syncytia formation. Here, we investigated the roles of wild-type fusion (F WT ) and attachment (H WT ) proteins in Vero cells expressing, or not, the canine SLAM receptor by transfection experiments and by studying recombinants viruses expressing different combinations of wild-type and OP-CDV glycoproteins. We show that low fusogenicity is not due to a defect of the envelope proteins to reach the cell surface and that H WT determines persistent infection in a receptor-dependent manner, emphasizing the role of SLAM as a potent enhancer of fusogenicity. However, importantly, F WT reduced cell-to-cell fusion independently of the cell surface receptor, thus demonstrating that the fusion protein of the neurovirulent A75/17-CDV strain plays a key role in determining persistent infection

Heterologous production of peptides in plants: fusion proteins and beyond.

Science.gov (United States)

Viana, Juliane Flávia Cançado; Dias, Simoni Campos; Franco, Octávio Luiz; Lacorte, Cristiano

2013-11-01

Recombinant DNA technology has allowed the ectopic production of proteins and peptides of different organisms leading to biopharmaceutical production in large cultures of bacterial, yeasts and mammalian cells. Otherwise, the expression of recombinant proteins and peptides in plants is an attractive alternative presenting several advantages over the commonly used expression systems including reduced production costs, easy scale-up and reduced risks of pathogen contamination. Different types of proteins and peptides have been expressed in plants, including antibodies, antigens, and proteins and peptides of medical, veterinary and industrial applications. However, apart from providing a proof of concept, the use of plants as platforms for heterologous protein and peptide production still depends on key steps towards optimization including the enhancement of expression levels, manipulation of post-transcriptional modifications and improvements in purification methods. In this review, strategies to increase heterologous protein and peptide stability and accumulation are discussed, focusing on the expression of peptides through the use of gene fusions.

IFMIF : International Fusion Materials Irradiation Facility Conceptual Design Activity: Executive summary

International Nuclear Information System (INIS)

1997-01-01

This report is a summary of the results of the Conceptual Design Activity (CDA) on the International Fusion Materials Irradiation Facility (IFMIF), conducted during 1995 and 1996. The activity is under the auspices of the International Energy Agency (IEA) Implementing Agreement for a Programme of Research and Development on Fusion Materials. An IEA Fusion Materials Executive Subcommittee was charged with overseeing the IFMIF-CDA work. Participants in the CDA are the European Union, Japan, and the United States, with the Russian Federation as an associate member

IFMIF : International Fusion Materials Irradiation Facility Conceptual Design Activity: Executive summary

Energy Technology Data Exchange (ETDEWEB)

NONE

1997-01-01

This report is a summary of the results of the Conceptual Design Activity (CDA) on the International Fusion Materials Irradiation Facility (IFMIF), conducted during 1995 and 1996. The activity is under the auspices of the International Energy Agency (IEA) Implementing Agreement for a Programme of Research and Development on Fusion Materials. An IEA Fusion Materials Executive Subcommittee was charged with overseeing the IFMIF-CDA work. Participants in the CDA are the European Union, Japan, and the United States, with the Russian Federation as an associate member.

An Updated Point Design for Heavy Ion Fusion

International Nuclear Information System (INIS)

Yu, S.S.; Meier, W.R.; Abbott, R.B.; Barnard, J.J.; Brown, T.; Callahan, D.A.; Heitzenroeder, P.; Latkowski, J.F.; Logan, B.G.; Pemberton, S.J.; Peterson, P.F.; Rose, D.V.; Sabbi, G.L.; Sharp, W.M.; Welch, D.R.

2002-01-01

An updated, self-consistent point design for a heavy ion fusion (HIF) power plant based on an induction linac driver, indirect-drive targets, and a thick liquid wall chamber has been completed. Conservative parameters were selected to allow each design area to meet its functional requirements in a robust manner, and thus this design is referred to as the Robust Point Design (RPD-2002). This paper provides a top-level summary of the major characteristics and design parameters for the target, driver, final focus magnet layout and shielding, chamber, beam propagation to the target, and overall power plant

Bulk-shield design for the Fusion Materials Irradiation Test facility

International Nuclear Information System (INIS)

Carter, L.L.; Mann, F.M.; Morford, R.J.; Johnson, D.L.; Huang, S.T.

1982-07-01

The accelerator-based Fusion Materials Irradiation Test (FMIT) facility will provide a high-fluence, fusion-like radiation environment for the testing of materials. While the neutron spectrum produced in the forward direction by the 35 MeV deuterons incident upon a flowing lithium target is characterized by a broad peak around 14 MeV, a high energy tail extends up to about 50 MeV. Some shield design considerations are reviewed

Delayed Toxicity Associated with Soluble Anthrax Toxin Receptor Decoy-Ig Fusion Protein Treatment

Science.gov (United States)

Cote, Christopher; Welkos, Susan; Manchester, Marianne; Young, John A. T.

2012-01-01

Soluble receptor decoy inhibitors, including receptor-immunogloubulin (Ig) fusion proteins, have shown promise as candidate anthrax toxin therapeutics. These agents act by binding to the receptor-interaction site on the protective antigen (PA) toxin subunit, thereby blocking toxin binding to cell surface receptors. Here we have made the surprising observation that co-administration of receptor decoy-Ig fusion proteins significantly delayed, but did not protect, rats challenged with anthrax lethal toxin. The delayed toxicity was associated with the in vivo assembly of a long-lived complex comprised of anthrax lethal toxin and the receptor decoy-Ig inhibitor. Intoxication in this system presumably results from the slow dissociation of the toxin complex from the inhibitor following their prolonged circulation. We conclude that while receptor decoy-Ig proteins represent promising candidates for the early treatment of B. anthracis infection, they may not be suitable for therapeutic use at later stages when fatal levels of toxin have already accumulated in the bloodstream. PMID:22511955

Overview of the Fusion Engineering Device (FED) design

International Nuclear Information System (INIS)

Steiner, D.; Flanagan, C.A.

1981-01-01

The device has a major radius of 5.0 m with a plasma minor radius of 1.3 m elongated by 1.6. Capability is provided for operating the toroidal field coils up to 10 T, but the bulk of the operations are designed for 8 T. At 8-T conditions the fusion power is approx. 180 MW (neutron wall loading approx. 0.4 MW/m 2 ) and a plasma Q of approx. 5 is expected. At 10-T conditions, which are expected to be limited to about 10% of the total operations, the fusion power is approx. 450 MW (approx. 1.0 MW/m 2 ) and ignition is expected

Overview of the fusion engineering device (FED) design

International Nuclear Information System (INIS)

Steiner, D.; Flanagan, C.A.

1981-10-01

The device has a major radius of 5.0 m with a plasma minor radius of 1.3 m elongated by 1.6. Capability is provided for operating the toroidal field coils up to 10 T, but the bulk of the operations are designed for 8 T. At 8-T conditions, the fusion power is approx. 180 MW (neutron wall loading approx. 0.4 MW/m 2 ) and a plasma Q of approx. 5 is expected. At 10-T conditions, which are expected to be limited to about 10% of the total operations, the fusion power is approx. 450 MW (approx. 1.0 MW/m 2 ) and ignition is expected

Designs of tandem-mirror fusion reactors

International Nuclear Information System (INIS)

Carlson, G.A.; Barr, W.L.; Boghosian, B.M.

1981-01-01

We have completed a comparative evaluation of several end plug configurations for tandem mirror fusion reactors with thermal barriers. The axi-cell configuration has been selected for further study and will be the basis for a detailed conceptual design study to be carried out over the next two years. The axi-cell end plug has a simple mirror cell produced by two circular coils followed by a transition coil and a yin-yang pair, which provides for MHD stability

The RCC-MR design code for LMFBR components. A useful basis for fusion reactor design tools development

International Nuclear Information System (INIS)

Acker, D.; Chevereau, G.

1986-01-01

LMFBR and fusion reactors exhibit common features with regard to structural materials, temperature service level, loading types. So, design and construction rules used in France for LMFBR, that is to say RCC-MR Code, can constitute a good basis for fusion reactors design. Some original aspects of RCC-MR design rules are described, relating to unsignificant creep, ratchetting effect, fatigue and creep damage limits, creep damage evaluation, fatigue damage evaluation, buckling. The main originality of RCC-MR consists to propose comprehensive simplified rules based on elastic calculations and extended from classical cold temperatures to the elevated temperature domain. (author)

Baculovirus display of fusion protein of Peste des petits ruminants virus and hemagglutination protein of Rinderpest virus and immunogenicity of the displayed proteins in mouse model

International Nuclear Information System (INIS)

Masmudur Rahman, Md.; Shaila, M.S.; Gopinathan, Karumathil P.

2003-01-01

Recombinant Bombyx mori nucleopolyhedroviruses (BmNPV) displaying the immunodominant ectodomains of fusion glycoprotein (F) of Peste des petitis ruminants virus (PPRV) and the hemagglutinin protein (H) of Rinderpest virus (RPV), on the budded virions as well as the surface of the infected host cells have been constructed. The F and H protein sequences were inserted in-frame within the amino-terminal region of BmNPV envelope glycoprotein GP64 expressing under the strong viral polyhedrin (polh) promoter. We improved the recombinant virus selection in BmNPV by incorporating the green fluorescent protein gene (gfp) as selection marker under a separate promoter within the transfer cassette harboring the desired genes. Following infection of the insect larvae or the host-derived BmN cells with these recombinant BmNPVs, the expressed GP64 fusion proteins were displayed on the host cell surface and the budded virions. The antigenic epitopes of the recombinant proteins were properly displayed and the recombinant virus particles induced immune response in mice against PPRV or RPV

Conceptual design of fusion experimental reactor (FER)

International Nuclear Information System (INIS)

1984-02-01

This report describes the engineering conceptual design of Fusion Experimental Reactor (FER) which is to be built as a next generation tokamak machine. This design covers overall reactor systems including MHD equilibrium analysis, mechanical configuration of reactor, divertor, pumped limiter, first wall/breeding blanket/shield, toroidal field magnet, poloidal field magnet, cryostat, electromagnetic analysis, vacuum system, power handling and conversion, NBI, RF heating device, tritium system, neutronics, maintenance, cooling system and layout of facilities. The engineering comparison of a divertor with pumped limiters and safety analysis of reactor systems are also conducted. (author)

Molecular dynamics analysis of conformational change of paramyxovirus F protein during the initial steps of membrane fusion

International Nuclear Information System (INIS)

Martín-García, Fernando; Mendieta-Moreno, Jesús Ignacio; Mendieta, Jesús; Gómez-Puertas, Paulino

2012-01-01

Highlights: ► Initial conformational change of paramyxovirus F protein is caused only by mechanical forces. ► HRA region undergoes a structural change from a beta + alpha conformation to an extended coil and then to an all-alpha conformation. ► HRS domains of F protein form three single α-helices prior to generation of the coiled coil. -- Abstract: The fusion of paramyxovirus to the cell membrane is mediated by fusion protein (F protein) present in the virus envelope, which undergoes a dramatic conformational change during the process. Unlike hemagglutinin in orthomyxovirus, this change is not mediated by an alteration of environmental pH, and its cause remains unknown. Steered molecular dynamics analysis leads us to suggest that the conformational modification is mediated only by stretching mechanical forces once the transmembrane fusion peptide of the protein is anchored to the cell membrane. Such elongating forces will generate major secondary structure rearrangement in the heptad repeat A region of the F protein; from β-sheet conformation to an elongated coil and then spontaneously to an α-helix. In addition, it is proposed that the heptad repeat A region adopts a final three-helix coiled coil and that this structure appears after the formation of individual helices in each monomer.

Flow cytometric immunobead assay for fast and easy detection of PML-RARA fusion proteins for the diagnosis of acute promyelocytic leukemia

NARCIS (Netherlands)

E.H.A. Dekking (E. H A); V.H.J. van der Velden (Vincent); A. Varro (Andras); H. Wai; S. Böttcher (Stephan); M. Kneba (Michael); E. Sonneveld (Edwin); A. Koning; N. Boeckx; N. Van Poecke; P. Lucio (Paulo); A. Mendonça; L. Sedek (Lukasz); T. Szczepanski (Tomasz); T. Kalina (Tomas); V. Kanderová (V.); P.G. Hoogeveen (Patricia); J. Flores-Montero (Juan); C. Chillón (Carmen); A. Orfao (Alberto); J.M.M. Almeida (Julia); P.A.S. Evans; C. Cullen; A.L. Noordijk; P.M. Vermeulen (P.); M.T. de Man (M.); E.P. Dixon (Eric); W.M. Comans-Bitter; J.J.M. van Dongen (Jacques)

2012-01-01

textabstractThe PML-RARA fusion protein is found in approximately 97% of patients with acute promyelocytic leukemia (APL). APL can be associated with life-threatening bleeding complications when undiagnosed and not treated expeditiously. The PML-RARA fusion protein arrests maturation of myeloid

Design study of electrical power supply system for tokamak fusion power reactor

International Nuclear Information System (INIS)

1977-01-01

Design study of the electrical power supply system for a 2000MWt Tokamak-type fusion reactor has been carried out. The purposes are to reveal and study problems in the system, leading to a plan of the research and development. Performed were study of the electrical power supply system and design of superconducting inductive energy storages and power switches. In study of the system, specification and capability of various power supplies for the fusion power reactor and design of the total system with its components were investigated. For the superconducting inductive energy storages, material choice, design calculation, and structural design were conducted, giving the size, weight and performance. For thyristor switches, circuit design in the parallel / series connection of element valves and cooling design were studied, providing the size and weight. (auth.)

Antibody-cytokine fusion proteins for treatment of cancer: engineering cytokines for improved efficacy and safety.

Science.gov (United States)

Young, Patricia A; Morrison, Sherie L; Timmerman, John M

2014-10-01

The true potential of cytokine therapies in cancer treatment is limited by the inability to deliver optimal concentrations into tumor sites due to dose-limiting systemic toxicities. To maximize the efficacy of cytokine therapy, recombinant antibody-cytokine fusion proteins have been constructed by a number of groups to harness the tumor-targeting ability of monoclonal antibodies. The aim is to guide cytokines specifically to tumor sites where they might stimulate more optimal anti-tumor immune responses while avoiding the systemic toxicities of free cytokine therapy. Antibody-cytokine fusion proteins containing interleukin (IL)-2, IL-12, IL-21, tumor necrosis factor (TNF)α, and interferons (IFNs) α, β, and γ have been constructed and have shown anti-tumor activity in preclinical and early-phase clinical studies. Future priorities for development of this technology include optimization of tumor targeting, bioactivity of the fused cytokine, and choice of appropriate agents for combination therapies. This review is intended to serve as a framework for engineering an ideal antibody-cytokine fusion protein, focusing on previously developed constructs and their clinical trial results. Copyright © 2014 Elsevier Inc. All rights reserved.

Synergies in the design and development of fusion and generation IV fission reactors

International Nuclear Information System (INIS)

Bogusch, E.; Carre, F.; Knebel, J.; Aoto, K.

2007-01-01

Future fusion reactors or systems and Generation IV fission reactors are designed and developed in worldwide programmes mostly involving the same partners to investigate and assess their potential for realisation and contribution to meet the future energy needs beyond 2030. Huge scientific and financial effort is necessary to meet these objectives. First programmes have been launched in Generation IV International Forum (GIF) for fission and in the Broader Approach for fusion reactor system development. Except the physics basis for the energy source, future fusion and fission reactors, in particular those with fast neutron core face similar design issues and development needs. Therefore the call for the identification of synergies became evident. Beyond ITER cooled by water, future fusion reactors or systems will be designed for helium and liquid metal cooling and higher temperatures similar to those proposed for some of the six fission reactor concepts in GIF with their diverse coolants. Beside materials developments which are not discussed in this paper, design and performance of components and systems related to the diverse coolants including lifetime and maintenance aspects might offer significant potentials for synergies. Furthermore, the use of process heat for applications in addition to electricity production as well as their safety approaches might create synergistic design and development programmes. Therefore an early identification of possible synergies in the relevant programmes should be endorsed to minimise the effort for future power plants in terms of investments and resources. In addition to a general overview of a possible synergistic work programme which promotes the interaction between fusion and fission programmes towards an integrated organisation of their design and R and D programmes, some specific remarks will be given for joint design tools, numerical code systems and joint experiments in support of common technologies. (orig.)

Synergies in the design and development of fusion and generation IV fission reactors

International Nuclear Information System (INIS)

Bogusch, E.; Carre, F.; Knebel, J.U.; Aoto, K.

2008-01-01

Future fusion reactor and Generation IV fission reactor systems are designed and developed in worldwide programmes to investigate and assess their potential for realisation and contribution to the future energy needs beyond 2030 mostly involving the same partners. Huge scientific and financial effort is necessary to meet these objectives. First programmes have been launched in Generation IV International Forum (GIF) for fission and in the Broader Approach for fusion reactor system development. Except for the physics basis for the energy source, future fusion and fission reactors, in particular those with fast neutron core, face similar design issues and development needs. Therefore, the call for the identification of synergies became evident. Beyond ITER cooled by water, future fusion reactor systems will be designed for high-temperature helium and liquid metal cooling but also water including supercritical water and molten salt similar to those proposed for some of the six fission reactor concepts in GIF with their diverse coolants. Beside materials developments which are not discussed in this paper, design and performance of components and systems related to the diverse coolants including lifetime and maintenance aspects might offer significant potentials for synergies. Furthermore, the use of process heat for applications in addition to electricity production as well as their safety approaches can create synergistic design and development programmes. Therefore, an early identification of possible synergies in the relevant programmes should be endorsed to minimise the effort for future power plants in terms of investments and resources. In addition to a general overview of a possible synergistic work programme which promotes the interaction between fusion and fission programmes towards an integrated organisation of their design and R and D programmes, some specific remarks will be given for joint design tools, numerical code systems and joint experiments in

Biochemical and biophysical characterization of the transmissible gastroenteritis coronavirus fusion core

International Nuclear Information System (INIS)

Ma Guangpeng; Feng Youjun; Gao Feng; Wang Jinzi; Liu Cheng; Li Yijing

2005-01-01

Transmissible gastroenteritis coronavirus (TGEV) is one of the most destructive agents, responsible for the enteric infections that are lethal for suckling piglets, causing enormous economic loss to the porcine fostering industry every year. Although it has been known that TGEV spiker protein is essential for the viral entry for many years, the detail knowledge of the TGEV fusion protein core is still very limited. Here, we report that TGEV fusion core (HR1-SGGRGG-HR2), in vitro expressed in GST prokaryotic expression system, shares the typical properties of the trimer of coiled-coil heterodimer (six α-helix bundle), which has been confirmed by a combined series of biochemical and biophysical evidences including size exclusion chromatography (gel-filtration), chemical crossing, and circular diagram. The 3D homologous structure model presents its most likely structure, extremely similar to those of the coronaviruses documented. Taken together, TGEV spiker protein belongs to the class I fusion protein, characterized by the existence of two heptad-repeat (HR) regions, HR1 and HR2, and the present knowledge about the truncated TGEV fusion protein core may facilitate in the design of the small molecule or polypeptide drugs targeting the membrane fusion between TGEV and its host

Targeted recombinant fusion proteins of IFNγ and mimetic IFNγ with PDGFβR bicyclic peptide inhibits liver fibrogenesis in vivo.

Directory of Open Access Journals (Sweden)

Ruchi Bansal

Full Text Available Hepatic stellate cells (HSCs, following transdifferentiation to myofibroblasts plays a key role in liver fibrosis. Therefore, attempts to attenuate this myofibroblastic phenotype would be a promising therapeutic approach. Interferon gamma (IFNγ is a potent anti-fibrotic cytokine, but its pleiotropic receptor expression leading to severe adverse effects has limited its clinical application. Since, activated HSC express high-level of platelet derived growth factor beta receptor (PDGFβR, we investigated the potential of PDGFβR-specific targeting of IFNγ and its signaling peptide that lacks IFNγR binding site (mimetic IFNγ or mimIFNγ in liver fibrosis. We prepared DNA constructs expressing IFNγ, mimIFNγ or BiPPB (PDGFβR-specific bicyclic peptide-IFNγ, BiPPB-mimIFNγ fusion proteins. Both chimeric proteins alongwith IFNγ and mimIFNγ were produced in E.coli. The expressed proteins were purified and analyzed for PDGFβR-specific binding and in vitro effects. Subsequently, these recombinant proteins were investigated for the liver uptake (pSTAT1α signaling pathway, for anti-fibrotic effects and adverse effects (platelet counts in CCl4-induced liver fibrogenesis in mice. The purified HSC-targeted IFNγ and mimIFNγ fusion proteins showed PDGFβR-specific binding and significantly reduced TGFβ-induced collagen-I expression in human HSC (LX2 cells, while mouse IFNγ and mimIFNγ did not show any effect. Conversely, mouse IFNγ and BiPPB-IFNγ induced activation and dose-dependent nitric oxide release in mouse macrophages (express IFNγR while lack PDGFβR, which was not observed with mimIFNγ and BiPPB-mimIFNγ, due to the lack of IFNγR binding sites. In vivo, targeted BiPPB-IFNγ and BiPPB-mimIFNγ significantly activated intrahepatic IFNγ-signaling pathway compared to IFNγ and mimIFNγ suggesting increased liver accumulation. Furthermore, the targeted fusion proteins ameliorated liver fibrogenesis in mice by significantly reducing

Fluorescent IgG fusion proteins made in E. coli

Science.gov (United States)

Luria, Yael; Raichlin, Dina; Benhar, Itai

2012-01-01

Antibodies are among the most powerful tools in biological and biomedical research and are presently the fastest growing category of new bio-pharmaceutics. The most common format of antibody applied for therapeutic, diagnostic and analytical purposes is the IgG format. For medical applications, recombinant IgGs are made in cultured mammalian cells in a process that is too expensive to be considered for producing antibodies for diagnostic and analytical purposes. Therefore, for such purposes, mouse monoclonal antibodies or polyclonal sera from immunized animals are used. While looking for an easier and more rapid way to prepare full-length IgGs for therapeutic purposes, we recently developed and reported an expression and purification protocol for full-length IgGs, and IgG-based fusion proteins in E. coli, called “Inclonals.” By applying the Inclonals technology, we could generate full-length IgGs that are genetically fused to toxins. The aim of the study described herein was to evaluate the possibility of applying the “Inclonals” technology for preparing IgG-fluorophore fusion proteins. We found that IgG fused to the green fluorescent proteins enhanced GFP (EGFP) while maintaining functionality in binding, lost most of its fluorescence during the refolding process. In contrast, we found that green fluorescent Superfolder GFP (SFGFP)-fused IgG and red fluorescent mCherry-fused IgG were functional in antigen binding and maintained fluorescence intensity. In addition, we found that we can link several SFGFPs in tandem to each IgG, with fluorescence intensity increasing accordingly. Fluorescent IgGs made in E. coli may become attractive alternatives to monoclonal or polyclonal fluorescent antibodies derived from animals. PMID:22531449

A Type-2 fuzzy data fusion approach for building reliable weighted protein interaction networks with application in protein complex detection.

Science.gov (United States)

Mehranfar, Adele; Ghadiri, Nasser; Kouhsar, Morteza; Golshani, Ashkan

2017-09-01

Detecting the protein complexes is an important task in analyzing the protein interaction networks. Although many algorithms predict protein complexes in different ways, surveys on the interaction networks indicate that about 50% of detected interactions are false positives. Consequently, the accuracy of existing methods needs to be improved. In this paper we propose a novel algorithm to detect the protein complexes in 'noisy' protein interaction data. First, we integrate several biological data sources to determine the reliability of each interaction and determine more accurate weights for the interactions. A data fusion component is used for this step, based on the interval type-2 fuzzy voter that provides an efficient combination of the information sources. This fusion component detects the errors and diminishes their effect on the detection protein complexes. So in the first step, the reliability scores have been assigned for every interaction in the network. In the second step, we have proposed a general protein complex detection algorithm by exploiting and adopting the strong points of other algorithms and existing hypotheses regarding real complexes. Finally, the proposed method has been applied for the yeast interaction datasets for predicting the interactions. The results show that our framework has a better performance regarding precision and F-measure than the existing approaches. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluation of divertor conceptual designs for a fusion power plant

International Nuclear Information System (INIS)

Ferrari, M.; Giancarli, L.; Kleefeldt, K.; Nardi, C.; Roedig, M.; Reimann, J.; Salavy, J.F.

2001-01-01

In the frame of the preliminary study of plants suitable for the energy production from the fusion power, particular emphasis has been given on the divertor studies. Since a significant percentage of the power generated from the fusion process is absorbed in the divertor, the thermal efficiency of the power conversion cycle requires a high coolant outlet temperature of the divertor, leading to solutions that are different from those adopted for the present experimental fusion plants. Therefore, copper alloys having extremely high thermal conductivity, cannot be used as structural material for this kind of devices. The most suitable coolants to be used in the divertor are water, helium and liquid metals. A conceptual design study has been developed for each of these three fluids, with the aim to evaluate the maximum allowable thermal flux at the divertor target plate and the R and D requirements for each solution. While a water-cooled divertor can be designed with a limited R and D effort, the development of helium or liquid metal cooled divertors requires a more engaging R and D program

The RCC-MR design code for LMFBR components. A useful basic for fusion reactor design tools development

International Nuclear Information System (INIS)

Acker, D.; Chevereau, G.

1985-11-01

LMFBR and fusion reactors exhibit common features with regard to structural materials (Stainless steels), temperature service level (550-600 0 C), loading types. So, design and construction rules used in France for LMFBR, that is to say RCC-MR Code, can constitute a good basis for fusion reactors design. Some original aspects of RCC-MR design rules are described, relating to unsignificant creep, ratchetting effect, fatigue and creep damage limits, creep damage evaluation, fatigue damage evaluation, buckling. The main originality of RCC-MR consists to propose comprehensive simplified rules based on elastic calculations and extended from classical cold temperatures to the elevated temperature domain

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.

Review of fission-fusion pellet designs and inertial confinement system studies at EIR

Energy Technology Data Exchange (ETDEWEB)

Seifriz, W [Eidgenoessisches Inst. fuer Reaktorforschung, Wuerenlingen (Switzerland)

1978-01-01

The article summarizes the work done so far at the Swiss Federal Institute for Reactor Research (EIR) in the field of the inertial confinement fusion technique. The following subjects are reviewed: a) fission fusion pellet designs using fissionable triggers, b) uranium tampered pellets, c) tampered pellets recycling unwanted actinide wastes from fission reactors in beam-driven micro-explosion reactors, and d) symbiotic fusion/fission reactor studies.

Overview of the TITAN-II reversed-field pinch aqueous fusion power core design

Energy Technology Data Exchange (ETDEWEB)

Wong, C.P.C.; Creedon, R.L.; Grotz, S.; Cheng, E.T.; Sharafat, S.; Cooke, P.I.H.

1988-03-01

TITAN-II is a compact, high power density Reversed-Field Pinch fusion power reactor design based on the aqueous lithium solution fusion power core concept. The selected breeding and structural materials are LiNO/sub 3/ and 9-C low activation ferritic steel, respectively. TITAN-II is a viable alternative to the TITAN-I lithium self-cooled design for the Reversed-Field Pinch reactor to operate at a neutron wall loading of 18 MWm/sup 2/. Submerging the complete fusion power core and the primary loop in a large pool of cool water will minimize the probability of radioactivity release. Since the protection of the large pool integrity is the only requirement for the protection of the public, TITAN-II is a passive safety assurance design. 13 refs., 3 figs., 1 tab.

Overview of the TITAN-II reversed-field pinch aqueous fusion power core design

Energy Technology Data Exchange (ETDEWEB)

Wong, C.P.C.; Creedon, R.L.; Cheng, E.T. (General Atomic Co., San Diego, CA (USA)); Grotz, S.P.; Sharafat, S.; Cooke, P.I.H. (California Univ., Los Angeles (USA). Dept. of Mechanical, Aerospace and Nuclear Engineering; California Univ., Los Angeles, CA (USA). Inst. for Plasma and Fusion Research); TITAN Research Group

1989-04-01

TITAN-II is a compact, high-power-density Reversed-Field Pinch fusion power reactor design based on the aqueous lithium solution fusion power core concept. The selected breeding and structural materials are LiNO/sub 3/ and 9-C low activation ferritic steel, respectively. TITAN-II is a viable alternative to the TITAN-I lithium self-cooled design for the Reversed-Field Pinch reactor to operate at a neutron wall loading of 18 MW/m/sup 2/. Submerging the complete fusion power core and the primary loop in a large pool of cool water will minimize the probability of radioactivity release. Since the protection of the large pool integrity is the only requirement for the protection of the public, TITAN-II is a level 2 of passive safety assurance design. (orig.).

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

Directory of Open Access Journals (Sweden)

Jolene Read

2015-06-01

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

Characterization of EBV gB indicates properties of both class I and class II viral fusion proteins

International Nuclear Information System (INIS)

Backovic, Marija; Leser, George P.; Lamb, Robert A.; Longnecker, Richard; Jardetzky, Theodore S.

2007-01-01

To gain insight into Epstein-Barr virus (EBV) glycoprotein B (gB), recombinant, secreted variants were generated. The role of putative transmembrane regions, the proteolytic processing and the oligomerization state of the gB variants were investigated. Constructs containing 2 of 3 C-terminal hydrophobic regions were secreted, indicating that these do not act as transmembrane anchors. The efficiency of cleavage of the gB furin site was found to depend on the nature of C-terminus. All of the gB constructs formed rosette structures reminiscent of the postfusion aggregates formed by other viral fusion proteins. However, substitution of putative fusion loop residues, WY 112-113 and WLIY 193-196 , with less hydrophobic amino acids from HSV-1 gB, produced trimeric protein and abrogated the ability of the EBV gB ectodomains to form rosettes. These data demonstrate biochemical features of EBV gB that are characteristic of other class I and class II viral fusion proteins, but not of HSV-1 gB

[Construction and expression of fusion protein TRX-hJagged1 in E.coli BL21].

Science.gov (United States)

Li, Guo-Hui; Fan, Yu-Zhen; Huang, Si-Yong; Liu, Qiang; Yin, Dan-Dan; Liu, Li; Chen, Ren-An; Hao, Miao-Wang; Liang, Ying-Min

2014-06-01

This study was purposed to construct prokaryotic expression vector and to investigate the expression of Notch ligand Jagged1 in E.coli. An expression vector pET-hJagged1 was constructed, which can be inserted in Jagged1 with different lengths, but the DSL domain of human Jagged1 should be contained. Then the recombinant plasmids were transformed into the competent cell of E.coli BL21, and the expression of the fusion protein was induced by IPTG. Fusion protein was purified from the supernatant of cell lysates via the Nickel affinity chromatography. The results showed that prokaryotic expression vectors pET-hJagged1 (Bgl II), pET-hJagged1 (Hind I) and pET-hJagged1 (Stu I) were successfully constructed, but only pET-hJagged1 (Stu I) could express the soluble TRX-hJagged1. The purified TRX-Jagged1 protein could be obtained via the Nickel affinity chromatography, and then confirmed by Western Blot. It is concluded that prokaryotic expression vector pET-hJagged1 is successfully constructed, but only pET-hJagged1 (Stu I) can express the soluble TRX-hJagged1 and the TRX-Jagged1 fusion protein is obtained through the prokaryotic expression system, which laid a solid foundation for further to explore the effects of Jagged1 in hematopoietic and lymphoid system.

Pooled-matrix protein interaction screens using Barcode Fusion Genetics.

Science.gov (United States)

Yachie, Nozomu; Petsalaki, Evangelia; Mellor, Joseph C; Weile, Jochen; Jacob, Yves; Verby, Marta; Ozturk, Sedide B; Li, Siyang; Cote, Atina G; Mosca, Roberto; Knapp, Jennifer J; Ko, Minjeong; Yu, Analyn; Gebbia, Marinella; Sahni, Nidhi; Yi, Song; Tyagi, Tanya; Sheykhkarimli, Dayag; Roth, Jonathan F; Wong, Cassandra; Musa, Louai; Snider, Jamie; Liu, Yi-Chun; Yu, Haiyuan; Braun, Pascal; Stagljar, Igor; Hao, Tong; Calderwood, Michael A; Pelletier, Laurence; Aloy, Patrick; Hill, David E; Vidal, Marc; Roth, Frederick P

2016-04-22

High-throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome-scale interaction mapping. Here, we report Barcode Fusion Genetics-Yeast Two-Hybrid (BFG-Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG-Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein-pair barcodes that can be quantified via next-generation sequencing. We applied BFG-Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG-Y2H increases the efficiency of protein matrix screening, with quality that is on par with state-of-the-art Y2H methods. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Conceptual design study of fusion experimental reactor (FY86 FER)

International Nuclear Information System (INIS)

Kobayashi, Takeshi; Yamada, Masao; Mizoguchi, Tadanori

1987-09-01

This report describes the results of the reactor configuration/structure design for the fusion experimental reactor (FER) performed in FY 1986. The design was intended to meet the physical and engineering mission of the next step device which was decided by the subcommittee on the next step device of the nuclear fusion council. The objectives of the design study in FY 1986 are to advance and optimize the design concept of the last year because the recommendation of the subcommittee was basically the same as the design philosophy of the last year. Six candidate reactor configurations which correspond to options C ∼ D presented by the subcommittee were extensively examined. Consequently, ACS reactor (Advanced Option-C with Single Null Divertor) was selected as the reference configuration from viewpoints of technical risks and cost performance. Regarding the reactor structure, the following items were investigated intensively: minimization of reactor size, protection of first wall against plasma disruption, simplification of shield structure, reactor configuration which enables optimum arrangement of poloidal field coils. (author)

Design of force-cooled conductors for large fusion magnets

Energy Technology Data Exchange (ETDEWEB)

Dresner, L.; Lue, J.W.

1977-01-01

Conductors cooled by supercritical helium in forced convection are under active consideration for large toroidal fusion magnets. One of the central problems in designing such force cooled conductors is to maintain an adequate stability margin while keeping the pumping power tolerably low. A method has been developed for minimizing the pumping power for fixed stability by optimally choosing the matrix-to-superconductor and the metal-to-helium ratios. Such optimized conductors reduce pumping power requirements for fusion size magnets to acceptable limits. Furthermore, the mass flow and hence pumping losses can be varied through a magnet according to the local magnetic field and magnitude of desired stability margin. Force cooled conductors give flexibility in operation, permitting, for example, higher fields to be obtained than originally intended by lowering the bath temperature or increasing the pumping power or both. This flexibility is only available if the pumping power is low to begin with. Scaling laws for the pumping requirement and stability margin as functions of operating current density, number of strands and such physical parameters as stabilizer resistivity and critical current density, have been proved. Numerical examples will be given for design of conductors intended for use in large toroidal fusion magnet systems.

Design of force-cooled conductors for large fusion magnets

International Nuclear Information System (INIS)

Dresner, L.; Lue, J.W.

1977-01-01

Conductors cooled by supercritical helium in forced convection are under active consideration for large toroidal fusion magnets. One of the central problems in designing such force cooled conductors is to maintain an adequate stability margin while keeping the pumping power tolerably low. A method has been developed for minimizing the pumping power for fixed stability by optimally choosing the matrix-to-superconductor and the metal-to-helium ratios. Such optimized conductors reduce pumping power requirements for fusion size magnets to acceptable limits. Furthermore, the mass flow and hence pumping losses can be varied through a magnet according to the local magnetic field and magnitude of desired stability margin. Force cooled conductors give flexibility in operation, permitting, for example, higher fields to be obtained than originally intended by lowering the bath temperature or increasing the pumping power or both. This flexibility is only available if the pumping power is low to begin with. Scaling laws for the pumping requirement and stability margin as functions of operating current density, number of strands and such physical parameters as stabilizer resistivity and critical current density, have been proved. Numerical examples will be given for design of conductors intended for use in large toroidal fusion magnet systems

Functional role of the cytoplasmic tail domain of the major envelope fusion protein of group II baculoviruses

NARCIS (Netherlands)

Long, G.; Pan, M.; Westenberg, M.; Vlak, J.M.

2006-01-01

F proteins from baculovirus nucleopolyhedrovirus (NPV) group II members are the major budded virus (BV) viral envelope fusion proteins. They undergo furin-like proteolysis processing in order to be functional. F proteins from different baculovirus species have a long cytoplasmic tail domain (CTD),

High-yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development.

Science.gov (United States)

Zhang, Ningning; Gonzalez, Maria; Savary, Brett; Xu, Jianfeng

2016-03-01

Low-yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline-O-glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY-2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of "Ser-Pro" dipeptide, or (SP)32 , to study cell growth and protein secretion, culture scale-up, and establishment of perfusion cultures for continuous production. The BY-2 cells accumulated low levels of cell biomass (~7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)32 -tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY-2 cells cultured in a 5-L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day(-1) , generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Design of a fusion reaction-history measurement system with high temporal resolution

International Nuclear Information System (INIS)

Peng Xiaoshi; Wang Feng; Liu Shenye; Jiang Xiaohua; Tang Qi

2010-01-01

In order to accurately measure the history of fusion reaction for experimental study of inertial confinement fusion, we advance the design of a fusion reaction-history measurement system with high temporal resolution. The diagnostic system is composed of plastic scintillator and nose cone, an optical imaging system and the system of optic streak camera. Analyzing the capability of the system indicated that the instrument measured fusion reaction history at temporal resolution as low as 55ps and 40ps correspond to 2.45MeV DD neutrons and 14.03MeV DT neutrons. The instrument is able to measure the fusion reaction history at yields 1.5 x 10 9 DD neutrons, about 4 x 10 8 DT neutrons are required for a similar quality signal. (authors)

Analysis of nuclear export using photoactivatable GFP fusion proteins and interspecies heterokaryons.

Science.gov (United States)

Nakrieko, Kerry-Ann; Ivanova, Iordanka A; Dagnino, Lina

2010-01-01

In this chapter, we review protocols for the analysis of nucleocytoplasmic shuttling of transcription factors and nuclear proteins, using two different approaches. The first involves the use of photoactivatable forms of the protein of interest by fusion to photoactivatable green fluorescent protein to follow its movement out of the nucleus by live-cell confocal microscopy. This methodology allows for the kinetic characterization of protein movements as well as measurement of steady-state levels. In a second procedure to assess the ability of a nuclear protein to move into and out of the nucleus, we describe the use of interspecies heterokaryon assays, which provide a measurement of steady-state distribution. These technologies are directly applicable to the analysis of nucleocytoplasmic movements not only of transcription factors, but also other nuclear proteins.

Intrafocal heterogeneity of ERG protein expression and gene fusion pattern in prostate cancer.

Science.gov (United States)

Suh, Ja Hee; Park, Jeong Hwan; Lee, Cheol; Moon, Kyung Chul

2017-10-01

Prostate cancer is considered to be highly heterogeneous, with various morphologic features and biologic behaviors. The TMPRSS2-ERG gene fusion is the most frequently observed genetic aberration in prostate cancer. The aim of this study was to elucidate the intrafocal heterogeneity of ERG gene fusion status. ERG immunohistochemistry (IHC) was performed in samples from 168 prostate cancer patients who had undergone radical prostatectomy, and 40 cases showing ERG-positive IHC staining were selected for tissue microarray (TMA) construction. Two to six representative cores were selected from each tumor focus. In the cases with heterogeneous ERG IHC staining intensity, the areas showing different intensities were separately selected. Using the TMA blocks, IHC and fluorescence in situ hybridization (FISH) were conducted to evaluate the heterogeneity of ERG protein expression and ERG fusion gene patterns, respectively, in a single tumor focus. Heterogeneity of ERG IHC staining was defined as the simultaneous presence of negative and positive cores in the same tumor focus. Heterogeneity of ERG FISH was defined by the presence of cores with positive and negative FISH signals or cores with break-apart and interstitial deletion FISH signals in the same tumor focus. A total of 202 TMA cores were isolated from 40 ERG-positive cases. Of the 202 total cores, 19 were negative for ERG IHC staining, and 46 showed 1+, 52 showed 2+, and 85 showed 3+ ERG staining intensity. Eleven cores were negative for ERG FISH signal, 119 cores showed ERG break-apart FISH signals, and the remaining 72 cores revealed interstitial deletion. Intrafocal heterogeneity of ERG IHC staining was found in 20% (8/40) of cases, and intrafocal heterogeneity of ERG gene fusion pattern was found in 32.5% (13/40) of cases. In summary, this study showed significantly frequent intrafocal heterogeneity of ERG protein expression, gene fusion status and fusion pattern. This heterogeneity can be caused by the development

Production of FMDV virus-like particles by a SUMO fusion protein approach in Escherichia coli

Directory of Open Access Journals (Sweden)

Liang Shu-Mei

2009-08-01

Full Text Available Abstract Virus-like particles (VLPs are formed by the self-assembly of envelope and/or capsid proteins from many viruses. Some VLPs have been proven successful as vaccines, and others have recently found applications as carriers for foreign antigens or as scaffolds in nanoparticle biotechnology. However, production of VLP was usually impeded due to low water-solubility of recombinant virus capsid proteins. Previous studies revealed that virus capsid and envelope proteins were often posttranslationally modified by SUMO in vivo, leading into a hypothesis that SUMO modification might be a common mechanism for virus proteins to retain water-solubility or prevent improper self-aggregation before virus assembly. We then propose a simple approach to produce VLPs of viruses, e.g., foot-and-mouth disease virus (FMDV. An improved SUMO fusion protein system we developed recently was applied to the simultaneous expression of three capsid proteins of FMDV in E. coli. The three SUMO fusion proteins formed a stable heterotrimeric complex. Proteolytic removal of SUMO moieties from the ternary complexes resulted in VLPs with size and shape resembling the authentic FMDV. The method described here can also apply to produce capsid/envelope protein complexes or VLPs of other disease-causing viruses.

Mimivirus reveals Mre11/Rad50 fusion proteins with a sporadic distribution in eukaryotes, bacteria, viruses and plasmids

Directory of Open Access Journals (Sweden)

Ogata Hiroyuki

2011-09-01

Full Text Available Abstract Background The Mre11/Rad50 complex and the homologous SbcD/SbcC complex in bacteria play crucial roles in the metabolism of DNA double-strand breaks, including DNA repair, genome replication, homologous recombination and non-homologous end-joining in cellular life forms and viruses. Here we investigated the amino acid sequence of the Mimivirus R555 gene product, originally annotated as a Rad50 homolog, and later shown to have close homologs in marine microbial metagenomes. Results Our bioinformatics analysis revealed that R555 protein sequence is constituted from the fusion of an N-terminal Mre11-like domain with a C-terminal Rad50-like domain. A systematic database search revealed twelve additional cases of Mre11/Rad50 (or SbcD/SbcC fusions in a wide variety of unrelated organisms including unicellular and multicellular eukaryotes, the megaplasmid of a bacterium associated to deep-sea hydrothermal vents (Deferribacter desulfuricans and the plasmid of Clostridium kluyveri. We also showed that R555 homologs are abundant in the metagenomes from different aquatic environments and that they most likely belong to aquatic viruses. The observed phyletic distribution of these fusion proteins suggests their recurrent creation and lateral gene transfers across organisms. Conclusions The existence of the fused version of protein sequences is consistent with known functional interactions between Mre11 and Rad50, and the gene fusion probably enhanced the opportunity for lateral transfer. The abundance of the Mre11/Rad50 fusion genes in viral metagenomes and their sporadic phyletic distribution in cellular organisms suggest that viruses, plasmids and transposons played a crucial role in the formation of the fusion proteins and their propagation into cellular genomes.

Conceptual design of laser fusion reactor, SENRI-I - 1. concept and system design

International Nuclear Information System (INIS)

Ido, S.; Naki, S.; Norimatsu, T.

1981-01-01

Design features of a laser fusion reactor concept SENRI-I and new concepts are reviewed and discussed. The unique feature is the utilization of a magnetic field to guide and control the inner liquid Li flow. Basic requirements and typical parameters used in the design are presented. Items to be discussed are constitution of the system, performance of liquid Li flow, neutronics, thermo-electric cycle, fuel cycle and new concepts

Maximal design basis accident of fusion neutron source DEMO-TIN

Energy Technology Data Exchange (ETDEWEB)

Kolbasov, B. N., E-mail: Kolbasov-BN@nrcki.ru [National Research Center Kurchatov Institute (Russian Federation)

2015-12-15

When analyzing the safety of nuclear (including fusion) facilities, the maximal design basis accident at which the largest release of activity is expected must certainly be considered. Such an accident is usually the failure of cooling systems of the most thermally stressed components of a reactor (for a fusion facility, it is the divertor or the first wall). The analysis of safety of the ITER reactor and fusion power facilities (including hybrid fission–fusion facilities) shows that the initial event of such a design basis accident is a large-scale break of a pipe in the cooling system of divertor or the first wall outside the vacuum vessel of the facility. The greatest concern is caused by the possibility of hydrogen formation and the inrush of air into the vacuum chamber (VC) with the formation of a detonating mixture and a subsequent detonation explosion. To prevent such an explosion, the emergency forced termination of the fusion reaction, the mounting of shutoff valves in the cooling systems of the divertor and the first wall or blanket for reducing to a minimum the amount of water and air rushing into the VC, the injection of nitrogen or inert gas into the VC for decreasing the hydrogen and oxygen concentration, and other measures are recommended. Owing to a continuous feed-out of the molten-salt fuel mixture from the DEMO-TIN blanket with the removal period of 10 days, the radioactivity release at the accident will mainly be determined by tritium (up to 360 PBq). The activity of fission products in the facility will be up to 50 PBq.

Osiris and SOMBRERO inertial confinement fusion power plant designs

International Nuclear Information System (INIS)

Meier, W.R.; Bieri, R.L.; Monsler, M.J.

1992-03-01

Conceptual designs and assessments have been completed for two inertial fusion energy (IFE) electric power plants. The detailed designs and results of the assessment studies are presented in this report. Osiris is a heavy-ion-beam (HIB) driven power plant and SOMBRERO is a Krypton-Fluoride (KrF) laser-driven power plant. Both plants are sized for a net electric power of 1000 MWe

Introduction of fusion driven subcritical system plasma design

International Nuclear Information System (INIS)

Bin Wu

2003-01-01

Fusion driven subcritical nuclear system (FDS) is a multifunctional hybrid reactor, which could breed nuclear fuel, transmute long-lived wastes, producing tritium and so on. This paper presents an introduction of FDS plasma design. Several different advance equilibrium configurations have been proposed and a 1.5-D discharge simulation of FDS was also present

Conceptual design of a fission-based integrated test facility for fusion reactor components

International Nuclear Information System (INIS)

Watts, K.D.; Deis, G.A.; Hsu, P.Y.S.; Longhurst, G.R.; Masson, L.S.; Miller, L.G.

1982-01-01

The testing of fusion materials and components in fission reactors will become increasingly important because of lack of fusion engineering test devices in the immediate future and the increasing long-term demand for fusion testing when a fusion reactor test station becomes available. This paper presents the conceptual design of a fission-based Integrated Test Facility (ITF) developed by EG and G Idaho. This facility can accommodate entire first wall/blanket (FW/B) test modules such as those proposed for INTOR and can also accommodate smaller cylindrical modules similar to those designed by Oak Ridge National laboratory (ORNL) and Westinghouse. In addition, the facility can be used to test bulk breeder blanket materials, materials for tritium permeation, and components for performance in a nuclear environment. The ITF provides a cyclic neutron/gamma flux as well as the numerous module and experiment support functions required for truly integrated tests

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

Science.gov (United States)

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

2013-03-01

Chimeric peptide MVF-EGFR(237-267), comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His(6)-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His(6)-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0 mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×10(4) titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides. Copyright © 2012 Elsevier Inc. All rights reserved.

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

Directory of Open Access Journals (Sweden)

Aiko Amagai

2012-01-01

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

Inhibition of CRM1-mediated nuclear export of transcription factors by leukemogenic NUP98 fusion proteins.

Science.gov (United States)

Takeda, Akiko; Sarma, Nayan J; Abdul-Nabi, Anmaar M; Yaseen, Nabeel R

2010-05-21

NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin and HIV-1 Rev. In vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG repeat motif in a Ran-GTP-dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in myeloid cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wild-type NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of a myeloid cell line and primary human CD34+ cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFkappaB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFkappaB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators.

Production and evaluation of cytotoxic effects of DT386-BR2 fusion protein as a novel anti-cancer agent.

Science.gov (United States)

Shafiee, Fatemeh; Rabbani, Mohammad; Jahanian-Najafabadi, Ali

2016-11-01

The aim of this study was to produce a fusion protein consisting of the catalytic and translocation domains of diphtheria toxin fused to BR2, a cancer specific cell penetrating peptide, and evaluation of its cytotoxic effects for targeted eradication of cancer cells. For this purpose, The DT386-BR2 structure was predicted using Modeller 9.14 and the best predicted model was selected based on the minimum DOPE score. A synthetic gene encoding DT386-BR2 was cloned in pET28a expression vector, expressed and purified by affinity chromatography. SDS-PAGE and Western blotting confirmed the expression of the DT386-BR2 fusion protein by revealing a band of about 47kDa after the induction of the expression. Finally, the purified protein was subjected to MTT assay for evaluation of its cyto-lethal effects on cancer and normal cell lines. Statistical analysis showed significant reduction in survival percent of HeLa and MCF-7 cancer cells in comparison to negative control (PBS), while the cytotoxic effect was not significant on the normal cells, i.e. HUVEC and HEK 293. The IC50 of DT386-BR2 for HeLa and MCF-7 was about 0.55 and 2.08μg/ml, respectively. In conclusion, the production and purification of DT386-BR2 fusion protein was successfully achieved and its cytotoxic effects on the studied cancer cell lines was established. The promising cytotoxic effects of this newly constructed fusion protein made it a suitable candidate for targeted therapy of cancer, and further in vitro and in vivo studies on this fusion protein is underway. Copyright © 2016 Elsevier B.V. All rights reserved.

Neutronics design for a spherical tokamak fusion-transmutation reactor

International Nuclear Information System (INIS)

Deng Meigen; Feng Kaiming; Yang Bangchao

2002-01-01

Based on studies of the spherical tokamak fusion reactors, a concept of fusion-transmutation reactor is put forward. By using the one-dimension transport and burn-up code BISON3.0 to process optimized design, a set of plasma parameters and blanket configuration suitable for the transmutation of MA (Minor Actinides) nuclear waste is selected. Based on the one-dimension calculation, two-dimension calculation has been carried out by using two-dimension neutronics code TWODANT. Combined with the neutron flux given by TWODANT calculation, burn-up calculation has been processed by using the one-dimension radioactivity calculation code FDKR and some useful and reasonable results are obtained

Safety methodology implementation in the conceptual design phase of a fusion reactor

International Nuclear Information System (INIS)

Rodriguez-Rodrigo, L.; Elbez-Uzan, J.

2007-01-01

The licensing of ITER in France represents the first process for licensing a fusion facility in the framework of an experimental device with a total Tritium inventory of 3 kg. The main ITER parameters are far from those expected in the future demonstration reactors where the fusion power will be at least 5 times higher and the additional heating power could also reach up to 5 times the one foreseen in ITER. Main safety requirements for these reactors are based, among other conditions, on their inherent features as low amount of fuel, very low impurity content of structural materials, minimum waste repository, no active systems for safe shut-down, and no need for evacuation of population after the most severe accident. The design of such reactors is at the stage of conceptual studies and is mainly dealing with plasma performances, tritium breeding, blanket/divertor designs and solution of engineering issues, as well as bounding accidents or classification of waste. The methodological approach for integrating safety analysis as a tool for optimizing the design of the overall fusion installation for future reactors in the conceptual design phase is sketched, including the machine itself and the different auxiliary nuclear buildings. (author)

Engineering design of the Nova Laser Facility for inertial-confinement fusion

International Nuclear Information System (INIS)

Simmons, W.W.; Godwin, R.O.; Hurley, C.A.

1982-01-01

The design of the Nova Laser Facility for inertial confinement fusion experiments at Lawrence Livermore National Laboratory is presented from an engineering perspective. Emphasis is placed upon design-to-performance requirements as they impact the various subsystems that comprise this complex experimental facility

Moult-inhibiting fusion protein augments while polyclonal antisera attenuate moult stages and duration in Penaeus monodon.