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Sample records for micelle-encapsulated recombinant baculovirus

  1. Pathogenesis induced by (recombinant) baculoviruses in insects

    NARCIS (Netherlands)

    Flipsen, H.

    1995-01-01

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

  2. Micelle-encapsulated fullerenes in aqueous electrolytes

    Energy Technology Data Exchange (ETDEWEB)

    Ala-Kleme, T., E-mail: timo.ala-kleme@utu.fi [Department of Chemistry, University of Turku, 20014 Turku (Finland); Maeki, A.; Maeki, R.; Kopperoinen, A.; Heikkinen, M.; Haapakka, K. [Department of Chemistry, University of Turku, 20014 Turku (Finland)

    2013-03-15

    Different micellar particles Mi(M{sup +}) (Mi=Triton X-100, Triton N-101 R, Triton CF-10, Brij-35, M{sup +}=Na{sup +}, K{sup +}, Cs{sup +}) have been prepared in different aqueous H{sub 3}BO{sub 3}/MOH background electrolytes. It has been observed that these particles can be used to disperse the highly hydrophobic spherical [60]fullerene (1) and ellipsoidal [70]fullerene (2). This dispersion is realised as either micelle-encapsulated monomers Mi(M{sup +})1{sub m} and Mi(M{sup +})2{sub m} or water-soluble micelle-bound aggregates Mi(M{sup +})1{sub agg} and Mi(M{sup +})2{sub agg}, where especially the hydration degree and polyoxyethylene (POE) thickness of the micellar particle seems to play a role of vital importance. Further, the encapsulation microenvironment of 1{sub m} was found to depend strongly on the selected monovalent electrolyte cation, i.e., the encapsulated 1{sub m} is accommodated in the more hydrophobic microenvironment the higher the cationic solvation number is. - Highlights: Black-Right-Pointing-Pointer Different micellar particles is used to disperse [60]fullerene and [70]fullerene. Black-Right-Pointing-Pointer Fullerene monomers or aggregates are dispersed encaging or bounding by micelles. Black-Right-Pointing-Pointer Effective facts are hydration degree and polyoxyethylene thickness of micelle.

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

    Science.gov (United States)

    2010-01-01

    Background Baculovirus comprise the largest group of insect viruses most studied worldwide, mainly because they efficiently kill agricutural insect pests. In this study, two recombinant baculoviruses containing the ScathL gene from Sarcophaga peregrina (vSynScathL), and the Keratinase gene from the fungus Aspergillus fumigatus (vSynKerat), were constructed. and their insecticidal properties analysed against Spodoptera frugiperda larvae. Results Bioassays of third-instar and neonate S. frugiperda larvae with vSynScathL and vSynKerat showed a decrease in the time needed to kill the infected insects when compared to the wild type virus. We have also shown that both recombinants were able to increase phenoloxidase activity in the hemolymph of S. frugiperda larvae. The expression of proteases in infected larvae resulted in destruction of internal tissues late in infection, which could be the reason for the increased viral speed of kill. Conclusions Baculoviruses and their recombinant forms constitute viable alternatives to chemical insecticides. Recombinant baculoviruses containing protease genes can be added to the list of engineered baculoviruses with great potential to be used in integrated pest management programs. PMID:20587066

  4. Characterization of canine herpesvirus glycoprotein C expressed by a recombinant baculovirus in insect cells.

    Science.gov (United States)

    Xuan, X; Maeda, K; Mikami, T; Otsuka, H

    1996-12-01

    The gene encoding the canine herpesvirus (CHV) glycoprotein C (gC) homologue has been identified by sequence homology analyses with other well studied herpesviruses. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gC, a recombinant baculovirus which contains the putative CHV gC structural gene under the baculovirus polyhedrin promoter was constructed. The recombinant baculovirus expressed gC-related polypeptides (44-62 kDa), which reacted only with MAbs against CHV gp80, indicating that the previously identified CHV gp80 is the translation product of the gC gene. The baculovirus expressed gC was glycosylated and transported to the surface of infected cells. At least seven neutralizing epitopes were conserved on the gC produced in insect cells. It was found that the recombinant baculovirus infected cells adsorbed murine erythrocytes as is the case for CHV-infected cells. The hemadsorption activity was inhibited by heparin, indicating that the CHV gC binds to heparan sulfate on the surface of murine erythrocytes. Mice immunized with the recombinant gC produced strong neutralizing antibodies. Our results suggest that CHV gC produced in insect cells may be useful as a subunit vaccine to control CHV infections.

  5. In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors.

    Science.gov (United States)

    Guijarro-Pardo, Eva; Gómez-Sebastián, Silvia; Escribano, José M

    2017-12-01

    Trichoplusia ni insect larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the insect larvae (Trichoplusia ni) by feeding, formulated in the insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in insect larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a

  6. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    Science.gov (United States)

    Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

    2015-01-01

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

  7. Error assessment in recombinant baculovirus titration: evaluation of different methods.

    Science.gov (United States)

    Roldão, António; Oliveira, Rui; Carrondo, Manuel J T; Alves, Paula M

    2009-07-01

    The success of baculovirus/insect cells system in heterologous protein expression depends on the robustness and efficiency of the production workflow. It is essential that process parameters are controlled and include as little variability as possible. The multiplicity of infection (MOI) is the most critical factor since irreproducible MOIs caused by inaccurate estimation of viral titers hinder batch consistency and process optimization. This lack of accuracy is related to intrinsic characteristics of the method such as the inability to distinguish between infectious and non-infectious baculovirus. In this study, several methods for baculovirus titration were compared. The most critical issues identified were the incubation time and cell concentration at the time of infection. These variables influence strongly the accuracy of titers and must be defined for optimal performance of the titration method. Although the standard errors of the methods varied significantly (7-36%), titers were within the same order of magnitude; thus, viral titers can be considered independent of the method of titration. A cost analysis of the baculovirus titration methods used in this study showed that the alamarblue, real time Q-PCR and plaque assays were the most expensive techniques. The remaining methods cost on average 75% less than the former methods. Based on the cost, time and error analysis undertaken in this study, the end-point dilution assay, microculture tetrazolium assay and flow cytometric assay were found to be the techniques that combine all these three main factors better. Nevertheless, it is always recommended to confirm the accuracy of the titration either by comparison with a well characterized baculovirus reference stock or by titration using two different methods and verification of the variability of results.

  8. Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production

    International Nuclear Information System (INIS)

    Jarvis, Donald L.

    2003-01-01

    The baculovirus-insect cell expression system is widely used to produce recombinant glycoproteins for many different biomedical applications. However, due to the fundamental nature of insect glycoprotein processing pathways, this system is typically unable to produce recombinant mammalian glycoproteins with authentic oligosaccharide side chains. This minireview summarizes our current understanding of insect protein glycosylation pathways and our recent efforts to address this problem. These efforts have yielded new insect cell lines and baculoviral vectors that can produce recombinant glycoproteins with humanized oligosaccharide side chains

  9. New baculovirus recombinants expressing Pseudorabies virus (PRV) glycoproteins protect mice against lethal challenge infection.

    Science.gov (United States)

    Grabowska, Agnieszka K; Lipińska, Andrea D; Rohde, Jörg; Szewczyk, Boguslaw; Bienkowska-Szewczyk, Krystyna; Rziha, Hanns-Joachim

    2009-06-02

    The present study demonstrates the protective potential of novel baculovirus recombinants, which express the glycoproteins gB, gC, or gD of Pseudorabies virus (PRV; Alphaherpesvirus of swine) and additionally contain the glycoprotein G of Vesicular Stomatitis Virus (VSV-G) in the virion (Bac-G-PRV). To evaluate the protective capacity, mixtures of equal amounts of the PRV gB-, gC-, and gD-expressing baculoviruses were used for immunization. Three intramuscular immunizations with that Bac-G-PRV mixture could protect mice against a lethal PRV challenge infection. To achieve complete protection high titers of Bac-G-PRV and three immunizations were necessary. This immunization with Bac-G-PRV resulted in the induction of high titers of PRV-specific serum antibodies of the IgG2a subclass and of interferon (IFN)-gamma, indicating a Th1-type immune response. Moreover, splenocytes of immunized mice exhibited natural killer cell activity accompanied by the production of IFN-alpha and IFN-gamma. Collectively, the presented data demonstrate for the first time that co-expression of VSV-G in baculovirus recombinant vaccines can improve the induction of a protective immune response against foreign antigens.

  10. Construction of a recombinant baculovirus expressing swine hepatitis E Virus ORF2 and preliminary research on its immune effect.

    Science.gov (United States)

    Yang, Z; Hu, Y; Yuan, P; Yang, Y; Wang, K; Xie, L Y; Huang, S L; Liu, J; Ran, L; Song, Z H

    2018-03-01

    In the swine hepatitis E virus (HEV), open reading frame 2 (ORF2) is rich in antigenic determinants and neutralizing epitopes that could induce immune protection. We chose the Bac-to-Bac® Baculovirus Expression System to express fragments containing the critical neutralizing antigenic sites within the HEV ORF2 protein of pigs to obtain a recombinant baculovirus. The fragment of swine HEV ORF2 region (1198-1881bp) was cloned into vector pFastBacTM. A recombinant baculovirus, rBacmid-ORF2, was obtained after transposition and transfection. The molecular mass of the recombinant protein was 26 kDa. Mice were immunized by the intraperitoneal and oral routes with cell lysates of recombinant baculovirus rBacmid-ORF2. Serum and feces of the mice were collected separately at 0, 14, 28, and 42 d after immunization and the antibody levels of IgG and secretory IgA against swine HEV were determined using an enzyme-linked immunosorbent assay. The results suggested that rBacmid-ORF2 induced antibodies of the humoral and mucosal immune responses in mice and that the oral route was significantly superior to the intraperitoneal route. This is the first study to demonstrate that that recombinant baculovirus swine HEV ORF2 could induce humoral and mucosal immune responses in mice. Copyright© by the Polish Academy of Sciences.

  11. Antigenic evaluation of a recombinant baculovirus-expressed Sarcocystis neurona SAG1 antigen.

    Science.gov (United States)

    Gupta, G D; Lakritz, J; Saville, W J; Livingston, R S; Dubey, J P; Middleton, J R; Marsh, A E

    2004-10-01

    Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neospora spp., Hammondia spp., and Toxoplasma gondii. Therefore, there is a need to develop an improved method for the detection of S. neurona-specific antibodies. The sera of infected horses react strongly to surface antigen 1 (SnSAG1), an approximately 29-kDa protein, in immunoblot analysis, suggesting that it is an immunodominant antigen. The SnSAG1 gene of S. neurona was cloned, and recombinant S. neurona SAG1 protein (rSnSAG1-Bac) was expressed with the use of a baculovirus system. By immunoblot analysis, the rSnSAG1-Bac antigen detected antibodies to S. neurona from naturally infected and experimentally inoculated equids, cats, rabbit, mice, and skunk. This is the first report of a baculovirus-expressed recombinant S. neurona antigen being used to detect anti-S. neurona antibodies in a variety of host species.

  12. ION-EXCHANGE IMMUNOAFFINITY PURIFICATION OF A RECOMBINANT BACULOVIRUS PLASMODIUM-FALCIPARUM APICAL MEMBRANE ANTIGEN, PF83/AMA-1

    NARCIS (Netherlands)

    NARUM, DL; WELLING, GW; THOMAS, AW

    1993-01-01

    A two-step purification regime has been developed for a quantitatively minor, putatively transmembrane, M(r) 83 000, apical membrane blood stage vaccine candidate antigen of Plasmodium falciparum (PF83/AMA-1), that has been expressed as a full-length baculovirus recombinant protein, PF83-FG8-1. The

  13. Vaccination with Recombinant Baculovirus Expressing Ranavirus Major Capsid Protein Induces Protective Immunity in Chinese Giant Salamander, Andrias davidianus

    Directory of Open Access Journals (Sweden)

    Xiaoyuan Zhou

    2017-07-01

    Full Text Available The Chinese giant salamander iridovirus (CGSIV, belonging to the genus Ranavirus in the family Iridoviridae, is the causative agent of an emerging infectious disease causing high mortality of more than 90% and economic losses in Chinese giant salamanders in China. In this study, a recombinant baculovirus-based vaccine expressing the CGSIV major capsid protein (MCP was developed and its protective immunity in Chinese giant salamanders was evaluated. The recombinant Autographa californica nucleopolyhedrosis virus (AcNPV, expressing CGSIV MCP, designated as AcNPV-MCP, was generated with the highest titers of 1 × 108 plaque forming units/mL (PFU/mL and confirmed by Western blot and indirect immunofluorescence (IIF assays. Western blot analysis revealed that the expressed MCP reacted with mouse anti-MCP monoclonal antibodies at the band of about 53 kDa. The results of IIF indicated that the MCP was expressed in the infected Spodoptera frugiperda 9 (Sf9 cells with the recombinant baculovirus, and the Chinese giant salamander muscle cells also transduced with the AcNPV-MCP. Immunization with the recombinant baculovirus of AcNPV-MCP elicited robust specific humoral immune responses detected by ELISA and neutralization assays and potent cellular immune responses in Chinese giant salamanders. Importantly, the effective immunization conferred highly protective immunity for Chinese giant salamanders against CGSIV challenge and produced a relative percent of survival rate of 84%. Thus, the recombinant baculovirus expressing CGSIV MCP can induce significant immune responses involving both humoral and cell-mediated immunity in Chinese giant salamanders and might represent a potential baculovirus based vaccine candidate for Chinese giant salamanders against CGSIV.

  14. Expression of the human multidrug transporter in insect cells by a recombinant baculovirus

    International Nuclear Information System (INIS)

    Germann, U.A.; Willingham, M.C.; Pastan, I.; Gottesman, M.M.

    1990-01-01

    The plasma membrane associated human multidrug resistance (MDR1) gene product, known as the 170-kDa P-glycoprotein or the multidrug transporter, acts as an ATP-dependent efflux pump for various cytotoxic agents. The authors expressed recombinant human multidrug transporter in a baculovirus expression system to obtain large quantities and further investigate its structure and mechanism of action. MDR1 cDNA was inserted into the genome of the Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter. Spodoptera frugiperda insect cells synthesized high levels of recombinant multidrug transporter 2-3 days after infection. The transporter was localized by immunocytochemical methods on the external surface of the plasma membranes, in the Golgi apparatus, and within the nuclear envelope. The human multidrug transporter expressed in insect cells is not susceptible to endoglycosidase F treatment and has a lower apparent molecular weight of 140,000, corresponding to the nonglycosylated precursor of its authentic counterpart expressed in multidrug-resistant cells. Labeling experiments showed that the recombinant multidrug transporter is phosphorylated and can be photoaffinity labeled by [ 3 H]azidopine, presumably at the same two sites as the native protein. Various drugs and reversing agents compete with the [ 3 H]azidopine binding reaction when added in excess, indicating that the recombinant human multidrug transporter expressed in insect cells is functionally similar to its authentic counterpart

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

    Directory of Open Access Journals (Sweden)

    Daniel Mendes Pereira Ardisson-Araújo

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

  16. Induction of antigen-specific immune responses in mice by recombinant baculovirus expressing premembrane and envelope proteins of West Nile virus

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    Zhu Bibo

    2012-07-01

    Full Text Available Abstract Background West Nile Virus (WNV is an emerging arthropod-born flavivirus with increasing distribution worldwide that is responsible for a large proportion of viral encephalitis in humans and horses. Given that there are no effective antiviral drugs available for treatment of the disease, efforts have been directed to develop vaccines to prevent WNV infection. Recently baculovirus has emerged as a novel and attractive gene delivery vehicle for mammalian cells. Results In the present study, recombinant baculoviruses expressing WNV premembrane (prM and envelope (E proteins under the cytomegalovirus (CMV promoter with or without vesicular stomatitis virus glycoprotein (VSV/G were constructed. The recombinant baculoviruses designated Bac-G-prM/E and Bac-prM/E, efficiently express E protein in mammalian cells. Intramuscular injection of the two recombinant baculoviruses (at doses of 108 or 109 PFU/mouse induced the production of WNV-specific antibodies, neutralizing antibodies as well as gamma interferon (IFN-γ in a dose-dependent pattern. Interestingly, the recombinant baculovirus Bac-G-prM/E was found to be a more efficient immunogen than Bac-prM/E to elicit a robust immune response upon intramuscular injection. In addition, inoculation of baculovirus resulted in the secretion of inflammatory cytokines, such as TNF-α, IL-2 and IL-6. Conclusions These recombinant baculoviruses are capable of eliciting robust humoral and cellular immune responses in mice, and may be considered as novel vaccine candidates for West Nile Virus.

  17. Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system.

    Science.gov (United States)

    Imai, Saki; Kusakabe, Takahiro; Xu, Jian; Li, Zhiqing; Shirai, Shintaro; Mon, Hiroaki; Morokuma, Daisuke; Lee, Jae Man

    2015-11-01

    Baculovirus expression vector system (BEVS) is widely used for production of recombinant eukaryotic proteins in insect larvae or cultured cells. BEVS has advantages over bacterial expression system in producing post-translationally modified secreted proteins. However, for some unknown reason, it is very difficult for insects to secrete sufficiently for certain proteins of interest. To understand the reasons why insect cells fail to secrete some kinds of recombinant proteins, we here employed three mammalian proteins as targets, EPO, HGF, and Wnt3A, with different secretion levels in BEVS and investigated their mRNA transcriptions from the viral genome, subcellular localizations, and interactions with silkworm ER chaperones. Moreover, we observed that no significantly influence on the secretion amounts of all three proteins when depleting or overexpressing most endogenous ER chaperone genes in cultured silkworm cells. However, among all detected ER chaperones, the depletion of BiP severely decreased the recombinant protein secretion in BEVS, indicating the possible central role of Bip in silkworm secretion pathway.

  18. Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System

    KAUST Repository

    Yamashita, Mami; Xu, Jian; Morokuma, Daisuke; Hirata, Kazuma; Hino, Masato; Mon, Hiroaki; Takahashi, Masateru; Hamdan, Samir; Sakashita, Kosuke; Iiyama, Kazuhiro; Banno, Yutaka; Kusakabe, Takahiro; Lee, Jae Man

    2017-01-01

    The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.

  19. Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System

    KAUST Repository

    Yamashita, Mami

    2017-05-08

    The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.

  20. Evaluation of the Insecticidal Efficacy of Wild Type and Recombinant Baculoviruses.

    Science.gov (United States)

    Popham, Holly J R; Ellersieck, Mark R; Li, Huarong; Bonning, Bryony C

    2016-01-01

    A considerable amount of work has been undertaken to genetically enhance the efficacy of baculovirus insecticides. Following construction of a genetically altered baculovirus, laboratory bioassays are used to quantify various parameters of insecticidal activity such as the median lethal concentration (or dose) required to kill 50 % of infected larvae (LC50 or LD50), median survival of larvae infected (ST50), and feeding damage incurred by infected larvae. In this chapter, protocols are described for a variety of bioassays and the corresponding data analyses for assessment of the insecticidal activity of baculovirus insecticides.

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

    NARCIS (Netherlands)

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

    2000-01-01

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

  2. Incorporation of adenylate cyclase into membranes of giant liposomes using membrane fusion with recombinant baculovirus-budded virus particles.

    Science.gov (United States)

    Mori, Takaaki; Kamiya, Koki; Tomita, Masahiro; Yoshimura, Tetsuro; Tsumoto, Kanta

    2014-06-01

    Recombinant transmembrane adenylate cyclase (AC) was incorporated into membranes of giant liposomes using membrane fusion between liposomes and baculovirus-budded virus (BV). AC genes were constructed into transfer vectors in a form fused with fluorescent protein or polyhistidine at the C-terminus. The recombinant BVs were collected by ultracentrifugation and AC expression was verified using western blotting. The BVs and giant liposomes generated using gentle hydration were fused under acidic conditions; the incorporation of AC into giant liposomes was demonstrated by confocal laser scanning microscopy through the emission of fluorescence from their membranes. The AC-expressing BVs were also fused with liposomes containing the substrate (ATP) with/without a specific inhibitor (SQ 22536). An enzyme immunoassay on extracts of the sample demonstrated that cAMP was produced inside the liposomes. This procedure facilitates direct introduction of large transmembrane proteins into artificial membranes without solubilization.

  3. Production of mink enteritis parvovirus empty capsids by expression in a baculovirus vector system: a recombinant vaccine for mink enteritis parvovirus in mink

    DEFF Research Database (Denmark)

    Christensen, J; Alexandersen, Søren; Bloch, B.

    1994-01-01

    The VP-2 gene of mink enteritis parvovirus (MEV) was amplified by the polymerase chain reaction using MEV DNA isolated from the faeces of a naturally infected mink. Subsequently the VP-2 gene was cloned into a baculovirus expression vector. Recombinant baculo-viruses were isolated and the MEV VP-2...... protein was able to form parvovirus-like particles, which had haemagglutinating properties comparable with the wild-type MEV. The cloned VP-2 gene was sequenced and only five nucleotide differences were found after alignment with the known sequences of the MEV type 1 and type 2 isolates. Surprisingly...

  4. Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system.

    Science.gov (United States)

    Maghodia, Ajay B; Geisler, Christoph; Jarvis, Donald L

    2016-06-01

    Cell lines derived from the fall armyworm, Spodoptera frugiperda (Sf), are widely used as hosts for recombinant protein production in the baculovirus-insect cell system (BICS). However, it was recently discovered that these cell lines are contaminated with a virus, now known as Sf-rhabdovirus [1]. The detection of this adventitious agent raised a potential safety issue that could adversely impact the BICS as a commercial recombinant protein production platform. Thus, we examined the properties of Sf-RVN, an Sf-rhabdovirus-negative Sf cell line, as a potential alternative host. Nested RT-PCR assays showed Sf-RVN cells had no detectable Sf-rhabdovirus over the course of 60 passages in continuous culture. The general properties of Sf-RVN cells, including their average growth rates, diameters, morphologies, and viabilities after baculovirus infection, were virtually identical to those of Sf9 cells. Baculovirus-infected Sf-RVN and Sf9 cells produced equivalent levels of three recombinant proteins, including an intracellular prokaryotic protein and two secreted eukaryotic glycoproteins, and provided similar N-glycosylation patterns. In fact, except for the absence of Sf-rhabdovirus, the only difference between Sf-RVN and Sf9 cells was SF-RVN produced higher levels of infectious baculovirus progeny. These results show Sf-RVN cells can be used as improved, alternative hosts to circumvent the potential safety hazard associated with the use of Sf-rhabdovirus-contaminated Sf cells for recombinant protein manufacturing with the BICS. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

    Directory of Open Access Journals (Sweden)

    B.M. Ribeiro

    1998-06-01

    Full Text Available The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs. Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV. The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

  6. Characterization of self-assembled virus-like particles of human polyomavirus BK generated by recombinant baculoviruses

    International Nuclear Information System (INIS)

    Li, T.-C.; Takeda, Naokazu; Kato, Kenzo; Nilsson, Josefina; Xing Li; Haag, Lars; Cheng, R. Holland; Miyamura, Tatsuo

    2003-01-01

    The major structural protein of the human polyomavirus BK (BKV), VP1, was expressed by using recombinant baculoviruses. A large amount of protein with a molecular mass of about 42 kDa was synthesized and identified by Western blotting. The protein was detected exclusively in the nuclei by immunofluorescent analysis and it was released into culture medium. The expressed BKV VP1 protein was self-assembled into virus-like particles (BK-VLPs) with two different sizes (50 and 26 nm in diameter), which migrated into four different bands in CsCl gradient with buoyant densities of 1.29, 1.30, 1.33, and 1.35 g/cm 3 . The immunological studies on the BK-VLPs suggested that they have similar antigenicity with those of authentic BKV particles. Cryoelectron microscopy and 3D image analysis further revealed that the larger BK-VLPs were composed of 72 capsomers which all were pentamers arranged in a T = 7 surface lattice. This system provides useful information for detailed studies of viral morphogenesis and the structural basis for the antigenicity of BKV

  7. Expression of deleted, atoxic atypical recombinant beta2 toxin in a baculovirus system and production of polyclonal and monoclonal antibodies.

    Science.gov (United States)

    Serroni, Anna; Magistrali, Chiara Francesca; Pezzotti, Giovanni; Bano, Luca; Pellegrini, Martina; Severi, Giulio; Di Pancrazio, Chiara; Luciani, Mirella; Tittarelli, Manuela; Tofani, Silvia; De Giuseppe, Antonio

    2017-05-25

    Clostridium perfringens is an important animal and human pathogen that can produce more than 16 different major and minor toxins. The beta-2 minor toxin (CPB2), comprising atypical and consensus variants, appears to be involved in both human and animal enterotoxaemia syndrome. The exact role of CPB2 in pathogenesis is poorly investigated, and its mechanism of action at the molecular level is still unknown because of the lack of specific reagents such as monoclonal antibodies against the CPB2 protein and/or the availability of a highly purified antigen. Previous studies have reported that purified wild-type or recombinant CPB2 toxin, expressed in a heterologous system, presented cytotoxic effects on human intestinal cell lines. Undoubtedly, for this reason, to date, these purified proteins have not yet been used for the production of monoclonal antibodies (MAbs). Recently, monoclonal antibodies against CPB2 were generated using peptides designed on predicted antigenic epitopes of this toxin. In this paper we report, for the first time, the expression in a baculovirus system of a deleted recombinant C-terminal 6xHis-tagged atypical CPB2 toxin (rCPB2 Δ1-25 -His 6 ) lacking the 25 amino acids (aa) of the N-terminal putative signal sequence. A high level of purified recombinant rCPB2 Δ1-25 -His 6 was obtained after purification by Ni 2+ affinity chromatography. The purified product showed no in vitro and in vivo toxicity. Polyclonal antibodies and twenty hybridoma-secreting Mabs were generated using purified rCPB2 Δ1-25 -His 6 . Finally, the reactivity and specificity of the new antibodies were tested against both recombinant and wild-type CPB2 toxins. The high-throughput of purified atoxic recombinant CPB2 produced in insect cells, allowed to obtain monoclonal and polyclonal antibodies. The availability of these molecules could contribute to develop immunoenzymatic methods and/or to perform studies about the biological activity of CPB2 toxin.

  8. Analysis of expression and glycosylation of avian metapneumovirus attachment glycoprotein from recombinant baculoviruses.

    Science.gov (United States)

    Luo, Lizhong; Nishi, Krista; MacLeod, Erin; Sabara, Marta I; Li, Yan

    2010-11-01

    Recently, we reported the expression and glycosylation of avian metapneumovirus attachment glycoprotein (AMPV/C G protein) in eukaryotic cell lines by a transient-expression method. In the present study, we investigated the biosynthesis and O-linked glycosylation of the AMPV/C G protein in a baculovirus expression system. The results showed that the insect cell-produced G protein migrated more rapidly in SDS-PAGE as compared to LLC-MK2 cell-derived G proteins owing to glycosylation differences. The fully processed, mature form of G protein migrated between 78 and 86 kDa, which is smaller than the 110 kDa mature form of G expressed in LLC-MK2 cells. In addition, several immature G gene products migrating at 40-48 and 60-70 kDa were also detected by SDS-PAGE and represented glycosylated intermediates. The addition of the antibiotic tunicamycin, which blocks early steps of glycosylation, to insect cell culture resulted in the disappearance of two glycosylated forms of the G protein and identified a 38 kDa unglycosylated precursor. The maturation of the G protein was completely blocked by monensin, suggesting that the O-linked glycosylation of G initiated in the trans-Golgi compartment. The presence of O-linked sugars on the mature protein was further confirmed by lectin Arachis hypogaea binding assay. Furthermore, antigenic features of the G protein expressed in insect cells were evaluated by ELISA. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

  9. Baculovirus Genomics

    NARCIS (Netherlands)

    Oers, van M.M.; Vlak, J.M.

    2007-01-01

    Baculovirus genomes are covalently closed circles of double stranded-DNA varying in size between 80 and 180 kilobase-pair. The genomes of more than fourty-one baculoviruses have been sequenced to date. The majority of these (37) are pathogenic to lepidopteran hosts; three infect sawflies

  10. Overproduction and partial purification of the Norrie disease gene product, norrin, from a recombinant baculovirus.

    Science.gov (United States)

    Shastry, Barkur S; Trese, Michael T

    2003-12-05

    Abnormal vascularization of the peripheral retina and retinal detachment are common clinical characteristics of Norrie disease (ND), familial exudative vitreoretinopathy, Coats' disease, and retinopathy of prematurity. Although little is known about the molecular basis of these diseases, studies have shown that all of these diseases are associated with mutations in the ND gene. In spite of this, little is known about norrin, its molecular mechanism of action, and its functional relationship with the development of abnormal retinal vasculature. To obtain a large quantity of norrin for structural and functional studies, we have overproduced it in insect cells. For this purpose, a cDNA fragment (869 bp) was isolated from a human retinal cDNA library by amplification and was cloned into an expression vector. The purified plasmid was co-transfected with wild-type linearized Bac-N-Blue DNA into S. frugiperda Sf21 insect cells. The recombinant virus plaques were purified and clones were selected based on the level of recombinant protein expressed in Sf21 cells infected with a purified recombinant virus. From these, a high-titer stock was generated and subsequently used to prepare a fused protein on a large scale. The protein was partially purified by the process of immobilized metal affinity chromatography and the use of ion exchange chromatography

  11. Characterization of self-assembled virus-like particles of dromedary camel hepatitis e virus generated by recombinant baculoviruses.

    Science.gov (United States)

    Zhou, Xianfeng; Kataoka, Michiyo; Liu, Zheng; Takeda, Naokazu; Wakita, Takaji; Li, Tian-Cheng

    2015-12-02

    Dromedary camel hepatitis E virus (DcHEV), a novel hepatitis E virus, has been identified in dromedary camels in Dubai, United Arab Emirates. The antigenicity, pathogenicity and epidemiology of this virus have been unclear. Here we first used a recombinant baculovirus expression system to express the 13 and 111 N-terminus amino-acid-truncated DcHEV ORF2 protein in insect Tn5 cells, and we obtained two types of virus-like particles (VLPs) with densities of 1.300 g/cm(3) and 1.285 g/cm(3), respectively. The small VLPs (Dc4sVLPs) were estimated to be 24 nm in diameter, and were assembled by a protein with the molecular mass 53 kDa. The large VLPs (Dc3nVLPs and Dc4nVLPs) were 35 nm in diameter, and were assembled by a 64-kDa protein. An antigenic analysis demonstrated that DcHEV was cross-reactive with G1, G3-G6, ferret and rat HEVs, and DcHEV showed a stronger cross-reactivity to G1 G3-G6 HEV than it did to rat and ferret HEV. In addition, the antibody against DcHEV-LPs neutralized G1 and G3 HEV in a cell culture system, suggesting that the serotypes of these HEVs are identical. We also found that the amino acid residue Met-358 affects the small DcHEV-LPs assembly. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. The Pacific White Shrimp β-actin Promoter: Functional Properties and the Potential Application for Transduction System Using Recombinant Baculovirus.

    Science.gov (United States)

    Shi, Yingli; Xiang, Jianhai; Zhou, Guangzhou; Ron, Tetsuzan Benny; Tong, Hsin-I; Kang, Wen; Sun, Si; Lu, Yuanan

    2016-06-01

    A newly isolated Pacific white shrimp (Litopenaeus vannamei) beta-actin promoter SbaP and its derivative compact construct SbaP (ENX) have recently been demonstrated to promote ectopic gene expression in vitro and in vivo. To further explore the potential transduction application, this newly isolated shrimp promoter SbaP was comparatively tested with cytomegalovirus (CMV), simian virus 40 (SV40), polyhedrin (Polh), and white spot syndrome virus immediate early gene 1 (WSSV ie1) four constitutive promoters and a beta-actin promoter (TbaP) from tilapia fish to characterize its promoting function in eight different cell lines. Luciferase quantitation assays revealed that SbaP can drive luciferase gene expression in all eight cell lines including sf21 (insect), PAC2 (zebrafish), EPC (carp), CHSE-214 (chinook salmon), GSTEF (green sea turtle), MS-1 (monk seal), 293T (human), and HeLa (human), but at different levels. Comparative analysis revealed that the promoting activity of SbaP was lower (≤10-fold) than CMV but higher (2-20 folds) than Polh in most of these cell lines tested. Whereas, SbaP mediated luciferase expression in sf21 cells was over 20-fold higher than CMV, SV40, Polh, and TbaP promoter. Compared to the SbaP, SbaP (ENX), which was constructed on the basis of SbaP by deletion of two "negative" regulatory elements, exhibited no significant change of promoting activity in EPC and PAC2 cells, but a 5 and 16 % lower promoting effect in 293T and HeLa cells, respectively. Additionally, a recombinant baculovirus was constructed under the control of SbaP (ENX), and efficient promoter activity of newly generated baculoviral vector was detected both in vitro of infected sf21 cells and in vivo of injected indicator shrimp. These results warrant the potential application of SbaP, particularly SbaP (ENX) in ectopic gene expression in future.

  13. Pluronic-based micelle encapsulation potentiates myricetin-induced cytotoxicity in human glioblastoma cells

    Directory of Open Access Journals (Sweden)

    Tang XJ

    2016-10-01

    Full Text Available Xiang-Jun Tang,1,* Kuan-Ming Huang,1,* Hui Gui,1,* Jun-Jie Wang,2 Jun-Ti Lu,1 Long-Jun Dai,1,3 Li Zhang,1 Gang Wang2 1Department of Neurosurgery, TaiHe Hospital, Hubei University of Medicine, Shiyan, 2Department of Pharmaceutics, Shanghai Eighth People’s Hospital, Jiangsu University, Shanghai, People’s Republic of China; 3Department of Surgery, University of British Columbia, Vancouver, BC, Canada *These authors contributed equally to this work Abstract: As one of the natural herbal flavonoids, myricetin has attracted much research interest, mainly owing to its remarkable anticancer properties and negligible side effects. It holds great potential to be developed as an ideal anticancer drug through improving its bioavailability. This study was performed to investigate the effects of Pluronic-based micelle encapsulation on myricetin-induced cytotoxicity and the mechanisms underlying its anticancer properties in human glioblastoma cells. Cell viability was assessed using a methylthiazol tetrazolium assay and a real-time cell analyzer. Immunoblotting and quantitative reverse transcriptase polymerase chain reaction techniques were used for determining the expression levels of related molecules in protein and mRNA. The results indicated that myricetin-induced cytotoxicity was highly potentiated by the encapsulation of myricetin. Mitochondrial apoptotic pathway was demonstrated to be involved in myricetin-induced glioblastoma cell death. The epidermal growth factor receptor (EGFR/PI3K/Akt pathway located in the plasma membrane and cytosol and the RAS-ERK pathway located in mitochondria served as upstream and downstream targets, respectively, in myricetin-induced apoptosis. MiR-21 inhibitors interrupted the expression of EGFR, p-Akt, and K-Ras in the same fashion as myricetin-loaded mixed micelles (MYR-MCs and miR-21 expression were dose-dependently inhibited by MYR-MCs, indicating the interaction of miR-21 with MYR-MCs. This study provided evidence

  14. Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose

    International Nuclear Information System (INIS)

    Ju, Huanyu; Wei, Na; Wang, Qian; Wang, Chunyuan; Jing, Zhiqiang; Guo, Lu; Liu, Dapeng; Gao, Mingchun; Ma, Bo; Wang, Junwei

    2011-01-01

    Highlights: → All three capsid proteins can be expressed in insect cells in baculovirus expression system. → All three recombinant proteins were spontaneously self-assemble into virus-like particles whose size and appearance were similar to those of native purified GPV virions. → The immunogenicity of GPV-VLPs was better than commercial inactivated vaccine and attenuated vaccine. -- Abstract: Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs.

  15. Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose

    Energy Technology Data Exchange (ETDEWEB)

    Ju, Huanyu; Wei, Na; Wang, Qian; Wang, Chunyuan; Jing, Zhiqiang; Guo, Lu; Liu, Dapeng; Gao, Mingchun; Ma, Bo [College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030 (China); Wang, Junwei, E-mail: jwwang@neau.edu.cn [College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030 (China)

    2011-05-27

    Highlights: {yields} All three capsid proteins can be expressed in insect cells in baculovirus expression system. {yields} All three recombinant proteins were spontaneously self-assemble into virus-like particles whose size and appearance were similar to those of native purified GPV virions. {yields} The immunogenicity of GPV-VLPs was better than commercial inactivated vaccine and attenuated vaccine. -- Abstract: Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs.

  16. Baculovirus display of functional antibody Fab fragments.

    Science.gov (United States)

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

    2015-08-01

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

  17. Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose.

    Science.gov (United States)

    Ju, Huanyu; Wei, Na; Wang, Qian; Wang, Chunyuan; Jing, Zhiqiang; Guo, Lu; Liu, Dapeng; Gao, Mingchun; Ma, Bo; Wang, Junwei

    2011-05-27

    Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. [Use of the recombinant baculovirus BacVP6C for the construction of an internal positive control of rotavirus C].

    Science.gov (United States)

    Abid-Ayadi, I; Guix, S; Pintó, R M; Bosch, A

    2011-06-01

    Unlike group A, a few studies have interested other groups of the rotavirus, especially in Tunisia. The role of rotavirus C (RVC) infection is underestimated because of its sporadic nature. The aim of our study was to develop rapid diagnostic procedures of RVC by using an internal positive control of reverse transcription PCR (RT-PCR). The internal positive control (386pb) was designed from the recombinant baculovirus BacVP6C containing the full length cDNA of the Cowden strain gene 5 (1353pb). A fragment of 596pb was amplified by PCR using the BacVP6C DNA ds as template. Then, a central part of 210pb was deleted and the remaining fragment (386pb) was cloned into pGEM-3Zf(+) plasmid between SP6 and T7 RNA polymerase promoters. The obtained recombinant plasmid "pIAM1" was then used for the generation of the internal positive control by in vitro transcription. The sensibility of the RT-PCR was about 3.66×10(5) molecules of RNA/μl. The use of a shorter positive control, as compared to the wild type, allows increased specificity of the RT-PCR reaction, and could be used for efficient diagnostic and surveillance of RVC-caused diseases. Copyright © 2009 Elsevier Masson SAS. All rights reserved.

  19. Baculovirus-mediated gene transfer and recombinant protein expression do not interfere with insulin dependent phosphorylation of PKB/Akt in human SHSY-5Y and C3A cells

    Directory of Open Access Journals (Sweden)

    Selander Martin

    2007-02-01

    Full Text Available Abstract Background Recombinant adenovirus vectors and transfection agents comprising cationic lipids are widely used as gene delivery vehicles for functional expression in cultured cells. Consequently, these tools are utilized to investigate the effects of functional over-expression of proteins on insulin mediated events. However, we have previously reported that cationic lipid reagents cause a state of insulin unresponsiveness in cell cultures. In addition, we have found that cultured cells often do not respond to insulin stimulation following adenovirus treatment. Infection with adenovirus compromises vital functions of the host cell leading to the activation of protein kinases central to insulin signalling, such as protein kinase B/Akt. Therefore, we investigated the effect of adenovirus infection on insulin unresponsiveness by means of Akt activation in cultured cells. Moreover, we investigated the use of baculovirus as a heterologous viral gene delivery vehicle to circumvent these phenomena. Since the finding that baculovirus can efficiently transduce mammalian cells, the applications of this viral system in gene delivery has greatly expanded and one advantage is the virtual absence of cytotoxicity in mammalian cells. Results We show that infection of human neuroblastoma SHSY-5Y and liver C3A cells with recombinant adenovirus results in the activation of Akt in a dose dependent manner. In addition, this activation makes treated cells unresponsive to insulin stimulation as determined by an apparent lack of differential phosphorylation of Akt on serine-473. Our data further indicate that the use of recombinant baculovirus does not increase the phosphorylation of Akt in SHSY-5Y and C3A cells. Moreover, following infection with baculovirus, SHSY-5Y and C3A cells respond to insulin by means of phosphorylation of Akt on serine-473 in the same manner as uninfected cells. Conclusion Widely-used adenovirus vectors for gene delivery cause a state of

  20. Development of an enzyme-linked immunoelectrotransfer blot (EITB) assay using two baculovirus expressed recombinant antigens for diagnosis of Taenia solium taeniasis.

    Science.gov (United States)

    Levine, Min Z; Lewis, Melissa M; Rodriquez, Silvia; Jimenez, Juan A; Khan, Azra; Lin, Sehching; Garcia, Hector H; Gonzales, Armando E; Gilman, Robert H; Tsang, Victor C W

    2007-04-01

    Taeniasis diagnosis is an important step in the control and elimination of both cysticercosis and taeniasis. We report the development of 2 serological taeniasis diagnostic tests using recombinant antigens rES33 and rES38 expressed by baculovirus in insect cells in an EITB format. In laboratory testing with defined sera from nonendemic areas, rES33 has a sensitivity of 98% (n = 167) and a specificity of 99% (n = 310) (J index: 0.97); rES38 has a sensitivity of 99% (n = 146) and a specificity of 97% (n = 275) (J index: 0.96). Independent field testing in Peru showed 97% (n = 203) of the taeniasis sera were positive with rES33, and 100% of the nontaeniasis sera (n = 272) were negative with rES33; 98% (n = 198) of taeniasis sera were positive with rES38, and 91% (n = 274) of the nontaeniasis sera were negative with rES38. Among the Peruvian sera tested, 17 of 26 Peruvian Taenia saginata sera were false positive with rES38 test. Both tests were also examined with cysticercosis sera, with a positive rate ranging from 21% to 46%. rES33 and rES38 tests offer sensitive and specific diagnosis of taeniasis and easy sample collection through finger sticks that can be used in large-scale studies. They are currently being used in cysticercosis elimination programs in Peru.

  1. Reverse micelles as a tool for probing solvent modulation of protein dynamics: Reverse micelle encapsulated hemoglobin

    Science.gov (United States)

    Roche, Camille J.; Dantsker, David; Heller, Elizabeth R.; Sabat, Joseph E.; Friedman, Joel M.

    2013-08-01

    Hydration waters impact protein dynamics. Dissecting the interplay between hydration waters and dynamics requires a protein that manifests a broad range of dynamics. Proteins in reverse micelles (RMs) have promise as tools to achieve this objective because the water content can be manipulated. Hemoglobin is an appropriate tool with which to probe hydration effects. We describe both a protocol for hemoglobin encapsulation in reverse micelles and a facile method using PEG and cosolvents to manipulate water content. Hydration properties are probed using the water-sensitive fluorescence from Hb bound pyranine and covalently attached Badan. Protein dynamics are probed through ligand recombination traces derived from photodissociated carbonmonoxy hemoglobin on a log scale that exposes the potential role of both α and β solvent fluctuations in modulating protein dynamics. The results open the possibility of probing hydration level phenomena in this system using a combination of NMR and optical probes.

  2. Membrane fusion between baculovirus budded virus-enveloped particles and giant liposomes generated using a droplet-transfer method for the incorporation of recombinant membrane proteins.

    Science.gov (United States)

    Nishigami, Misako; Mori, Takaaki; Tomita, Masahiro; Takiguchi, Kingo; Tsumoto, Kanta

    2017-07-01

    Giant proteoliposomes are generally useful as artificial cell membranes in biochemical and biophysical studies, and various procedures for their preparation have been reported. We present here a novel preparation technique that involves the combination of i) cell-sized lipid vesicles (giant unilamellar vesicles, GUVs) that are generated using the droplet-transfer method, where lipid monolayer-coated water-in-oil microemulsion droplets interact with oil/water interfaces to form enclosed bilayer vesicles, and ii) budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus) that express recombinant transmembrane proteins on their envelopes. GP64, a fusogenic glycoprotein on viral envelopes, is activated by weak acids and is thought to cause membrane fusion with liposomes. Using confocal laser scanning microscopy (CLSM), we observed that the single giant liposomes fused with octadecyl rhodamine B chloride (R18)-labeled wild-type BV envelopes with moderate leakage of entrapped soluble compounds (calcein), and the fusion profile depended on the pH of the exterior solution: membrane fusion occurred at pH ∼4-5. We further demonstrated that recombinant transmembrane proteins, a red fluorescent protein (RFP)-tagged GPCR (corticotropin-releasing hormone receptor 1, CRHR1) and envelope protein GP64 could be partly incorporated into membranes of the individual giant liposomes with a reduction of the pH value, though there were also some immobile fluorescent spots observed on their circumferences. This combination may be useful for preparing giant proteoliposomes containing the desired membranes and inner phases. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Baculovirus expression vector system: An efficient tool for the ...

    African Journals Online (AJOL)

    Baculovirus expression vector system is considered one of the most successful and widely acceptable means for the production of recombinant proteins in extremely large quantities. Proper posttranslational modifications of the expressed proteins in insect cells, the usual host of baculoviruses, get them soluble, correctly ...

  4. Initiative to manufacture and characterize Baculovirus Reference Material

    NARCIS (Netherlands)

    Kamen, A.; Aucoin, M.; Merten, O.W.; Alves, P.C.M.; Hashimoto, Y.; Airenne, K.; Hu, Y.C.; Mezzina, M.; Oers, van M.M.

    2011-01-01

    This letter to the editor brings to the attention of researchers an initiative to develop a baculovirus reference material repository. To be successful this initiative needs the support of a broad panel of researchers working with baculovirus vectors for recombinant protein production and gene

  5. Baculovirus DNA replication

    OpenAIRE

    Kool, M.

    1994-01-01

    Baculoviruses are attractive biological agents for the control of insect pests. They are highly specific for insects and cause a fatal disease (Granados and Federici, 1986). in addition, baculoviruses are successfully exploited as expression vectors for the production of heterologous proteins for various applications (Luckow and Summers, 1988; Luckow, 1991). In both cases large-scale systems for the production of baculoviruses are important. Production in insect larvae is difficult t...

  6. Baculovirus DNA replication

    NARCIS (Netherlands)

    Kool, M.

    1994-01-01

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

  7. Baculoviruses and nucleosome management

    International Nuclear Information System (INIS)

    Volkman, Loy E.

    2015-01-01

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

  8. Baculoviruses and nucleosome management

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-02-15

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

  9. Comparison of the Protective Efficacy of DNA and Baculovirus-Derived Protein Vaccines for EBOLA Virus in Guinea Pigs

    National Research Council Canada - National Science Library

    Mellquist-Riemenschneider, Jenny L; Garrison, Aura R; Geisbert, Joan B; Saikh, Kamal U; Heidebrink, Kelli D

    2003-01-01

    .... Previously, a priming dose of a DNA vaccine expressing the glycoprotein (GP) gene of MARV followed by boosting with recombinant baculovirus-derived GP protein was found to confer protective immunity to guinea pigs (Hevey et al., 2001...

  10. Budded baculovirus particle structure revisited

    NARCIS (Netherlands)

    Wang, Qiushi; Bosch, Berend-Jan; Vlak, Just M; van Oers, Monique M; Rottier, Peter J; van Lent, Jan W M

    2015-01-01

    Baculoviruses are a group of enveloped, double-stranded DNA insect viruses with budded (BV) and occlusion-derived (ODV) virions produced during their infection cycle. BVs are commonly described as rod shaped particles with a high apical density of protein extensions (spikes) on the lipid envelope

  11. Budded baculovirus particle structure revisited

    NARCIS (Netherlands)

    Wang, Qiushi; Bosch, Berend Jan; Vlak, J.M.; Oers, van M.M.; Rottier, P.J.; Lent, van J.W.M.

    2016-01-01

    Baculoviruses are a group of enveloped, double-stranded DNA insect viruses with budded (BV) and occlusion-derived (ODV) virions produced during their infection cycle. BVs are commonly described as rod shaped particles with a high apical density of protein extensions (spikes) on the lipid envelope

  12. Utilizing the virus-induced blocking of apoptosis in an easy baculovirus titration method.

    Science.gov (United States)

    Niarchos, Athanasios; Lagoumintzis, George; Poulas, Konstantinos

    2015-10-22

    Baculovirus-mediated protein expression is a robust experimental technique for producing recombinant higher-eukaryotic proteins because it combines high yields with considerable post-translational modification capabilities. In this expression system, the determination of the titer of recombinant baculovirus stocks is important to achieve the correct multiplicity of infection for effective amplification of the virus and high expression of the target protein. To overcome the drawbacks of existing titration methods (e.g., plaque assay, real-time PCR), we present a simple and reliable assay that uses the ability of baculoviruses to block apoptosis in their host cells to accurately titrate virus samples. Briefly, after incubation with serial dilutions of baculovirus samples, Sf9 cells were UV irradiated and, after apoptosis induction, they were viewed via microscopy; the presence of cluster(s) of infected cells as islets indicated blocked apoptosis. Subsequently, baculovirus titers were calculated through the determination of the 50% endpoint dilution. The method is simple, inexpensive, and does not require unique laboratory equipment, consumables or expertise; moreover, it is versatile enough to be adapted for the titration of every virus species that can block apoptosis in any culturable host cells which undergo apoptosis under specific conditions.

  13. Recombiner

    International Nuclear Information System (INIS)

    Kikuchi, Nobuo.

    1983-01-01

    Purpose: To shorten the pre-heating time for a recombiner and obtain a uniform temperature distribution for the charged catalyst layer in a BWR type reactor. Constitution: A pre-heating heater is disposed to the outer periphery of a vessel for a recombiner packed with catalysts for recombining hydrogen and oxygen in gases flowing through a radioactive gaseous wastes processing system. Heat pipes for transmitting the heat applied to said container to the catalyst are disposed vertically and horizontally within the container. Different length of the heat pipes are combined. In this way, pre-heating time for the recombiner before the operation start and before the system switching can be shortened and the uniform pre-heating for the inside of the recombiner is also made possible. Further, heater control in the pre-heating can be carried out effectively and with ease. (Moriyama, K.)

  14. Evolution in Oryctes baculovirus: rate and types of genomic change.

    Science.gov (United States)

    Crawford, A M; Zelazny, B

    1990-01-01

    Three cloned strains of Oryctes baculovirus were released into a previously unexposed population of the host insect, the coconut palm rhinoceros beetle, Oryctes rhinoceros. The experiment was conducted on Meemu Atoll in the Maldive Islands. Viruses were isolated from the beetle population at 1 year, 1.75 years, and 4 years after release. No changes in genotype were observed in viruses isolated after 1 and 1.75 years. After 4 years, however, three types of genomic change had occurred. A recombinant derived from two of the released strains, an isolate containing a 100-bp insert, and one example of a point mutation were found in the 22 isolates examined.

  15. Recombiner

    International Nuclear Information System (INIS)

    Osumi, Morimichi.

    1979-01-01

    Purpose: To provide a recombiner which is capable of converting hydrogen gas into water by use of high-frequency heating at comparatively low temperatures and is safe and cheap in cost. Constitution: Hydrogen gas is introduced from an outer pipeline to the main structure of a recombiner, and when it passes through the vicinity of the central part of the recombiner, it is reacted with copper oxide (CuO 2 ) heated to a temperature more than 300 0 C by a high-frequency heater, and converted gently into water by reduction operation (2H 2 + CuO 2 → Cu + 2H 2 O). The thus prepared water is exhausted through the outer pipeline to a suppression pool. A part of hydrogen gas which has not been converted completely into water by the reaction and is remaining as hydrogen is recovered through exhaust nozzles and again introduced into the main structure of the recombiner. (Yoshino, Y.)

  16. Quantitative proteomics of Spodoptera frugiperda cells during growth and baculovirus infection.

    Directory of Open Access Journals (Sweden)

    Nuno Carinhas

    Full Text Available Baculovirus infection of Spodoptera frugiperda cells is a system of choice to produce a range of recombinant proteins, vaccines and, potentially, gene therapy vectors. While baculovirus genomes are well characterized, the genome of S. frugiperda is not sequenced and the virus-host molecular interplay is sparsely known. Herein, we describe the application of stable isotope labeling by amino acids in cell culture (SILAC to obtain the first comparative proteome quantitation of S. frugiperda cells during growth and early baculovirus infection. The proteome coverage was maximized by compiling a search database with protein annotations from insect species. Of interest were differentially proteins related to energy metabolism, endoplasmic reticulum and oxidative stress, yet not investigated in the scope of baculovirus infection. Further, the reduced expression of key viral-encoded proteins early in the infection cycle is suggested to be related with decreased viral replication at high cell density culture. These findings have implications for virological research and improvement of baculovirus-based bioprocesses.

  17. Properties of Oryctes Baculovirus Isolated in Indonesia

    Directory of Open Access Journals (Sweden)

    Jun Kobayashi

    1995-12-01

    Full Text Available An Indonesian isolate of Oryctes baculovirus was purified from infected midguts of the rhinoceros beetle (Oryctes rhinoceros by centrifugation on a 10–40% (w/v sucroese gradient. Morphological features of  nucleocapsid including a tail-like projection were very same as those previously reported. Both protein components of purified particles and restriction fragment electrophoresis profiles of viral DNA were similar to those of their isolates of Oryctes baculovirus, although there were some differences. Key words: Baculovirus oryctes, electrophoresis

  18. Recombiner

    International Nuclear Information System (INIS)

    Saalfrank, H.

    1985-01-01

    Air containing hydrogen can be oxidized by heating in a container called a recombiner, in order to avoid the collection of hydrogen. The container is long and a large number of straight heating bars are arranged in parallel in it and they are flanged to a lid. The heating bars are surrounded by tubes, in order to obtain good heat transfer by a narrow annular gap. (orig.) [de

  19. Propagation and Purification of Baculovirus oryctes Huger

    Directory of Open Access Journals (Sweden)

    Susamto Somowiyarjo

    1995-12-01

    Full Text Available An isolate of Baculovirus oryctes, a possible biological control agent for coconut beetle (Oryctes rhinoceros Huger from East Java was propagated and purified. The virus could be transmitted by feeding the imago with 10% sucrose containing virus from homogenate of infected beetles. Effectivity of virus to 9 healthy females by sexual copulation. Virus be succesfully purified by a method of Payne. Key words: Baculovirus oryctes, transmission, purification

  20. Localization of VP28 on the baculovirus envelope and its immunogenicity against white spot syndrome virus in Penaeus monodon

    International Nuclear Information System (INIS)

    Syed Musthaq, S.; Madhan, Selvaraj; Sahul Hameed, A.S.; Kwang, Jimmy

    2009-01-01

    White spot syndrome virus (WSSV) is a large dsDNA virus responsible for white spot disease in shrimp and other crustaceans. VP28 is one of the major envelope proteins of WSSV and plays a crucial role in viral infection. In an effort to develop a vaccine against WSSV, we have constructed a recombinant baculovirus with an immediate early promoter 1 which expresses VP28 at an early stage of infection in insect cells. Baculovirus expressed rVP28 was able to maintain its structural and antigenic conformity as indicated by immunofluorescence assay and western blot analysis. Interestingly, our results with confocal microscopy revealed that rVP28 was able to localize on the plasma membrane of insect cells infected with recombinant baculovirus. In addition, we demonstrated with transmission electron microscopy that baculovirus successfully acquired rVP28 from the insect cell membrane via the budding process. Using this baculovirus displaying VP28 as a vaccine against WSSV, we observed a significantly higher survival rate of 86.3% and 73.5% of WSSV-infected shrimp at 3 and 15 days post vaccination respectively. Quantitative real-time PCR also indicated that the WSSV viral load in vaccinated shrimp was significantly reduced at 7 days post challenge. Furthermore, our RT-PCR and immunohistochemistry results demonstrated that the recombinant baculovirus was able to express VP28 in vivo in shrimp tissues. This study will be of considerable significance in elucidating the morphogenesis of WSSV and will pave the way for new generation vaccines against WSSV.

  1. Probability to produce animal vaccines in insect baculovirus ...

    African Journals Online (AJOL)

    Administrator

    2011-09-07

    Sep 7, 2011 ... The insect baculovirus expression system is a valuable tool for the production of vaccine. .... vaccine expression/delivery vehicle (Yu-Chen et al., ... baculoviruses are applied in cell-based assays for drug ... Intramuscular.

  2. Genomic support for speciation and specificity of baculoviruses

    NARCIS (Netherlands)

    Jakubowska, A.K.

    2010-01-01

    Keywords: baculovirus, insects, speciation, genomics, phylogeny, host specificity

    The Baculoviridae comprise a large family of double-stranded DNA viruses infecting
    arthropods. In this thesis two baculoviruses, Leucoma salicis nucleopolyhedrovirus
    (LesaNPV) and Agrotis

  3. Baculovirus virions displaying Plasmodium berghei circumsporozoite protein protect mice against malaria sporozoite infection

    International Nuclear Information System (INIS)

    Yoshida, Shigeto; Kondoh, Daisuke; Arai, Eriko; Matsuoka, Hiroyuki; Seki, Chisato; Tanaka, Takao; Okada, Masaji; Ishii, Akira

    2003-01-01

    The display of foreign proteins on the surface of baculovirus virions has provided a tool for the analysis of protein-protein interactions and for cell-specific targeting in gene transfer applications. To evaluate the baculovirus display system as a vaccine vehicle, we have generated a recombinant baculovirus (AcNPV-CSPsurf) that displays rodent malaria Plasmodium berghei circumsporozoite protein (PbCSP) on the virion surface as a fusion protein with the major baculovirus envelope glycoprotein gp64. The PbCSP-gp64 fusion protein was incorporated and oligomerized on the virion surface and led to a 12-fold increase in the binding activity of AcNPV-CSPsurf virions to HepG2 cells. Immunization with adjuvant-free AcNPV-CSPsurf virions induced high levels of antibodies and gamma interferon-secreting cells against PbCSP and protected 60% of mice against sporozoite challenge. These data demonstrate that AcNPV-CSPsurf displays sporozoite-like PbCSP on the virion surface and possesses dual potentials as a malaria vaccine candidate and a liver-directed gene delivery vehicle

  4. Baculovirus enhancins and their role in viral pathogenicity. Chapter 9

    Science.gov (United States)

    James M. Slavicek

    2012-01-01

    Baculoviruses are a large group of viruses pathogenic to arthropods, primarily insects from the order Lepidoptera and also insects in the orders Hymenoptera and Diptera. Baculoviruses have been used to control insect pests on agricultural crops and forests around the world. Efforts have been ongoing for the last two decades to develop strains of baculoviruses with...

  5. Chapter 15. transforming lepidopteran insect cells for continuous recombinant protein expression

    Science.gov (United States)

    The baculovirus expression vector system (BEVS) is widely used to produce large quantities of recombinant proteins. However, yields of extracellular and membrane-bound proteins obtained with this system often are very low, possibly due to the adverse effects of baculovirus infection on the host ins...

  6. Cloning and baculovirus expression of a desiccation stress gene from the beetle, Tenebrio molitor.

    Science.gov (United States)

    Graham, L A; Bendena, W G; Walker, V K

    1996-02-01

    The cDNA sequence encoding a novel desiccation stress protein (dsp28) found in the hemolymph of the common yellow mealworm beetle, Tenebrio molitor, has been determined. The sequence encodes a 225 amino acid protein containing a 20 amino acid signal peptide. Dsp28 shows no significant similarity to any known nucleic acid or protein sequence. Levels of dsp28 mRNA were found to increase approx 5-fold following desiccation. Dsp28 cDNA has been cloned into a baculovirus expression vector and the expressed protein was compared to native dsp28. Both dsp28 expressed by recombinant baculovirus and native dsp28 are glycosylated and N-terminally processed. Although dsp28 is induced by cold in addition to desiccation stress, it does not contribute to the freezing point depression (thermal hysteresis) observed in Tenebrio hemolymph.

  7. Radioimmunoassay analysis of baculovirus granulins and polyhedrins

    International Nuclear Information System (INIS)

    Summers, M.D.; Hoops, P.

    1980-01-01

    Granulin and polyhedrin proteins were purified by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis from the baculoviruses Autographa californica, Rachiplusia ou, Heliothis zea, Heliothis armigera. Trichoplusia ni, and Spodoptera frugiperda. Antisera were raised against Autographa californica (Ac) polyhedrin and Trichoplusia ni (Tn) granulin and analyzed for homologous and heterologous immunoreactivity by immunodiffusion and radioimmunoassay (RIA). Ac polyhedrin and Tn granulin antisera recognized antigenic determinants on several baculovirus polyhedrin and granulin proteins even though the heterologous proteins had different immunoreactivities when compared by competition radioimmunoassay. Antigenic differences among granulin and polyhedrin proteins were also detected by altered slopes of the competition reaction curves. Antiserum raised against Ac polyhedrin which was purified in the presence of SDS was tested by competition RIA for its ability to detect and react with native polyhedrin produced in the infected TN-368 cells. Ac polyhedrin antiserum had similar if not identical ability to bind to native polyhedrin and to polyhedrin purified in the presence of SDS

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-04-15

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

  9. A pseudotype baculovirus expressing the capsid protein of foot-and-mouth disease virus and a T-Cell immunogen shows enhanced immunogenicity in mice

    Directory of Open Access Journals (Sweden)

    Liu Xiangtao

    2011-02-01

    Full Text Available Abstract Background Foot-and-mouth disease (FMD is a highly contagious disease of livestock which causes severe economic loss in cloven-hoofed animals. Vaccination is still a major strategy in developing countries to control FMD. Currently, inactivated vaccine of FMDV has been used in many countries with limited success and safety concerns. Development of a novel effective vaccine is must. Methods In the present study, two recombinant pseudotype baculoviruses, one expressing the capsid of foot-and-mouth disease virus (FMDV under the control of a cytomegalovirus immediate early enhancer/promoter (CMV-IE, and the other the caspid plus a T-cell immunogen coding region under a CAG promoter were constructed, and their expression was characterized in mammalian cells. In addition, their immunogenicity in a mouse model was investigated. The humoral and cell-mediated immune responses induced by pseudotype baculovirus were compared with those of inactivated vaccine. Results Indirect immunofluorescence assay (IFA and indirect sandwich-ELISA (IS-ELISA showed both recombinant baculoviruses (with or without T-cell epitopes were transduced efficiently and expressed target proteins in BHK-21 cells. In mice, intramuscular inoculation of recombinants with 1 × 109 or 1 × 1010 PFU/mouse induced the production of FMDV-specific neutralizing antibodies and gamma interferon (IFN-γ. Furthermore, recombinant baculovirus with T-cell epitopes had better immunogenicity than the recombinant without T-cell epitopes as demonstrated by significantly enhanced IFN-γ production (P P Conclusions These results indicate that pseudotype baculovirus-mediated gene delivery could be a alternative strategy to develop a new generation of vaccines against FMDV infection.

  10. A baculovirus (Bombyx mori nuclear polyhedrosis virus) repeat element functions as a powerful constitutive enhancer in transfected insect cells.

    Science.gov (United States)

    Lu, M; Farrell, P J; Johnson, R; Iatrou, K

    1997-12-05

    It has been previously reported that baculovirus homologous regions, the regions of baculovirus genomes that contain the origins of DNA replication, can augment the expression of a small number of baculovirus genes in vitro. We are now reporting that a region of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) containing the homologous region 3 (HR3) acts as an enhancer for the promoter of a nonviral gene, the cytoplasmic actin gene of the silkmoth B. mori. Incorporation of the HR3 sequences of BmNPV into an actin promoter-based expression cassette results in an augmentation of transgene expression in transfected cells by two orders of magnitude relative to the control recombinant expression cassette. This increase is due to a corresponding increase in the rate of transcription from the actin promoter and not to replication of the expression cassette and occurs only when the HR3 element is linked to the expression cassette in cis. A comparable degree of enhancement in the activity of the silkworm actin promoter occurs also in heterologous lepidopteran cells. Concomitant supplementation of transfected cells with the BmIE1 trans-activator, which was previously shown to be capable of functioning in vitro as a transcriptional co-activator of the cytoplasmic actin gene promoter, results in more than a 1,000-fold increase in the level of expression of recombinant proteins placed under the control of the actin gene promoter. These findings provide the foundation for the development of a nonlytic insect cell expression system for continuous high-level expression of recombinant proteins. Such a system should provide levels of expression of recombinant proteins comparable to those obtained from baculovirus expression systems and should also have the additional advantage of continuous production in a cellular environment that, in contrast to that generated by a baculovirus infection, supports continuously proper posttranslational modifications of recombinant

  11. Baculovirus PTP2 functions as a pro-apoptotic protein

    NARCIS (Netherlands)

    Han, Yue; Houte, van Stineke; Oers, van Monique M.; Ros, Vera I.D.

    2018-01-01

    The family Baculoviridae encompasses a large number of invertebrate viruses, mainly infecting caterpillars of the order Lepidoptera. The baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) induces physiological and behavioral changes in its host Spodoptera exigua, as well as

  12. Baculovirus-mediated gene transfer in butterfly wings in vivo: an efficient expression system with an anti-gp64 antibody.

    Science.gov (United States)

    Dhungel, Bidur; Ohno, Yoshikazu; Matayoshi, Rie; Otaki, Joji M

    2013-03-25

    Candidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo. A recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules. Our baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer, express, and functionally

  13. The silencing suppressor (NSs) protein of the plant virus Tomato spotted wilt virus enhances heterologous protein expression and baculovirus pathogenicity in cells and lepidopteran insects.

    Science.gov (United States)

    de Oliveira, Virgínia Carla; da Silva Morgado, Fabricio; Ardisson-Araújo, Daniel Mendes Pereira; Resende, Renato Oliveira; Ribeiro, Bergmann Morais

    2015-11-01

    In this work, we showed that cell death induced by a recombinant (vAcNSs) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressing the silencing suppressor (NSs) protein of Tomato spotted wilt virus (TSWV) was enhanced on permissive and semipermissive cell lines. The expression of a heterologous gene (firefly luciferase) during co-infection of insect cells with vAcNSs and a second recombinant baculovirus (vAgppolhfluc) was shown to increase when compared to single vAgppolhfluc infections. Furthermore, the vAcNSs mean time-to-death values were significantly lower than those for wild-type AcMNPV on larvae of Spodoptera frugiperda and Anticarsia gemmatalis. These results showed that the TSWV-NSs protein could efficiently increase heterologous protein expression in insect cells as well as baculovirus pathogenicity and virulence, probably by suppressing the gene-silencing machinery in insects.

  14. Gene Acquisition Convergence between Entomopoxviruses and Baculoviruses

    Directory of Open Access Journals (Sweden)

    Julien Thézé

    2015-04-01

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

  15. Elektron Mikroskopi dan Imunogenisitas Baculovirus oryctes Isolat Yogyakarta

    Directory of Open Access Journals (Sweden)

    YB Sumardiyono

    1995-12-01

    Full Text Available Palm rhinoceros beetle (Oryctes rhinoceros was infected per os with Yogyakarta isolate of Baculovirus oryctes in laboratory condition. Midguts of infected beetle obtained were then extracted for further nucleoprotein purification by centrifugation method. Electron microscopy studies on purified nucleoprotein revealed rod-shape viruses with rounded end measured 190×94 nm in average. One end of the particle showed tail-like structure. Antibodies against the virus were obtained by immunization to rabbit, and reacted against either purified virus or extract of infected beetle, but not against extract of healthy beetle. Key words: Baculovirus oryctes, polyclonal antibody

  16. Genetically-engineered baculovirus pesticides and their environmental safety

    Science.gov (United States)

    H. Alan Wood; Yu Zailin

    1991-01-01

    Baculoviruses such as the Lymantria dispar nuclear polyhedrosis virus (LdMNPV) are ecologically attractive alternatives to chemical insect pesticides but have a slow rate of control. To overcome this we have developed and are field testing an environmentally acceptable strategy which can be used for the introduction and expression of pesticide-...

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

    NARCIS (Netherlands)

    Oers, van M.M.

    2006-01-01

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

  18. BACULOVIRUS REPLICATION ALTERS HORMONE-REGULATED HOST DEVELOPMENT.

    Science.gov (United States)

    The baculovirus Lymantria dispar nuclear polyhedrosis virus interferes with insect larval development by altering the host's hormonal system. The level of haemolymph ecdysteroids, the insect moulting hormone, was found to be higher in virus-infected larvae than in uninfected cont...

  19. Probability to produce animal vaccines in insect baculovirus ...

    African Journals Online (AJOL)

    The insect baculovirus expression system is a valuable tool for the production of vaccine. Many subunit vaccines have been expressed in this system. The first vaccine produced in insect cells for animal use is now in the market. In this study, we reviewed recent progress of animal's vaccine production for different expression ...

  20. Review of Vaccinia Virus and Baculovirus Viability Versus Virucides

    Science.gov (United States)

    2008-03-01

    25 6.4 Lignin ......................................................................................... 25 6.5...a lower pH (4.83 - 5.22), the virus rapidly inactivated over a month (Tomas et al., 1973). 16 The effects of alkalis on baculoviruses are important...of antioxidant and oxidative enzymes on UV inactivation by inhibiting the generation of highly reactive free radicals within HzSNPV. Water suspensions

  1. Expression and purification of recombinant nattokinase in Spodoptera frugiperda cells.

    Science.gov (United States)

    Li, Xiaoxiang; Wang, Xiaoli; Xiong, Shaoling; Zhang, Jing; Cai, Litao; Yang, Yanyan

    2007-10-01

    A recombinant baculovirus, rv-egfp-NK, containing a reporter gene encoding the enhanced green fluorescent protein (EGFP), was used to express nattokinase (NK), a fibrinolytic enzyme, in Spodoptera frugiperda (SF-9) cells. The recombinant protein also included a histidine tag for purification using Ni(2+) resins. The recombinant NK, approximately 30 kDa, retained fibrinolytic activity (60 U/ml). The integration of the EGFP expression cassette in the Bac-to-Bac system is thus an effective method for the expression and purification of recombinant NK protein in Spodoptera frugiperda insect cells.

  2. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems.

    Directory of Open Access Journals (Sweden)

    Tamer Z Salem

    Full Text Available The simian virus 40 polyadenylation signal (SV40 polyA has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS. In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV, the polyhedrin promoter (very late promoter transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt, and gp37. In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications.

  3. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems

    Science.gov (United States)

    Salem, Tamer Z.; Seaborn, Craig P.; Turney, Colin M.; Xue, Jianli; Shang, Hui; Cheng, Xiao-Wen

    2015-01-01

    The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications). PMID:26659470

  4. The Current Status of Baculovirus and Their Implication for Insect Pest Control

    Directory of Open Access Journals (Sweden)

    Arman Wijonarko

    2001-07-01

    Full Text Available Baculovirus have been promoted as the promising bioinsecticides for their pest control potential for more than half a century. But only a few have been successful as biological control agent, and almost none has been proven as commercial success, or widely used for large-scale insect pest control. The bioinsecticides currently represent only a small fraction of the world pesticide market. The successful of the Bt crop marked a special achievement in the bioinsecticide market growth. How about the baculoviruses? The main hurdle for baculovirus to be developed as bioinsecticide is its poor performance compare to synthetic chemical ones, include the speed of kill, and host range. It is important to understand the nature of baculovirus, and explore the possibilities to develop new way in applying the baculovirus as bioinsecticides. Key words: current status, baculovirus, insect control

  5. Expression of tomato yellow leaf curl virus coat protein using baculovirus expression system and evaluation of its utility as a viral antigen.

    Science.gov (United States)

    Elgaied, Lamiaa; Salem, Reda; Elmenofy, Wael

    2017-08-01

    DNA encoding the coat protein (CP) of an Egyptian isolate of tomato yellow leaf curl virus (TYLCV) was inserted into the genome of Autographa californica nucleopolyhedrovirus (AcNPV) under the control of polyhedrin promoter. The generated recombinant baculovirus construct harboring the coat protein gene was characterized using PCR analysis. The recombinant coat protein expressed in infected insect cells was used as a coating antigen in an indirect Enzyme-linked immunosorbent assay (ELISA) and dot blot to test its utility for the detection of antibody generated against TYLCV virus particles. The results of ELISA and dot blot showed that the TYLCV-antibodies reacted positively with extracts of infected cells using the recombinant virus as a coating antigen with strong signals as well as the TYLCV infected tomato and beat plant extracts as positive samples. Scanning electron microscope examination showed that the expressed TYLCV coat protein was self-assembled into virus-like particles (VLPs) similar in size and morphology to TYLCV virus particles. These results concluded that, the expressed coat protein of TYLCV using baculovirus vector system is a reliable candidate for generation of anti-CP antibody for inexpensive detection of TYLCV-infected plants using indirect CP-ELISA or dot blot with high specificity.

  6. On the role of baculovirus photolyases in DNA repair upon UV damage of occlusion bodies

    NARCIS (Netherlands)

    Biernat, M.A.; Caballero, P.; Vlak, J.M.; Oers, van M.M.

    2013-01-01

    The use of baculoviruses in insect biocontrol is hampered by their sensitivity to ultraviolet (UV) light. This irradiation induces cyclobutane pyrimidine dimers (CPDs) in DNA. CPD-photolyases repair CPDs using visible light. Plusiine baculoviruses encode photolyases, which could potentially repair

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

    Science.gov (United States)

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

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

    International Nuclear Information System (INIS)

    Murphy, C.I.; Lennick, M.; Lehar, S.M.; Beltz, G.A.; Young, E.

    1990-01-01

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

  9. A silencing suppressor protein (NSs) of a tospovirus enhances baculovirus replication in permissive and semipermissive insect cell lines.

    Science.gov (United States)

    Oliveira, Virgínia Carla; Bartasson, Lorrainy; de Castro, Maria Elita Batista; Corrêa, José Raimundo; Ribeiro, Bergmann Morais; Resende, Renato Oliveira

    2011-01-01

    The nonstructural protein (NSs) of the Tomato spotted wilt virus (TSWV) has been identified as an RNAi suppressor in plant cells. A recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) designated vAcNSs, containing the NSs gene under the control of the viral polyhedrin (polh) gene promoter, was constructed and the effects of NSs in permissive, semipermissive and nonpermissive insect cells to vAcNSs infection were evaluated. vAcNSs produced more budded virus when compared to wild type in semipermissive cells. Co-infection of vAcNSs with wild type baculoviruses clearly enhanced polyhedra production in all host cells. Confocal microscopy analysis showed that NSs accumulated in abundance in the cytoplasm of permissive and semipermissive cells. In contrast, high amounts of NSs were detected in the nuclei of nonpermissive cells. Co-infection of vAcNSs with a recombinant AcMNPV containing the enhanced green fluorescent protein (egfp) gene, significantly increased EGFP expression in semipermissive cells and in Anticarsia gemmatalis-hemocytes. Absence of small RNA molecules of egfp transcripts in this cell line and in a permissive cell line indicates the suppression of gene silencing activity. On the other hand, vAcNSs was not able to suppress RNAi in a nonpermissive cell line. Our data showed that NSs protein of TSWV facilitates baculovirus replication in different lepidopteran cell lines, and these results indicate that NSs could play a similar role during TSWV-infection in its thrips vector. Copyright © 2010 Elsevier B.V. All rights reserved.

  10. Baculovirus potential for agricultural pests management in Cuba

    Directory of Open Access Journals (Sweden)

    Jorge Luis Ayala Sifontes

    2017-10-01

    Full Text Available Cuba has an international reputation for implementing widespread biological control of pests, and microbial biocontrol is an integral component of most pest management programs. One class of microbial pesticides however, has not been developed in Cuba, bio-insecticides based on the Baculoviridae. This class of safe and environmentally protective microbial pesticides is used ever more commonly worldwide as an alternative to chemical pesticides. The characteristics of the viruses of this family, particularly their high host specificity, safety to non-target organisms, capacity to persist in nature and create epizootics, and the economy with which they can be produced "in vivo", all make them attractive for incorporation into pest management programs along with other pesticides developed in Cuba. The mass production technology is well understood in Cuba and biofactories already exist for a number of microbial biocontrol products. In the province of Sancti Spíritus, the Plant Protection Laboratory of the Ministry of Agriculture, with the cooperation of the Institute for Sustainable Horticulture, Kwantlen Polytechnic University, are resuming the work which began in the 90´s to develop baculovirus products in support of sustainable agriculture in Cuba. This work is being carried out with the participation of young Canadian and Cuban students and professionals. The program includes research with the multicapsid nuclear polyhedrosis viruses of Spodoptera frugiperda (SfMNPV and S. exigua (SeMNPV and the search for native isolates of Baculovirus in Plutella xylostella, three priority pests in Cuba. In other jurisdictions they are well controlled by baculoviruses, and the expectation is that this same result is possible in Cuba.

  11. N-glycan sialylation in a silkworm-baculovirus expression system.

    Science.gov (United States)

    Suganuma, Masatoshi; Nomura, Tsuyoshi; Higa, Yukiko; Kataoka, Yukiko; Funaguma, Shunsuke; Okazaki, Hironobu; Suzuki, Takeo; Fujiyama, Kazuhito; Sezutsu, Hideki; Tatematsu, Ken-Ichiro; Tamura, Toshiki

    2018-02-09

    A silkworm-baculovirus system is particularly effective for producing recombinant proteins, including glycoproteins. However, N-glycan structures in silkworm differ from those in mammals. Glycoproteins in silkworm are secreted as pauci-mannose type N-glycans without sialic acid or galactose residues. Sialic acid on N-glycans plays important roles in protein functions. Therefore, we developed pathways for galactosylation and sialylation in silkworm. Sialylated N-glycans on proteins were successfully produced in silkworm by co-expressing galactosyltransferase and sialyltransferase and providing an external supply of a sialylation-related substrate. α2,3/α2,6 Sialylation to N-glycans was controlled by changing the type of sialyltransferase expressed in silkworm. Furthermore, the co-expression of N-acetylglucosaminyltransferase II facilitated the formation of additional di-sialylated N-glycan structures. Our results provide new information on the control of N-glycosylation in silkworm. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  12. Future vital prospect of gene expression factors of lef-7 (baculovirus expression: Old body, young cherub

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    Md. Reyad-ul-ferdous

    2018-04-01

    Full Text Available Background Baculovirus; late expression factors (Lef-7 have potential roles for protein expression in insect and mammalian cells; Efficient expression of recombinant proteins to facilitate the practical and structural investigation. Aims Lef-7 might play crucial roles in transcription and translation reactions of insect cell lines. Methods Materials and Methods: All required information regards Lef-7 was generated by exploring the internet search engine like as (PubMed, Wiley, ScienceDirect, CNKI, ACS, Google Scholar, Web of Science, SciFinder, and Baidu Scholar and libraries. Results These properties issue crucial scope for DNA cloning and act as a vital vector for insect and mammalian cells. Left-7 could be the significant site in the development of the vaccine for a couple of chronic diseases. Further investigation needs to study on therapeutic vaccines with few immunologic advantages over proteins derived from mammalian sources, and animal sources. Lef-7 demonstrates the significant impact in the fields of DNA immunology research to insight into the mechanistic and utilitarian link between autoimmunity, infectious diseases, and cancer. Conclusion This review reveals Lef-7 gene function offers a workable strategy for the expression of whole viral protomers as the future prospect of Lef-7.

  13. Cloning of fusion protein gene of Newcastle disease virus into a baculovirus derived bacmid shuttle vector, in order to express it in insect cell line

    Directory of Open Access Journals (Sweden)

    Hashemzadeh MS

    2015-05-01

    Full Text Available Abstract Background: Newcastle disease virus (NDV is one of the major pathogens in poultry and vaccination is intended to control the disease, as an effective solution, yet. Fusion protein (F on surface of NDV, has a fundamental role in virus pathogenicity and can induce protective immunity, alone. With this background, here our aim was to construct a baculovirus derived recombinant bacmid shuttle vector (encoding F-protein in order to express it in insect cell line. Materials and Methods: In this experimental study, at first complete F gene from avirulent strain La Sota of NDV was amplified by RT-PCR to produce F cDNA. The amplicon was cloned into T/A cloning vector and afterwards into pFastBac Dual donor plasmid. After the verification of cloning process by two methods, PCR and enzymatic digestion analysis, the accuracy of F gene sequence was confirmed by sequencing. Finally, F-containing recombinant bacmid was subsequently generated in DH10Bac cell and the construct production was confirmed by a special PCR panel, using F specific primers and M13 universal primers. Results: Analysis of confirmatory tests showed that the recombinant bacmid, expressing of F-protein gene in correct sequence and framework, has been constructed successfully. Conclusion: The product of this F-containing recombinant bacmid, in addition to its independent application in the induction of protective immunity, can be used with the other individual recombinant baculoviruses, expressing HN and NP genes to produce NDV-VLPs in insect cell line.

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

    Directory of Open Access Journals (Sweden)

    Chang Myint OO

    2009-10-01

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

  15. Pemanfaatan Antibodi Monoklonal dalam Immunoassay untuk Deteksi Baculovirus oryctes

    Directory of Open Access Journals (Sweden)

    Susamto Somowiyarjo

    2000-12-01

    Full Text Available The application of non-precoated Indirect Enzyme-Linked Immunosorbent Assay (ELISA and Dot Immunobinding Assay (DIBA employing monoclonal antibodies (MCA against Yogyakarta isolate of Baculovirus oryctes Huger. was described. The MCA-Bv-4 having subclass of IgG2a and titer in vitro of 10^4 - 10^5 proved to be useful antibody for virus detection. The great potential of I-ELISA using MCA-Bv-4 has been it's specificity being able to discriminate between healthy an virus-infected coconut beetle (Oryctes rhinoceros L.. The assay could also differentiate between B. oryctes and Monodon baculovirus, the pathogen of shrimp disease. The best tissue for preparing virus antigen from crude extract was found to be the midgut of the beetle. Head, thorax, abdomen and tibia were not suitable for preparing the test antigen. The crude extract of beetle showed high endogenous enzyme activity to the substrate of DIBA, which precluded the detection of B. oryctes using DIBA. The MCA-Bv-4 could be used to improve the monitoring of the virus to support the program of biological control of coconut beetle using B. oryctes.

  16. A novel recombinant virus-like particle vaccine for prevention of porcine parvovirus-induced reproductive failure

    NARCIS (Netherlands)

    Antonis, A.F.G.; Bruschke, C.J.M.; Rueda, P.; Maranga, L.; Casal, J.; Vela, C.; Hilgers, L.A.T.; Belt, P.B.G.M.; Weerdmeester, K.; Carrondo, M.J.; Langeveld, J.P.M.

    2006-01-01

    A novel vaccine against porcine parvovirus (PPV), composed of recombinant virus-like particles (PPV-VLPs) produced with the baculovirus expression vector system (BEVS) at industrial scale, was tested for its immunogenicity and protective potency. A formulation of submicrogram amounts of PPV-VLPs in

  17. 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

  18. Characterization of viral proteins of Oryctes baculovirus and comparison between two geographical isolates.

    Science.gov (United States)

    Mohan, K S; Gopinathan, K P

    1989-01-01

    Bacilliform Oryctes baculovirus particles have been visualized in electron micrographs of midgut sections from virus infected Oryctes rhinoceros beetles. Morphologically the Indian isolate (Oryctes baculovirus, KI) resembled the previously reported Oryctes baculovirus, isolate PV505. The constituent proteins of baculovirus KI have been analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blots using polyclonal antibodies raised against the complete viral particles, as probes. A total of forty eight viral proteins have been identified. Fourteen viral proteins were located on the viral envelope. Among the proteins constituting the nucleocapsid, three were located internally within the capsid. A 23.5 kDa protein was tightly associated with viral DNA in the nucleocapsid core. Two envelope and seven capsid proteins of KI and PV505 revealed differences in SDS-PAGE profiles and glycosylation patterns. Immunoblotting of KI and PV505 proteins with anti KI antiserum demonstrated antigenic differences between the two viral isolates.

  19. DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines.

    Science.gov (United States)

    Yamagishi, J; Isobe, R; Takebuchi, T; Bando, H

    2003-03-01

    We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef- 6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV.

  20. Expression of the ’Bacillus anthracis’ Protective Antigen Gene by Baculovirus and Vaccinia Virus Recombinants

    Science.gov (United States)

    1990-02-01

    procaryotic systems (12. 45). Certain eucaryotic ically cleaved by a trypsin-like proteas: ito produce a recep- viruses are currently being explored as...19847. Proteolytic activation of anthrax toxin bound to cellular recep- ACKN()WEIX;NMNTS tor%.. p. 111-112. In F. Fehrenbach et al. ifed.). Bacterial

  1. Baculovirus-mediated expression and isolation of human ribosomal phosphoprotein P0 carrying a GST-tag in a functional state

    International Nuclear Information System (INIS)

    Abo, Yohichi; Hagiya, Akiko; Naganuma, Takao; Tohkairin, Yukiko; Shiomi, Kunihiro; Kajiura, Zenta; Hachimori, Akira; Uchiumi, Toshio; Nakagaki, Masao

    2004-01-01

    We constructed an overexpression system for human ribosomal phosphoprotein P0, together with P1 and P2, which is crucially important for translation. Genes for these proteins, fused with the glutathione S-transferase (GST)-tag at the N-terminus, were inserted into baculovirus and introduced to insect cells. The fusion proteins, but not the proteins without the tag, were efficiently expressed into cells as soluble forms. The fusion protein GST.P0 as well as GST.P1/GST.P2 was phosphorylated in cells as detected by incorporation of 32 P and reactivity with monoclonal anti-phosphoserine antibody. GST.P0 expressed in insect cells, but not the protein obtained in Escherichia coli, had the ability to form a complex with P1 and P2 proteins and to bind to 28S rRNA. Moreover, the GST.P0-P1-P2 complex participated in high eEF-2-dependent GTPase activity. Baculovirus expression systems appear to provide recombinant human P0 samples that can be used for studies on the structure and function

  2. Recombinant Programming

    OpenAIRE

    Pawlak , Renaud; Cuesta , Carlos; Younessi , Houman

    2004-01-01

    This research report presents a promising new approach to computation called Recombinant Programming. The novelty of our approach is that it separates the program into two layers of computation: the recombination and the interpretation layer. The recombination layer takes sequences as inputs and allows the programmer to recombine these sequences through the definition of cohesive code units called extensions. The output of such recombination is a mesh that can be used by the interpretation la...

  3. Global Screening of Antiviral Genes that Suppress Baculovirus Transgene Expression in Mammalian Cells.

    Science.gov (United States)

    Wang, Chia-Hung; Naik, Nenavath Gopal; Liao, Lin-Li; Wei, Sung-Chan; Chao, Yu-Chan

    2017-09-15

    Although baculovirus has been used as a safe and convenient gene delivery vector in mammalian cells, baculovirus-mediated transgene expression is less effective in various mammalian cell lines. Identification of the negative regulators in host cells is necessary to improve baculovirus-based expression systems. Here, we performed high-throughput shRNA library screening, targeting 176 antiviral innate immune genes, and identified 43 host restriction factor genes in a human A549 lung carcinoma cell line. Among them, suppression of receptor interaction protein kinase 1 (RIP1, also known as RIPK1) significantly increased baculoviral transgene expression without resulting in significant cell death. Silencing of RIP1 did not affect viral entry or cell viability, but it did inhibit nuclear translocation of the IRF3 and NF-κB transcription factors. Also, activation of downstream signaling mediators (such as TBK1 and IRF7) was affected, and subsequent interferon and cytokine gene expression levels were abolished. Further, Necrostatin-1 (Nec-1)-an inhibitor of RIP1 kinase activity-dramatically increased baculoviral transgene expression in RIP1-silenced cells. Using baculovirus as a model system, this study presents an initial investigation of large numbers of human cell antiviral innate immune response factors against a "nonadaptive virus." In addition, our study has made baculovirus a more efficient gene transfer vector for some of the most frequently used mammalian cell systems.

  4. Baculovirus LEF-11 nuclear localization signal is important for viral DNA replication.

    Science.gov (United States)

    Chen, Tingting; Dong, Zhanqi; Hu, Nan; Hu, Zhigang; Dong, Feifan; Jiang, Yaming; Li, Jun; Chen, Peng; Lu, Cheng; Pan, Minhui

    2017-06-15

    Baculovirus LEF-11 is a small nuclear protein that is involved in viral late gene transcription and DNA replication. However, the characteristics of its nuclear localization signal and its impact on viral DNA replication are unknown. In the present study, systemic bioinformatics analysis showed that the baculovirus LEF-11 contains monopartite and bipartite classical nuclear localization signal sequences (cNLSs), which were also detected in a few alphabaculovirus species. Localization of representative LEF-11 proteins of four baculovirus genera indicated that the nuclear localization characteristics of baculovirus LEF-11 coincided with the predicted results. Moreover, Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 could be transported into the nucleus during viral infection in the absence of a cNLSs. Further investigations demonstrated that the NLS of BmNPV LEF-11 is important for viral DNA replication. The findings of the present study indicate that the characteristics of the baculovirus LEF-11 protein and the NLS is essential to virus DNA replication and nuclear transport mechanisms. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. 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),

  6. Characterization of A Baculovirus of Spodoptera litura (Lepidoptera : Noctuidae Isolated from Yogyakarta

    Directory of Open Access Journals (Sweden)

    Arman Wijonarko

    2007-07-01

    Full Text Available A Baculovirus has been isolated from cadaver of larvae of Spodoptera litura, a Noctuidae of agricultural pest, importance due to its wide-range hosts and the damage to their respective host. Phase contrast light microscopy observation from infected larvae showed that the fat body, hemocyte cells, and cells surrounding the trachea or tracheolus were the most tissue invaded by polyhedra. Transmission electron microscopy analysis of the occlusion body purified from diseased larva showed that the baculovirus envelope containing multiple nucleocapsid. Digestion of viral DNA with three restriction enzymes showed that the genome pattern of baculovirus isolated from Bantul were close to SpliNPV isolated from Japan and those of Spodoptera littoralis and quite distinct from those isolated from Southeast Asia region. Bioassay test performed on first to fifth instar larvae showed that the virus effectively control the young larvae, but showed some level of resistance against older larvae of Spodoptera litura.

  7. In vivo study of immunogenicity and kinetic characteristics of a quantum dot-labelled baculovirus.

    Science.gov (United States)

    Wang, Meng; Zheng, Zhenhua; Meng, Jin; Wang, Han; He, Man; Zhang, Fuxian; Liu, Yan; Hu, Bin; He, Zike; Hu, Qinxue; Wang, Hanzhong

    2015-09-01

    Nanomaterials conjugated with biomacromolecules, including viruses, have great potential for in vivo applications. Therefore, it is important to evaluate the safety of nanoparticle-conjugated macromolecule biomaterials (Nano-mbio). Although a number of studies have assessed the risks of nanoparticles and macromolecule biomaterials in living bodies, only a few of them investigated Nano-mbios. Here we evaluated the in vivo safety profile of a quantum dot-conjugated baculovirus (Bq), a promising new Nano-mbio, in mice. Each animal was injected twice intraperitoneally with 50 μg virus protein labelled with around 3*10(-5)nmol conjugated qds. Control animals were injected with PBS, quantum dots, baculovirus, or a mixture of quantum dots and baculovirus. Blood, tissues and body weight were analysed at a series of time points following both the first and the second injections. It turned out that the appearance and behaviour of the mice injected with Bq were similar to those injected with baculovirus alone. However, combination of baculovirus and quantum dot (conjugated or simply mixed) significantly induced stronger adaptive immune responses, and lead to a faster accumulation and longer existence of Cd in the kidneys. Thus, despite the fact that both quantum dot and baculovirus have been claimed to be safe in vivo, applications of Bq in vivo should be cautious. To our knowledge, this is the first study examining the interaction between a nanoparticle-conjugated virus and a living body from a safety perspective, providing a basis for in vivo application of other Nano-mbios. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Santiago Haase

    2015-04-01

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

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

    NARCIS (Netherlands)

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

    2011-01-01

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

  10. Identification of genes involved in DNA replication of the Autographa californica baculovirus

    NARCIS (Netherlands)

    Kool, M.; Ahrens, C. H.; Goldbach, R. W.; Rohrmann, G. F.; Vlak, J. M.

    1994-01-01

    By use of a transient replication assay, nine genes involved in DNA replication were identified in the genome of the Autographa californica baculovirus. Six genes encoding helicase, DNA polymerase, IE-1, LEF-1, LEF-2, and LEF-3 are essential for DNA replication while three genes encoding P35, IE-2,

  11. Contributions of immune responses to developmental resistance in Lymantria dispar challenged with baculovirus

    Science.gov (United States)

    James McNeil; Diana Cox-Foster; James Slavicek; Kelli. Hoover

    2010-01-01

    How the innate immune system functions to defend insects from viruses is an emerging field of study. We examined the impact of melanized encapsulation, a component of innate immunity that integrates both cellular and humoral immune responses, on the success of the baculovirus Lymantria dispar multiple nucleocapsid nucleopolyhedrovirus (LdMNPV) in its...

  12. Feasibility of baculovirus-mediated reporter gene delivery for efficient monitoring of islet transplantation in vivo

    International Nuclear Information System (INIS)

    Liu, Shuai; Pan, Yu; Lv, Jing; Wu, Haifei; Tian, Jingyan; Zhang, Yifan

    2014-01-01

    Objective: The objective of this study was to explore the feasibility of baculovirus vector-mediated sodium iodide symporter (NIS) gene delivery to monitor islet transplantation. Methods: Baculovirus vectors expressing green fluorescent protein (GFP) or NIS (Bac-GFP and Bac-NIS) were established using the Bac-to-Bac baculovirus expression system. The GFP expression of Bac-GFP-infected rat islets was observed in vitro by fluorescence microscopy. Iodine uptake and inhibition of iodine uptake by NaClO 4 in Bac-NIS-infected islets were dynamically monitored in vitro. Bac-GFP- or Bac-NIS-infected islets were implanted into the left axillary cavity of NOD-SCID mice, and fluorescence imaging and 125 I NanoSPECT/CT imaging were subsequently performed in vivo. Results: Bac-GFP efficiently infected rat islets (over 95% infected at MOI = 40), and the expression of GFP lasted approximately two weeks. NaClO 4 could inhibit iodine uptake by Bac-NIS-infected islets. In vivo imaging revealed that the fluorescence intensity of the transplant sites in Bac-GFP-infected groups was significantly higher than in the non-infected group. Grafts could be clearly observed by 125 I NanoSPECT/CT imaging for up to 8 h. Conclusion: Baculovirus vectors are powerful vehicles for studying rat islets in gene delivery. It is feasible to use a baculovirus vector to delivery an NIS gene for non-invasive monitoring transplanted islets in vivo by the expression of the target gene

  13. Sleeping Beauty-baculovirus hybrid vectors for long-term gene expression in the eye.

    Science.gov (United States)

    Turunen, Tytteli Anni Kaarina; Laakkonen, Johanna Päivikki; Alasaarela, Laura; Airenne, Kari Juhani; Ylä-Herttuala, Seppo

    2014-01-01

    A baculovirus vector is capable of efficiently transducing many nondiving and diving cell types. However, the potential of baculovirus is restricted for many gene delivery applications as a result of the transient gene expression that it mediates. The plasmid-based Sleeping Beauty (SB) transposon system integrates transgenes into target cell genome efficiently with a genomic integration pattern that is generally considered safer than the integration of many other integrating vectors; yet efficient delivery of therapeutic genes into cells of target tissues in vivo is a major challenge for nonviral gene therapy. In the present study, SB was introduced into baculovirus to obtain novel hybrid vectors that would combine the best features of the two vector systems (i.e. effective gene delivery and efficient integration into the genome), thus circumventing the major limitations of these vectors. We constructed and optimized SB-baculovirus hybrid vectors that bear either SB100x transposase or SB transposon in the forward or reverse orientations with respect to the viral backbone The functionality of the novel hybrid vectors was investigated in cell cultures and in a proof-of-concept study in the mouse eye. The hybrid vectors showed high and sustained transgene expression that remained stable and demonstrated no signs of decline during the 2 months follow-up in vitro. These results were verified in the mouse eye where persistent transgene expression was detected two months after intravitreal injection. Our results confirm that (i) SB-baculovirus hybrid vectors mediate long-term gene expression in vitro and in vivo, and (ii) the hybrid vectors are potential new tools for the treatment of ocular diseases. Copyright © 2014 John Wiley & Sons, Ltd.

  14. Genetic Recombination

    Science.gov (United States)

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  15. Feasibility of a novel positive feedback effect of 131I-promoted Bac-Egr1-hNIS expression in malignant glioma via baculovirus

    International Nuclear Information System (INIS)

    Guo Rui; Tian Lipeng; Han Bing; Xu Haoping; Zhang Miao; Li Biao

    2011-01-01

    Purpose: As intracellular iodine is released rapidly, increased expression of sodium/iodide symporter (NIS) is required for effective radioiodine treatment of tumor. As Egr1 promoter is activated by 131 I and may promote human NIS (hNIS) expression, hNIS also induces 131 I uptake and activates Egr1, so the existence of a positive feedback effect of 131 I-promoted Egr1-hNIS expression is possible. Our purpose was to investigate the possible existence of this positive feedback effect through a series of in vitro pioneer studies. Method: Recombinant baculovirus (Bac-Egr1-hNIS) encoding the hNIS gene under the control of a radiation-inducible Egrl promoter was constructed. To test 131 I-promoted hNIS expression, human malignant glioma U87 cells were transfected with Bac-Egr1-hNIS, stimulated with or without 131 I; the expression of hNIS protein was detected by immunofluorescence and flow cytometry test. In addition, the uptake and efflux of 131 I were determined after the incubation of Bac-Egr1-hNIS-transfected U87 cells with or without 131 I. Results: Immunocytochemical staining and flow cytometry test showed a higher hNIS protein expression in Bac-Egr1-hNIS-transfected U87 cells with 131 I stimulation than in cells without stimulation. Bac-Egr1-hNIS-transfected U87 cells accumulated up to about 4.05 times of 131 I after 131 I stimulation. The amount of 131 I uptake in both groups showed a baculovirus dose-dependent manner. However, rapid efflux of radioactivity was observed in both groups, with 50% lost during the first 2 min after the 131 I-containing medium had been replaced by a nonradioactive medium. Conclusion: Our results indicated that an improved transgene expression of 131 I-stimulated hNIS in U87 cells using a baculovirus vector containing the Egr1 promoter is possible, and the increased expression of hNIS is responsible for a higher 131 I uptake. It might provide a reference for the existence of a positive feedback effect in 131 I-promoted Bac-Egr1-h

  16. Insect Larvae: A New Platform to Produce Commercial Recombinant Proteins.

    Science.gov (United States)

    Targovnik, Alexandra M; Arregui, Mariana B; Bracco, Lautaro F; Urtasun, Nicolas; Baieli, Maria F; Segura, Maria M; Simonella, Maria A; Fogar, Mariela; Wolman, Federico J; Cascone, Osvaldo; Miranda, Maria V

    2016-01-01

    In Biotechnology, the expression of recombinant proteins is a constantly growing field and different hosts are used for this purpose. Some valuable proteins cannot be produced using traditional systems. Insects from the order Lepidoptera infected with recombinant baculovirus have appeared as a good choice to express high levels of proteins, especially those with post-translational modifications. Lepidopteran insects, which are extensively distributed in the world, can be used as small protein factories, the new biofactories. Species like Bombyx mori (silkworm) have been analyzed in Asian countries to produce a great number of recombinant proteins for use in basic and applied science and industry. Many proteins expressed in this larva have been commercialized. Several recombinant proteins produced in silkworms have already been commercialized. On the other hand, species like Spodoptera frugiperda, Heliothis virescens, Rachiplusia nu, Helicoverpa zea and Trichoplusia ni are widely distributed in both the occidental world and Europe. The expression of recombinant proteins in larvae has the advantage of its low cost in comparison with insect cell cultures. A wide variety of recombinant proteins, including enzymes, hormones and vaccines, have been efficiently expressed with intact biological activity. The expression of pharmaceutically proteins, using insect larvae or cocoons, has become very attractive. This review describes the use of insect larvae as an alternative to produce commercial recombinant proteins.

  17. 寨卡病毒E蛋白在重组杆状病毒中的表达%Expression of envelope protein of Zika virus in baculovirus expression system

    Institute of Scientific and Technical Information of China (English)

    高寒春; 姚立红; 王超; 郑丽舒

    2017-01-01

    目的 在杆状病毒中表达寨卡病毒(Zika virus,ZIKV)包膜蛋白(Envelope protein,E蛋白).方法 人工合成ZIKV E基因全长序列(1 518 bp),并克隆到pFastBac1载体上,得到重组杆状病毒转移载体pFB1-E,转化含DH10Bac感受态细胞,获得骨架质粒rBacmid-E,转染sf9细胞,得到重组杆状病毒rBac-E.检测病毒滴度,PCR法检测E基因的插入,间接免疫荧光和Western blot法检测E蛋白的表达.结果 经PCR方法鉴定,重组杆状病毒骨架质粒构建成功,重组病毒rBac-E(第3代)病毒滴度为2.58×105pfu/ml.提取感染rBac-E的sf9细胞基因组,经PCR扩增得到3 830 bp的条带,间接免疫荧光检测出现特异性绿色荧光.Western blot鉴定可见感染重组杆状病毒rBac-E的细胞沉淀中在相对分子质量55×103处有特异性条带.结论 在杆状病毒表达系统中重组表达了ZIKV E蛋白,为ZIKV E蛋白功能研究及疫苗的开发奠定了基础.%Objective To express envelope protein of ZIKA virus in baculovirus expression system.Methods Full-length E gene of ZIKA virus was obtained by DNA synthesis and inserted into vector pFastBac1.The constructed recombinant baculovirus transfer vector pFB1-E was transformed to competent DH10Bac cells.The obtained skeleton plasmid rBacmid-E was transfected to sf9 cells,and the constructed recombinant baculovirus rBac-E was determined for titer,for insertion of E gene by PCR,and for expression of E protein by IFA and Western blotting.Results PCR proved that skeleton plasmid rBacmid-E was constructed correctly.The titer of rBac-E of passage 3 was 2.58 × 105 pfu/ ml.The genome of infected cells virus was extracted,the gene band at length of 3 830 bp was observed after PCR amplification.Indirect immunofluorescence of the infected cells showed the specific green fluorescence,55 × 103 specific band was determined by Western blotting identification in the cell pellet of the infected recombinant baculovirus rBacE.Conclusions The recombinant

  18. The baculovirus core gene ac83 is required for nucleocapsid assembly and per os infectivity of Autographa californica nucleopolyhedrovirus.

    Science.gov (United States)

    Zhu, Shimao; Wang, Wei; Wang, Yan; Yuan, Meijin; Yang, Kai

    2013-10-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac83 is a baculovirus core gene whose function in the AcMNPV life cycle is unknown. In the present study, an ac83-knockout AcMNPV (vAc83KO) was constructed to investigate the function of ac83 through homologous recombination in Escherichia coli. No budded virions were produced in vAc83KO-transfected Sf9 cells, although viral DNA replication was unaffected. Electron microscopy revealed that nucleocapsid assembly was aborted due to the ac83 deletion. Domain-mapping studies revealed that the expression of Ac83 amino acid residues 451 to 600 partially rescued the ability of AcMNPV to produce infectious budded virions. Bioassays indicated that deletion of the chitin-binding domain of Ac83 resulted in the failure of oral infection of Trichoplusia ni larvae by AcMNPV, but AcMNPV remained infectious following intrahemocoelic injection, suggesting that the domain is involved in the binding of occlusion-derived virions to the peritrophic membrane and/or to other chitin-containing insect tissues. It has been demonstrated that Ac83 is the only component with a chitin-binding domain in the per os infectivity factor complex on the occlusion-derived virion envelope. Interestingly, a functional inner nuclear membrane sorting motif, which may facilitate the localization of Ac83 to the envelopes of occlusion-derived virions, was identified by immunofluorescence analysis. Taken together, these results demonstrate that Ac83 plays an important role in nucleocapsid assembly and the establishment of oral infection.

  19. Display of a Maize cDNA library on baculovirus infected insect cells

    Directory of Open Access Journals (Sweden)

    Jones Ian M

    2008-08-01

    Full Text Available Abstract Background Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. Results We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 × 105 independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1, was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. Conclusion The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

  20. Towards a molecular identification and classification system of lepidopteran-specific baculoviruses

    International Nuclear Information System (INIS)

    Lange, Martin; Wang Hualin; Hu Zhihong; Jehle, Johannes A.

    2004-01-01

    Virus genomics provides novel approaches for virus identification and classification. Based on the comparative analyses of sequenced lepidopteran-specific baculovirus genomes, degenerate oligonucleotides were developed that allow the specific amplification of several regions of the genome using polymerase chain reaction (PCR) followed by DNA sequencing. The DNA sequences within the coding regions of three highly conserved genes, namely polyhedrin/granulin (polh/gran), late expression factor 8 (lef-8), and late expression factor 9 (lef-9), were targeted for amplification. The oligonucleotides were tested on viral DNAs isolated from historical field samples, and amplification products were generated from 12 isolated nucleopolyhedrovirus (NPV) and 8 granulovirus (GV) DNAs. The PCR products were cloned or directly sequenced, and phylogenetic trees were inferred from individual and combined data sets of these three genes and compared to a phylogeny, which includes 22 baculoviruses using a combined data set of 30 core genes. This method allows a fast and reliable detection and identification of lepidopteran-specific NPVs and GVs. Furthermore, a strong correlation of the base composition of these three genome areas with that of the complete virus genome was observed and used to predict the base composition of uncharacterized baculovirus genomes. These analyses suggested that GVs have a significantly higher AT content than NPVs

  1. Proteomics computational analyses suggest that baculovirus GP64 superfamily proteins are class III penetrenes

    Directory of Open Access Journals (Sweden)

    Garry Robert F

    2008-02-01

    Full Text Available Abstract Background Members of the Baculoviridae encode two types of proteins that mediate virus:cell membrane fusion and penetration into the host cell. Alignments of primary amino acid sequences indicate that baculovirus fusion proteins of group I nucleopolyhedroviruses (NPV form the GP64 superfamily. The structure of these viral penetrenes has not been determined. The GP64 superfamily includes the glycoprotein (GP encoded by members of the Thogotovirus genus of the Orthomyxoviridae. The entry proteins of other baculoviruses, group II NPV and granuloviruses, are class I penetrenes. Results Class III penetrenes encoded by members of the Rhabdoviridae and Herpesviridae have an internal fusion domain comprised of beta sheets, other beta sheet domains, an extended alpha helical domain, a membrane proximal stem domain and a carboxyl terminal anchor. Similar sequences and structural/functional motifs that characterize class III penetrenes are located collinearly in GP64 of group I baculoviruses and related glycoproteins encoded by thogotoviruses. Structural models based on a prototypic class III penetrene, vesicular stomatitis virus glycoprotein (VSV G, were established for Thogoto virus (THOV GP and Autographa california multiple NPV (AcMNPV GP64 demonstrating feasible cysteine linkages. Glycosylation sites in THOV GP and AcMNPV GP64 appear in similar model locations to the two glycosylation sites of VSV G. Conclusion These results suggest that proteins in the GP64 superfamily are class III penetrenes.

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

    Directory of Open Access Journals (Sweden)

    Aurélien Chateigner

    2015-07-01

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

  3. Baculovirus display of single chain antibody (scFv using a novel signal peptide

    Directory of Open Access Journals (Sweden)

    Gonzalez Gaëlle

    2010-11-01

    Full Text Available Abstract Background Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17, was found to exert an inhibitory effect on HIV-1 replication. Results Two versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP assembly and egress, and both failed to be encapsidated in VLP. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2 to another scFv recognizing CD147 (scFv-M6-1B9 conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6. Conclusion Expression of scFvE2/p17 in insect cells using a BV

  4. Fisetin and polymeric micelles encapsulating fisetin exhibit potent cytotoxic effects towards ovarian cancer cells.

    Science.gov (United States)

    Xiao, Xue; Zou, Juan; Fang, Yin; Meng, Yibo; Xiao, Chao; Fu, Jiaxin; Liu, Shiyu; Bai, Peng; Yao, Yuan

    2018-03-15

    The anti-tumor activities of Natural compounds and their derivatives are of great interest to pharmaceutical industries. Fisetin is one of prospective natural compounds in this regard but unfortunately with poor hydrophilicity. The effects of unmodified and modified fisetin in cultured ovarian cancer cells were compared by transmission electronmicroscopy to determine apoptotic bodies, MTT assay to quantitate cell numbers, and fluorescence activated cell sorting analyse of various markers to determine the apoptotic state. In addition, the efficacy of fisetin and fisetin-micelles in vivo was determined by using immunocompromised mice. Apoptosis was measured by established markers using both western blot analysis and immunochemistry. Angiogenesis in a xenograft mouse model carring SKOV3 cells was evaluated by color Doppler ultrasound and immunohistochemistry. Multiple lines of evidence indicated that fisetin and fisetin micelles induce apoptosis in ovarian cancer cells in a dose-dependent manner. Histological analysis, terminal deoxynucleotidyltransferase-mediated nick-end labeling assay, western blot, immunohistochemical detection and microvessel density detection demonstrated that fisetin and fisetin micelles induced increased tumor apoptosis, proliferation suppression and antiangiogenesis activities. As far as we know, the present study is the first time to demonstrate the potency of both fisetin and fisetin micelles inducing apoptosis in ovarian cancer cells. Further studies will be needed to validate the therapeutic potential of fisetin and fisetin micelles in ovarian cancer treatment.

  5. Polymeric micelles encapsulating fisetin improve the therapeutic effect in colon cancer.

    Science.gov (United States)

    Chen, Yishan; Wu, Qinjie; Song, Linjiang; He, Tao; Li, Yuchen; Li, Ling; Su, Weijun; Liu, Lei; Qian, Zhiyong; Gong, Changyang

    2015-01-14

    The natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) was discovered to possess antitumor activity, revealing its potential value in future chemotherapy. However, its poor water solubility makes it difficult for intravenous administration. In this study, the monomethyl poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) copolymer was applied to prepare nanoassemblies of fisetin by a self-assembly procedure. The prepared fisetin micelles gained a mean particle size of 22 ± 3 nm, polydisperse index of 0.163 ± 0.032, drug loading of 9.88 ± 0.14%, and encapsulation efficiency of 98.53 ± 0.02%. Compared with free fisetin, fisetin micelles demonstrated a sustained and prolonged in vitro release behavior, as well as enhanced cytotoxicity, cellular uptake, and fisetin-induced apoptosis in CT26 cells. As for in vivo studies, fisetin micelles were more competent for suppressing tumor growth and prolonging survival time than free fisetin in the subcutaneous CT26 tumor model. Furthermore, histological analysis, terminal deoxynucleotidyl transferase-mediated nick-end labeling assay, immunohistochemical detection of Ki-67, and microvessel density detection were conducted, demonstrating that fisetin micelles gained increased tumor apoptosis induction, proliferation suppression, and antiangiogenesis activities. In conclusion, we have successfully produced a MPEG-PCL-based nanocarrier encapsulating fisetin with enhanced antitumor activity.

  6. Reverse micelles as a tool for probing solvent modulation of protein dynamics: Reverse micelle encapsulated hemoglobin☆

    OpenAIRE

    Roche, Camille J.; Dantsker, David; Heller, Elizabeth R.; Sabat, Joseph E.; Friedman, Joel M.

    2013-01-01

    Hydration waters impact protein dynamics. Dissecting the interplay between hydration waters and dynamics requires a protein that manifests a broad range of dynamics. Proteins in reverse micelles (RMs) have promise as tools to achieve this objective because the water content can be manipulated. Hemoglobin is an appropriate tool with which to probe hydration effects. We describe both a protocol for hemoglobin encapsulation in reverse micelles and a facile method using PEG and cosolvents to mani...

  7. Expression and Characterization of Human β-1, 4-Galactosyltransferase 1 (β4GalT1) Using Silkworm–Baculovirus Expression System

    KAUST Repository

    Morokuma, Daisuke

    2017-03-24

    Baculovirus expression vector system (BEVS) is widely known as a mass-production tool to produce functional recombinant glycoproteins except that it may not be always suitable for medical practice due to the differences in the structure of N-linked glycans between insects and mammalian. Currently, various approaches have been reported to alter N-linked glycan structures of glycoproteins derived from insects into terminally sialylated complex-type N-glycans. In the light of those studies, we also proposed in vitro maturation of N-glycan with mass-produced and purified glycosyltransferases by silkworm–BEVS. β-1,4-Galactosyltransferase 1 (β4GalT1) is known as one of type II transmembrane enzymes that transfer galactose in a β-1, 4 linkage to accepter sugars, and a key enzyme for further sialylation of N-glycans. In this study, we developed a large-scale production of recombinant human β4GalT1 (rhβ4GalT1) with N- or C-terminal tags in silkworm–BEVS. We demonstrated that rhβ4GalT1 is N-glycosylated and without mucin-type glycosylation. Interestingly, we found that purified rhβ4GalT1 from silkworm serum presented higher galactosyltransferase activity than that expressed from cultured mammalian cells. We also validated the UDP-galactose transferase activity of produced rhβ4GalT1 proteins by using protein subtracts from silkworm silk gland. Taken together, rhβ4GalT1 from silkworms can become a valuable tool for producing high-quality recombinant glycoproteins with mammalian-like N-glycans.

  8. Spectrum Recombination.

    Science.gov (United States)

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  9. MacroBac: New Technologies for Robust and Efficient Large-Scale Production of Recombinant Multiprotein Complexes.

    Science.gov (United States)

    Gradia, Scott D; Ishida, Justin P; Tsai, Miaw-Sheue; Jeans, Chris; Tainer, John A; Fuss, Jill O

    2017-01-01

    Recombinant expression of large, multiprotein complexes is essential and often rate limiting for determining structural, biophysical, and biochemical properties of DNA repair, replication, transcription, and other key cellular processes. Baculovirus-infected insect cell expression systems are especially well suited for producing large, human proteins recombinantly, and multigene baculovirus systems have facilitated studies of multiprotein complexes. In this chapter, we describe a multigene baculovirus system called MacroBac that uses a Biobricks-type assembly method based on restriction and ligation (Series 11) or ligation-independent cloning (Series 438). MacroBac cloning and assembly is efficient and equally well suited for either single subcloning reactions or high-throughput cloning using 96-well plates and liquid handling robotics. MacroBac vectors are polypromoter with each gene flanked by a strong polyhedrin promoter and an SV40 poly(A) termination signal that minimize gene order expression level effects seen in many polycistronic assemblies. Large assemblies are robustly achievable, and we have successfully assembled as many as 10 genes into a single MacroBac vector. Importantly, we have observed significant increases in expression levels and quality of large, multiprotein complexes using a single, multigene, polypromoter virus rather than coinfection with multiple, single-gene viruses. Given the importance of characterizing functional complexes, we believe that MacroBac provides a critical enabling technology that may change the way that structural, biophysical, and biochemical research is done. © 2017 Elsevier Inc. All rights reserved.

  10. MicroRNAome of Spodoptera frugiperda cells (Sf9) and its alteration following baculovirus infection.

    Science.gov (United States)

    Mehrabadi, Mohammad; Hussain, Mazhar; Asgari, Sassan

    2013-06-01

    MicroRNAs (miRNAs) as small non-coding RNAs play important roles in many biological processes such as development, cell signalling and immune response. Studies also suggest that miRNAs are important in host-virus interactions where the host limits virus infection by differentially expressing miRNAs that target essential viral genes. Here, we identified conserved and new miRNAs from Spodoptera frugiperda cells (Sf9) using a combination of deep sequencing and bioinformatics as well as experimental approaches. S. frugiperda miRNAs share common features of miRNAs in other organisms, such as uracil (U) at the 5' end of miRNA. The 5' ends of the miRNAs were more conserved than the 3' ends, revealing evolutionary protection of the seed region in miRNAs. The predominant miRNAs were found to be conserved among arthropods. The majority of homologous miRNAs were found in Bombyx mori, with 76 of the 90 identified miRNAs. We found that seed shifting and arm switching have happened in this insect's miRNAs. Expression levels of the majority of miRNAs changed following baculovirus infection. Results revealed that baculovirus infection mainly led to an overall suppression of cellular miRNAs. We found four different genes being regulated by sfr-miR-184 at the post-transcriptional level. The data presented here further support conservation of miRNAs in insects and other organisms. In addition, the results reveal a differential expression of host miRNAs upon baculovirus infection, suggesting their potential roles in host-virus interactions. Seed shifting and arm switching happened during evolution of miRNAs in different insects and caused miRNA diversification, which led to changes in the target repository of miRNAs.

  11. High-yield production of canine parvovirus virus-like particles in a baculovirus expression system.

    Science.gov (United States)

    Jin, Hongli; Xia, Xiaohong; Liu, Bing; Fu, Yu; Chen, Xianping; Wang, Huihui; Xia, Zhenqiang

    2016-03-01

    An optimized VP2 gene from the current prevalent CPV strain (new CPV-2a) in China was expressed in a baculovirus expression system. It was found that the VP2 proteins assembled into virus-like particles (VLPs) with antigenic properties similar to those of natural CPV and with an especially high hemagglutination (HA) titer (1:2(20)). Dogs intramuscularly or orally immunized with VLPs produced antibodies against CPV with >1:80 hemagglutination inhibition (HI) units for at least 3 months. The CPV VLPs could be considered for use as a vaccine against CPV or as a platform for research on chimeric VLP vaccines against other diseases.

  12. Ultrastructural and pathogenesis of Monodon baculovirus in SPF shrimp, Litopenaeus vannamei imported to Iran

    OpenAIRE

    Bahari-Meimandi, S.A.; Afsharnasab, M.; Motallebi Moghanjoghi, A.A.; Azaritakami, G.; Sharifrohani, M.

    2014-01-01

    Viral pathogens are major causes of outbreaks in shrimp farms throughout the world. Monodon baculovirus has been known to be invasive in 85-100% of the shrimp hatcheries, in early or late stages of shrimp. Three-hundred and sixty juvenile of Litopenaeus vannamei with average (±SD) size of 7.99±0.54 g and 3600 post larvae 10-15 were prepared from Shrimp Research Station located in Helleh and 3 hatcheries from Bushehr Province, southern part of Iran, respectively. They were allocated to 9 glass...

  13. Experiment study with baculovirus-mediated transfer of the thyroid sodium/iodide symporter gene into thyroid cancer for a targeted radiotherapy

    International Nuclear Information System (INIS)

    Zhang Yifan; Li Biao; Zhao Long; You Bei; Yin Guizhi; Zhu Chengmo

    2004-01-01

    Objective: To explore the feasibility of thyroid cancers for radiotherapy by using baculoviral vector to deliver the NIS gene into the tumor cells. Method: Constructed a recombinant baculovirus encoding the human NIS gene under the control of the cytomegalovirus promoter. Using a mouse monoclonal antibody and a FITC-labeled antimouse antibody to confirm expression of the NIS protein of infected tumor cells by immunofluorescence. In vitro iodide uptake experiments were carded out on BacNIS-infected tumor cells to further characterize the BacNIS virus, and cell killing with 131I and clonogenic assay were performed on BacNIS-infected cell to observe the selective killing effect of 1311 on NIS-expressing cells. Results: Infection of thyroidcancer cells (FTC-133, W3) with BacNIS resulted in perchlorate-sensitive 125I uptake by these cells to a higher level than that in noninfected cells. But 1251 uptake of 8505C is very low. Demonstrating that the BacNIS vector can function in tumor cells. In addition, AdNIS-infected tumor cells were selectively killed by exposure to 1311, as revealed by clonogenicassays, higher than that in nontreated tumors. Conclusions: AdNIS is very efficient in triggering iodide uptake by infected tumor cell, outlining the potential of this novel cancer gene therapy approach for a targeted radiotherapy. (authors)

  14. Expression of foot-and-mouth disease virus capsid proteins in silkworm-baculovirus expression system and its utilization as a subunit vaccine.

    Directory of Open Access Journals (Sweden)

    Zhiyong Li

    Full Text Available BACKGROUND: Foot-and-mouth disease (FMD is a highly contagious disease of livestock that causes severe economic loss in susceptible cloven-hoofed animals. Although the traditional inactivated vaccine has been proved effective, it may lead to a new outbreak of FMD because of either incomplete inactivation of FMDV or the escape of live virus from vaccine production workshop. Thus, it is urgent to develop a novel FMDV vaccine that is safer, more effective and more economical than traditional vaccines. METHODOLOGY AND PRINCIPAL FINDINGS: A recombinant silkworm baculovirus Bm-P12A3C which contained the intact P1-2A and 3C protease coding regions of FMDV Asia 1/HNK/CHA/05 was developed. Indirect immunofluorescence test and sandwich-ELISA were used to verify that Bm-P12A3C could express the target cassette. Expression products from silkworm were diluted to 30 folds and used as antigen to immunize cattle. Specific antibody was induced in all vaccinated animals. After challenge with virulent homologous virus, four of the five animals were completely protected, and clinical symptoms were alleviated and delayed in the remaining one. Furthermore, a PD(50 (50% bovine protective dose test was performed to assess the bovine potency of the subunit vaccine. The result showed the subunit vaccine could achieve 6.34 PD(50 per dose. CONCLUSION: The results suggest that this strategy might be used to develop the new subunit FMDV vaccine.

  15. Isolation of a Spodoptera exigua baculovirus recombinant with retained biological activity despite a 10.6 kb deletion

    NARCIS (Netherlands)

    Dai, X.; Hajos, J.P.; Joosten, N.N.; Oers, van M.M.; IJkel, W.F.J.; Zuidema, D.; Yi, P.

    2000-01-01

    When Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) is grown in insect cell culture, defective viruses are generated. These viruses lack about 25 kbp of sequence information and are no longer infectious for insects. This makes the engineering of SeMNPV for improved insecticidal activity

  16. Expression of a male accessory gland peptide of Leptinotarsa decemlineata in insect cells infected with a recombinant baculovirus.

    NARCIS (Netherlands)

    Smid, H.M.; Schooneveld, H.; Deserno, M.L.L.G.; Put, B.; Vlak, J.M.

    1998-01-01

    The male accessory glands (MAGs) of Leptinotarsa decemlineata produce an 8kDa peptide, designated Led-MAGP, that is recognized by monoclonal antibody MAC-18. The site of synthesis, amino acid sequence and the gene encoding this peptide have been documented (). The primary structure is homologous to

  17. How baculovirus polyhedra fit square pegs into round holes to robustly package viruses.

    Science.gov (United States)

    Ji, Xiaoyun; Sutton, Geoff; Evans, Gwyndaf; Axford, Danny; Owen, Robin; Stuart, David I

    2010-01-20

    Natural protein crystals (polyhedra) armour certain viruses, allowing them to survive for years under hostile conditions. We have determined the structure of polyhedra of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), revealing a highly symmetrical covalently cross-braced robust lattice, the subunits of which possess a flexible adaptor enabling this supra-molecular assembly to specifically entrap massive baculoviruses. Inter-subunit chemical switches modulate the controlled release of virus particles in the unusual high pH environment of the target insect's gut. Surprisingly, the polyhedrin subunits are more similar to picornavirus coat proteins than to the polyhedrin of cytoplasmic polyhedrosis virus (CPV). It is, therefore, remarkable that both AcMNPV and CPV polyhedra possess identical crystal lattices and crystal symmetry. This crystalline arrangement must be particularly well suited to the functional requirements of the polyhedra and has been either preserved or re-selected during evolution. The use of flexible adaptors to generate a powerful system for packaging irregular particles is characteristic of the AcMNPV polyhedrin and may provide a vehicle to sequester a wide range of objects such as biological nano-particles.

  18. Sunlight stability and rain-fastness of formulations of Baculovirus heliothis

    International Nuclear Information System (INIS)

    Ignoffo, C.M.; Garcia, C.; Saathoff, S.G.

    1997-01-01

    Sunlight-Ultraviolet, with an activity spectrum from 290 to 400 nm, is the most destructive factor affecting the persistence of baculoviruses. Benzopurpurin (a disazo dye) and carbon provided the best protection when polyhedral inclusion bodies (PIB) of Baculovirus heliothis were exposed to an artificial spectrum simulating sunlight-UV (UV). Greater than 75% of the original PIB activity was still present after 48 h of sunlight-UV. When sprayed on soybeans and exposed to natural sunlight, only formulations with carbon provided significant protection of PIB. The half-life of formulations were PIB-only 4.9 +/- 1.4 h (mean +/- SE), PIB + polymer (pyrrolidone-based sticker) 3.3 +/- 0.6 h, PIB + polymer + benzopurpurin 3.4 +/- 0.7 h, and PIB + polymer + carbon 27.7 +/- 5.2 h. PIB of B. heliothis tenaciously adhere to soybean, Glycine max (L.) Merrill, leaflets after spraying and drying. Less than 6% of the PIB activity of nonformulated PIB was lost after a drenching, simulated rainfall. More than 97% of the original PIB activity of carbon formulations was still present on soybean leaflets after 10 h of exposure to sunlight-UV. In contrast, 20% was present for formulations without carbon

  19. Crystallization and preliminary crystallographic studies of the metalloglycoprotein esterase A4 using a baculovirus expression system

    Energy Technology Data Exchange (ETDEWEB)

    Hiraki, Toshiki [Protein Design Laboratory, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama 230-0045 (Japan); Shibayama, Naoya [Department of Physiology, Division of Biophysics, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498 (Japan); Yoon, Young-Ho [Protein Design Laboratory, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama 230-0045 (Japan); Yun, Kyung-Mook [Department of Physiology, Division of Biophysics, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498 (Japan); Hamamoto, Toshiro [Department of Biochemistry, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498 (Japan); Tame, Jeremy R. H.; Park, Sam-Yong, E-mail: park@tsurumi.yokohama-cu.ac.jp [Protein Design Laboratory, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama 230-0045 (Japan)

    2007-09-01

    Esterase A4 (EA4) is a timer protein found in diapause eggs of the silkworm Bombyx mori. The gene for this metalloglycoprotein was cloned from B. mori eggs and expressed using a baculovirus expression system in silkworm pupae. Crystals of the purified protein have been grown that diffract to beyond 2.1 Å resolution at 100 K using synchrotron radiation. Esterase A4 (EA4) is a timer protein found in diapause eggs of the silkworm Bombyx mori. The gene for this metalloglycoprotein was cloned from B. mori eggs and expressed using a baculovirus expression system in silkworm pupae. Crystals of the purified protein have been grown that diffract to beyond 2.1 Å resolution at 100 K using synchrotron radiation. The protein crystals belong to space group P2{sub 1}, with unit-cell parameters a = 47.1, b = 73.9, c = 47.4 Å, β = 104.1°. With one dimer per asymmetric unit, the crystal volume per unit protein weight (V{sub M}) is 2.3 Å{sup 3} Da{sup −1} and the solvent content is 47%.

  20. Expression of Clonorchis sinensis GIIIsPLA2 protein in baculovirus-infected insect cells and its overexpression facilitating epithelial-mesenchymal transition in Huh7 cells via AKT pathway.

    Science.gov (United States)

    Shang, Mei; Xie, Zhizhi; Tang, Zeli; He, Lei; Wang, Xiaoyun; Wang, Caiqin; Wu, Yinjuan; Li, Ye; Zhao, Lu; Lv, Zhiyue; Wu, Zhongdao; Huang, Yan; Yu, Xinbing; Li, Xuerong

    2017-04-01

    Although prior studies confirmed that group III secretory phospholipase A 2 of Clonorchis sinensis (CsGIIIsPLA 2 ) had stimulating effect on liver fibrosis by binding to LX-2 cells, large-scale expression of recombinant protein and its function in the progression of hepatoma are worth exploring. Because of high productivity and low lipopolysaccharides (LPS) in the Sf9-baculovirus expression system, we firstly used this system to express the coding region of CsGIIIsPLA 2 . The molecular weight of recombinant CsGIIIsPLA 2 protein was about 34 kDa. Further investigation showed that most of the recombinant protein presented intracellular expression in Sf9 insect cell nucleus and could be detected only into cell debris, which made the protein purification and further functional study difficult. Therefore, to study the role of CsGIIIsPLA 2 in hepatocellular carcinoma (HCC) progression, CsGIIIsPLA 2 overexpression Huh7 cell model was applied. Cell proliferation, migration, and the expression level of epithelial-mesenchymal transition (EMT)-related molecules (E-cadherin, N-cadherin, α-catenin, Vimentin, p300, Snail, and Slug) along with possible mechanism were measured. The results indicated that CsGIIIsPLA 2 overexpression not only inhibited cell proliferation and promoted migration and EMT but also enhanced the phosphorylation of AKT in HCC cells. In conclusion, this study supported that CsGIIIsPLA 2 overexpression suppressed cell proliferation and induced EMT through the AKT pathway.

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

    NARCIS (Netherlands)

    Sun, X.

    2005-01-01

    Keywords:   Helicoverpa armigera , baculovirus, genetic modification, cotton,transmission

  2. Development of an influenza virus vaccine using the baculovirus-insect cell expression system : implications for pandemic preparedness

    NARCIS (Netherlands)

    Cox, M.M.J.

    2009-01-01

    Key word

    Influenza, rHA, vaccine, baculovirus, insect cells, production, pandemic preparedness

    Influenza (or flu) is a highly contagious, acute viral respiratory disease that occurs seasonally in most parts of the world and is caused by influenza viruses. Influenza

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

    NARCIS (Netherlands)

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

    2006-01-01

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

  4. Expression of Na,K-ATPase and H,K-ATPase Isoforms with the Baculovirus Expression System

    NARCIS (Netherlands)

    Koenderink, J.B.; Swarts, H.G.

    2016-01-01

    P-type ATPases can be expressed in several cell systems. The baculovirus expressions system uses an insect virus to enter and express proteins in Sf9 insect cells. This expression system is a lytic system in which the cells will die a few days after viral infection. Subsequently, the expressed

  5. The role of baculovirus apoptotic suppressors in AcMNPV-mediated translation arrest in Ld652Y cells

    International Nuclear Information System (INIS)

    Thiem, Suzanne M.; Chejanovsky, Nor

    2004-01-01

    Infecting the insect cell line IPLB-Ld652Y with the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) results in global translation arrest, which correlates with the presence of the AcMNPV apoptotic suppressor, p35. In this study, we investigated the role of apoptotic suppression on AcMNPV-induced translation arrest. Infecting cells with AcMNPV bearing nonfunctional mutant p35 did not result in global translation arrest. In contrast, global translation arrest was observed in cells infected with AcMNPV in which p35 was replaced with Opiap, Cpiap, or p49, baculovirus apoptotic suppressors that block apoptosis by different mechanisms than p35. These results indicated that suppressing apoptosis triggered translation arrest in AcMNPV-infected Ld652Y cells. Experiments using the DNA synthesis inhibitor aphidicolin and temperature shift experiments, using the AcMNPV replication mutants ts8 and ts8Δp35, indicated that translation arrest initiated during the early phase of infection, but events during the late phase were required for global translation arrest. Peptide caspase inhibitors could not substitute for baculovirus apoptotic suppressors to induce translation arrest in Ld652Y cells infected with a p35-null virus. However, if the p35-null-AcMNPV also carried hrf-1, a novel baculovirus host range gene, progeny virus was produced and treatment with peptide caspase inhibitors enhanced translation of a late viral gene transcript. Together, these results indicate that translation arrest in AcMNPV-infected Ld652Y cells is due to the anti-apoptotic function of p35, but suggests that rather than simply preventing caspase activation, its activity enhances signaling to a separate translation arrest pathway, possibly by stimulating the late stages of the baculovirus infection cycle

  6. Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning.

    Science.gov (United States)

    Tung, Hsuan; Wei, Sung-Chan; Lo, Huei-Ru; Chao, Yu-Chan

    2016-01-01

    Baculoviruses have gained popularity as pest control agents and for protein production in insect systems. These viruses are also becoming popular for gene expression, tissue engineering and gene therapy in mammalian systems. Baculovirus infection triggers a heat shock response, and this response is crucial for its successful infection of host insect cells. However, the viral protein(s) or factor(s) that trigger this response are not yet clear. Previously, we revealed that IE2-an early gene product of the baculovirus-could form unique nuclear bodies for the strong trans-activation of various promoters in mammalian cells. Here, we purified IE2 nuclear bodies from Vero E6 cells and investigated the associated proteins by using mass spectrometry. Heat shock proteins (HSPs) were found to be one of the major IE2-associated proteins. Our experiments show that HSPs are greatly induced by IE2 and are crucial for the trans-activation function of IE2. Interestingly, blocking both heat shock protein expression and the proteasome pathway preserved the IE2 protein and its nuclear body structure, and revived its function. These observations reveal that HSPs do not function directly to assist the formation of the nuclear body structure, but may rather protect IE2 from proteasome degradation. Aside from functional studies in mammalian cells, we also show that HSPs were stimulated and required to determine IE2 protein levels, in insect cells infected with baculovirus. Upon inhibiting the expression of heat shock proteins, baculovirus IE2 was substantially suppressed, resulting in a significantly suppressed viral titer. Thus, we demonstrate a unique feature in that IE2 can function in both insect and non-host mammalian cells to stimulate HSPs, which may be associated with IE2 stabilization and lead to the protection of the its strong gene activation function in mammalian cells. On the other hand, during viral infection in insect cells, IE2 could also strongly stimulate HSPs and

  7. Immune responses to baculovirus-displayed enterovirus 71 VP1 antigen.

    Science.gov (United States)

    Kiener, Tanja K; Premanand, Balraj; Kwang, Jimmy

    2013-04-01

    The increased distribution and neurovirulence of enterovirus 71 is an important health threat for young children in Asia Pacific. Vaccine design has concentrated on inactivated virus with the most advanced undergoing Phase III clinical trials. By using a subunit vaccine approach, production costs could be reduced by lowering the need for biocontainment. In addition, novel mutations could be rapidly incorporated to reflect the emergence of new enterovirus 71 subgenogroups. To circumvent the problems associated with conventional subunit vaccines, the antigen can be displayed on a viral vector that conveys stability and facilitates purification. Additional advantages of viral-vectored subunit vaccines are their ability to stimulate the innate immune system by transducing cells and the possibility of oral or nasal delivery, which dispenses with the need for syringes and medical personnel. Baculovirus-displayed VP1 combines all these benefits with protection that is as efficient as inactivated virus.

  8. Assembly of recombinant Israeli Acute Paralysis Virus capsids.

    Directory of Open Access Journals (Sweden)

    Junyuan Ren

    Full Text Available The dicistrovirus Israeli Acute Paralysis Virus (IAPV has been implicated in the worldwide decline of honey bees. Studies of IAPV and many other bee viruses in pure culture are restricted by available isolates and permissive cell culture. Here we show that coupling the IAPV major structural precursor protein ORF2 to its cognate 3C-like processing enzyme results in processing of the precursor to the individual structural proteins in a number of insect cell lines following expression by a recombinant baculovirus. The efficiency of expression is influenced by the level of IAPV 3C protein and moderation of its activity is required for optimal expression. The mature IAPV structural proteins assembled into empty capsids that migrated as particles on sucrose velocity gradients and showed typical dicistrovirus like morphology when examined by electron microscopy. Monoclonal antibodies raised to recombinant capsids were configured into a diagnostic test specific for the presence of IAPV. Recombinant capsids for each of the many bee viruses within the picornavirus family may provide virus specific reagents for the on-going investigation of the causes of honeybee loss.

  9. Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells.

    Science.gov (United States)

    Fabrick, Jeffrey A; Hull, J Joe

    2017-04-20

    Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

  10. An eight-year epidemiologic study based on baculovirus-expressed type-specific spike proteins for the differentiation of type I and II feline coronavirus infections

    Science.gov (United States)

    2014-01-01

    Background Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV). FCoVs are divided into two serotypes with markedly different infection rates among cat populations around the world. A baculovirus-expressed type-specific domain of the spike proteins of FCoV was used to survey the infection of the two viruses over the past eight years in Taiwan. Results An immunofluorescence assay based on cells infected with the recombinant viruses that was capable of distinguishing between the two types of viral infection was established. A total of 833 cases from a teaching hospital was surveyed for prevalence of different FCoV infections. Infection of the type I FCoV was dominant, with a seropositive rate of 70.4%, whereas 3.5% of cats were infected with the type II FCoV. In most cases, results derived from serotyping and genotyping were highly agreeable. However, 16.7% (4/24) FIP cats and 9.8% (6/61) clinically healthy cats were found to possess antibodies against both viruses. Moreover, most of the cats (84.6%, 22/26) infected with a genotypic untypable virus bearing a type I FCoV antibody. Conclusion A relatively simple serotyping method to distinguish between two types of FCoV infection was developed. Based on this method, two types of FCoV infection in Taiwan was first carried out. Type I FCoV was found to be predominant compared with type II virus. Results derived from serotyping and genotyping support our current understanding of evolution of disease-related FCoV and transmission of FIP. PMID:25123112

  11. Characterization of recombinant human HBP/CAP37/azurocidin, a pleiotropic mediator of inflammation-enhancing LPS-induced cytokine release from monocytes.

    Science.gov (United States)

    Rasmussen, P B; Bjørn, S; Hastrup, S; Nielsen, P F; Norris, K; Thim, L; Wiberg, F C; Flodgaard, H

    1996-07-15

    Neutrophil-derived heparin-binding protein (HBP) is a strong chemoattractant for monocytes. We report here for the first time the expression of recombinant HBP. A baculovirus containing the human HBP cDNA mediated in insect cells the secretion of a 7-residue N-terminally extended HBP form (pro-HBP). Deletion of the pro-peptide-encoding cDNA sequence resulted in correctly processed HBP at the N-terminus. Electrospray mass spectrum analysis of recombinant HBP yielded a molecular weight of 27.237 +/- 3 amu. Consistent with this mass is a HBP form of 225 amino acids (mature part +3 amino acid C-terminal extension). The biological activity of recombinant HBP was confirmed by its chemotactic action towards monocytes. Furthermore, we have shown that recombinant HBP stimulates in a dose-dependent manner the lipopolysaccharide (LPS)-induced cytokine release from human monocytes.

  12. Baculovirus p35 gene is oppositely regulated by P53 and AP-1 like factors in Spodoptera frugiperda

    International Nuclear Information System (INIS)

    Mohareer, Krishnaveni; Sahdev, Sudhir; Hasnain, Seyed E.

    2011-01-01

    Highlights: ► Baculovirus p35 is regulated by both viral and host factors. ► Baculovirus p35 is negatively regulated by SfP53-like factor. ► Baculovirus p35 is positively regulated by SfAP-1-like factor. -- Abstract: Baculovirus p35 belongs to the early class of genes of AcMNPV and requires viral factors like Immediate Early protein-1 for its transcription. To investigate the role of host factors in regulating p35 gene expression, the putative transcription factor binding sites were examined in silico and the role of these factors in influencing the transcription of p35 gene was assessed. We focused our studies on AP-1 and P53-like factors, which are activated under oxidative stress conditions. The AP-1 motif is located at −1401 while P53 motif is at −1912 relative to p35 translation start site. The predicted AP-1 and P53 elements formed specific complexes with Spodoptera frugiperda nuclear extracts. Both AP-1 and P53 motif binding proteins were down regulated as a function of AcMNPV infection in Spodoptera cells. To address the question whether during an oxidative outburst, the p35 transcription is enhanced; we investigated the role of these oxidative stress induced host transcription factors in influencing p35 gene transcription. Reporter assays revealed that AP-1 element enhances the transcription of p35 by a factor of two. Interestingly, P53 element appears to repress the transcription of p35 gene.

  13. Baculovirus p35 gene is oppositely regulated by P53 and AP-1 like factors in Spodoptera frugiperda

    Energy Technology Data Exchange (ETDEWEB)

    Mohareer, Krishnaveni [Laboratory of Molecular and Cell Biology, Center for DNA Fingerprinting and Diagnostics, Hyderabad 500001 (India); Institute of Life Sciences, University of Hyderabad Campus, Prof. C.R. Rao Road, Gachibowli, Hyderabad 500046 (India); Sahdev, Sudhir [Laboratory of Molecular and Cell Biology, Center for DNA Fingerprinting and Diagnostics, Hyderabad 500001 (India); Ranbaxy Pharmaceuticals, Gurgaon, New Delhi (India); Hasnain, Seyed E., E-mail: seh@bioschool.iitd.ac.in [Institute of Life Sciences, University of Hyderabad Campus, Prof. C.R. Rao Road, Gachibowli, Hyderabad 500046 (India); Kusuma School of Biological Sciences, IIT Delhi, New Delhi 110016 (India); ILBS, Vasant Kunj, New Delhi (India); King Saud University, Riyadh, KSA (Saudi Arabia)

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Baculovirus p35 is regulated by both viral and host factors. Black-Right-Pointing-Pointer Baculovirus p35 is negatively regulated by SfP53-like factor. Black-Right-Pointing-Pointer Baculovirus p35 is positively regulated by SfAP-1-like factor. -- Abstract: Baculovirus p35 belongs to the early class of genes of AcMNPV and requires viral factors like Immediate Early protein-1 for its transcription. To investigate the role of host factors in regulating p35 gene expression, the putative transcription factor binding sites were examined in silico and the role of these factors in influencing the transcription of p35 gene was assessed. We focused our studies on AP-1 and P53-like factors, which are activated under oxidative stress conditions. The AP-1 motif is located at -1401 while P53 motif is at -1912 relative to p35 translation start site. The predicted AP-1 and P53 elements formed specific complexes with Spodoptera frugiperda nuclear extracts. Both AP-1 and P53 motif binding proteins were down regulated as a function of AcMNPV infection in Spodoptera cells. To address the question whether during an oxidative outburst, the p35 transcription is enhanced; we investigated the role of these oxidative stress induced host transcription factors in influencing p35 gene transcription. Reporter assays revealed that AP-1 element enhances the transcription of p35 by a factor of two. Interestingly, P53 element appears to repress the transcription of p35 gene.

  14. The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase

    International Nuclear Information System (INIS)

    Long, C.M.; Rohrmann, G.F.; Merrill, G.F.

    2009-01-01

    Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involved in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.

  15. Molecular biology of baculovirus and its use in biological control in Brazil Biologia molecular de baculovírus e seu uso no controle biológico de pragas no Brasil

    Directory of Open Access Journals (Sweden)

    Maria Elita Batista de Castro

    1999-10-01

    Full Text Available Baculoviruses are insect viruses found mainly in Lepidoptera. The family Baculoviridae is taxonomically divided in two genera, Nucleopolyhedrovirus and Granulovirus, which differ by occlusion body morphology. NPVs (Nucleopolyhedroviruses have polyhedrical inclusion bodies (PIBs containing multiple viral particles, while GVs (Granuloviruses appear to be generally single particles occluded in oval shaped occlusion bodies. During the life cycle, two different viral progenies are produced: BV (Budded Virus and PDV (Polyhedra Derived Virus, which are essential for the infectious process and virus propagation in host cells. Baculoviruses are being used for pest control and they are especially safe due to their specificity and invertebrate-restricted host range. Baculoviruses have been used as vectors for high level protein expression ofheterologous genes from prokaryotic and eukaryotic organisms. Also, recombinant DNA techniques have allowed the production of genetically modified viral insecticides. This study is a review on the taxonomy, structure, replication and molecular biology of baculoviruses, as well as their use as bioinsecticides in Brazil.Os baculovírus são vírus patogênicos a insetos, encontrados principalmente na ordem Lepidoptera. A família Baculoviridae é taxonomicamente dividida em dois gêneros: Nucleopolyhedrovirus e Granulovirus, que diferem pela morfologia do corpo de oclusão. Os nucleopoliedrovírus (NPV possuem corpos de inclusão poliédrica (PIB, contendo múltiplas partículas virais, enquanto os granulovírus (GV contêm, em geral, partículas únicas, ocluídas em corpos protéicos de forma ovóide. Durante o ciclo de vida são produzidos dois tipos de progênies virais: BV ("Budded Virus" e PDV ("Polyhedra Derived Virus", que são essenciais para o processo de infecção e propagação do vírus. Os baculovírus têm sido empregados no controle de pragas e, por serem específicos e restritos a invertebrados, s

  16. Production of recombinant Bombyx mori nucleopolyhedrovirus in silkworm by intrahaemocoelic injection with invasive diaminopimelate auxotrophic Escherichia coli containing BmNPV-Bacmid.

    Science.gov (United States)

    Sun, Jingchen; Yao, Lunguang; Yao, Ning; Xu, Hua; Jin, Pengfei; Kan, Yunchao

    2010-12-01

    The present study elaborates a cost-effective and transfectant-free method for generating recombinant Bombyx mori (silkworm) nucleopolyhedrovirus in silkworm larvae and pupae by injecting invasive Escherichia coli carrying BmBacmid [BmNPV (B. mori nucleopolyhedrovirus)-Bacmid] into larval haemocoel. Up to 109 PFU (plaque-forming units)/ml of infective recombinant baculovirus was generated in the silkworm by intrahaemocoelic injection with 106 DAP (diaminopimelic acid) auxotrophic and BmBacmid containing E. coli cells expressing both invasin and listeriolysin. Thus 1 ml of overnight culture of E. coli is sufficient to inject more than 2000 larvae, while DAP costing up to $1 is enough to inject about 4000 larvae. Recombinant proteins can be controlled to be expressed mainly in pupae by adjusting the injection dose, too. In this new method, many original manipulations have been eliminated, including BmBacmid preparation and the subsequent complex transfection procedures. Hence it is a time- and cost-saving means for large-scale injection of B. mori for recombinant baculovirus production in comparison with the traditional transfection methods, which may play an important role in the industrial development of the BmNPV-silkworm bioreactor.

  17. Pharmacology of Recombinant Adeno-associated Virus Production

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    Magalie Penaud-Budloo

    2018-03-01

    Full Text Available Recombinant adeno-associated viral (rAAV vectors have been used in more than 150 clinical trials with a good safety profile and significant clinical benefit in many genetic diseases. In addition, due to their ability to infect non-dividing and dividing cells and to serve as efficient substrate for homologous recombination, rAAVs are being used as a tool for gene-editing approaches. However, manufacturing of these vectors at high quantities and fulfilling current good manufacturing practices (GMP is still a challenge, and several technological platforms are competing for this niche. Herein, we will describe the most commonly used upstream methods to produce rAAVs, paying particular attention to the starting materials (input used in each platform and which related impurities can be expected in final products (output. The most commonly found impurities in rAAV stocks include defective particles (i.e., AAV capsids that do contain the therapeutic gene or are not infectious, residual proteins from host cells and helper viruses (adenovirus, herpes simplex virus, or baculoviruses, and illegitimate DNA from plasmids, cells, or helper viruses that may be encapsidated into rAAV particles. Given the role that impurities may play in immunotoxicity, this article reviews the impurities inherently associated with each manufacturing platform.

  18. Characterization of a baculovirus nuclear localization signal domain in the late expression factor 3 protein

    International Nuclear Information System (INIS)

    Au, Victoria; Yu Mei; Carstens, Eric B.

    2009-01-01

    The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) single-stranded DNA binding protein LEF-3 is a multi-functional protein that is required to transport the helicase protein P143 into the nucleus of infected cells where they function to replicate viral DNA. The N-terminal 56 amino acid region of LEF-3 is required for nuclear transport. In this report, we analyzed the effect of site-specific mutagenesis of LEF-3 on its intracellular distribution. Fluorescence microscopy of expression plasmid-transfected cells demonstrated that the residues 28 to 32 formed the core nuclear localization signal, but other adjacent positively-charged residues augmented these sequences. Comparison with other group I Alphabaculoviruses suggested that this core region functionally duplicated residues including 18 and 19. This was demonstrated by the loss of nuclear localization when the equivalent residues (18 to 20) in Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) LEF-3 were mutated. The AcMNPV LEF-3 nuclear localization domain was also shown to drive nuclear transport in mammalian cells indicating that the protein nuclear import systems in insect and mammalian cells are conserved. We also demonstrated by mutagenesis that two conserved cysteine residues located at 82 and 106 were not essential for nuclear localization or for interaction with P143. However, by using a modified construct of P143 that localized on its own to the nucleus, we demonstrated that a functional nuclear localization domain on LEF-3 was required for interaction between LEF-3 and P143

  19. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  20. Photoionization and Recombination

    Science.gov (United States)

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  1. A novel recombinant virus-like particle vaccine for prevention of porcine parvovirus-induced reproductive failure.

    Science.gov (United States)

    Antonis, Adriaan F G; Bruschke, Christianne J M; Rueda, Paloma; Maranga, Luis; Casal, J Ignacio; Vela, Carmen; Hilgers, Luuk A Th; Belt, Peter B G M; Weerdmeester, Klaas; Carrondo, Manuel J T; Langeveld, Jan P M

    2006-06-29

    A novel vaccine against porcine parvovirus (PPV), composed of recombinant virus-like particles (PPV-VLPs) produced with the baculovirus expression vector system (BEVS) at industrial scale, was tested for its immunogenicity and protective potency. A formulation of submicrogram amounts of PPV-VLPs in a water-in-mineral oil adjuvant evoked high serum antibody titres in both guinea pigs, used as reference model, and target species, pigs. A single immunisation with 0.7microg of this antigen yielded complete foetal protection against PPV infection after challenge with a virulent strain of this virus. Furthermore, also in the presence of mild adjuvants the protective action of these PPV-VLPs is excellent. This recombinant subunit vaccine overcomes some of the drawbacks of classical PPV vaccines.

  2. Aromatic/heterocyclic amino acids and the simulated sunlight-ultraviolet inactivation of the Heliothis/Helicoverpa baculovirus

    International Nuclear Information System (INIS)

    Ignoffo, C.M.; Garcia, C.

    1995-01-01

    Tryptophan, of five aromatic/heterocyclic amino acids (tyrosine, phenylalanine, proline, histidine) provided significant protection of the Heliothis baculovirus (HzSNPV) from inactivation by simulated ultraviolet (SUV). Fifty percent of SUV protection of HzSNPV with tryptophan or tyrosine was obtained at 0.03 mg/ml and 0.5 mg/ml, respectively. Rates as high as 100.0 mg/ml of phenylalanine, histidine, or proline provided <50% protection. The extent of tryptophan protection was correlated with its absorption in the sunlight UV-B spectra. 16 refs., 2 tabs

  3. Baculovirus p35 increases pancreatic β-cell resistance to apoptosis

    International Nuclear Information System (INIS)

    Hollander, Kenneth; Bar-Chen, Michal; Efrat, Shimon

    2005-01-01

    β-cells die by apoptosis in type 1 diabetes as a result of autoimmune attack mediated by cytokines, and in type 2 diabetes by various perpetrators including human islet amyloid polypeptide (hIAPP). The cascade of apoptotic events induced by cytokines and hIAPP is mediated through caspases and reactive oxygen species. The baculovirus p35 protein is a potent anti-apoptotic agent shown to be effective in a variety of species and able to inhibit a number of apoptotic pathways. Here, we aimed at determining the protective potential of p35 in β-cells exposed to cytokines and hIAPP, as well as the effects of p35 on β-cell function. The p35 gene was introduced into βTC-tet cells, a differentiated murine β-cell line capable of undergoing inducible growth-arrest. Both proliferating and growth-arrested cells expressing p35 manifested increased resistance to cytokines and hIAPP, compared with control cells, as judged by cell viability, DNA fragmentation, and caspase-3 activity assays. p35 was significantly more protective in growth-arrested, compared with proliferating, cells. No significant differences were observed in proliferation and insulin content between cells expressing p35 and control cells. In contrast, p35 manifested a perturbing effect on glucose-induced insulin secretion. These findings suggest that p35 could be incorporated as part of a multi-pronged approach of immunoprotective strategies to provide protection from recurring autoimmunity for transplanted β-cells, as well as in preventive gene therapy in type 1 diabetes. p35 may also be protective from β-cell damage caused by hIAPP in type 2 diabetes

  4. [Construction and immunogenicity of recombinant porcine parvovirus-like particles with somatostatin].

    Science.gov (United States)

    Zhang, Xuehua; Zheng, Qisheng; Chen, Jin; Xue, Gang; Hou, Hongyan; Hou, Jibo

    2010-08-01

    In order to obtain a virus-like particle vaccine both for porcine parvovirus (PPV) prevention and growth-promotion, VP2 gene of PPV NJ-a strain was amplified with PCR, and four copies of synthetic somatostatin gene were fused to the N-terminal of VP2 gene. The fused gene was cloned into pFast-HT A to construct the recombinant plasmid pFast-SS4-VP2, then the pFast-SS4-VP2 was transformed into DH10Bac competent cells and recombined with shuttle vector Bacmid, followed by identification with blue-white screening and PCR analysis for three cycles, and the positive recombinant was named as rBacmid-SS4-VP2. The positive Sf-9 cells were transfected with rBacmid-SS4-VP2 by Lipofectamine to produce recombinant baculovirus. When the cytopathic effect (CPE) was obvious, the transfected Sf-9 cell was harvested, and the positive recombinant virus was named as rBac-SS4-VP2. The insertion for the target gene into baculovirus genome was confirmed with PCR. SDS-PAGE and Western blotting revealed that the calculated protein of approximately 68 kDa was in the expressed in the insect cells. The Sf-9 cells infected with rBac-SS4-VP2 were stained positive against PPV antibody using the indirect immunofluorescence assay (IFA). Moreover, the virus particle self-assembly was observed under electron microscopy. 90 four-week-old mice were immunized by the recombinant protein coupled with different adjuvants alhydrogel, IMS and oil. VP2-specific ELISA antibodies, PPV-specific neutralizing antibody, somatostatin antibody and growth hormone levels were examined to evaluate the immunogenicity of this virus like particle. Results indicated that mice groups immunized rSS4-VP2 protein with alhydrogel and IMS developed similar humoral immune response comparing with inactived PPV vaccine. Mice group immunized with rSS4-VP2 generated higher level of SS antibody and growth hormone comparing with negative control, mice receiving rSS4-VP2 with alhydrogel developed the highest antibody titre than all

  5. Reconstitution of the yeast RNA polymerase III transcription system with all recombinant factors.

    Science.gov (United States)

    Ducrot, Cécile; Lefebvre, Olivier; Landrieux, Emilie; Guirouilh-Barbat, Josée; Sentenac, André; Acker, Joel

    2006-04-28

    Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, tauA and tauB, using the baculovirus expression system. The B block-binding module, rtauB, was reconstituted with rtau138, rtau91, and rtau60 subunits. rtau131, rtau95, and rtau55 formed also a stable complex, rtauA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B'' fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system.

  6. Recombination of cluster ions

    Science.gov (United States)

    Johnsen, Rainer

    1993-01-01

    Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.

  7. Chemotherapeutic Effect of CD147 Antibody-labeled Micelles Encapsulating Doxorubicin Conjugate Targeting CD147-Expressing Carcinoma Cells.

    Science.gov (United States)

    Asakura, Tadashi; Yokoyama, Masayuki; Shiraishi, Koichi; Aoki, Katsuhiko; Ohkawa, Kiyoshi

    2018-03-01

    CD147 (basigin/emmprin) is expressed on the surface of carcinoma cells. For studying the efficacy of CD147-targeting medicine on CD147-expressing cells, we studied the effect of anti-CD147-labeled polymeric micelles (CD147ab micelles) that encapsulated a conjugate of doxorubicin with glutathione (GSH-DXR), with specific accumulation and cytotoxicity against CD147-expressing A431 human epidermoid carcinoma cells, Ishikawa human endometrial adenocarcinoma cells, and PC3 human prostate carcinoma cells. By treatment of each cell type with CD147ab micelles for 1 h, a specific accumulation of CD147ab micelles in CD147-expressing cells was observed. In addition, the cytotoxicity of GSH-DXR-encapsulated micelles against each cell type was measured by treatment of the micelles for 1 h. The cytotoxic effect of CD147ab micelles carrying GSH-DXR was 3- to 10-fold higher for these cells than that of micelles without GSH-DXR. These results suggest that GSH-DXR-encapsulated CD147ab micelles could serve as an effective drug delivery system to CD147-expressing carcinoma cells. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  8. High molecular weight chitosan derivative polymeric micelles encapsulating superparamagnetic iron oxide for tumor-targeted magnetic resonance imaging

    Directory of Open Access Journals (Sweden)

    Xiao Y

    2015-02-01

    Full Text Available Yunbin Xiao,1,* Zuan Tao Lin,2,* Yanmei Chen,1 He Wang,1 Ya Li Deng,2 D Elizabeth Le,3 Jianguo Bin,1 Meiyu Li,1 Yulin Liao,1 Yili Liu,1 Gangbiao Jiang,2 Jianping Bin1 1State Key Laboratory of Organ Failure Research, Division of Cardiology, Nanfang Hospital, Southern Medical University, Guangzhou, People’s Republic of China; 2Department of Pharmaceutical Engineering, South China Agricultural University, Guangzhou, People’s Republic of China; 3Cardiovascular Division, Oregon Health and Science University, Portland, OR, USA *These authors contributed equally to this work Abstract: Magnetic resonance imaging (MRI contrast agents based on chitosan derivatives have great potential for diagnosing diseases. However, stable tumor-targeted MRI contrast agents using micelles prepared from high molecular weight chitosan derivatives are seldom reported. In this study, we developed a novel tumor-targeted MRI vehicle via superparamagnetic iron oxide nanoparticles (SPIONs encapsulated in self-aggregating polymeric folate-conjugated N-palmitoyl chitosan (FAPLCS micelles. The tumor-targeting ability of FAPLCS/SPIONs was demonstrated in vitro and in vivo. The results of dynamic light scattering experiments showed that the micelles had a relatively narrow size distribution (136.60±3.90 nm and excellent stability. FAPLCS/SPIONs showed low cytotoxicity and excellent biocompatibility in cellular toxicity tests. Both in vitro and in vivo studies demonstrated that FAPLCS/SPIONs bound specifically to folate receptor-positive HeLa cells, and that FAPLCS/SPIONs accumulated predominantly in established HeLa-derived tumors in mice. The signal intensities of T2-weighted images in established HeLa-derived tumors were reduced dramatically after intravenous micelle administration. Our study indicates that FAPLCS/SPION micelles can potentially serve as safe and effective MRI contrast agents for detecting tumors that overexpress folate receptors. Keywords: superparamagnetic iron oxide, magnetic resonance imaging, polymeric micelles, folate receptors, tumor-targeted MRI, N-palmitoyl chitosan

  9. Initial preclinical safety of non-replicating human endogenous retrovirus envelope protein-coated baculovirus vector-based vaccines against human papillomavirus.

    Science.gov (United States)

    Han, Su-Eun; Kim, Mi-Gyeong; Lee, Soondong; Cho, Hee-Jeong; Byun, Youngro; Kim, Sujeong; Kim, Young Bong; Choi, Yongseok; Oh, Yu-Kyoung

    2013-12-01

    Human endogenous retrovirus (HERV) envelope protein-coated, baculovirus vector-based HPV 16 L1 (AcHERV-HPV16L1) is a non-replicating recombinant baculoviral vaccine. Here, we report an initial evaluation of the preclinical safety of AcHERV-HPV16L1 vaccine. In an acute toxicity study, a single administration of AcHERV-HPV16L1 DNA vaccine given intramuscularly (i.m.) to mice at a dose of 1 × 10(8) plaque-forming units (PFU) did not cause significant changes in body weight compared with vehicle-treated controls. It did cause a brief increase in the weights of some organs on day 15 post-treatment, but by day 30, all organ weights were not significantly different from those in the vehicle-treated control group. No hematological changes were observed on day 30 post-treatment. In a range-finding toxicity study with three doses of 1 × 10(7) , 2 × 10(7) and 5 × 10(7) PFU once daily for 5 days, the group treated with 5 × 10(7) PFU showed a transient decrease in the body weights from day 5 to day 15 post-treatment, but recovery to the levels similar to those in the vehicle-treated control group by post-treatment day 20. Organ weights were slightly higher for lymph nodes, spleen, thymus and liver after repeated dosing with 5 × 10(7) PFU on day 15, but had normalized by day 30. Moreover, repeated administration of AcHERV-HPV16L1 did not induce myosin-specific autoantibody in serum, and did not cause immune complex deposition or tissue damage at injection sites. Taken together, these results provide preliminary evidence of the preclinical safety of AcHERV-based HPV16L1 DNA vaccines in mice. Copyright © 2012 John Wiley & Sons, Ltd.

  10. Intranasal immunization of baculovirus displayed hemagglutinin confers complete protection against mouse adapted highly pathogenic H7N7 reassortant influenza virus.

    Directory of Open Access Journals (Sweden)

    Subaschandrabose Rajesh Kumar

    Full Text Available BACKGROUND: Avian influenza A H7N7 virus poses a pandemic threat to human health because of its ability for direct transmission from domestic poultry to humans and from human to human. The wide zoonotic potential of H7N7 combined with an antiviral immunity inhibition similar to pandemic 1918 H1N1 and 2009 H1N1 influenza viruses is disconcerting and increases the risk of a putative H7N7 pandemic in the future, underlining the urgent need for vaccine development against this virus. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we developed a recombinant vaccine by expressing the H7N7-HA protein on the surface of baculovirus (Bac-HA. The protective efficacy of the live Bac-HA vaccine construct was evaluated in a mouse model by challenging mice immunized intranasally (i.n. or subcutaneously (s.c. with high pathogenic mouse adapted H7N7 reassorted strain. Although s.c. injection of live Bac-HA induced higher specific IgG than i.n. immunization, the later resulted in an elevated neutralization titer. Interestingly, 100% protection from the lethal viral challenge was only observed for the mice immunized intranasally with live Bac-HA, whereas no protection was achieved in any other s.c. or i.n. immunized mice groups. In addition, we also observed higher mucosal IgA as well as increased IFN-γ and IL-4 responses in the splenocytes of the surviving mice coupled with a reduced viral titer and diminished histopathological signs in the lungs. CONCLUSION: Our results indicated that protection from high pathogenic H7N7 (NL/219/03 virus requires both mucosal and systemic immune responses in mice. The balance between Th1 and Th2 cytokines is also required for the protection against the H7N7 pathogen. Intranasal administration of live Bac-HA induced all these immune responses and protected the mice from lethal viral challenge. Therefore, live Bac-HA is an effective vaccine candidate against H7N7 viral infections.

  11. MacoNPV baculovirus midgut-specific gene expression during infection of the bertha armyworm, Mamestra configurata

    Energy Technology Data Exchange (ETDEWEB)

    Donly, B. Cameron, E-mail: Cam.Donly@agr.gc.ca [London Research and Development Centre, AAFC, London, ON (Canada); Kaplanoglu, Emine [London Research and Development Centre, AAFC, London, ON (Canada); Theilmann, David A. [Summerland Research and Development Centre, AAFC, Summerland, BC (Canada); Baldwin, Doug; Sieminska, Edyta; Hegedus, Dwayne D.; Erlandson, Martin A. [Saskatoon Research and Development Centre, AAFC, Saskatoon, SK (Canada)

    2016-12-15

    Baculoviruses have two forms, occlusion derived virus (ODV) which is responsible for primary infection in host midgut tissue and budded virus (BV), which infects all other host tissues during secondary infection. This study examined the primary infection by ODV of midgut cells of bertha armyworm Mamestra configurata fourth instar larvae and measured the expression of viral genes over a time course of infection. Both digital PCR and RNA sequencing methods showed the profile of transcription to be different from those produced by AcMNPV BV infection of in vitro cell cultures. This included having unique collections of genes expressed early, as well as much greater late gene expression of p6.9 and much reduced expression of polh and p10. These differences likely reflect characteristics unique to the critical step of in vivo midgut cell infection, and provide insights into the processes that regulate viral gene expression in different host tissues. -- Highlights: •The transcriptome of MacoNPV ODV in larval midgut was measured by RNA-seq and digital PCR. •The earliest genes expressed included fusion protein, hoar, and me53. •p6.9 was highly expressed late but polH and p10 were less so. •These patterns are unique from BV of other baculoviruses in tissue culture cells.

  12. A Role for the Anti-Viral Host Defense Mechanism in the Phylogenetic Divergence in Baculovirus Evolution.

    Directory of Open Access Journals (Sweden)

    Toshihiro Nagamine

    Full Text Available Although phylogenic analysis often suggests co-evolutionary relationships between viruses and host organisms, few examples have been reported at the microevolutionary level. Here, we show a possible example in which a species-specific anti-viral response may drive phylogenic divergence in insect virus evolution. Two baculoviruses, Autographa californica multiple nucleopolyhedrovirus (AcMNPV and Bombyx mori nucleopolyhedrovirus (BmNPV, have a high degree of DNA sequence similarity, but exhibit non-overlapping host specificity. In our study of their host-range determination, we found that BmNPV replication in B. mori cells was prevented by AcMNPV-P143 (AcP143, but not BmNPV-P143 (BmP143 or a hybrid P143 protein from a host-range expanded phenotype. This suggests that AcMNPV resistance in B. mori cells depends on AcP143 recognition and that BmNPV uses BmP143 to escapes this recognition. Based on these data, we propose an insect-baculovirus co-evolution scenario in which an ancestor of silkworms exploited an AcMNPV-resistant mechanism; AcMNPV counteracted this resistance via P143 mutations, resulting in the birth of BmNPV.

  13. Baculovirus proteins IE-1, LEF-3, and P143 interact with DNA in vivo: a formaldehyde cross-linking study

    International Nuclear Information System (INIS)

    Ito, Emma; Sahri, Daniela; Knippers, Rolf; Carstens, Eric B.

    2004-01-01

    IE-1, LEF-3, and P143 are three of six proteins encoded by Autographa californica nucleopolyhedrovirus (AcMNPV) essential for baculovirus DNA replication in transient replication assays. IE-1 is the major baculovirus immediate early transcription regulator. LEF-3 is a single-stranded DNA binding protein (SSB) and P143 is a DNA helicase protein. To investigate their interactions in vivo, we treated AcMNPV-infected Spodoptera frugiperda cells with formaldehyde and separated soluble proteins from chromatin by cell fractionation and cesium chloride equilibrium centrifugation. Up to 70% of the total LEF-3 appeared in the fraction of soluble, probably nucleoplasmic proteins, while almost all P143 and IE-1 were associated with viral chromatin in the nucleus. This suggests that LEF-3 is produced in quantities that are higher than needed for the coverage of single stranded regions that arise during viral DNA replication and is consistent with the hypothesis that LEF-3 has other functions such as the localization of P143 to the nucleus. Using a chromatin immunoprecipitation procedure, we present the first direct evidence of LEF-3, P143, and IE-1 proteins binding to closely linked sites on viral chromatin in vivo, suggesting that they may form replication complexes on viral DNA in infected cells

  14. MacoNPV baculovirus midgut-specific gene expression during infection of the bertha armyworm, Mamestra configurata

    International Nuclear Information System (INIS)

    Donly, B. Cameron; Kaplanoglu, Emine; Theilmann, David A.; Baldwin, Doug; Sieminska, Edyta; Hegedus, Dwayne D.; Erlandson, Martin A.

    2016-01-01

    Baculoviruses have two forms, occlusion derived virus (ODV) which is responsible for primary infection in host midgut tissue and budded virus (BV), which infects all other host tissues during secondary infection. This study examined the primary infection by ODV of midgut cells of bertha armyworm Mamestra configurata fourth instar larvae and measured the expression of viral genes over a time course of infection. Both digital PCR and RNA sequencing methods showed the profile of transcription to be different from those produced by AcMNPV BV infection of in vitro cell cultures. This included having unique collections of genes expressed early, as well as much greater late gene expression of p6.9 and much reduced expression of polh and p10. These differences likely reflect characteristics unique to the critical step of in vivo midgut cell infection, and provide insights into the processes that regulate viral gene expression in different host tissues. -- Highlights: •The transcriptome of MacoNPV ODV in larval midgut was measured by RNA-seq and digital PCR. •The earliest genes expressed included fusion protein, hoar, and me53. •p6.9 was highly expressed late but polH and p10 were less so. •These patterns are unique from BV of other baculoviruses in tissue culture cells.

  15. THE THEORETICAL AND PRACTICAL ASPECTS OF THE RECOMBINANT ANTIGENS FOR THE DIAGNOSIS OF BOVINE LEUKEMIA PRODUCTION

    Directory of Open Access Journals (Sweden)

    Shapovalova OV

    2016-12-01

    Full Text Available Introduction. Nowadays the problem of bovine leukemia (EBL effective diagnosis in countries where EBL is registered and the disease-free areas remains actual. The main diagnostic tests are immunodiffusion reaction (AGID and enzyme-linked immunosorbent assay (ELISA, which allow the identification of infected animals by the presence of antibodies to bovine leukemia virus (BLV both in serum and in milk samples. The effectiveness of these methods depends on the quality of diagnostic test systems used and determines by the cultural and recombinant virus antigens specificity. EBL recombinant antigens have certain advantages as they are more active and cheap. Purpose of the work. The analysis of theoretical and practical approaches in the bovine leukemia virus recombinant antigens development and its diagnostic potential evaluation. The article contains data from the literature on the recombinant antigens of bovine leukemia virus construction and use. Analysis of the literature showed that the recombinant proteins are widely used in the serological diagnosis of bovine leukemia. Numerous protocols of BLV gp51 and p24 immunodominant antigens preparation has been developed in heterologous systems (Saccharomyces cerevisiae, E. coli, vaccinia virus, baculovirus. In order to obtain recombinant antigens, the BLV provirus genome regions isolated from FLK-BLV cell culture, lymphocytes or tumor cells from naturally infected cattle are typically used. For the recombinant antigens labeled by hexahistidine or Srept II purification one-step immobilized-metal affinity chromatography IMAC and highly selective Strep-Tactin affinity chromatography methods are carried out. The end products activity and specificity are studied in the immunoblotting, ELISA and AGID diagnostic reactions. The ukrainian scientists’ publications are devoted to the clone E. coli HB101-2 transformed by the recombinant plasmid containing fully functional BLV env and gag genes nucleotide

  16. Haemonchus contortus: Characterization of the baculovirus expressed form of aminopeptidase H11

    NARCIS (Netherlands)

    Reszka, N.; Rijsewijk, F.A.M.; Zelnik, V.; Moskwa, B.; Bienkowska-Szewczyk, K.

    2007-01-01

    Recombinant form of Haemonchus contortus aminopeptidase H11, an intestinal membrane glycoprotein considered to be in its native form the most promising vaccine candidate, was produced in insect cells, characterised and tested in pilot vaccination-challenge trial on sheep. The sequence of the cloned

  17. Recombinant Cry1Ia protein is highly toxic to cotton boll weevil (Anthonomus grandis Boheman) and fall armyworm (Spodoptera frugiperda).

    Science.gov (United States)

    Martins, E S; Aguiar, R W D S; Martins, N F; Melatti, V M; Falcão, R; Gomes, A C M M; Ribeiro, B M; Monnerat, R G

    2008-05-01

    To evaluate the activity of cry1Ia gene against cotton pests, Spodoptera frugiperda and Anthonomus grandis. Had isolated and characterized a toxin gene from the Bacillus thuringiensis S1451 strain which have been previously shown to be toxic to S. frugiperda and A. grandis. The toxin gene (cry1Ia) was amplified by PCR, sequenced, and cloned into the genome of a baculovirus. The Cry1Ia protein was expressed in baculovirus infected insect cells, producing protein inclusions in infected cells. The Cry1Ia protein has used in bioassays against to S. frugiperda and A. grandis. Bioassays using the purified recombinant protein showed high toxicity to S. frugiperda and A. grandis larvae. Molecular modelling of the Cry1Ia protein translated from the DNA sequence obtained in this work, showed that this protein possibly posses a similar structure to the Cry3A protein. Ultrastructural analysis of midgut cells from A. grandis incubated with the Cry1Ia toxin, showed loss of microvilli integrity. The results indicate that the cry1Ia is a good candidate for the construction of transgenic plants resistant to these important cotton pests.

  18. Activated recombinant adenovirus proteinases

    Science.gov (United States)

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  19. Hadron correlations from recombination

    Energy Technology Data Exchange (ETDEWEB)

    Fries, Rainer J [School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455 (United States)

    2005-01-01

    Quark recombination is a successful model to describe the hadronization of a deconfined quark gluon plasma. Jet-like dihadron correlations measured at RHIC provide a challenge for this picture. We discuss how correlations between hadrons can arise from correlations between partons before hadronization. An enhancement of correlations through the recombination process, similar to the enhancement of elliptic flow is found. Hot spots from completely or partially quenched jets are a likely source of such parton correlations.

  20. Regulation of Meiotic Recombination

    Energy Technology Data Exchange (ETDEWEB)

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  1. Use of a fragment of glycoprotein G-2 produced in the baculovirus expression system for detecting herpes simplex virus type 2-specific antibodies

    NARCIS (Netherlands)

    Ikoma, M; Liljeqvist, JA; Glazenburg, KL; The, TH; Welling-Wester, S; Groen, J.

    Fragments of glycoprotein G (gG-2(281-594His)), comprising residues 281 to 594 of herpes simplex virus type 2 (HSV-2), glycoprotein G of HSV-1 (gG-1(t26-189His)), and glycoprotein D of HSV-1 (gD-1(1-313)), were expressed in the baculovirus expression system to develop an assay for the detection of

  2. Recombinational repair: workshop summary

    International Nuclear Information System (INIS)

    Howard-Flanders, P.

    1983-01-01

    Recombinational repair may or may not be synonymous with postreplication repair. Considerable progress has been made in the study of the relevant enzymes, particularly those from bacteria. In this workshop we focus on the recombination enzyme RecA protein. What structural changes take place in the protein and in DNA during repair. How does homologous pairing take place. How is ATP hydrolysis coupled to the stand exchange reaction and the formation of heteroduplx DNA. Turning to another enzyme needed for certain kinds of bacterial recombination, we will ask whether the purified recB protein and recC protein complement each other and are sufficient for exonuclease V activity. In higher cells, we would like to know whether sister exchanges, which occur in bacteria after uv irradiation, are also seen in animal cells

  3. Technology transfer and scale-up of the Flublok recombinant hemagglutinin (HA) influenza vaccine manufacturing process.

    Science.gov (United States)

    Buckland, Barry; Boulanger, Robert; Fino, Mireli; Srivastava, Indresh; Holtz, Kathy; Khramtsov, Nikolai; McPherson, Clifton; Meghrous, Jamal; Kubera, Paul; Cox, Manon M J

    2014-09-22

    Multiple different hemagglutinin (HA) protein antigens have been reproducibly manufactured at the 650L scale by Protein Sciences Corporation (PSC) based on an insect cell culture with baculovirus infection. Significantly, these HA protein antigens were produced by the same Universal Manufacturing process as described in the biological license application (BLA) for the first recombinant influenza vaccine approved by the FDA (Flublok). The technology is uniquely designed so that a change in vaccine composition can be readily accommodated from one HA protein antigen to another one. Here we present a vaccine candidate to combat the recently emerged H7N9 virus as an example starting with the genetic sequence for the required HA, creation of the baculovirus and ending with purified protein antigen (or vaccine component) at the 10L scale accomplished within 38 days under GMP conditions. The same process performance is being achieved at the 2L, 10L, 100L, 650L and 2500L scale. An illustration is given of how the technology was transferred from the benchmark 650L scale facility to a retrofitted microbial facility at the 2500L scale within 100 days which includes the time for facility engineering changes. The successful development, technology transfer and scale-up of the Flublok process has major implications for being ready to make vaccine rapidly on a worldwide scale as a defense against pandemic influenza. The technology described does not have the same vulnerability to mutations in the egg adapted strain, and resulting loss in vaccine efficacy, faced by egg based manufacture. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Process optimization of large-scale production of recombinant adeno-associated vectors using dielectric spectroscopy.

    Science.gov (United States)

    Negrete, Alejandro; Esteban, Geoffrey; Kotin, Robert M

    2007-09-01

    A well-characterized manufacturing process for the large-scale production of recombinant adeno-associated vectors (rAAV) for gene therapy applications is required to meet current and future demands for pre-clinical and clinical studies and potential commercialization. Economic considerations argue in favor of suspension culture-based production. Currently, the only feasible method for large-scale rAAV production utilizes baculovirus expression vectors and insect cells in suspension cultures. To maximize yields and achieve reproducibility between batches, online monitoring of various metabolic and physical parameters is useful for characterizing early stages of baculovirus-infected insect cells. In this study, rAAVs were produced at 40-l scale yielding ~1 x 10(15) particles. During the process, dielectric spectroscopy was performed by real time scanning in radio frequencies between 300 kHz and 10 MHz. The corresponding permittivity values were correlated with the rAAV production. Both infected and uninfected reached a maximum value; however, only infected cell cultures permittivity profile reached a second maximum value. This effect was correlated with the optimal harvest time for rAAV production. Analysis of rAAV indicated the harvesting time around 48 h post-infection (hpi), and 72 hpi produced similar quantities of biologically active rAAV. Thus, if operated continuously, the 24-h reduction in the production process of rAAV gives sufficient time for additional 18 runs a year corresponding to an extra production of ~2 x 10(16) particles. As part of large-scale optimization studies, this new finding will facilitate the bioprocessing scale-up of rAAV and other bioproducts.

  5. Parton recombination model

    International Nuclear Information System (INIS)

    Hwa, R.C.

    1978-08-01

    Low P/sub T/ meson production in hadronic collisions is described in the framework of the parton model. The recombination of quark and antiquark is suggested as the dominant mechanism in the large x region. Phenomenological evidences for the mechanism are given. The application to meson initiated reactions yields the quark distribution in mesons. 21 references

  6. Molecular design for recombinant adeno-associated virus (rAAV) vector production.

    Science.gov (United States)

    Aponte-Ubillus, Juan Jose; Barajas, Daniel; Peltier, Joseph; Bardliving, Cameron; Shamlou, Parviz; Gold, Daniel

    2018-02-01

    Recombinant adeno-associated virus (rAAV) vectors are increasingly popular tools for gene therapy applications. Their non-pathogenic status, low inflammatory potential, availability of viral serotypes with different tissue tropisms, and prospective long-lasting gene expression are important attributes that make rAAVs safe and efficient therapeutic options. Over the last three decades, several groups have engineered recombinant AAV-producing platforms, yielding high titers of transducing vector particles. Current specific productivity yields from different platforms range from 10 3 to 10 5 vector genomes (vg) per cell, and there is an ongoing effort to improve vector yields in order to satisfy high product demands required for clinical trials and future commercialization.Crucial aspects of vector production include the molecular design of the rAAV-producing host cell line along with the design of AAV genes, promoters, and regulatory elements. Appropriately, configuring and balancing the expression of these elements not only contributes toward high productivity, it also improves process robustness and product quality. In this mini-review, the rational design of rAAV-producing expression systems is discussed, with special attention to molecular strategies that contribute to high-yielding, biomanufacturing-amenable rAAV production processes. Details on molecular optimization from four rAAV expression systems are covered: adenovirus, herpesvirus, and baculovirus complementation systems, as well as a recently explored yeast expression system.

  7. 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

  8. Site directed recombination

    Science.gov (United States)

    Jurka, Jerzy W.

    1997-01-01

    Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

  9. Quantitative phosphoproteome on the silkworm (Bombyx mori) cells infected with baculovirus.

    Science.gov (United States)

    Shobahah, Jauharotus; Xue, Shengjie; Hu, Dongbing; Zhao, Cui; Wei, Ming; Quan, Yanping; Yu, Wei

    2017-06-19

    Bombyx mori has become an important model organism for many fundamental studies. Bombyx mori nucleopolyhedrovirus (BmNPV) is a significant pathogen to Bombyx mori, yet also an efficient vector for recombinant protein production. A previous study indicated that acetylation plays many vital roles in several cellular processes of Bombyx mori while global phosphorylation pattern upon BmNPV infection remains elusive. Employing tandem mass tag (TMT) labeling and phosphorylation affinity enrichment followed by high-resolution LC-MS/MS analysis and intensive bioinformatics analysis, the quantitative phosphoproteome in Bombyx mori cells infected by BmNPV at 24 hpi with an MOI of 10 was extensively examined. Totally, 6480 phosphorylation sites in 2112 protein groups were identified, among which 4764 sites in 1717 proteins were quantified. Among the quantified proteins, 81 up-regulated and 25 down-regulated sites were identified with significant criteria (the quantitative ratio above 1.3 was considered as up-regulation and below 0.77 was considered as down-regulation) and with significant p-value (p < 0.05). Some proteins of BmNPV were also hyperphosphorylated during infection, such as P6.9, 39 K, LEF-6, Ac58-like protein, Ac82-like protein and BRO-D. The phosphorylated proteins were primary involved in several specific functions, out of which, we focused on the binding activity, protein synthesis, viral replication and apoptosis through kinase activity.

  10. Characterization of recombinant Trypanosoma brucei gambiense Translationally Controlled Tumor Protein (rTbgTCTP) and its interaction with Glossina midgut bacteria.

    Science.gov (United States)

    Bossard, Géraldine; Bartoli, Manon; Fardeau, Marie-Laure; Holzmuller, Philippe; Ollivier, Bernard; Geiger, Anne

    2017-09-03

    In humans, sleeping sickness (i.e. Human African Trypanosomiasis) is caused by the protozoan parasites Trypanosoma brucei gambiense (Tbg) in West and Central Africa, and T. b. rhodesiense in East Africa. We previously showed in vitro that Tbg is able to excrete/secrete a large number of proteins, including Translationally Controlled Tumor Protein (TCTP). Moreover, the tctp gene was described previously to be expressed in Tbg-infected flies. Aside from its involvement in diverse cellular processes, we have investigated a possible alternative role within the interactions occurring between the trypanosome parasite, its tsetse fly vector, and the associated midgut bacteria. In this context, the Tbg tctp gene was synthesized and cloned into the baculovirus vector pAcGHLT-A, and the corresponding protein was produced using the baculovirus Spodoptera frugicola (strain 9) / insect cell system. The purified recombinant protein rTbgTCTP was incubated together with bacteria isolated from the gut of tsetse flies, and was shown to bind to 24 out of the 39 tested bacteria strains belonging to several genera. Furthermore, it was shown to affect the growth of the majority of these bacteria, especially when cultivated under microaerobiosis and anaerobiosis. Finally, we discuss the potential for TCTP to modulate the fly microbiome composition toward favoring trypanosome survival.

  11. Nonradiative recombination in semiconductors

    CERN Document Server

    Abakumov, VN; Yassievich, IN

    1991-01-01

    In recent years, great progress has been made in the understandingof recombination processes controlling the number of excessfree carriers in semiconductors under nonequilibrium conditions. As a result, it is now possible to give a comprehensivetheoretical description of these processes. The authors haveselected a number of experimental results which elucidate theunderlying physical problems and enable a test of theoreticalmodels. The following topics are dealt with: phenomenological theory ofrecombination, theoretical models of shallow and deep localizedstates, cascade model of carrier captu

  12. Recombination epoch revisited

    International Nuclear Information System (INIS)

    Krolik, J.H.

    1989-01-01

    Previous studies of cosmological recombination have shown that this process produces as a by-product a highly superthermal population of Ly-alpha photons which retard completion of recombination. Cosmological redshifting was thought to determine the frequency distribution of the photons, while two-photon decay of hydrogen's 2s state was thought to control their numbers. It is shown here that frequency diffusion due to photon scattering dominate the cosmological redshift in the frequency range near line center which fixes the ratio of ground state to excited state population, while incoherent scattering into the far-red damping wing effectively destroys Ly-alpha photons as a rate which is competitive with two-photon decay. The former effect tends to hold back recombination, while the latter tends to accelerate it; the net results depends on cosmological parameters, particularly the combination Omega(b) h/sq rt (2q0), where Omega(b) is the fraction of the critical density provided by baryons. 18 references

  13. Dielectronic recombination theory

    International Nuclear Information System (INIS)

    LaGattuta, K.J.

    1991-01-01

    A theory now in wide use for the calculation of dielectronic recombination cross sections (σ DR ) and rate coefficients (α DR ) was one introduced originally by Feshbach for nuclear physics applications, and then later adapted for atomic scattering problems by Hahn. In the following, we briefly review this theory in a very general form, which allows one to account for the effects of overlapping and interacting resonances, as well as continuum-continuum coupling. An extension of our notation will then also allow for the inclusion of the effects of direct radiative recombination, along with a treatment of the interference between radiative and dielectronic recombination. Other approaches to the calculation of σ DR have been described by Fano and by Seaton. We will not consider those theories here. Calculations of α DR have progressed considerably over the last 25 years, since the early work of Burgess. Advances in the reliability of theoretical predictions have also been promoted recently b a variety of direct laboratory measurements of σ DR . While the measurements of σ DR for δn ≠ 0 excitations have tended to agree very well with calculations, the case of δn = 0 has been much problematic. However, by invoking a mechanism originally proposed by Jacobs, which takes into account the effect of stray electric fields on high Rydberg states (HRS) participating in the DR process, new calculations have improved the agreement between theory and experiment for these cases. Nevertheless, certain discrepancies still remain

  14. Recombinant Collagenlike Proteins

    Science.gov (United States)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  15. Recombinant Innovation and Endogenous Transitions

    OpenAIRE

    Koen Frenken; Luis R. Izquierdo; Paolo Zeppini

    2012-01-01

    We propose a model of technological transitions based on two different types of innovations. Branching innovations refer to technological improvements along a particular path, while recombinant innovations represent fusions of multiple paths. Recombinant innovations create “short-cuts” which reduce switching costs allowing agents to escape a technological lock-in. As a result, recombinant innovations speed up technological progress allowing transitions that are impossible with only branching ...

  16. Expression of recombinant Antibodies

    Directory of Open Access Journals (Sweden)

    André eFrenzel

    2013-07-01

    Full Text Available Recombinant antibodies are highly specific detection probes in research, diagnostics and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines and transgenic plants are promising to obtain antibodies with human-like post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  17. On the relict recombination lines

    International Nuclear Information System (INIS)

    Bershtejn, I.N.; Bernshtejn, D.N.; Dubrovich, V.K.

    1977-01-01

    Accurate numerical calculation of intensities and profiles of hydrogen recombination lines of cosmological origin is made. Relie radiation distortions stipulated by recombination quantum release at the irrevocable recombination are investigated. Mean number calculation is given for guantums educing for one irrevocably-lost electron. The account is taken of the educed quantums interraction with matter. The main quantum-matter interrraction mechanisms are considered: electronic blow broadening; free-free, free-bound, bound-bound absorptions Recombination dynamics is investigated depending on hydrogen density and total density of all the matter kinds in the Universe

  18. High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization

    Directory of Open Access Journals (Sweden)

    Tanaka Shigeyasu

    2009-06-01

    Full Text Available Abstract Background Budded baculoviruses are utilized for vaccine, the production of antibody and functional analysis of transmembrane proteins. In this study, we tried to produce and purify the recombinant Bombyx mori nucleopolyhedrovirus (rBmNPV-hPRR that displayed human (prorenin receptor (hPRR connected with FLAG peptide sequence on its own surface. These particles were used for further binding analysis of hPRR to human prorenin. The rBmNPV-hPRR was produced in silkworm larvae and purified from its hemolymph using size exclusion chromatography (SEC. Results A rapid method of BmNPV titer determination in hemolymph was performed using quantitative real-time PCR (Q-PCR. A correlation coefficient of BmNPV determination between end-point dilution and Q-PCR methods was found to be 0.99. rBmNPV-hPRR bacmid-injected silkworm larvae produced recombinant baculovirus of 1.31 × 108 plaque forming unit (pfu in hemolymph, which was 2.8 × 104 times higher than transfection solution in Bm5 cells. Its purification yield by Sephacryl S-1000 SF column chromatography was 264 fold from larval hemolymph at 4 days post-injection (p.i., but 35 or 39 fold at 4.5 or 5 days p.i., respectively. Protein patterns of rBmNPV-hPRR purified at 4 and 5 days were the same and ratio of envelope proteins (76, 45 and 35 kDa to VP39, one of nucleocapsid proteins, increased at 5 days p.i. hPRR was detected in only purified rBmNPV-hPRR at 5 days p.i.. Conclusion The successful purification of rBmNPV-hPRR indicates that baculovirus production using silkworm larvae and its purification from hemolymph by Sephacryl S-1000 SF column chromatography can provide an economical approach in obtaining the purified BmNPV stocks with high titer for large-scale production of hPRR. Also, it can be utilized for further binding analysis and screening of inhibitors of hPRR.

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

    Directory of Open Access Journals (Sweden)

    Dulce María Meneses-Ruiz

    2015-01-01

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

  20. Dissociative recombination of dications

    International Nuclear Information System (INIS)

    Seiersen, K.; Heber, O.; Jensen, M.J.; Safvan, C.P.; Andersen, L. H.

    2003-01-01

    Dissociative recombination (DR) of doubly-charged positive ions has been studied at the heavy ion storage ring ASTRID. Low-energy electrons were scattered on the dication of the N 2 molecule, and the absolute cross section was measured in the energy range of 10 -4 -50 eV. From the measured cross section, a thermal rate coefficient of 5.8x10 -7 cm 3 s -1 at 300 K was extracted. Furthermore, we present new results on the CO 2+ DR rate, and a summary and comparison of measured DR rate coefficients for both the singly and doubly-charged ions of CO, CO 2 , and N 2 is presented

  1. Cell biology of mitotic recombination

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2015-01-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as w...

  2. Hadron Correlations and Parton Recombination

    Energy Technology Data Exchange (ETDEWEB)

    Fries, R.J. [School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455 (United States)]. E-mail: rjfries@comp.tamu.edu

    2007-02-15

    Parton recombination has been found to be an extremely useful model to understand hadron production at the Relativistic Heavy Ion Collider. It is particularly important to explore its connections with hard processes. This article reviews some of the aspects of the quark recombination model and places particular emphasis on hadron correlations.

  3. Molecular characterization of calreticulin from Anopheles stephensi midgut cells and functional assay of the recombinant calreticulin with Plasmodium berghei ookinetes.

    Science.gov (United States)

    Borhani Dizaji, Nahid; Basseri, Hamid Reza; Naddaf, Saied Reza; Heidari, Mansour

    2014-10-25

    Transmission blocking vaccines (TBVs) that target the antigens on the midgut epithelium of Anopheles mosquitoes are among the promising tools for the elimination of the malaria parasite. Characterization and analysis of effective antigens is the first step to design TBVs. Calreticulin (CRT), a lectin-like protein, from Anopheles albimanus midgut, has shown antigenic features, suggesting a promising and novel TBV target. CRT is a highly conserved protein with similar features in vertebrates and invertebrates including anopheline. We cloned the full-length crt gene from malaria vector, Anopheles stephensi (AsCrt) and explored the interaction of recombinant AsCrt protein, expressed in a prokaryotic system (pGEX-6p-1), with surface proteins of Plasmodium berghei ookinetes by immunofluorescence assay. The cellular localization of AsCrt was determined using the baculovirus expression system. Sequence analysis of the whole cDNA of AsCrt revealed that AsCrt contains an ORF of 1221 bp. The amino acid sequence of AsCrt protein obtained in this study showed 64% homology with similar protein in human. The AsCrt shares the most common features of CRTs from other species. This gene encodes a 406 amino-acid protein with a molecular mass of 46 kDa, which contains a predicted 16 amino-acid signal peptides, conserved cysteine residues, a proline-rich region, and highly acidic C-terminal domain with endoplasmic reticulum retrieval sequence HDEL. The production of GST-AsCrt recombinant protein was confirmed by Western blot analysis using an antibody against the GST protein. The FITC-labeled GST-AsCrt exhibited a significant interaction with P. berghei ookinete surface proteins. Purified recombinant GST-AsCrt, labeled with FITC, displayed specific binding to the surface of P. berghei ookinetes in comparison with control. Moreover, the expression of AsCrt in baculovirus expression system indicated that AsCrt was localized on the surface of Sf9 cells. Our results suggest that AsCrt could

  4. Auger recombination in sodium iodide

    Science.gov (United States)

    McAllister, Andrew; Kioupakis, Emmanouil; Åberg, Daniel; Schleife, André

    2014-03-01

    Scintillators are an important tool used to detect high energy radiation - both in the interest of national security and in medicine. However, scintillator detectors currently suffer from lower energy resolutions than expected from basic counting statistics. This has been attributed to non-proportional light yield compared to incoming radiation, but the specific mechanism for this non-proportionality has not been identified. Auger recombination is a non-radiative process that could be contributing to the non-proportionality of scintillating materials. Auger recombination comes in two types - direct and phonon-assisted. We have used first-principles calculations to study Auger recombination in sodium iodide, a well characterized scintillating material. Our findings indicate that phonon-assisted Auger recombination is stronger in sodium iodide than direct Auger recombination. Computational resources provided by LLNL and NERSC. Funding provided by NA-22.

  5. Introduction of the anti-apoptotic baculovirus p35 gene in passion fruit induces herbicide tolerance, reduced bacterial lesions, but does not inhibits passion fruit woodiness disease progress induced by cowpea aphid-borne mosaic virus (CABMV

    NARCIS (Netherlands)

    Freitas, D.S.; Coelho, M.C.F.; Souza, M.T.; Marques, A.; Ribeiro, B.M.

    2007-01-01

    The introduction of anti-apoptotic genes into plants leads to resistance to environmental stress and broad-spectrum disease resistance. The anti-apoptotic gene (p35) from a baculovirus was introduced into the genome of passion fruit plants by biobalistics. Eleven regenerated plants showed the

  6. Virus-Like Particles of Chimeric Recombinant Porcine Circovirus Type 2 as Antigen Vehicle Carrying Foreign Epitopes

    Directory of Open Access Journals (Sweden)

    Huawei Zhang

    2014-12-01

    Full Text Available Virus-like particles (VLPs of chimeric porcine circovirus type 2 (PCV2 were generated by replacing the nuclear localization signal (NLS; at 1–39 aa of PCV2 capsid protein (Cap with classical swine fever virus (CSFV T-cell epitope (1446–1460 aa, CSFV B-cell epitope (693–716 aa and CSFV T-cell epitope conjugated with B-cell epitope. The recombinant proteins were expressed using the baculovirus expression system and detected by immunoblotting and indirect immunofluorescence assay. The abilities to form PCV2 VLPs were confirmed by transmission electron microscopy. Immunogenicities of the three recombinant proteins were evaluated in mice. Our Results indicated that Cap protein NLS deletion or substitution with CSFV epitopes did not affect the VLPs assembly. Three chimeric Cap proteins could form VLPs and induce efficient humoral and cellular immunity against PCV2 and CSFV in mice. Results show that PCV2 VLPs can be used as an efficient antigen carrier for delivery of foreign epitopes, and a potential novel vaccine.

  7. Recombinant EPF/chaperonin 10 promotes the survival of O4-positive pro-oligodendrocytes prepared from neonatal rat brain.

    Science.gov (United States)

    McCombe, P A

    2008-12-01

    Chaperonin 10 (cpn 10) is a small heat-shock protein that is usually intracellular. Early pregnancy factor (EPF), a biologically active protein that was first described in the serum of pregnant mammals, is homologous to cpn 10. EPF/cpn 10 has been reported to have effects on immunomodulation and cell survival and to inhibit activation of toll-like receptors by lipopolysaccharide. We found that recombinant EPF/cpn 10 was able to suppress experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, which is a disease causing inflammation and demyelination of the brain and spinal cord. This beneficial effect could be due to anti-inflammatory and/or cell survival properties of EPF/cpn 10. We aimed to assess the effects of cpn 10 on cells of the oligodendrocyte lineage because oligodendrocytes are the brain cells that produce myelin and that are depleted in multiple sclerosis. Two forms of recombinant EPF/cpn 10 were prepared in the pGEX expression system and in the baculovirus expression system. Purified O4(+) pro-oligodendrocytes were prepared from the brains of day-old Wistar rats and isolated by cell sorting with flow cytometry. Single cells were dispensed into micro-well plates and tested for survival in the presence of a range of concentrations of the two forms of cpn 10. We also studied the effects of bFGF, PDGF, IGF-1 and insulin as controls. With cpn 10 present, there was enhanced survival of O4(+) cells.

  8. Analysis of antigenic cross-reactivity between subgroup C avian pneumovirus and human metapneumovirus by using recombinant fusion proteins.

    Science.gov (United States)

    Luo, L; Sabara, M I; Li, Y

    2009-10-01

    Avian pneumovirus subgroup C (APV/C) has recently been reported to be more closely related to human metapneumovirus (hMPV) as determined by sequence analysis. To examine the antigenic relationship between APV/C and hMPV, the APV/C fusion (F) gene was cloned and expressed as an uncleaved glycoprotein in a baculovirus system. The reactivity of the APV/C F protein with antibodies against APV subgroups A, B, C, and hMPV was examined by Western blot analysis. The results showed that the expressed APV/C F protein was not only recognized by APV/C-specific antibodies but also by antibodies raised against hMPV. Previously expressed recombinant hMPV F protein also reacted with APV/C-specific antibodies, suggesting that there was significant antigenic cross-reactivity and a potential evolutionary relationship between hMPV and APV/C. Interestingly, the recombinant F proteins from APV/C and hMPV were not recognized by polyclonal antibodies specific to APV subgroups A and B.

  9. Recombinant Rhipicephalus appendiculatus gut (Ra86 and salivary gland cement (Trp64 proteins as candidate antigens for inclusion in tick vaccines: protective effects of Ra86 on infestation with adult R. appendiculatus

    Directory of Open Access Journals (Sweden)

    Saimo M

    2011-11-01

    Full Text Available Margaret Saimo1,2,*, David O Odongo3,4,*, Stephen Mwaura3, Just M Vlak1, Anthony J Musoke5, George W Lubega2, Richard P Bishop3, Monique M van Oers11Laboratory of Virology, Wageningen University, Wageningen, The Netherlands; 2School of Veterinary Medicine, Makerere University, Kampala, Uganda; 3International Livestock Research Institute, Nairobi, Kenya; 4School of Biological Sciences, University of Nairobi, Nairobi, Kenya; 5Onderstepoort Veterinary Institute, Onderstepoort, Pretoria, South Africa *These two authors made an equal contribution to this workAbstract: Rhipicephalus appendiculatus gut protein Ra86 (variants Ra85A and Ra92A and the salivary gland cement protein (Trp64 were expressed in the baculovirus-insect cell system. The recombinant gut proteins expressed as soluble proteins and the recombinant cement protein, as insoluble inclusion bodies, were used to immunize rabbits, which were then challenged with larval, nymphal, and adult stages of R. appendiculatus ticks. High tick mortality (23.3% occurred on adult ticks that fed on rabbits vaccinated with the gut proteins, compared with 1.9% mortality in ticks that fed on unvaccinated naïve control rabbits. The mean weight of engorged female ticks was significantly reduced by 31.5% in rabbits vaccinated with the Ra86 recombinant protein compared with controls, as was egg production. Marked effects on these parameters were also observed in adult ticks as a result from vaccination using Trp64, but these were not statistically significant. For both antigens, there was no demonstrable effect on larval or nymphal ticks. This study demonstrates for the first time the protective efficacy of a homolog of Boophilus microplus Bm86 in reducing tick infestation by the adult stage of the three-host tick R. appendiculatus. The results demonstrate the potential of Ra86 for vaccine development against this tick and for the control of East Coast fever.Keywords: baculovirus, Ra85A, Ra92A, Boophilus

  10. Oxygen-hydrogen recombination system

    International Nuclear Information System (INIS)

    Sato, Shuichiro; Takejima, Masaki.

    1981-01-01

    Purpose: To avoid reduction in the performance of catalyst used for an oxygen-hydrogen recombiner in the off gas processing system of a nuclear reactor. Constitution: A thermometer is provided for the detection of temperature in an oxygen-hydrogen recombiner. A cooling pipe is provided in the recombiner and cooling medium is introduced externally. The cooling medium may be water or air. In accordance with the detection value from the thermometer, ON-OFF control is carried out for a valve to control the flow rate of the cooling medium thereby rendering the temperature in the recombiner to a predetermined value. This can prevent the catalyst from being exposed to high temperature and avoid the reduction in the performance of the catalyst. (Ikeda, J.)

  11. Controlled Release from Recombinant Polymers

    Science.gov (United States)

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-01-01

    Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed. PMID:24956486

  12. Hydrogen recombiner development at AECL

    International Nuclear Information System (INIS)

    Dewit, W.A.; Koroll, G.W.; Loesel Sitar, J.; Graham, W.R.C.

    1997-01-01

    Catalytic recombiners have been developed at AECL for the purpose of hydrogen removal in post-accident nuclear containment buildings. The recombiners are based on a particular catalyst designed by AECL which has extraordinary resistance to fouling from water and water vapour and a large thermodynamic range of operation. The catalysts were developed, originally, for the purpose of heavy water manufacturing by way of a catalytic exchange process. Application of these catalyst materials in recombiners for containment applications began in the late 1980's. The first application was a passive recombiner, qualified for use in control of radiolytic hydrogen in the headspace of a pool-type experimental reactor of AECL design in 1988. The passive, or natural convection recombiner concept has continued development to commercial stage for application in power reactor containments. This paper reviews the AECL recombiner development, describes the current model and shows results from tests of full-scale recombiners in the Large Scale Vented Combustion Test Facility at AECL-WL. The AECL recombiner is designed for compactness and ease of engineering into containment. The design is a simple, open-ended rectangular enclosure with catalyst elements arranged inside to promote optimum convective flow driven by heat of recombination at the catalyst surface. Self start, as evidenced by catalyst heating and initiation of flow, is achieved in less than 1% hydrogen, with available oxygen, at room temperature and 100% relative humidity. This low temperature start-up in condensing atmospheres is viewed as the most challenging condition for wet-proofing effectiveness. Cold start-up is a vital performance requirement in containments, such as CANDU, where engineered air-cooling systems are operating and where long-term hydrogen control is required, after containment atmospheres have cooled. Once started, the removal capacity scales linearly with the inlet cross-section area and the partial

  13. Review of Parton Recombination Models

    International Nuclear Information System (INIS)

    Bass, Steffen A

    2006-01-01

    Parton recombination models have been very successful in explaining data taken at RHIC on hadron spectra and emission patterns in Au+Au collisions at transverse momenta above 2 GeV/c, which have exhibited features which could not be understood in the framework of basic perturbative QCD. In this article I will review the current status on recombination models and outline which future challenges need to be addressed by this class of models

  14. Recombinant snake venom prothrombin activators

    OpenAIRE

    L?vgren, Ann

    2012-01-01

    Three prothrombin activators; ecarin, which was originally isolated from the venom of the saw-scaled viper Echis carinatus, trocarin from the rough-scaled snake Tropidechis carinatus, and oscutarin from the Taipan snake Oxyuranus scutellatus, were expressed in mammalian cells with the purpose to obtain recombinant prothrombin activators that could be used to convert prothrombin to thrombin. We have previously reported that recombinant ecarin can efficiently generate thrombin without the need ...

  15. Delayed recombination and cosmic parameters

    International Nuclear Information System (INIS)

    Galli, Silvia; Melchiorri, Alessandro; Bean, Rachel; Silk, Joseph

    2008-01-01

    Current cosmological constraints from cosmic microwave background anisotropies are typically derived assuming a standard recombination scheme, however additional resonance and ionizing radiation sources can delay recombination, altering the cosmic ionization history and the cosmological inferences drawn from the cosmic microwave background data. We show that for recent observations of the cosmic microwave background anisotropy, from the Wilkinson microwave anisotropy probe satellite mission (WMAP) 5-year survey and from the arcminute cosmology bolometer array receiver experiment, additional resonance radiation is nearly degenerate with variations in the spectral index, n s , and has a marked effect on uncertainties in constraints on the Hubble constant, age of the universe, curvature and the upper bound on the neutrino mass. When a modified recombination scheme is considered, the redshift of recombination is constrained to z * =1078±11, with uncertainties in the measurement weaker by 1 order of magnitude than those obtained under the assumption of standard recombination while constraints on the shift parameter are shifted by 1σ to R=1.734±0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: ε α i <0.058 at 95% c.l.. Although delayed recombination limits the precision of parameter estimation from the WMAP satellite, we demonstrate that this should not be the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.

  16. Near infrared imaging-guided photodynamic therapy under an extremely low energy of light by galactose targeted amphiphilic polypeptide micelle encapsulating BODIPY-Br2.

    Science.gov (United States)

    Liu, Le; Ruan, Zheng; Li, Tuanwei; Yuan, Pan; Yan, Lifeng

    2016-10-18

    Near infrared (NIR) imaging-guided photodynamic therapy (PDT) is attractive, especially the utilization of one dye as both a photosensitizer and fluorescent probe, and the as-synthesized BODIPY-Br 2 molecule is a candidate. Here, a galactose targeted amphiphilic copolymer of a polypeptide was synthesized and its micelles work as nanocarriers for BODIPY for targeting the NIR imaging-guided PDT of hepatoma cancer cells. At the same time, BODIPY could light up the cytoplasm for real-time imaging and kill cancer cells when the light was switched on. In vitro tests performed on both HepG2 and HeLa cells confirmed that the as-prepared PMAGP-POEGMA-PLys-B micelles showed efficient cell suppression of the cells with galactose receptors in the presence of light under an extremely low energy density (6.5 J cm -2 ). This protocol highlights the potential of polypeptides as biodegradable carriers for NIR image-guided and confined targeting photodynamic therapy.

  17. PROGENITORS OF RECOMBINING SUPERNOVA REMNANTS

    Energy Technology Data Exchange (ETDEWEB)

    Moriya, Takashi J., E-mail: takashi.moriya@ipmu.jp [Kavli Institute for the Physics and Mathematics of the Universe, Todai Institutes for Advanced Study, University of Tokyo, Kashiwanoha 5-1-5, Kashiwa, Chiba 277-8583 (Japan)

    2012-05-01

    Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with ionization temperature higher than the electron temperature, has been recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the time of the supernova explosion. If the circumstellar medium is dense enough, collisional ionization equilibrium can be established in the early stage of the evolution of the supernova remnant and subsequent adiabatic cooling, which occurs after the shock wave gets out of the dense circumstellar medium, makes the electron temperature lower than the ionization temperature. We study the circumstellar medium around several supernova progenitors and show which supernova progenitors can have a circumstellar medium dense enough to establish collisional ionization equilibrium soon after the explosion. We find that the circumstellar medium around red supergiants (especially massive ones) and the circumstellar medium dense enough to make Type IIn supernovae can establish collisional ionization equilibrium soon after the explosion and can evolve to become recombining supernova remnants. Wolf-Rayet stars and white dwarfs have the possibility to be recombining supernova remnants but the fraction is expected to be very small. As the occurrence rate of the explosions of red supergiants is much higher than that of Type IIn supernovae, the major progenitors of recombining supernova remnants are likely to be red supergiants.

  18. Meiotic recombination in human oocytes.

    Directory of Open Access Journals (Sweden)

    Edith Y Cheng

    2009-09-01

    Full Text Available Studies of human trisomies indicate a remarkable relationship between abnormal meiotic recombination and subsequent nondisjunction at maternal meiosis I or II. Specifically, failure to recombine or recombination events located either too near to or too far from the centromere have been linked to the origin of human trisomies. It should be possible to identify these abnormal crossover configurations by using immunofluorescence methodology to directly examine the meiotic recombination process in the human female. Accordingly, we initiated studies of crossover-associated proteins (e.g., MLH1 in human fetal oocytes to analyze their number and distribution on nondisjunction-prone human chromosomes and, more generally, to characterize genome-wide levels of recombination in the human female. Our analyses indicate that the number of MLH1 foci is lower than predicted from genetic linkage analysis, but its localization pattern conforms to that expected for a crossover-associated protein. In studies of individual chromosomes, our observations provide evidence for the presence of "vulnerable" crossover configurations in the fetal oocyte, consistent with the idea that these are subsequently translated into nondisjunctional events in the adult oocyte.

  19. Electric hydrogen recombiner special tests

    International Nuclear Information System (INIS)

    Wilson, J.F.

    1975-12-01

    Westinghouse has produced an electric hydrogen recombiner to control hydrogen levels in reactor containments following a postulated loss-of-coolant accident. The recombiner underwent extensive testing for NRC qualification (see WCAP 7709-L and Supplements 1, 2, 3, 4). As a result, WCAP 7709-L and Supplements 1, 2, 3, and 4 have been accepted by the NRC for reference in applications not committed to IEEE-323-1974. Supplement 5 and the next supplement will demonstrate conformance to IEEE-323-1974. This supplement describes additional tests, beyond those necessary to qualify the system, which will be referenced in supplement 6. Each test has demonstrated a considerable margin of safety over required performance. Concurrently, the test results increased the fund of technical information on the electric hydrogen recombiner

  20. Production and recombination of gluons

    International Nuclear Information System (INIS)

    Temiraliev, A.T.

    2006-01-01

    Full text: Nonlinear Markov process of parton production has been considered. The Kolmogorov equation is applied for the evolution equation based on the approximation of independent gluons production in every decay act. We introduced a 'crossing' parameter and used the combination relations to obtain nonlinear recombination equation for the evolution of gluon structure function. (author)

  1. Recombinator of hydrogen and oxygen

    International Nuclear Information System (INIS)

    Stejskal, J.; Klein, O.; Scholtz, G.; Schmidt, P.; Olaussson, A.

    1976-01-01

    Improvements are proposed for the well known reactors for the catalytic recombination of hydrogen and oxygen, which should permit this being used in contiuous operation in nuclear reactors (BWRs). The improvements concern the geometric arrangement of gas-inlet and -outlet pipes, the inclination of the axis of the catalyst container and the introduction of remote operation. (UWI) [de

  2. Improving recombinant protein purification yield

    Science.gov (United States)

    Production of adequate amounts of recombinant proteins is essential for antibody production, biochemical activity study, and structural determination during the post-genomic era. It’s technologically challenging and a limiting factor for tung oil research because analytical reagents such as high qua...

  3. Recombination in hepatitis C virus.

    Science.gov (United States)

    González-Candelas, Fernando; López-Labrador, F Xavier; Bracho, María Alma

    2011-10-01

    Hepatitis C virus (HCV) is a Flavivirus with a positive-sense, single-stranded RNA genome of about 9,600 nucleotides. It is a major cause of liver disease, infecting almost 200 million people all over the world. Similarly to most RNA viruses, HCV displays very high levels of genetic diversity which have been used to differentiate six major genotypes and about 80 subtypes. Although the different genotypes and subtypes share basic biological and pathogenic features they differ in clinical outcomes, response to treatment and epidemiology. The first HCV recombinant strain, in which different genome segments derived from parentals of different genotypes, was described in St. Petersburg (Russia) in 2002. Since then, there have been only a few more than a dozen reports including descriptions of HCV recombinants at all levels: between genotypes, between subtypes of the same genotype and even between strains of the same subtype. Here, we review the literature considering the reasons underlying the difficulties for unequivocally establishing recombination in this virus along with the analytical methods necessary to do it. Finally, we analyze the potential consequences, especially in clinical practice, of HCV recombination in light of the coming new therapeutic approaches against this virus.

  4. Live recombinant BHV/BRSV vaccine

    NARCIS (Netherlands)

    Keil, G.M.; Rijsewijk, F.A.M.

    1998-01-01

    The present invention refers to synthetic Bovine Respiratory Syncytium virus genes. Also the invention relates to live attenuated Bovine Herpesvirus recombinants carrying such synthetic genes. Furthermore, the invention relates to vaccines based on these live attenuated recombinants, for the

  5. Hadron production at RHIC: recombination of quarks

    Energy Technology Data Exchange (ETDEWEB)

    Fries, Rainer J [School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455 (United States)

    2005-01-01

    We discuss quark recombination applied to the hadronization of a quark gluon plasma. It has been shown that the quark recombination model can explain essential features of hadron production measured in high energy heavy ion collisions.

  6. Affinity purification of recombinant human plasminogen activator ...

    African Journals Online (AJOL)

    Affinity purification of recombinant human plasminogen activator from ... Screening antibody was performed using rhPA milk in an ELISA-elution assay. ... useful for purifying other tPA mutants or other novel recombinant milkderived proteins.

  7. Graded Recombination Layers for Multijunction Photovoltaics

    KAUST Repository

    Koleilat, Ghada I.; Wang, Xihua; Sargent, Edward H.

    2012-01-01

    it to achieve multicolor and spectrally tunable behavior. In series-connected current-matched multijunction devices, the recombination layers must allow the hole current from one cell to recombine, with high efficiency and low voltage loss, with the electron

  8. Recombinant innovation and endogenous technological transitions

    NARCIS (Netherlands)

    Frenken, K.; Izquierdo, L.R.; Zeppini, P.

    2012-01-01

    We propose a model of technological transitions based on two different types of innovations. Branching innovations refer to technological improvements along a particular path, while recombinant innovations represent fusions of multiple paths. Recombinant innovations create "short-cuts" which reduce

  9. Baculoviral expression and characterization of human recombinant PGCP in the form of an active mature dimer and an inactive precursor protein.

    Science.gov (United States)

    Zajc, Tajana; Suban, Dejan; Rajković, Jelena; Dolenc, Iztok

    2011-02-01

    The human-blood plasma glutamate carboxypeptidase (PGCP) is a proteinase that acts on the unsubstituted N- and C-termini of dipeptides. It has been suggested that this PGCP is involved in the release of thyroxine. Furthermore, research has suggested that its activity is up-regulated in hepatitis-C-virus-infected patients with hepatocellular carcinoma. In this study expressed human PGCP in the baculovirus expression system was produced by a Sf9 insect cell line with aim to prepare sufficient amounts of active recombinant enzyme for a subsequent biological characterization. Recombinant PGCP was expressed and secreted into the medium in the form of an inactive proenzyme. It was gradually converted into an active form in the medium after three days, with the highest expression of the active form on day six. The protein was sequentially purified by a combination of various liquid chromatographies, such as hydroxyapatite, ion exchange, and gel chromatography, and as final step with affinity chromatography on Phe-Leu-Sepharose. The human PGCP was purified as an active enzyme in the dimer form and as inactive precursor protein. The dipeptidase activity was confirmed by measuring the hydrolysis of the Ser-Met dipeptide at a slightly acidic pH. Copyright © 2010 Elsevier Inc. All rights reserved.

  10. Vaccination of dogs with six different candidate leishmaniasis vaccines composed of a chimerical recombinant protein containing ribosomal and histone protein epitopes in combination with different adjuvants.

    Science.gov (United States)

    Poot, J; Janssen, L H M; van Kasteren-Westerneng, T J; van der Heijden-Liefkens, K H A; Schijns, V E J C; Heckeroth, A

    2009-07-16

    Chimerical protein "Q", composed of antigenic ribosomal and histone sequences, in combination with live BCG is a promising canine leishmaniasis vaccine candidate; one of the few vaccine candidates that have been tested successfully in dogs. Unfortunately, live BCG is not an appropriate adjuvant for commercial application due to safety problems in dogs. In order to find a safe adjuvant with similar efficacy to live BCG, muramyl dipeptide, aluminium hydroxide, Matrix C and killed Propionibacterium acnes in combination with either E. coli- or baculovirus-produced recombinant JPCM5_Q protein were tested. Groups of five or seven dogs were vaccinated with six different adjuvant-antigen combinations and challenged with a high dose intravenous injection of Leishmania infantum JPC strain promastigotes. All candidate vaccines proved to be safe, and both humoral and cellular responses to the recombinant proteins were detected at the end of the prime-boost vaccination scheme. However, clinical and parasitological data obtained during the 10 month follow-up period indicated that protection was not induced by either of the six candidate vaccines. Although no direct evidence was obtained, our data suggest that live BCG may have a significant protective effect against challenge with L. infantum in dogs.

  11. Population inversion in recombining hydrogen plasma

    International Nuclear Information System (INIS)

    Furukane, Utaro; Yokota, Toshiaki; Oda, Toshiatsu.

    1978-11-01

    The collisional-radiative model is applied to a recombining hydrogen plasma in order to investigate the plasma condition in which the population inversion between the energy levels of hydrogen can be generated. The population inversion is expected in a plasma where the three body recombination has a large contribution to the recombining processes and the effective recombination rate is beyond a certain value for a given electron density and temperature. Calculated results are presented in figures and tables. (author)

  12. Regulation of homologous recombination in eukaryotes

    OpenAIRE

    Heyer, Wolf-Dietrich; Ehmsen, Kirk T.; Liu, Jie

    2010-01-01

    Homologous recombination is required for accurate chromosome segregation during the first meiotic division and constitutes a key repair and tolerance pathway for complex DNA damage including DNA double-stranded breaks, interstrand crosslinks, and DNA gaps. In addition, recombination and replication are inextricably linked, as recombination recovers stalled and broken replication forks enabling the evolution of larger genomes/replicons. Defects in recombination lead to genomic instability and ...

  13. Expression and purification of functional JNK2beta2: perspectives on high-level production of recombinant MAP kinases.

    Science.gov (United States)

    Savopoulos, John W; Dowd, Stephen; Armour, Carolyn; Carter, Paul S; Greenwood, Catherine J; Mills, David; Powell, David; Pettman, Gary R; Jenkins, Owen; Walsh, Frank S; Philpott, Karen L

    2002-02-01

    The mitogen-activated protein (MAP) kinases are a group of serine/threonine kinases that mediate intracellular signal transduction in response to environmental stimuli including stress, growth factors, and various cytokines. Of this family, the c-Jun N-terminal kinases (JNKs) are members which, depending on cell type, have been shown to activate the transcription of genes involved in the inflammatory response, apoptosis, and hypertrophy. Here we report the use Baculovirus/Sf9 cells to produce milligram quantities of recombinant JNK2beta2 substrate which could be purified to >90% as judged by SDS-PAGE. In addition, we report a novel method for the site-specific biotinylation for this enzyme and demonstrate that the biotinylated product is an authentic substrate of the upstream kinases MKK4 and 7 and can phosphorylate a downstream target, ATF-2. We also show that the phosphorylated product can be captured efficiently on streptavidin-coated beads for use in scintillation proximity assays. Copyright 2002 Elsevier Science (USA).

  14. The effect of a single recombination event

    DEFF Research Database (Denmark)

    Schierup, Mikkel Heide; Jensen, Thomas Mailund; Wiuf, Carsten

    We investigate the variance in how visible a single recombination event is in a SNP data set as a function of the type of recombination event and its age. Data is simulated under the coalescent with recombination and inference is by the popular composite likelihood methods. The major determinant...

  15. Recombination Catalysts for Hypersonic Fuels

    Science.gov (United States)

    Chinitz, W.

    1998-01-01

    The goal of commercially-viable access to space will require technologies that reduce propulsion system weight and complexity, while extracting maximum energy from the products of combustion. This work is directed toward developing effective nozzle recombination catalysts for the supersonic and hypersonic aeropropulsion engines used to provide such access to space. Effective nozzle recombination will significantly reduce rk=le length (hence, propulsion system weight) and reduce fuel requirements, further decreasing the vehicle's gross lift-off weight. Two such catalysts have been identified in this work, barium and antimony compounds, by developing chemical kinetic reaction mechanisms for these materials and determining the engine performance enhancement for a typical flight trajectory. Significant performance improvements are indicated, using only 2% (mole or mass) of these compounds in the combustor product gas.

  16. Mechanisms of sister chromatid recombination

    International Nuclear Information System (INIS)

    Nakai, Sayaka; Machida, Isamu; Tsuji, Satsuki

    1985-01-01

    Studies using T948 as a model system have been carried out aimed at elucidating the mechanism of sister chromatid recombination (SCR). Characterization of U.V. light- and x-ray-induced SCR, the relationiship between SCR induction and DNA repair using rad mutations, and the relationship between SCR induction and the time of cell division using cdc mutations are presented. It has been supposed that SCR is induced at the phase of S-G 2 following DNA replication, that postreplication break of DNA strands is strongly involved in the induction of SCR, and that induction type of SCR, i.e., conversion type or recombination type, is dependent upon the type of molecular damage of DNA. (Namekawa, K.)

  17. Interface recombination influence on carrier transport

    International Nuclear Information System (INIS)

    Konin, A

    2013-01-01

    A theory of interface recombination in the semiconductor–semiconductor junction is developed. The interface recombination rate dependence on the nonequilibrium carrier densities is derived on the basis of a model in which the interface recombination occurs through the mechanism of trapping. The general relation between the interface recombination parameters at small carrier density deviation from the equilibrium ones is obtained. The validity of this relation is proved considering the generation of the Hall electric field in the extrinsic semiconductor sample. The anomalous Hall electromotive force in a weak magnetic field was investigated and interpreted by means of a new interface recombination model. The experimental data corroborate the developed theory. (paper)

  18. Recombinant Cyclophilins Lack Nuclease Activity

    OpenAIRE

    Manteca, Angel; Sanchez, Jesus

    2004-01-01

    Several single-domain prokaryotic and eukaryotic cyclophilins have been identified as also being unspecific nucleases with a role in DNA degradation during the lytic processes that accompany bacterial cell death and eukaryotic apoptosis. Evidence is provided here that the supposed nuclease activity of human and bacterial recombinant cyclophilins is due to contamination of the proteins by the host Escherichia coli endonuclease and is not an intrinsic property of these proteins.

  19. Workshop on Radio Recombination Lines

    CERN Document Server

    1980-01-01

    Since their first detection 15 years ago, radio recombination lines from several elements have been observed in a wide variety of objects including HII regions, planetary nebulae, molecular clouds, the diffuse interstellar medium, and recently, other galaxies. The observations span almost the entire range from 0.1 to 100 GHz, and employ both single­ djsh and aperture synthesis techniques. The theory of radio recombination lines has also advanced strongly, to the point where it is perhaps one of the best-understood in astro­ physics. In a parallel development, it has become possible over the last decade to study these same highly-excited atoms in the laboratory; this work provides further confirmation of the theoretical framework. However there has been continuing controversy over the astrophysical interpre­ tation of radio recombination line observations, especially regarding the role of stimulated emission. A workshop was held in Ottawa on 24-25 August, 1979, bringing together many of the active scientist...

  20. Consequences of recombination on traditional phylogenetic analysis

    DEFF Research Database (Denmark)

    Schierup, M H; Hein, J

    2000-01-01

    We investigate the shape of a phylogenetic tree reconstructed from sequences evolving under the coalescent with recombination. The motivation is that evolutionary inferences are often made from phylogenetic trees reconstructed from population data even though recombination may well occur (mt......DNA or viral sequences) or does occur (nuclear sequences). We investigate the size and direction of biases when a single tree is reconstructed ignoring recombination. Standard software (PHYLIP) was used to construct the best phylogenetic tree from sequences simulated under the coalescent with recombination....... With recombination present, the length of terminal branches and the total branch length are larger, and the time to the most recent common ancestor smaller, than for a tree reconstructed from sequences evolving with no recombination. The effects are pronounced even for small levels of recombination that may...

  1. EFECTO DE LA DENSIDAD CELULAR Y LA MULTIPLICIDAD DE INFECCIÓN SOBRE LA PRODUCCIÓN DE BACULOVIRUS RECOMBINANTES EN CULTIVOS DE CÉLULAS DE INSECTO

    Directory of Open Access Journals (Sweden)

    Cuitláhuac Chávez-Peña

    2010-01-01

    célula con CCI de 1 y 2 x106 cél/mL. Se siguieron las cinéticas de crecimiento de las células infectadas, la concentración de proteína total y de la proteína gp64 en el sobrenadante de los cultivos y los títulos virales al momento de la cosecha. Se encontró que una MDI de 0.1 ufp/cél y una concentración celular de 1x106 cél/mL resultaron en el mayor título viral, 3.8 ± 2 x108 ufp/mL. Los mayores títulos virales estuvieron acompañados por un menor crecimiento celular. Los resultados de este trabajo pueden ser utilizados para producir resguardos de baculovirus recombinantes con títulos virales altos y mejorar la eficiencia del proceso.

  2. Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome.

    Science.gov (United States)

    Denier, Colette C; Brisson-Lougarre, Andrée A; Biasini, Ghislaine G; Grozdea, Jean J; Fournier, Didier D

    2002-01-01

    In humans, there are four alkaline phosphatases, and each form exhibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnant with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60-80% of activity. Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome.

  3. Recombinant mouse PAP has pH-dependent ectonucleotidase activity and acts through A(1-adenosine receptors to mediate antinociception.

    Directory of Open Access Journals (Sweden)

    Nathaniel A Sowa

    Full Text Available Prostatic acid phosphatase (PAP is expressed in nociceptive neurons and functions as an ectonucleotidase. When injected intraspinally, the secretory isoforms of human and bovine PAP protein have potent and long-lasting antinociceptive effects that are dependent on A(1-adenosine receptor (A(1R activation. In this study, we purified the secretory isoform of mouse (mPAP using the baculovirus expression system to determine if recombinant mPAP also had antinociceptive properties. We found that mPAP dephosphorylated AMP, and to a much lesser extent, ADP at neutral pH (pH 7.0. In contrast, mPAP dephosphorylated all purine nucleotides (AMP, ADP, ATP at an acidic pH (pH 5.6. The transmembrane isoform of mPAP had similar pH-dependent ectonucleotidase activity. A single intraspinal injection of mPAP protein had long-lasting (three day antinociceptive properties, including antihyperalgesic and antiallodynic effects in the Complete Freund's Adjuvant (CFA inflammatory pain model. These antinociceptive effects were transiently blocked by the A(1R antagonist 8-cyclopentyl-1, 3-dipropylxanthine (CPX, suggesting mPAP dephosphorylates nucleotides to adenosine to mediate antinociception just like human and bovine PAP. Our studies indicate that PAP has species-conserved antinociceptive effects and has pH-dependent ectonucleotidase activity. The ability to metabolize nucleotides in a pH-dependent manner could be relevant to conditions like inflammation where tissue acidosis and nucleotide release occur. Lastly, our studies demonstrate that recombinant PAP protein can be used to treat chronic pain in animal models.

  4. Fine-Scale Recombination Maps of Fungal Plant Pathogens Reveal Dynamic Recombination Landscapes and Intragenic Hotspots.

    Science.gov (United States)

    Stukenbrock, Eva H; Dutheil, Julien Y

    2018-03-01

    Meiotic recombination is an important driver of evolution. Variability in the intensity of recombination across chromosomes can affect sequence composition, nucleotide variation, and rates of adaptation. In many organisms, recombination events are concentrated within short segments termed recombination hotspots. The variation in recombination rate and positions of recombination hotspot can be studied using population genomics data and statistical methods. In this study, we conducted population genomics analyses to address the evolution of recombination in two closely related fungal plant pathogens: the prominent wheat pathogen Zymoseptoria tritici and a sister species infecting wild grasses Z. ardabiliae We specifically addressed whether recombination landscapes, including hotspot positions, are conserved in the two recently diverged species and if recombination contributes to rapid evolution of pathogenicity traits. We conducted a detailed simulation analysis to assess the performance of methods of recombination rate estimation based on patterns of linkage disequilibrium, in particular in the context of high nucleotide diversity. Our analyses reveal overall high recombination rates, a lack of suppressed recombination in centromeres, and significantly lower recombination rates on chromosomes that are known to be accessory. The comparison of the recombination landscapes of the two species reveals a strong correlation of recombination rate at the megabase scale, but little correlation at smaller scales. The recombination landscapes in both pathogen species are dominated by frequent recombination hotspots across the genome including coding regions, suggesting a strong impact of recombination on gene evolution. A significant but small fraction of these hotspots colocalize between the two species, suggesting that hotspot dynamics contribute to the overall pattern of fast evolving recombination in these species. Copyright © 2018 Stukenbrock and Dutheil.

  5. Effects of nuclear mutations for recombination and repair functions and of caffeine on mitochondrial recombination

    International Nuclear Information System (INIS)

    Fraenkel, A.H.M.

    1974-01-01

    Studies of both prokaryotic and eukaryotic organisms indicate that pathways governing repair of damage to nuclear DNA caused by x-ray or ultraviolet irradiation overlap with those controlling recombination. Fourteen nuclear mutants of Saccharomyces cerevisiae were tested in order to determine whether these mutant genes affected mitochondrial recombination. None of the mutations studied significantly affected mitochondrial recombination. The nuclear recombination and repair pathways studied do not overlap with the nuclear pathway which controls recombination of mitochondrial DNA. A second set of experiments was designed to test the effect of caffeine on both nuclear and mitochondrial recombination in Saccharomyces cerevisiae. (U.S.)

  6. Vaccine platform recombinant measles virus.

    Science.gov (United States)

    Mühlebach, Michael D

    2017-10-01

    The classic development of vaccines is lengthy, tedious, and may not necessarily be successful as demonstrated by the case of HIV. This is especially a problem for emerging pathogens that are newly introduced into the human population and carry the inherent risk of pandemic spread in a naïve population. For such situations, a considerable number of different platform technologies are under development. These are also under development for pathogens, where directly derived vaccines are regarded as too complicated or even dangerous due to the induction of inefficient or unwanted immune responses causing considerable side-effects as for dengue virus. Among platform technologies are plasmid-based DNA vaccines, RNA replicons, single-round infectious vector particles, or replicating vaccine-based vectors encoding (a) critical antigen(s) of the target pathogens. Among the latter, recombinant measles viruses derived from vaccine strains have been tested. Measles vaccines are among the most effective and safest life-attenuated vaccines known. Therefore, the development of Schwarz-, Moraten-, or AIK-C-strain derived recombinant vaccines against a wide range of mostly viral, but also bacterial pathogens was quite straightforward. These vaccines generally induce powerful humoral and cellular immune responses in appropriate animal models, i.e., transgenic mice or non-human primates. Also in the recent first clinical phase I trial, the results have been quite encouraging. The trial indicated the expected safety and efficacy also in human patients, interestingly independent from the level of prevalent anti-measles immunity before the trial. Thereby, recombinant measles vaccines expressing additional antigens are a promising platform for future vaccines.

  7. CRMAGE: CRISPR Optimized MAGE Recombineering

    DEFF Research Database (Denmark)

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Sommer, Morten Otto Alexander

    2016-01-01

    A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli...... that are assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the λ Red...

  8. Atomic excitation and recombination in external fields

    International Nuclear Information System (INIS)

    Nayfeh, M.H.; Clark, C.W.

    1985-01-01

    This volume offers a timely look at Rydberg states of atoms in external fields and dielectronic recombination. Each topic provides authoritative coverage, presents a fresh account of a flourishing field of current atomic physics and introduces new opportunities for discovery and development. Topics considered include electron-atom scattering in external fields; observations of regular and irregular motion as exemplified by the quadratic zeeman effect and other systems; Rydberg atoms in external fields and the Coulomb geometry; crossed-field effects in the absorption spectrum of lithium in a magnetic field; precise studies of static electric field ionization; widths and shapes of stark resonances in sodium above the saddle point; studies of electric field effects and barium autoionizing resonances; autoionization and dielectronic recombination in plasma electric microfields; dielectronic recombination measurements on multicharged ions; merged beam studies of dielectronic recombination; Rydberg atoms and dielectronic recombination in astrophysics; and observations on dielectronic recombination

  9. Density dependence of dielectronic recombination in selenium

    International Nuclear Information System (INIS)

    Hagelstein, P.L.; Rosen, M.D.; Jacobs, V.L.

    1986-01-01

    Dielectronic recombination has been found to be the dominant recombination process in the determination of the ionization balance of selenium near the Ne-like sequence under conditions relevant to the exploding-foil EUV laser plasmas. The dielectronic recombination process tends to populate excited levels, and these levels in turn are more susceptible to subsequent excitation and ionization than are the ground-state ions. If one defines an effective recombination rate which includes, in addition to the primary recombination, the subsequent excitation and ionization of the additional excited-state population due to the primary recombination, then this effective recombination rate can be density-sensitive at relatively low electron density. We present results for this effective dielectronic recombination rate at an electron density of 3 x 10/sup 20/ electrons/cm 3 for recombination from Ne-like to Na-like selenium and from F-like to Ne-like selenium. In the former case, the effective recombination rate coefficient is found to be 1.8 x 10/sup -11/ cm 3 /sec at 1.0 keV, which is to be compared with the zero-density value of 2.8 x 10/sup -11/ cm 3 /sec. In the latter case (F-like to Ne-like), the effective recombination rate coefficient is found to be 1.3 x 10/sup -11/ cm 3 /sec, which is substantially reduced from the zero-density result of 3.3 x 10/sup -11/ cm 3 /sec. We have examined the effects of dielectronic recombination on the laser gain of the dominant Ne-like 3p-3s transitions and have compared our results with those presented by Whitten et al. [Phys. Rev. A 33, 2171 (1986)

  10. Rapid purification of recombinant histones.

    Science.gov (United States)

    Klinker, Henrike; Haas, Caroline; Harrer, Nadine; Becker, Peter B; Mueller-Planitz, Felix

    2014-01-01

    The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method "rapid histone purification" (RHP) as it circumvents isolation of inclusion bodies and thereby cuts out the most time-consuming step of traditional purification protocols. Instead of inclusion body isolation, whole cell extracts are prepared under strongly denaturing conditions that directly solubilize inclusion bodies. By ion exchange chromatography, the histones are purified from the extracts. The protocol has been successfully applied to all four canonical Drosophila and human histones. RHP histones and histones that were purified from isolated inclusion bodies had similar purities. The different purification strategies also did not impact the quality of octamers reconstituted from these histones. We expect that the RHP protocol can be readily applied to the purification of canonical histones from other species as well as the numerous histone variants.

  11. The extent and importance of intragenic recombination

    Directory of Open Access Journals (Sweden)

    de Silva Eric

    2004-11-01

    Full Text Available Abstract We have studied the recombination rate behaviour of a set of 140 genes which were investigated for their potential importance in inflammatory disease. Each gene was extensively sequenced in 24 individuals of African descent and 23 individuals of European descent, and the recombination process was studied separately in the two population samples. The results obtained from the two populations were highly correlated, suggesting that demographic bias does not affect our population genetic estimation procedure. We found evidence that levels of recombination correlate with levels of nucleotide diversity. High marker density allowed us to study recombination rate variation on a very fine spatial scale. We found that about 40 per cent of genes showed evidence of uniform recombination, while approximately 12 per cent of genes carried distinct signatures of recombination hotspots. On studying the locations of these hotspots, we found that they are not always confined to introns but can also stretch across exons. An investigation of the protein products of these genes suggested that recombination hotspots can sometimes separate exons belonging to different protein domains; however, this occurs much less frequently than might be expected based on evolutionary studies into the origins of recombination. This suggests that evolutionary analysis of the recombination process is greatly aided by considering nucleotide sequences and protein products jointly.

  12. AcMNPV ac143 (odv-e18) is essential for mediating budded virus production and is the 30th baculovirus core gene

    International Nuclear Information System (INIS)

    McCarthy, Christina B.; Theilmann, David A.

    2008-01-01

    Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac143 (odv-e18) is a late gene that encodes for a predicted 9.6 kDa structural protein that locates to the occlusion derived viral envelope and viral induced intranuclear microvesicles [Braunagel, S.C., He, H., Ramamurthy, P., and Summers, M.D. (1996). Transcription, translation, and cellular localization of three Autographa californica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35, and ODV-EC27. Virology 222, 100-114.]. In this study we demonstrate that ac143 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To examine the role of ac143 in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac143 knockout (KO) virus (AcBAC ac142REP-ac143KO ). Fluorescence and light microscopy showed that infection by AcBAC ac142REP-ac143KO is limited to a single cell and titration assays confirmed that AcBAC ac142REP-ac143KO was unable to produce budded virus (BV). Progression to very late phases of the viral infection was evidenced by the development of occlusion bodies in the nuclei of transfected cells. This correlated with the fact that viral DNA replication was unaffected in AcBAC ac142REP-ac143KO transfected cells. The entire ac143 promoter, which includes three late promoter motifs, is contained within the ac142 open reading frame. Different deletion mutants of this region showed that the integrity of the ac142-ac143 core gene cluster was required for the bacmids to display wild-type patterns of viral replication, BV production and RNA transcription

  13. Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome

    Science.gov (United States)

    Denier, Colette C; Brisson-Lougarre, Andrée A; Biasini, Ghislaine G; Grozdea, Jean J; Fournier, Didier D

    2002-01-01

    Background In humans, there are four alkaline phosphatases, and each form exibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnent with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. Results To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60–80% of activity. Conclusion Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome. PMID:11818032

  14. Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome

    Directory of Open Access Journals (Sweden)

    Grozdea Jean J

    2002-01-01

    Full Text Available Abstract Background In humans, there are four alkaline phosphatases, and each form exibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnent with a trisomy 21 fetus (Down's syndrome displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. Results To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate, allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60–80% of activity. Conclusion Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome.

  15. Proteolytic Processing and Assembly of gag and gag-pol Proteins of TED, a Baculovirus-Associated Retrotransposon of the Gypsy Family

    Science.gov (United States)

    Hajek, Kathryn L.; Friesen, Paul D.

    1998-01-01

    TED (transposable element D) is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This lepidopteran (moth) retroelement contains gag and pol genes that encode proteins capable of forming viruslike particles (VLP) with reverse transcriptase. Since VLP are likely intermediates in TED transposition, we investigated the roles of gag and pol in TED capsid assembly and maturation. By using constructed baculovirus vectors and TED Gag-specific antiserum, we show that the principal translation product of gag (Pr55gag) is cleaved to produce a single VLP structural protein, p37gag. Replacement of Asp436 within the retrovirus-like active site of the pol-encoded protease (PR) abolished Pr55gag cleavage and demonstrated the requirement for PR in capsid processing. As shown by expression of an in-frame fusion of TED gag and pol, PR is derived from the Gag-Pol polyprotein Pr195gag-pol. The PR cleavage site within Pr55gag was mapped to a position near the junction of a basic, nucleocapsid-like domain and a C-terminal acidic domain. Once released by cleavage, the C-terminal fragment was not detected. This acidic fragment was dispensable for VLP assembly, as demonstrated by the formation of VLP by C-terminal Pr55gag truncation proteins and replacement of the acidic domain with a heterologous protein. In contrast, C-terminal deletions that extended into the adjacent nucleocapsid-like domain of Pr55gag abolished VLP recovery and demonstrated that this central region contributes to VLP assembly or stability, or both. Collectively, these data suggest that the single TED protein p37gag provides both capsid and nucleocapsid functions. TED may therefore use a simple processing strategy for VLP assembly and genome packaging. PMID:9765414

  16. HUMAN PAPILLOMA VIRUS IMMUNOGEN CREATION ON THE BASE OF CHIMERIC RECOMBINANT PROTEIN L2E7

    Directory of Open Access Journals (Sweden)

    I. S. Malakhov

    2016-01-01

    Full Text Available The cervical cancer is one of the most common diseases in world. This malignancy is the seventh highest prevalence oncological disease worldwide and the second highest prevalence oncological disease of women in the world. Meanwhile women need to be infected by human papilloma virus (HPV is absolutely necessary for it further evolution, HPV DNA was found in 99.97% cases of disease. Except cervical cancer, HPV cause 85% of rectal cancer, 50% of the vulva, vagina and penis cancers, 20% of oropharyngeal cancer and 10% of larynx and esophagus cancers. In 2009, 14 000 women were diagnosed with cervical cancer in Russia. The growth in morbidity was 19% (in comparison with 1999. The most effective recognised measure for almost each infection prophylaxis is a vaccination. Two human papilloma virus vaccines are available in Russia nowadays — Gardasil and Cervarix, produced in Belgium and the Netherlands respectively. Cervarix is a bivalent vaccine based on virus-like particles (VLP of two types. Recombinant major capsid proteins L1 HPV 16 and HPV 18 express in baculovirus expression system and self-assembled into virus-like particles (about 70 percent of cervical cancers are caused by HPV 16 and HPV 18. VLP of each strain produced in different baculovirus vectors and then combined in single drug. Gardasil is like Cervarix with few exceptions. Producing organisms are fungi S. cerevisiae in this case, and this vaccine contains low-risk HPV 6 and HPV 11 VLP. Thus, Gardasil is quadrivalent HPV-6/11/16/18 vaccine. These vaccines are very effective in averting infection of disease and don’t have significant side-effects, however they have some disadvantages. Firstly, they have a high price because of necessity of their expression in eukaryotic cells. Secondly, they are strain-specific, so vaccines are completely effective only for virus’s strains which are represented in the vaccine. Thirdly, it`s the absence of therapeutic (treatment of established

  17. Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila

    Science.gov (United States)

    Smukowski Heil, Caiti S.; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A.F.

    2015-01-01

    Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human–chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. PMID:26430062

  18. Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila.

    Science.gov (United States)

    Smukowski Heil, Caiti S; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A F

    2015-10-01

    Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human-chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  19. Fundamental Studies of Recombinant Hydrogenases

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Michael W. [Univ. of Georgia, Athens, GA (United States)

    2014-01-25

    This research addressed the long term goals of understanding the assembly and organization of hydrogenase enzymes, of reducing them in size and complexity, of determining structure/function relationships, including energy conservation via charge separation across membranes, and in screening for novel H2 catalysts. A key overall goal of the proposed research was to define and characterize minimal hydrogenases that are produced in high yields and are oxygen-resistant. Remarkably, in spite of decades of research carried out on hydrogenases, it is not possible to readily manipulate or design the enzyme using molecular biology approaches since a recombinant form produced in a suitable host is not available. Such resources are essential if we are to understand what constitutes a “minimal” hydrogenase and design such catalysts with certain properties, such as resistance to oxygen, extreme stability and specificity for a given electron donor. The model system for our studies is Pyrococcus furiosus, a hyperthermophile that grows optimally at 100°C, which contains three different nickel-iron [NiFe-] containing hydrogenases. Hydrogenases I and II are cytoplasmic while the other, MBH, is an integral membrane protein that functions to both evolve H2 and pump protons. Three important breakthroughs were made during the funding period with P. furiosus soluble hydrogenase I (SHI). First, we produced an active recombinant form of SHI in E. coli by the co-expression of sixteen genes using anaerobically-induced promoters. Second, we genetically-engineered P. furiosus to overexpress SHI by an order of magnitude compared to the wild type strain. Third, we generated the first ‘minimal’ form of SHI, one that contained two rather than four subunits. This dimeric form was stable and active, and directly interacted with a pyruvate-oxidizing enzyme with any intermediate electron carrier. The research resulted in five peer-reviewed publications.

  20. Electron-ion recombination at low energy

    International Nuclear Information System (INIS)

    Andersen, L.H.

    1993-01-01

    The work is based on results obtained with a merged-beams experiment. A beam of electronics with a well characterized density and energy distribution was merged with a fast, monoenergetic ion beam. Results have been obtained for radiative recombination and dielectronic recombination at low relative energies (0 to ∼70eV). The obtained energy resolution was improved by about a factor of 30. High vacuum technology was used to suppress interactions with electrons from the environments. The velocity distribution of the electron beam was determined. State-selective dielectronic-recombination measurements were performable. Recombination processes were studied. The theoretical background for radiative recombination and Kramers' theory are reviewed. The quantum mechanical result and its relation to the semiclassical theory is discussed. Radiative recombination was also measured with several different non-bare ions, and the applicability of the semiclassical theory to non-bare ions was investigated. The use of an effective charge is discussed. For dielectronic recombination, the standard theoretical approach in the isolated resonance and independent-processes approximation is debated. The applicability of this method was tested. The theory was able to reproduce most of the experimental data except when the recombination process was sensitive to couplings between different electronic configurations. The influence of external perturbing electrostatic fields is discussed. (AB) (31 refs.)

  1. Recombination rate plasticity: revealing mechanisms by design

    Science.gov (United States)

    Sefick, Stephen; Rushton, Chase

    2017-01-01

    For over a century, scientists have known that meiotic recombination rates can vary considerably among individuals, and that environmental conditions can modify recombination rates relative to the background. A variety of external and intrinsic factors such as temperature, age, sex and starvation can elicit ‘plastic’ responses in recombination rate. The influence of recombination rate plasticity on genetic diversity of the next generation has interesting and important implications for how populations evolve. Further, many questions remain regarding the mechanisms and molecular processes that contribute to recombination rate plasticity. Here, we review 100 years of experimental work on recombination rate plasticity conducted in Drosophila melanogaster. We categorize this work into four major classes of experimental designs, which we describe via classic studies in D. melanogaster. Based on these studies, we highlight molecular mechanisms that are supported by experimental results and relate these findings to studies in other systems. We synthesize lessons learned from this model system into experimental guidelines for using recent advances in genotyping technologies, to study recombination rate plasticity in non-model organisms. Specifically, we recommend (1) using fine-scale genome-wide markers, (2) collecting time-course data, (3) including crossover distribution measurements, and (4) using mixed effects models to analyse results. To illustrate this approach, we present an application adhering to these guidelines from empirical work we conducted in Drosophila pseudoobscura. This article is part of the themed issue ‘Evolutionary causes and consequences of recombination rate variation in sexual organisms’. PMID:29109222

  2. Electron-ion recombination in merged beams

    International Nuclear Information System (INIS)

    Wolf, A.; Habs, D.; Lampert, A.; Neumann, R.; Schramm, U.; Schuessler, T.; Schwalm, D.

    1993-01-01

    Detailed studies of recombination processes between electrons and highly charged ions have become possible by recent improvements of merged-beams experiments. We discuss in particular measurements with stored cooled ion beams at the Test Storage Ring (TSR) in Heidelberg. The cross section of dielectronic recombination was measured with high energy resolution for few-electron systems up to the nuclear charge of Cu at a relative energy up to 2.6 keV. At low energy (∼0.1 eV) total recombination rates of several ions were measured and compared with calculated radiative recombination rates. Laser-stimulated recombination of protons and of C 6+ ions was investigated as a function of the photon energy using visible radiation. Both the total recombination rates and the stimulated recombination spectra indicate that in spite of the short interaction time in merged beams, also collisional capture of electrons into weakly bound levels (related to three-body recombination) could be important

  3. Electronic recombination in some physics problems

    International Nuclear Information System (INIS)

    Guzman, O.

    1988-01-01

    This work is related to calculations of electronic recombination rates, as a function of electronic density, electronic temperature, and ion nuclear charge. Recombination times can be calculated and compared to cooling time, in cooling processes of ion beans by electrons from storage rings. (A.C.A.S.) [pt

  4. Generation of Modified Pestiviruses by Targeted Recombination

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Friis, Martin Barfred; Risager, Peter Christian

    involves targeted modification of viral cDNA genomes, cloned within BACs, by Red/ET recombination-mediated mutagenesis in E.coli DH10B cells. Using recombination-mediated mutagenesis for the targeted design, the work can be expedited and focused in principal on any sequence within the viral genome...

  5. Cell biology of homologous recombination in yeast

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine Valerie; Rothstein, Rodney; Lisby, Michael

    2011-01-01

    Homologous recombination is an important pathway for error-free repair of DNA lesions, such as single- and double-strand breaks, and for rescue of collapsed replication forks. Here, we describe protocols for live cell imaging of single-lesion recombination events in the yeast Saccharomyces...

  6. Recombinant Vaccinia Virus: Immunization against Multiple Pathogens

    Science.gov (United States)

    Perkus, Marion E.; Piccini, Antonia; Lipinskas, Bernard R.; Paoletti, Enzo

    1985-09-01

    The coding sequences for the hepatitis B virus surface antigen, the herpes simplex virus glycoprotein D, and the influenza virus hemagglutinin were inserted into a single vaccinia virus genome. Rabbits inoculated intravenously or intradermally with this polyvalent vaccinia virus recombinant produced antibodies reactive to all three authentic foreign antigens. In addition, the feasibility of multiple rounds of vaccination with recombinant vaccinia virus was demonstrated.

  7. Recombinant organisms for production of industrial products

    OpenAIRE

    Adrio, Jose-Luis; Demain, Arnold L

    2009-01-01

    A revolution in industrial microbiology was sparked by the discoveries of ther double-stranded structure of DNA and the development of recombinant DNA technology. Traditional industrial microbiology was merged with molecular biology to yield improved recombinant processes for the industrial production of primary and secondary metabolites, protein biopharmaceuticals and industrial enzymes. Novel genetic techniques such as metabolic engineering, combinatorial biosynthesis and molecular breeding...

  8. 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

  9. Molecular requirements for radiation-activated recombination

    International Nuclear Information System (INIS)

    Stevens, Craig W.; Zeng Ming; Stamato, Thomas; Cerniglia, George

    1997-01-01

    Purpose/Objective: The major stumbling block to successful gene therapy today is poor gene transfer. We hypothesized that ionizing radiation might activate cellular recombination, and so improve stable gene transfer. We further hypothesized that known DNA-damage-repair proteins might also be important in radiation-activated recombination. Materials and Methods: The effect of irradiation on stable gene transfer efficiency was determined in human (A549 and 39F) and rodent (NIH/3T3) cell lines. Continuous low dose rate and multiple radiation fractions were also tested. Nuclear extracts were made and the effect of irradiation on inter-plasmid recombination/ligation determined. Multiple DNA damage-repair deficient cell lines were tested for radiation-activated recombination. Results: A significant radiation dose-dependent improvement in stable plasmid transfection (by as much as 1300 fold) is demonstrated in neoplastic and primary cells. An improvement in transient plasmid transfection is also seen, with as much as 85% of cells transiently expressing b-galactosidase (20-50 fold improvement). Stable transfection is only improved for linearized or nicked plasmids. Cells have improved gene transfer for at least 96 hours after irradiation. Both fractionated and continuous low dose rate irradiation are effective at improving stable gene transfer in mammalian cells, thus making relatively high radiation dose delivery clinically feasible. Inter-plasmid recombination is radiation dose dependent in nuclear extract assays, and the type of overhang (3', 5' or blunt end) significantly affects recombination efficiency and the type of product. The most common end-joining activity involves filling-in of the overhang followed by blunt end ligation. Adenovirus is a linear, double stranded DNA virus. We demonstrate that adenoviral infection efficiency is increased by irradiation. The duration of transgene expression is lengthened because the virus integrates with high efficiency (∼10

  10. RNAi and heterochromatin repress centromeric meiotic recombination

    DEFF Research Database (Denmark)

    Ellermeier, Chad; Higuchi, Emily C; Phadnis, Naina

    2010-01-01

    During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essen......During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes....... Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between...... types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis....

  11. BIOTECHNOLOGY OF RECOMBINANT HORMONES IN DOPING

    Directory of Open Access Journals (Sweden)

    Biljana Vitošević

    2011-09-01

    Full Text Available Recombinant DNA technology has allowed rapid progress in creating biosynthetic gene products for the treatment of many diseases. In this way it can produce large amounts of hormone, which is intended for the treatment of many pathological conditions. Recombinant hormones that are commonly used are insulin, growth hormone and erythropoietin. Precisely because of the availability of these recombinant hormones, it started their abuse by athletes. Experiments in animal models confirmed the potential effects of some of these hormones in increasing physical abilities, which attracted the attention of athletes who push the limits of their competitive capability by such manipulation. The risks of the use of recombinant hormones in doping include serious consequences for the health of athletes. Methods of detection of endogenous hormones from recombined based on the use of a monoclonal antibodies, capillary zone electrophoresis and protein biomarkers

  12. Effects of UV radiation on genetic recombination

    International Nuclear Information System (INIS)

    Vlahovic, K.; Zahradka, D.; Petranovic, M.; Petranovic, D.

    1996-01-01

    We have used the model consisting of Escherichia coli cells and l phage to study the effects of UV radiation on genetic recombination. We found two radiation induced processes that reduce or inhibit genetic recombination. One such process leads to the inability of prophage to excise itself from the irradiated bacterial chromosome by the site-specific recombination. The other process was shown to inhibit a type of general recombination by which the prophage transfers one of its genetic markers to the infecting homologous phage. Loss of the prophage ability to take part in both site-specific and general recombination was shown to develop in recB + but not in recB cells. From this we infer that the loss of prophage recombinogenicity in irradiated cells is a consequence of one process in which RecBCD enzyme (the product of recB, recC and recD genes) plays an essential role. (author)

  13. Containment air circulation for optimal hydrogen recombination

    International Nuclear Information System (INIS)

    Spinks, N.; Krause, M.

    1997-01-01

    An accepted first-line defense for hydrogen mitigation is to design for the hydrogen to be rapidly mixed with the containment atmosphere and diluted to below flammability concentrations. Then, as hydrogen continues to be produced in the longer term, recombiners can be used to remove hydrogen: recombiners can be located in forced-air ducts or passive recombiners can be distributed within containment and the heat of recombination used to promote local air circulation. However, this principle does not eliminate the possibility of high hydrogen concentrations at locations removed from the recombiners. An improvement on this strategy is to arrange for a specific, buoyancy-driven, overall circulation of the containment atmosphere such that the recombiners can be located within the recirculation flow, immediately downstream of the hydrogen source. This would make the mixing process more predictable and solve the mass-transfer problem associated with distributed recombiners. Ideally, the recombiners would be located just above the hydrogen source so that the heat of recombination would assist the overall circulation. In this way, the hydrogen would be removed as close as possible to the source, thereby minimizing the amount of hydrogen immediately downstream of the source and reducing the hydrogen concentration to acceptable levels at other locations. Such a strategy requires the containment volume to be divided into an upflow path, past the hydrogen source and the recombiner, and a downflow path to complete the circuit. The flow could be generated actively using fans or passively using buoyancy forces arising from the difference in density of gases in the upfiow and downflow paths; the gases in the downflow path being cooled at an elevated heat sink. (author)

  14. The unconventional xer recombination machinery of Streptococci/Lactococci

    NARCIS (Netherlands)

    Le Bourgeois, Pascal; Bugarel, Marie; Campo, Nathalie; Daveran-Mingot, Marie-Line; Labonte, Jessica; Lanfranchi, Daniel; Lautier, Thomas; Pages, Carine; Ritzenthaler, Paul

    Homologous recombination between circular sister chromosomes during DNA replication in bacteria can generate chromosome dimers that must be resolved into monomers prior to cell division. In Escherichia coli, dimer resolution is achieved by site-specific recombination, Xer recombination, involving

  15. Neutralizing antibodies induced by recombinant virus-like particles of enterovirus 71 genotype C4 inhibit infection at pre- and post-attachment steps.

    Directory of Open Access Journals (Sweden)

    Zhiqiang Ku

    Full Text Available BACKGROUND: Enterovirus 71 (EV71 is a major causative agent of hand, foot and mouth disease, which has been prevalent in Asia-Pacific regions, causing significant morbidity and mortality in young children. Antibodies elicited by experimental EV71 vaccines could neutralize infection in vitro and passively protect animal models from lethal challenge, indicating that neutralizing antibodies play an essential role in protection. However, how neutralizing antibodies inhibit infection in vitro remains unclear. METHODS/FINDINGS: In the present study, we explored the mechanisms of neutralization by antibodies against EV71 virus-like particles (VLPs. Recombinant VLPs of EV71 genotype C4 were produced in insect cells using baculovirus vectors. Immunization with the VLPs elicited a high-titer, EV71-specific antibody response in mice. Anti-VLP mouse sera potently neutralized EV71 infection in vitro. The neutralizing antibodies in the anti-VLP mouse sera were found to target mainly an extremely conserved epitope (FGEHKQEKDLEYGAC located at the GH loop of the VP1 protein. The neutralizing anti-VLP antisera were able to inhibit virus binding to target cells efficiently. In addition, post-attachment treatment of virus-bound cells with the anti-VLP antisera also neutralized virus infection, although the antibody concentration required was higher than that of the pre-attachment treatment. CONCLUSIONS: Collectively, our findings represent a valuable addition to the understanding of mechanisms of EV71 neutralization and have strong implications for EV71 vaccine development.

  16. Electron-ion recombination rates for merged-beams experiments

    International Nuclear Information System (INIS)

    Pajek, M.

    1994-01-01

    Energy dependence of the electron-ion recombination rates are studied for different recombination processes (radiative recombination, three-body recombination, dissociative recombination) for Maxwellian relative velocity distribution of arbitrary asymmetry. The results are discussed in context of the electron-ion merged beams experiments in cooling ion storage rings. The question of indication of a possible contribution of the three-body recombination to the measured recombination rates versus relative energy is particularly addressed. Its influence on the electron beam temperature derived from the energy dependence of recombination rate is discussed

  17. First-principles study of Frenkel pair recombination in tungsten

    International Nuclear Information System (INIS)

    Qin, Shi-Yao; Jin, Shuo; Li, Yu-Hao; Zhou, Hong-Bo; Zhang, Ying; Lu, Guang-Hong

    2017-01-01

    The recombination of one Frenkel pair in tungsten has been investigated through first-principles simulation. Two different recombination types have been identified: instantaneous and thermally activated. The small recombination barriers for thermally activated recombination cases indicate that recombination can occur easily with a slightly increased temperature. For both of the two recombination types, recombination occurs through the self-interstitial atom moving towards the vacancy. The recombination process can be direct or through replacement sequences, depending on the vertical distance between the vacancy and the 〈1 1 1〉 line of self-interstitial atom pair.

  18. Induction of homologous recombination in Saccharomyces cerevisiae.

    Science.gov (United States)

    Simon, J R; Moore, P D

    1988-09-01

    We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, or the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.

  19. Recombination every day: abundant recombination in a virus during a single multi-cellular host infection.

    Directory of Open Access Journals (Sweden)

    Remy Froissart

    2005-03-01

    Full Text Available Viral recombination can dramatically impact evolution and epidemiology. In viruses, the recombination rate depends on the frequency of genetic exchange between different viral genomes within an infected host cell and on the frequency at which such co-infections occur. While the recombination rate has been recently evaluated in experimentally co-infected cell cultures for several viruses, direct quantification at the most biologically significant level, that of a host infection, is still lacking. This study fills this gap using the cauliflower mosaic virus as a model. We distributed four neutral markers along the viral genome, and co-inoculated host plants with marker-containing and wild-type viruses. The frequency of recombinant genomes was evaluated 21 d post-inoculation. On average, over 50% of viral genomes recovered after a single host infection were recombinants, clearly indicating that recombination is very frequent in this virus. Estimates of the recombination rate show that all regions of the genome are equally affected by this process. Assuming that ten viral replication cycles occurred during our experiment-based on data on the timing of coat protein detection-the per base and replication cycle recombination rate was on the order of 2 x 10(-5 to 4 x 10(-5. This first determination of a virus recombination rate during a single multi-cellular host infection indicates that recombination is very frequent in the everyday life of this virus.

  20. Recombinant DNA production of spider silk proteins.

    Science.gov (United States)

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-11-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. © 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  1. Advances in recombinant antibody manufacturing.

    Science.gov (United States)

    Kunert, Renate; Reinhart, David

    2016-04-01

    Since the first use of Chinese hamster ovary (CHO) cells for recombinant protein expression, production processes have steadily improved through numerous advances. In this review, we have highlighted several key milestones that have contributed to the success of CHO cells from the beginning of their use for monoclonal antibody (mAb) expression until today. The main factors influencing the yield of a production process are the time to accumulate a desired amount of biomass, the process duration, and the specific productivity. By comparing maximum cell densities and specific growth rates of various expression systems, we have emphasized the limiting parameters of different cellular systems and comprehensively described scientific approaches and techniques to improve host cell lines. Besides the quantitative evaluation of current systems, the quality-determining properties of a host cell line, namely post-translational modifications, were analyzed and compared to naturally occurring polyclonal immunoglobulin fractions from human plasma. In summary, numerous different expression systems for mAbs are available and also under scientific investigation. However, CHO cells are the most frequently investigated cell lines and remain the workhorse for mAb production until today.

  2. Baculovirus-expressed constructs induce immunoglobulin G that recognizes VAR2CSA on Plasmodium falciparum-infected erythrocytes

    DEFF Research Database (Denmark)

    Barfod, Lea; Nielsen, Morten A; Turner, Louise

    2006-01-01

    We raised specific antisera against recombinant VAR2CSA domains produced in Escherichia coli and in insect cells. All were reactive in enzyme-linked immunosorbent assay, but only insect cell-derived constructs induced immunoglobulin G (IgG) that was reactive with native VAR2CSA on the surface...

  3. Development and validation of a genotype 3 recombinant protein-based immunoassay for hepatitis E virus serology in swine

    Directory of Open Access Journals (Sweden)

    W.H.M. van der Poel

    2014-04-01

    Full Text Available Hepatitis E virus (HEV is classified within the family Hepeviridae, genus Hepevirus. HEV genotype 3 (Gt3 infections are endemic in pigs in Western Europe and in North and South America and cause zoonotic infections in humans. Several serological assays to detect HEV antibodies in pigs have been developed, at first mainly based on HEV genotype 1 (Gt1 antigens. To develop a sensitive HEV Gt3 ELISA, a recombinant baculovirus expression product of HEV Gt3 open reading frame-2 was produced and coated onto polystyrene ELISA plates. After incubation of porcine sera, bound HEV antibodies were detected with anti-porcine anti-IgG and anti-IgM conjugates. For primary estimation of sensitivity and specificity of the assay, sets of sera were used from pigs experimentally infected with HEV Gt3. For further validation of the assay and to set the cutoff value, a batch of 1100 pig sera was used. All pig sera were tested using the developed HEV Gt3 assay and two other serologic assays based on HEV Gt1 antigens. Since there is no gold standard available for HEV antibody testing, further validation and a definite setting of the cutoff of the developed HEV Gt3 assay were performed using a statistical approach based on Bayes' theorem. The developed and validated HEV antibody assay showed effective detection of HEV-specific antibodies. This assay can contribute to an improved detection of HEV antibodies and enable more reliable estimates of the prevalence of HEV Gt3 in swine in different regions.

  4. The Challenges of Recombinant Endostatin in Clinical Application: Focus on the Different Expression Systems and Molecular Bioengineering

    Directory of Open Access Journals (Sweden)

    Abbas Mohajeri

    2017-04-01

    Full Text Available Angiogenesis plays an essential role in rapid growing and metastasis of the tumors. Inhibition of angiogenesis is a putative strategy for cancer therapy. Endostatin (Es is an attractive anti-angiogenesis protein with some clinical application challenges including; short half-life, instability in serum and requirement to high dosage. Therefore, production of recombinant endostatin (rEs is necessary in large scale. The production of rEs is difficult because of its structural properties and is high-cost. Therefore, this review focused on the different expression systems that involved in rEs production including; mammalian, baculovirus, yeast, and Escherichia coli (E. coli expression systems. The evaluating of the results of different expression systems declared that none of the mentioned systems can be considered to be generally superior to the other. Meanwhile with considering the advantages and disadvantage of E. coli expression system compared with other systems beside the molecular properties of Es, E. coli expression system can be a preferred expression system for expressing of the Es in large scale. Also, the molecular bioengineering and sustained release formulations that lead to improving of its stability and bioactivity will be discussed. Point mutation (P125A of Es, addition of RGD moiety or an additional zinc biding site to N-terminal of Es , fusing of Es to anti-HER2 IgG or heavy-chain of IgG, and finally loading of the endostar by PLGA and PEG- PLGA nanoparticles and gold nano-shell particles are the effective bioengineering methods to overcome to clinical changes of endostatin.

  5. Laser-induced electron--ion recombination used to study enhanced spontaneous recombination during electron cooling

    International Nuclear Information System (INIS)

    Schramm, U.; Wolf, A.; Schuess ler, T.; Habs, D.; Schwalm, D.; Uwira, O.; Linkemann, J.; Mueller, A.

    1997-01-01

    Spontaneous recombination of highly charged ions with free electrons in merged velocity matched electron and ion beams has been observed in earlier experiments to occur at rates significantly higher than predicted by theoretical estimates. To study this enhanced spontaneous recombination, laser induced recombination spectra were measured both in velocity matched beams and in beams with well defined relative velocities, corresponding to relative electron-ion detuning energies ranging from 1 meV up to 6.5 meV where the spontaneous recombination enhancement was found to be strongly reduced. Based on a comparison with simplified calculations, the development of the recombination spectra for decreasing detuning energies indicates additional contributions at matched velocities which could be related to the energy distribution of electrons causing the spontaneous recombination rate enhancement

  6. Recombinant Human Papillomavirus (HPV) Bivalent Vaccine

    Science.gov (United States)

    This page contains brief information about recombinant human papillomavirus (HPV) bivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  7. Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine

    Science.gov (United States)

    This page contains brief information about recombinant human papillomavirus (HPV) nonavalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  8. Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine

    Science.gov (United States)

    This page contains brief information about recombinant human papillomavirus (HPV) quadrivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.

  9. Ultramicroscopic observation of recombinant adenoassociated virus ...

    African Journals Online (AJOL)

    Ultramicroscopic observation of recombinant adenoassociated virus type 2 on the surface of formvarcarbon coated copper grids under different relative humidity and incubation time using negative stain transmission electron microscopy.

  10. Recombinant vaccines: experimental and applied aspects

    DEFF Research Database (Denmark)

    Lorenzen, Niels

    1999-01-01

    Development of vaccines for aquaculture fish represent an important applied functional aspect of fish immunology research. Particularly in the case of recombinant vaccines, where a single antigen is usually expected to induce immunity to a specific pathogen, knowledge of mechanisms involved...... in induction of a protective immune response may become vital. The few recombinant vaccines licensd so far, despite much research during the last decade, illustrate that this is not a straightforward matter. However, as vaccine technology as well as our knowledge of the fish immune system is steadily improved......, these fields will open up a number of interesting research objectives of mutual benefit. Recent aspects of recombinant protein vaccines, live recombinant vaccines and DNA vaccines are discussed....

  11. New perspectives on recombinant human antibodies

    NARCIS (Netherlands)

    J. de Kruif (John); A.-R. van der Vuurst de Vries (Anne); L. Cilenti (L.); E. Boel (E.); W. van Ewijk (Willem); T. Logtenberg (Ton)

    1996-01-01

    textabstractThe limited potential of murine monoclonal antibodies for human immunotherapy has driven recent progress in recombinant antibody technology. Here, de Kruif and colleagues report on advances in the development and use of phage-antibody-display libraries.

  12. Construction of retroviral recombinant containing human tissue ...

    African Journals Online (AJOL)

    USER

    2010-03-29

    Mar 29, 2010 ... Recombinant retroviral vector containing human tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) gene was ..... heavy metal ions, the protein could be express in an .... involves adhesion, degradation and movement. To.

  13. Generation, characterization and epitope mapping of two neutralizing and protective human recombinant antibodies against influenza A H5N1 viruses.

    Directory of Open Access Journals (Sweden)

    Lina Sun

    Full Text Available BACKGROUND: The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI H5N1 virus infection in humans is urgently needed. Broadly cross-neutralizing recombinant human antibodies obtained from the survivors of H5N1 avian influenza provide an important role in immunotherapy for human H5N1 virus infection and definition of the critical epitopes for vaccine development. METHODOLOGY/PRINCIPAL FINDINGS: We have characterized two recombinant baculovirus-expressed human antibodies (rhAbs, AVFluIgG01 and AVFluIgG03, generated by screening a Fab antibody phage library derived from a patient recovered from infection with a highly pathogenic avian influenza A H5N1 clade 2.3 virus. AVFluIgG01 cross-neutralized the most of clade 0, clade 1, and clade 2 viruses tested, in contrast, AVFluIgG03 only neutralized clade 2 viruses. Passive immunization of mice with either AVFluIgG01 or AVFluIgG03 antibody resulted in protection from a lethal H5N1 clade 2.3 virus infection. Furthermore, through epitope mapping, we identify two distinct epitopes on H5 HA molecule recognized by these rhAbs and demonstrate their potential to protect against a lethal H5N1 virus infection in a mouse model. CONCLUSIONS/SIGNIFICANCE: Importantly, localization of the epitopes recognized by these two neutralizing and protective antibodies has provided, for the first time, insight into the human antibody responses to H5N1 viruses which contribute to the H5 immunity in the recovered patient. These results highlight the potential of a rhAbs treatment strategy for human H5N1 virus infection and provide new insight for the development of effective H5N1 pandemic vaccines.

  14. Live recombinant BHV/BRSV vaccine

    OpenAIRE

    Keil, G.M.; Rijsewijk, F.A.M.

    1998-01-01

    The present invention refers to synthetic Bovine Respiratory Syncytium virus genes. Also the invention relates to live attenuated Bovine Herpesvirus recombinants carrying such synthetic genes. Furthermore, the invention relates to vaccines based on these live attenuated recombinants, for the protection of cattle against both Bovine herpesvirus infection and against Bovine Respiratory Syncytium virus infection. Also the invention relates to methods for the preparation of such live attenuated r...

  15. Co-factor activated recombinant adenovirus proteinases

    Science.gov (United States)

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  16. Hadron correlations from recombination and fragmentation

    Energy Technology Data Exchange (ETDEWEB)

    Fries, Rainer J [School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455 (United States)

    2005-04-01

    We review the formalism of quark recombination applied to the hadronization of a quark-gluon plasma. Evidence in favour of the quark recombination model is outlined. Recent work on parton correlations, leading to detectable correlations between hadrons, is discussed. Hot spots from completely quenched jets are a likely source of such correlations which appear to be jet like. It will be discussed how such a picture compares with measurement of associated hadron yields at RHIC.

  17. Recombination of a fast expanding plasma

    International Nuclear Information System (INIS)

    Salvat, M.

    1979-05-01

    The goal of the following calculations is to determine numerically the recombination of dense plasmas (for instance of laser-produced plasmas). The recombination is computed for plasmas with initial densities of 10 24 27 [m -3 ] and with initial temperatures >= 50 eV. The ionization of the plasma remains essentially constant during the early phase of expansion. The time for which the ionization is 'frozen-in' grows with decreasing initial density and with increasing initial temperature. (orig.) [de

  18. Dissociation of recombinant prion autocatalysis from infectivity.

    Science.gov (United States)

    Noble, Geoffrey P; Supattapone, Surachai

    2015-01-01

    Within the mammalian prion field, the existence of recombinant prion protein (PrP) conformers with self-replicating (ie. autocatalytic) activity in vitro but little to no infectious activity in vivo challenges a key prediction of the protein-only hypothesis of prion replication--that autocatalytic PrP conformers should be infectious. To understand this dissociation of autocatalysis from infectivity, we recently performed a structural and functional comparison between a highly infectious and non-infectious pair of autocatalytic recombinant PrP conformers derived from the same initial prion strain. (1) We identified restricted, C-terminal structural differences between these 2 conformers and provided evidence that these relatively subtle differences prevent the non-infectious conformer from templating the conversion of native PrP(C) substrates containing a glycosylphosphatidylinositol (GPI) anchor. (1) In this article we discuss a model, consistent with these findings, in which recombinant PrP, lacking post-translational modifications and associated folding constraints, is capable of adopting a wide variety of autocatalytic conformations. Only a subset of these recombinant conformers can be adopted by post-translationally modified native PrP(C), and this subset represents the recombinant conformers with high specific infectivity. We examine this model's implications for the generation of highly infectious recombinant prions and the protein-only hypothesis of prion replication.

  19. Mitigating Mitochondrial Genome Erosion Without Recombination.

    Science.gov (United States)

    Radzvilavicius, Arunas L; Kokko, Hanna; Christie, Joshua R

    2017-11-01

    Mitochondria are ATP-producing organelles of bacterial ancestry that played a key role in the origin and early evolution of complex eukaryotic cells. Most modern eukaryotes transmit mitochondrial genes uniparentally, often without recombination among genetically divergent organelles. While this asymmetric inheritance maintains the efficacy of purifying selection at the level of the cell, the absence of recombination could also make the genome susceptible to Muller's ratchet. How mitochondria escape this irreversible defect accumulation is a fundamental unsolved question. Occasional paternal leakage could in principle promote recombination, but it would also compromise the purifying selection benefits of uniparental inheritance. We assess this tradeoff using a stochastic population-genetic model. In the absence of recombination, uniparental inheritance of freely-segregating genomes mitigates mutational erosion, while paternal leakage exacerbates the ratchet effect. Mitochondrial fusion-fission cycles ensure independent genome segregation, improving purifying selection. Paternal leakage provides opportunity for recombination to slow down the mutation accumulation, but always at a cost of increased steady-state mutation load. Our findings indicate that random segregation of mitochondrial genomes under uniparental inheritance can effectively combat the mutational meltdown, and that homologous recombination under paternal leakage might not be needed. Copyright © 2017 by the Genetics Society of America.

  20. Electron - ion recombination processes - an overview

    International Nuclear Information System (INIS)

    Hahn, Yukap

    1997-01-01

    Extensive theoretical and experimental studies have been carried out for the past 20 years on electron - ion recombination processes, as they are applied to the analysis of astrophysical and laboratory plasmas. We review the basic understanding gained through these efforts, with emphasis on some of the more recent progress made in recombination theory as the recombining system is affected by time-dependent electric fields and plasma particles at low temperature. Together with collisional ionization and excitation processes, recombination is important in determining ionization balance and excited-state population in non-equilibrium plasmas. The radiation emitted by plasmas is usually the principal medium with which to study the plasma condition, as it is produced mainly during the recombination and decay of excited states of ions inside the plasma. This is especially true when the plasma under study is not readily accessible by direct probes, as in astrophysical plasmas. Moreover, external probes may sometimes cause undesirable disturbances of the plasma. Electron-ion recombination proceeds in several different modes. The direct modes include three-body recombination (TBR) and one-step radiative recombination (RR), all to the ground- and singly-excited states of the target ions. By contrast, the indirect resonant mode is a two-step dielectronic recombination (DR), which proceeds first with the formation of doubly-excited states by radiationless excitation/capture. The resonant states thus formed may relax by autoionization and/or radiative cascades. For more exotic modes of recombination, we consider off-shell dielectronic recombination (radiative DR = RDR), in which an electron capture is accompanied by simultaneous radiative emission and excitation of the target ion. Some discussion on attachment of electrons to neutral atoms, resulting in the formation of negative ions, is also given. When resonance states involve one or more electrons in high Rydberg states

  1. Oligonucleotide recombination enabled site-specific mutagenesis in bacteria

    Science.gov (United States)

    Recombineering refers to a strategy for engineering DNA sequences using a specialized mode of homologous recombination. This technology can be used for rapidly constructing precise changes in bacterial genome sequences in vivo. Oligo recombination is one type of recombineering that uses ssDNA olig...

  2. Recombination analysis based on the complete genome of bocavirus

    Directory of Open Access Journals (Sweden)

    Chen Shengxia

    2011-04-01

    Full Text Available Abstract Bocavirus include bovine parvovirus, minute virus of canine, porcine bocavirus, gorilla bocavirus, and Human bocaviruses 1-4 (HBoVs. Although recent reports showed that recombination happened in bocavirus, no systematical study investigated the recombination of bocavirus. The present study performed the phylogenetic and recombination analysis of bocavirus over the complete genomes available in GenBank. Results confirmed that recombination existed among bocavirus, including the likely inter-genotype recombination between HBoV1 and HBoV4, and intra-genotype recombination among HBoV2 variants. Moreover, it is the first report revealing the recombination that occurred between minute viruses of canine.

  3. Genetic recombination of the hepatitis C virus: clinical implications.

    Science.gov (United States)

    Morel, V; Fournier, C; François, C; Brochot, E; Helle, F; Duverlie, G; Castelain, S

    2011-02-01

    Genetic recombination is a well-known feature of RNA viruses that plays a significant role in their evolution. Although recombination is well documented for Flaviviridae family viruses, the first natural recombinant strain of hepatitis C virus (HCV) was identified as recently as 2002. Since then, a few other natural inter-genotypic, intra-genotypic and intra-subtype recombinant HCV strains have been described. However, the frequency of recombination may have been underestimated because not all known HCV recombinants are screened for in routine practice. Furthermore, the choice of treatment regimen and its predictive outcome remain problematic as the therapeutic strategy for HCV infection is genotype dependent. HCV recombination also raises many questions concerning its mechanisms and effects on the epidemiological and physiopathological features of the virus. This review provides an update on recombinant HCV strains, the process that gives rise to recombinants and clinical implications of recombination. © 2010 Blackwell Publishing Ltd.

  4. Heterogeneous recombination among Hepatitis B virus genotypes.

    Science.gov (United States)

    Castelhano, Nadine; Araujo, Natalia M; Arenas, Miguel

    2017-10-01

    The rapid evolution of Hepatitis B virus (HBV) through both evolutionary forces, mutation and recombination, allows this virus to generate a large variety of adapted variants at both intra and inter-host levels. It can, for instance, generate drug resistance or the diverse viral genotypes that currently exist in the HBV epidemics. Concerning the latter, it is known that recombination played a major role in the emergence and genetic diversification of novel genotypes. In this regard, the quantification of viral recombination in each genotype can provide relevant information to devise expectations about the evolutionary trends of the epidemic. Here we measured the amount of this evolutionary force by estimating global and local recombination rates in >4700 HBV complete genome sequences corresponding to nine (A to I) HBV genotypes. Counterintuitively, we found that genotype E presents extremely high levels of recombination, followed by genotypes B and C. On the other hand, genotype G presents the lowest level, where recombination is almost negligible. We discuss these findings in the light of known characteristics of these genotypes. Additionally, we present a phylogenetic network to depict the evolutionary history of the studied HBV genotypes. This network clearly classified all genotypes into specific groups and indicated that diverse pairs of genotypes are derived from a common ancestor (i.e., C-I, D-E and, F-H) although still the origin of this virus presented large uncertainty. Altogether we conclude that the amount of observed recombination is heterogeneous among HBV genotypes and that this heterogeneity can influence on the future expansion of the epidemic. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus

    DEFF Research Database (Denmark)

    Sørensen, K.J.; Madsen, K.G.; Madsen, E.S.

    1998-01-01

    The baculovirus expression system was found to be efficient at expressing the 3D, the 3AB and the 3ABC non-structural proteins (NSP) of foot-and-mouth disease virus (FMDV) as antigens recognised by immune sera in ELISA. ELISA's using 3D, 3AB and 3ABC detected antibodies from day 8 and 10 after...... experimental infection of susceptible cattle and sheep and cattle remained seropositive for more than 395 days. The ELISA's detected antibodies against any of the seven serotypes of FMDV. The 3D ELISA was specific and precise and as sensitive as established ELISA's which measure antibody to structural proteins....... The assay may be used as a resource saving alternative to established ELISA's for the detection of antibodies against any of the seven serotypes. The 3AB and the 3ABC ELISA were also specific and precise. FMDV infected cattle could be differentiated from those that had been merely vaccinated as they gave...

  6. Differentiation of foot-and-mouth disease virus infected animals from vaccinated animals using a blocking ELISA based on baculovirus expressed FMDV 3ABC antigen and a 3ABC monoclonal antibody

    DEFF Research Database (Denmark)

    Sørensen, K.J.; de Stricker, K.; Dyrting, K.C.

    2005-01-01

    A blocking ELISA that differentiated foot-and-mouth disease virus (FMDV) infected animals from vaccinated animals was developed which uses baculovirus expressed FMDV 3ABC non-structural protein as antigen and monoclonal antibody against FMDV 3ABC non-structural protein as capture and detector...... infected with all seven serotypes of FMDV. The test detected antibodies from days 7 or 9 following experimental infection of non-vaccinated cattle and sheep, and in cattle strong positive reactions persisted for up to 395 days after infection. In vaccinated cattle that became carriers after challenge...... with homologous FMDV, positive reactions were obtained in all but one case. In some of these cattle the antibody response was detected late in comparison to the non-vaccinated infected cattle. The test gave results that compared favourably with two commercial ELISA's when used to test sera from cattle, pigs...

  7. Large scale production and downstream processing of a recombinant porcine parvovirus vaccine

    NARCIS (Netherlands)

    Maranga, L.; Rueda, P.; Antonis, A.F.G.; Vela, C.; Langeveld, J.P.M.; Casal, J.I.; Carrondo, M.J.T.

    2002-01-01

    Porcine parvovirus (PPV) virus-like particles (VLPs) constitute a potential vaccine for prevention of parvovirus-induced reproductive failure in gilts. Here we report the development of a large scale (25 l) production process for PPV-VLPs with baculovirus-infected insect cells. A low multiplicity of

  8. Expression of recombinant proteinase 3, the autoantigen in Wegener's granulomatosis, in insect cells

    NARCIS (Netherlands)

    Van der Geld, YM; Smook, MLF; Huitema, MG; Harmsen, MC; Limburg, PC; Kallenberg, CGM

    2002-01-01

    Proteinase 3 (PR3) is the major autoantigen for anti-neutrophil cytoplasmic antibodies (ANCA) in patients with Wegener's granulomatosis. Little is known about the major antigenic sites on PR3. To facilitate epitope mapping, PR3 was cloned in insect cells using a baculovirus expression system. Four

  9. Graded Recombination Layers for Multijunction Photovoltaics

    KAUST Repository

    Koleilat, Ghada I.

    2012-06-13

    Multijunction devices consist of a stack of semiconductor junctions having bandgaps tuned across a broad spectrum. In solar cells this concept is used to increase the efficiency of photovoltaic harvesting, while light emitters and detectors use it to achieve multicolor and spectrally tunable behavior. In series-connected current-matched multijunction devices, the recombination layers must allow the hole current from one cell to recombine, with high efficiency and low voltage loss, with the electron current from the next cell. We recently reported a tandem solar cell in which the recombination layer was implemented using a progression of n-type oxides whose doping densities and work functions serve to connect, with negligible resistive loss at solar current densities, the constituent cells. Here we present the generalized conditions for design of efficient graded recombination layer solar devices. We report the number of interlayers and the requirements on work function and doping of each interlayer, to bridge an work function difference as high as 1.6 eV. We also find solutions that minimize the doping required of the interlayers in order to minimize optical absorption due to free carriers in the graded recombination layer (GRL). We demonstrate a family of new GRL designs experimentally and highlight the benefits of the progression of dopings and work functions in the interlayers. © 2012 American Chemical Society.

  10. Polyploidization increases meiotic recombination frequency in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Rehmsmeier Marc

    2011-04-01

    Full Text Available Abstract Background Polyploidization is the multiplication of the whole chromosome complement and has occurred frequently in vascular plants. Maintenance of stable polyploid state over generations requires special mechanisms to control pairing and distribution of more than two homologous chromosomes during meiosis. Since a minimal number of crossover events is essential for correct chromosome segregation, we investigated whether polyploidy has an influence on the frequency of meiotic recombination. Results Using two genetically linked transgenes providing seed-specific fluorescence, we compared a high number of progeny from diploid and tetraploid Arabidopsis plants. We show that rates of meiotic recombination in reciprocal crosses of genetically identical diploid and autotetraploid Arabidopsis plants were significantly higher in tetraploids compared to diploids. Although male and female gametogenesis differ substantially in meiotic recombination frequency, both rates were equally increased in tetraploids. To investigate whether multivalent formation in autotetraploids was responsible for the increased recombination rates, we also performed corresponding experiments with allotetraploid plants showing strict bivalent pairing. We found similarly increased rates in auto- and allotetraploids, suggesting that the ploidy effect is independent of chromosome pairing configurations. Conclusions The evolutionary success of polyploid plants in nature and under domestication has been attributed to buffering of mutations and sub- and neo-functionalization of duplicated genes. Should the data described here be representative for polyploid plants, enhanced meiotic recombination, and the resulting rapid creation of genetic diversity, could have also contributed to their prevalence.

  11. SequenceLDhot: detecting recombination hotspots.

    Science.gov (United States)

    Fearnhead, Paul

    2006-12-15

    There is much local variation in recombination rates across the human genome--with the majority of recombination occurring in recombination hotspots--short regions of around approximately 2 kb in length that have much higher recombination rates than neighbouring regions. Knowledge of this local variation is important, e.g. in the design and analysis of association studies for disease genes. Population genetic data, such as that generated by the HapMap project, can be used to infer the location of these hotspots. We present a new, efficient and powerful method for detecting recombination hotspots from population data. We compare our method with four current methods for detecting hotspots. It is orders of magnitude quicker, and has greater power, than two related approaches. It appears to be more powerful than HotspotFisher, though less accurate at inferring the precise positions of the hotspot. It was also more powerful than LDhot in some situations: particularly for weaker hotspots (10-40 times the background rate) when SNP density is lower (maths.lancs.ac.uk/~fearnhea/Hotspot.

  12. Genome-wide recombination rate variation in a recombination map of cotton.

    Science.gov (United States)

    Shen, Chao; Li, Ximei; Zhang, Ruiting; Lin, Zhongxu

    2017-01-01

    Recombination is crucial for genetic evolution, which not only provides new allele combinations but also influences the biological evolution and efficacy of natural selection. However, recombination variation is not well understood outside of the complex species' genomes, and it is particularly unclear in Gossypium. Cotton is the most important natural fibre crop and the second largest oil-seed crop. Here, we found that the genetic and physical maps distances did not have a simple linear relationship. Recombination rates were unevenly distributed throughout the cotton genome, which showed marked changes along the chromosome lengths and recombination was completely suppressed in the centromeric regions. Recombination rates significantly varied between A-subgenome (At) (range = 1.60 to 3.26 centimorgan/megabase [cM/Mb]) and D-subgenome (Dt) (range = 2.17 to 4.97 cM/Mb), which explained why the genetic maps of At and Dt are similar but the physical map of Dt is only half that of At. The translocation regions between A02 and A03 and between A04 and A05, and the inversion regions on A10, D10, A07 and D07 indicated relatively high recombination rates in the distal regions of the chromosomes. Recombination rates were positively correlated with the densities of genes, markers and the distance from the centromere, and negatively correlated with transposable elements (TEs). The gene ontology (GO) categories showed that genes in high recombination regions may tend to response to environmental stimuli, and genes in low recombination regions are related to mitosis and meiosis, which suggested that they may provide the primary driving force in adaptive evolution and assure the stability of basic cell cycle in a rapidly changing environment. Global knowledge of recombination rates will facilitate genetics and breeding in cotton.

  13. Recombinant human erythropoietin in sports: a review

    Directory of Open Access Journals (Sweden)

    Rafael Maia de Almeida Bento

    2003-06-01

    Full Text Available Erythropoietin is an endogenous hormone of glicoproteic nature secreted by the kidneys and is the main regulator of the erythropoiesis. An alteration in its production generates a disturbance in the plasmatic concentration giving rise to several types of pathologies related to the hematopoietic system. The recombinant forms of erythropoietin have indiscriminately been used by athletes, mainly in endurance sports, by increasing the erythrocytes concentration, generating a better delivery of oxygen to the muscle tissue. The administration of recombinant erythropoietin was prohibited by the International Olympic Committee and its use considered as doping. This review has the intention to describe the physical, biological and pharmacokinetic properties of the endogenous erythropoietin, as well as its recombinant form, describing also its use in sports and the process of searching methodologies for its detection in doping control.

  14. Regulation of Homologous Recombination by SUMOylation

    DEFF Research Database (Denmark)

    Pinela da Silva, Sonia Cristina

    factors such as the homologous recombination (HR) machinery. HR constitutes the main DSB repair pathway in Saccharomyces cerevisiae and despite being largely considered an error-free process and essential for genome stability, uncontrolled recombination can lead to loss of heterozygosity, translocations......, deletions, and genome rearrangements that can lead to cell death or cancer in humans. The post-translational modification by SUMO (small ubiquitinlike modifier) has proven to be an important regulator of HR and genome integrity, but the molecular mechanisms responsible for these roles are still unclear....... In this study I present new insights for the role of SUMOylation in regulating HR by dissecting the role of SUMO in the interaction between the central HR-mediator protein Rad52 and its paralogue Rad59 and the outcome of recombination. This data provides evidence for the importance of SUMO in promoting protein...

  15. Determination of recombination in Mycoplasma hominis

    DEFF Research Database (Denmark)

    Jacobsen, Iben Søgaard; Boesen, Thomas; Mygind, Tina

    2002-01-01

    disequilibrium and distance between the segregating sites, by the homoplasy ratio (H ratio), and by compatibility matrices. The gap gene showed well-supported evidence for high levels of recombination, whereas recombination was less frequent and not significant within the other genes. The analysis revealed......B-hitL, excinuclease ABC subunit A (uvrA) and glyceraldehyde-3-phosphate dehydrogenase (gap) genes. The level of variability of these M. hominis genes was low compared with the housekeeping genes from Helicobacter pylori and Neisseria meningitidis, but only few M. hominis isolates had identical sequences in all genes...... intergenic and intragenic recombination in M. hominis and this may explain the high intraspecies variability. The results obtained in the present study may be of importance for future population studies of Mycoplasma species....

  16. Constraints from jet calculus on quark recombination

    International Nuclear Information System (INIS)

    Jones, L.M.; Lassila, K.E.; Willen, D.

    1979-01-01

    Within the QCD jet calculus formalism, we deduce an equation describing recombination of quarks and antiquarks into mesons within a quark or gluon jet. This equation relates the recombination function R(x 1 ,x 2 ,x) used in current literature to the fragmentation function for producing that same meson out of the parton initiating the jet. We submit currently used recombination functions to our consistency test, taking as input mainly the u-quark fragmentation data into π + mesons, but also s-quark fragmentation into K - mesons. The constraint is well satisfied at large Q 2 for large moments. Our results depend on one parameter, Q 0 2 , the constraint equation being satisfied for small values of this parameter

  17. Recombination Processes and Nonlinear Markov Chains.

    Science.gov (United States)

    Pirogov, Sergey; Rybko, Alexander; Kalinina, Anastasia; Gelfand, Mikhail

    2016-09-01

    Bacteria are known to exchange genetic information by horizontal gene transfer. Since the frequency of homologous recombination depends on the similarity between the recombining segments, several studies examined whether this could lead to the emergence of subspecies. Most of them simulated fixed-size Wright-Fisher populations, in which the genetic drift should be taken into account. Here, we use nonlinear Markov processes to describe a bacterial population evolving under mutation and recombination. We consider a population structure as a probability measure on the space of genomes. This approach implies the infinite population size limit, and thus, the genetic drift is not assumed. We prove that under these conditions, the emergence of subspecies is impossible.

  18. Transcription and recombination: when RNA meets DNA.

    Science.gov (United States)

    Aguilera, Andrés; Gaillard, Hélène

    2014-08-01

    A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription-replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  19. Exceptionally high levels of recombination across the honey bee genome.

    Science.gov (United States)

    Beye, Martin; Gattermeier, Irene; Hasselmann, Martin; Gempe, Tanja; Schioett, Morten; Baines, John F; Schlipalius, David; Mougel, Florence; Emore, Christine; Rueppell, Olav; Sirviö, Anu; Guzmán-Novoa, Ernesto; Hunt, Greg; Solignac, Michel; Page, Robert E

    2006-11-01

    The first draft of the honey bee genome sequence and improved genetic maps are utilized to analyze a genome displaying 10 times higher levels of recombination (19 cM/Mb) than previously analyzed genomes of higher eukaryotes. The exceptionally high recombination rate is distributed genome-wide, but varies by two orders of magnitude. Analysis of chromosome, sequence, and gene parameters with respect to recombination showed that local recombination rate is associated with distance to the telomere, GC content, and the number of simple repeats as described for low-recombining genomes. Recombination rate does not decrease with chromosome size. On average 5.7 recombination events per chromosome pair per meiosis are found in the honey bee genome. This contrasts with a wide range of taxa that have a uniform recombination frequency of about 1.6 per chromosome pair. The excess of recombination activity does not support a mechanistic role of recombination in stabilizing pairs of homologous chromosome during chromosome pairing. Recombination rate is associated with gene size, suggesting that introns are larger in regions of low recombination and may improve the efficacy of selection in these regions. Very few transposons and no retrotransposons are present in the high-recombining genome. We propose evolutionary explanations for the exceptionally high genome-wide recombination rate.

  20. Thermal recombination: Beyond the valence quark approximation

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, B. [Department of Physics, Duke University, Durham, NC 27708 (United States); Fries, R.J. [School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455 (United States)]. E-mail: fries@physics.umn.edu; Bass, S.A. [Department of Physics, Duke University, Durham, NC 27708 (United States); RIKEN BNL Research Center, Brookhaven National Laboratory, Upton, NY 11973 (United States)

    2005-07-07

    Quark counting rules derived from recombination models agree well with data on hadron production at intermediate transverse momenta in relativistic heavy-ion collisions. They convey a simple picture of hadrons consisting only of valence quarks. We discuss the inclusion of higher Fock states that add sea quarks and gluons to the hadron structure. We show that, when recombination occurs from a thermal medium, hadron spectra remain unaffected by the inclusion of higher Fock states. However, the quark number scaling for elliptic flow is somewhat affected. We discuss the implications for our understanding of data from the Relativistic Heavy Ion Collider.

  1. The recombination of a helium plasma

    International Nuclear Information System (INIS)

    Hollenstein, C.; Sayasov, Y.; Schneider, H.

    1975-01-01

    A helium plasma (Tsub(e) 15 cm -3 ) in the afterglow without magnetic field was investigated. The measurements of the electron density and temperature are presented. Laser interferometry and radiowave diagnostics were used. The measured exponential decay of the electron density and temperature was explained with the collisional-radiative recombination and the thermal conduction of the electrons towards the wall of the discharge vessel. The measured recombination coefficients were compared with measurements and calculations of other authors. The best agreement was found with the calculations by Drawin. (Auth.)

  2. Theoretical models for recombination in expanding gas

    International Nuclear Information System (INIS)

    Avron, Y.; Kahane, S.

    1978-09-01

    In laser isotope separation of atomic uranium, one is confronted with the theoretical problem of estimating the concentration of thermally ionized uranium atoms. To investigate this problem theoretical models for recombination in an expanding gas and in the absence of local thermal equilibrium have been constructed. The expansion of the gas is described by soluble models of the hydrodynamic equation, and the recombination by rate equations. General results for the freezing effect for the suitable ranges of the gas parameters are obtained. The impossibility of thermal equilibrium in expanding two-component systems is proven

  3. Recombination coefficients in extrinsic n-InSb

    International Nuclear Information System (INIS)

    Schneider, W.; Groh, H.; Huebner, K.

    1976-01-01

    The bulk recombination coefficients for linear recombination via recombination centers as well as for direct recombination have been determined measuring the conductivity decay after two-photon absorption with a CO 2 laser. The Suhl effect was applied to measure the surface recombination velocity. The corresponding literature is discussed and compared with our results. We conclude that two different kinds of recombination centers are possible in n-InSb, with energy levels (0.1-0.12)eV above the valence band, or (0.14-0.2)eV respectively. (orig.) [de

  4. Recombination Modulates How Selection Affects Linked Sites in Drosophila

    Science.gov (United States)

    McGaugh, Suzanne E.; Heil, Caiti S. S.; Manzano-Winkler, Brenda; Loewe, Laurence; Goldstein, Steve; Himmel, Tiffany L.; Noor, Mohamed A. F.

    2012-01-01

    One of the most influential observations in molecular evolution has been a strong association between local recombination rate and nucleotide polymorphisms across the genome. This is interpreted as evidence for ubiquitous natural selection. The alternative explanation, that recombination is mutagenic, has been rejected by the absence of a similar association between local recombination rate and nucleotide divergence between species. However, many recent studies show that recombination rates are often very different even in closely related species, questioning whether an association between recombination rate and divergence between species has been tested satisfactorily. To circumvent this problem, we directly surveyed recombination across approximately 43% of the D. pseudoobscura physical genome in two separate recombination maps and 31% of the D. miranda physical genome, and we identified both global and local differences in recombination rate between these two closely related species. Using only regions with conserved recombination rates between and within species and accounting for multiple covariates, our data support the conclusion that recombination is positively related to diversity because recombination modulates Hill–Robertson effects in the genome and not because recombination is predominately mutagenic. Finally, we find evidence for dips in diversity around nonsynonymous substitutions. We infer that at least some of this reduction in diversity resulted from selective sweeps and examine these dips in the context of recombination rate. PMID:23152720

  5. Catalytic hydrogen recombination for nuclear containments

    International Nuclear Information System (INIS)

    Koroll, G.W.; Lau, D.W.P.; Dewit, W.A.; Graham, W.R.C.

    1994-01-01

    Catalytic recombiners appear to be a credible option for hydrogen mitigation in nuclear containments. The passive operation, versatility and ease of back fitting are appealing for existing stations and new designs. Recently, a generation of wet-proofed catalyst materials have been developed at AECL which are highly specific to H 2 -O 2 , are active at ambient temperatures and are being evaluated for containment applications. Two types of catalytic recombiners were evaluated for hydrogen removal in containments based on the AECL catalyst. The first is a catalytic combustor for application in existing air streams such as provided by fans or ventilation systems. The second is an autocatalytic recombiner which uses the enthalpy of reaction to produce natural convective flow over the catalyst elements. Intermediate-scale results obtained in 6 m 3 and 10 m 3 spherical and cylindrical vessels are given to demonstrate self-starting limits, operating limits, removal capacity, scaling parameters, flow resistance, mixing behaviour in the vicinity of an operating recombiner and sensitivity to poisoning, fouling and radiation. (author). 13 refs., 10 figs

  6. Recombinant protein blends: silk beyond natural design.

    Science.gov (United States)

    Dinjaski, Nina; Kaplan, David L

    2016-06-01

    Recombinant DNA technology and new material concepts are shaping future directions in biomaterial science for the design and production of the next-generation biomaterial platforms. Aside from conventionally used synthetic polymers, numerous natural biopolymers (e.g., silk, elastin, collagen, gelatin, alginate, cellulose, keratin, chitin, polyhydroxyalkanoates) have been investigated for properties and manipulation via bioengineering. Genetic engineering provides a path to increase structural and functional complexity of these biopolymers, and thereby expand the catalog of available biomaterials beyond that which exists in nature. In addition, the integration of experimental approaches with computational modeling to analyze sequence-structure-function relationships is starting to have an impact in the field by establishing predictive frameworks for determining material properties. Herein, we review advances in recombinant DNA-mediated protein production and functionalization approaches, with a focus on hybrids or combinations of proteins; recombinant protein blends or 'recombinamers'. We highlight the potential biomedical applications of fibrous protein recombinamers, such as Silk-Elastin Like Polypeptides (SELPs) and Silk-Bacterial Collagens (SBCs). We also discuss the possibility for the rationale design of fibrous proteins to build smart, stimuli-responsive biomaterials for diverse applications. We underline current limitations with production systems for these proteins and discuss the main trends in systems/synthetic biology that may improve recombinant fibrous protein design and production. Copyright © 2016. Published by Elsevier Ltd.

  7. Algae-based oral recombinant vaccines

    Science.gov (United States)

    Specht, Elizabeth A.; Mayfield, Stephen P.

    2014-01-01

    Recombinant subunit vaccines are some of the safest and most effective vaccines available, but their high cost and the requirement of advanced medical infrastructure for administration make them impractical for many developing world diseases. Plant-based vaccines have shifted that paradigm by paving the way for recombinant vaccine production at agricultural scale using an edible host. However, enthusiasm for “molecular pharming” in food crops has waned in the last decade due to difficulty in developing transgenic crop plants and concerns of contaminating the food supply. Microalgae could be poised to become the next candidate in recombinant subunit vaccine production, as they present several advantages over terrestrial crop plant-based platforms including scalable and contained growth, rapid transformation, easily obtained stable cell lines, and consistent transgene expression levels. Algae have been shown to accumulate and properly fold several vaccine antigens, and efforts are underway to create recombinant algal fusion proteins that can enhance antigenicity for effective orally delivered vaccines. These approaches have the potential to revolutionize the way subunit vaccines are made and delivered – from costly parenteral administration of purified protein, to an inexpensive oral algae tablet with effective mucosal and systemic immune reactivity. PMID:24596570

  8. Correlations in the Parton Recombination Model

    Energy Technology Data Exchange (ETDEWEB)

    Bass, S.A. [Department of Physics, Duke University, Durham, NC 27708-0305 (United States); RIKEN BNL Research Center, Brookhaven Nat. Lab., Upton, NY 11973 (United States); Fries, R.J. [School of Physics and Astronomy, Univ. of Minnesota, Minneapolis, MN 55455 (United States); Mueller, B. [Department of Physics, Duke University, Durham, NC 27708-0305 (United States)

    2006-08-07

    We describe how parton recombination can address the recent measurement of dynamical jet-like two particle correlations. In addition we discuss the possible effect realistic light-cone wave-functions including higher Fock-states may have on the well-known elliptic flow valence-quark number scaling law.

  9. Radiative recombination of excitons in amorphous semiconductors

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Jai [School of Engineering and Logistics, Faculty Technology, B-41, Charles Darwin University, Darwin, NT 0909 (Australia)]. E-mail: jai.singh@cdu.edu.au

    2005-04-15

    A theory for calculating the radiative lifetime of excitons in amorphous semiconductors is presented. Four possibilities of excitonic radiative recombination are considered and the corresponding rates are derived at thermal equilibrium. The radiative lifetime is calculated from the inverse of the maximum rate for all the four possibilities. Results agree very well with experiments.

  10. Precise genotyping and recombination detection of Enterovirus

    Science.gov (United States)

    2015-01-01

    Enteroviruses (EV) with different genotypes cause diverse infectious diseases in humans and mammals. A correct EV typing result is crucial for effective medical treatment and disease control; however, the emergence of novel viral strains has impaired the performance of available diagnostic tools. Here, we present a web-based tool, named EVIDENCE (EnteroVirus In DEep conception, http://symbiont.iis.sinica.edu.tw/evidence), for EV genotyping and recombination detection. We introduce the idea of using mixed-ranking scores to evaluate the fitness of prototypes based on relatedness and on the genome regions of interest. Using phylogenetic methods, the most possible genotype is determined based on the closest neighbor among the selected references. To detect possible recombination events, EVIDENCE calculates the sequence distance and phylogenetic relationship among sequences of all sliding windows scanning over the whole genome. Detected recombination events are plotted in an interactive figure for viewing of fine details. In addition, all EV sequences available in GenBank were collected and revised using the latest classification and nomenclature of EV in EVIDENCE. These sequences are built into the database and are retrieved in an indexed catalog, or can be searched for by keywords or by sequence similarity. EVIDENCE is the first web-based tool containing pipelines for genotyping and recombination detection, with updated, built-in, and complete reference sequences to improve sensitivity and specificity. The use of EVIDENCE can accelerate genotype identification, aiding clinical diagnosis and enhancing our understanding of EV evolution. PMID:26678286

  11. Theory of dielectronic recombination and plasma effects

    International Nuclear Information System (INIS)

    Yukap Hahn

    2000-01-01

    Current status of the various theoretical approaches to calculation of dielectronic recombination rates is summarized, with emphasis on the available data base and on the plasma effects of both the plasma ion (and external) fields and plasma electron collisional effects which seriously affect the rates and complicate compilation of data. (author)

  12. Optimization, purification and characterization of recombinant L ...

    African Journals Online (AJOL)

    We studied optimal L-asparaginase sequence from GenBank accession number X12746 encoding for Lasparaginase from Erwinia chrysanthemi NCPPB1125. The expression level of recombinant Lasparaginase was determined as 78% of the total proteins. The purified L-asparaginase had a molecular mass of 37 kDa with ...

  13. Meiotic recombination hotspots - a comparative view.

    Science.gov (United States)

    Choi, Kyuha; Henderson, Ian R

    2015-07-01

    During meiosis homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover. Meiotic recombination has a profound effect on patterns of genetic variation and is an important tool during crop breeding. Crossovers initiate from programmed DNA double-stranded breaks that are processed to form single-stranded DNA, which can invade a homologous chromosome. Strand invasion events mature into double Holliday junctions that can be resolved as crossovers. Extensive variation in the frequency of meiotic recombination occurs along chromosomes and is typically focused in narrow hotspots, observed both at the level of DNA breaks and final crossovers. We review methodologies to profile hotspots at different steps of the meiotic recombination pathway that have been used in different eukaryote species. We then discuss what these studies have revealed concerning specification of hotspot locations and activity and the contributions of both genetic and epigenetic factors. Understanding hotspots is important for interpreting patterns of genetic variation in populations and how eukaryotic genomes evolve. In addition, manipulation of hotspots will allow us to accelerate crop breeding, where meiotic recombination distributions can be limiting. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  14. Asthma and Therapeutics: Recombinant Therapies in Asthma

    Directory of Open Access Journals (Sweden)

    Cockcroft Donald W

    2005-03-01

    Full Text Available Abstract Numerous recombinant therapies are being investigated for the treatment of asthma. This report reviews the current status of several of these novel agents. Anti-immunoglobulin (IgE (omalizumab, Xolair markedly inhibits all aspects of the allergen challenge in subjects who have reduction of free serum IgE to undetectable levels. Several clinical studies in atopic asthma have demonstrated benefit by improved symptoms and lung function and a reduction in corticosteroid requirements. Early use in atopic asthmatics may be even more effective. Several approaches target interleukin (IL-4. Soluble IL-4 receptor has been shown to effectively replace inhaled corticosteroid; further studies are under way. Recombinant anti-IL-5 and recombinant IL-12 inhibit blood and sputum eosinophils and allergen-induced eosinophilia without any effect on airway responsiveness, allergen-induced airway responses, or allergen-induced airway hyperresponsiveness. Efalizumab, a recombinant antibody that inhibits lymphocyte trafficking, is effective in psoriasis. A bronchoprovocation study showed a reduction in allergen-induced late asthmatic response and allergen-induced eosinophilia, which suggests that it should be effective in clinical asthma. These exciting novel therapies provide not only promise of new therapies for asthma but also valuable tools for investigation of asthma mechanisms.

  15. Recombination in disordered regions at semiconductors

    International Nuclear Information System (INIS)

    Artem'ev, V.A.; Mikhnovich, V.V.

    1987-01-01

    Theoretical estimates indicate the need to allow for the heating of carriers by the electrostatic field in disordered regions when studies are made of recombination properties. An analysis is made of the experiments in which the influence of heating on the properties of disordered regions may be manifested and experimentally verifiable effects of this influence are considered

  16. Dielectronic recombination of highly ionized iron

    International Nuclear Information System (INIS)

    Griffin, D.C.; Pindzola, M.S.

    1987-01-01

    Dielectronic recombination of the iron ions Fe/sup 15+/, Fe/sup 23+/, and Fe/sup 25+/ has been studied in the isolated-resonance, distorted-wave approximation. The cross-section calculations include the dielec- tronic transitions associated with the 3s→3l and 3s→4l excitations in Fe/sup 15+/, the 2s→2p and 2s→3l excitations in Fe/sup 23+/, and the 1s→2l excitations in Fe/sup 25+/. The effects of external electric fields have been included by employing intermediate-coupled, field-mixed eigenvectors for the doubly excited Rydberg states, determined by diagonalizing a Hamiltonian matrix which includes the internal electrostatic and spin-orbit terms, as well as the Stark matrix elements. The field effects are found to be quite large in Fe/sup 15+/, relatively small in Fe/sup 23+/, and negligible in Fe/sup 25+/. The calculations indicate that there are large resonances near threshold in Fe/sup 23+/ that are unaffected by external fields and may be measurable in new experiments currently being designed. In addition, the contributions of radiative recombination and the possible interference between radiative and dielectronic recombination in low-lying resonances are considered. Even though the radiative recombination cross sections may be appreciable near threshold in Fe/sup 15+/ and Fe/sup 23+/, the interference between these processes appears to be completely negligible

  17. Expression of recombinant Streptokinase from local Egyptian ...

    African Journals Online (AJOL)

    We reported for the first time the expression of a recombinant SK from a local Streptococcus strain. When produced on industrial scale this r-SK may substantially contribute to reducing the costs of thrombolytic therapy in developing countries. In this study, a highly purified r-SK from Streptococcus sp. isolated from Egyptian ...

  18. Algae-based oral recombinant vaccines

    Directory of Open Access Journals (Sweden)

    Elizabeth A Specht

    2014-02-01

    Full Text Available Recombinant subunit vaccines are some of the safest and most effective vaccines available, but their high cost and the requirement of advanced medical infrastructure for administration make them impractical for many developing world diseases. Plant-based vaccines have shifted that paradigm by paving the way for recombinant vaccine production at agricultural scale using an edible host. However, enthusiasm for molecular pharming in food crops has waned in the last decade due to difficulty in developing transgenic crop plants and concerns of contaminating the food supply. Microalgae are poised to become the next candidate in recombinant subunit vaccine production, and they present several advantages over terrestrial crop plant-based platforms including scalable and contained growth, rapid transformation, easily obtained stable cell lines, and consistent transgene expression levels. Algae have been shown to accumulate and properly fold several vaccine antigens, and efforts are underway to create recombinant algal fusion proteins that can enhance antigenicity for effective orally-delivered vaccines. These approaches have the potential to revolutionize the way subunit vaccines are made and delivered – from costly parenteral administration of purified protein, to an inexpensive oral algae tablet with effective mucosal and system immune reactivity.

  19. Resonances in dissociative recombination: Trends and patterns

    Energy Technology Data Exchange (ETDEWEB)

    Orel, A E; Ngassam, V; Royal, J [Department of Applied Science, University of California, Davis (United States); Roos, J B; Larson, A, E-mail: aeorel@ucdavis.ed [Department of Theoretical Chemistry, Royal Institute of Technology, Stockholm (Sweden)

    2009-11-15

    In dissociative recombination, the kinetic energy of the incident electron is transferred into excitation of the electrons of the target ion and then into kinetic energy of the fragments. In general, this proceeds via a resonance where the electron is temporarily trapped by the ion, leading to efficient energy transfer. The study of dissociative recombination is the study of these resonances, Rydberg states converging to the ground and excited states of the ion. For a number of systems, we have studied the electronic states involved in dissociative recombination, including the ground and excited states of the ion, the resonant states and the bound Rydberg states of the system, by combining electron scattering calculations with multi-reference configuration interaction quantum chemistry calculations. We will report on trends and patterns in these resonance states. We will discuss studies of dissociative recombination of the rare-gas ions, moving down the periodic table from He{sup +}{sub 2} to Ne{sup +}{sub 2} to Ar{sup +}{sub 2}, where the ground electronic state of the ion is constant, but its polarizability increases. We will also present results on isoelectronic polyatomic systems, such as HCO{sup +} and HCNH{sup +}, as well as the effects of changing the electronic structure slightly such as HCN{sup +}/HNC{sup +} and H{sub 2}CO{sup +}.

  20. Expression and characterization of recombinant ecarin.

    NARCIS (Netherlands)

    Jonebring, A.; Lange, U.; Bucha, E.; Deinum, J.; Elg, M.; Lovgren, A.

    2012-01-01

    The snake venom protease ecarin from Echis carinatus was expressed in stable transfected CHO-S cells grown in animal component free cell culture medium. Recombinant ecarin (r-ecarin) was secreted from the suspension adapted Chinese Hamster Ovary (CHO-S) host cells as a pro-protein and activation to

  1. Recombination times in germanium under high pressure

    International Nuclear Information System (INIS)

    Kuyt, J.H.

    1975-01-01

    The influence of pressure on a well defined recombination process was studied. The centres were introduced by γirradiation and the lifetime determined by the decay time of photoconductivity. An optical pressure vessel is described which allows for a hydrostatic variation of 3000 bars. The diffusion constant and lifetime measurements are presented and analysed. (V.J.C.)

  2. Gas recombination assembly for electrochemical cells

    Science.gov (United States)

    Levy, Isaac; Charkey, Allen

    1989-01-01

    An assembly for recombining gases generated in electrochemical cells wherein a catalyst strip is enveloped within a hydrophobic, gas-porous film which, in turn, is encased between gas-porous, metallic layers. The sandwich construction of metallic layers and film is formed into a spiral with a tab for connection to the cell.

  3. Production, purification and characterization of two recombinant ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-06-17

    Jun 17, 2008 ... Two recombinant DNA-derived variants of ovine growth hormone were produced, purified, characterized and compared with the authentic pituitary derived GH. The variants oGH3 and oGH5 were isolated by differential centrifugation method and were purified after refolding by ion-exchange.

  4. Managing meiotic recombination in plant breeding

    NARCIS (Netherlands)

    Wijnker, T.G.; Jong, de J.H.S.G.M.

    2008-01-01

    Crossover recombination is a crucial process in plant breeding because it allows plant breeders to create novel allele combnations on chromosomes that can be used for breeding superior F1 hybrids. Gaining control over this process, in terms of increasing crossover incidence, altering crossover

  5. Radiative recombination of excitons in amorphous semiconductors

    International Nuclear Information System (INIS)

    Singh, Jai

    2005-01-01

    A theory for calculating the radiative lifetime of excitons in amorphous semiconductors is presented. Four possibilities of excitonic radiative recombination are considered and the corresponding rates are derived at thermal equilibrium. The radiative lifetime is calculated from the inverse of the maximum rate for all the four possibilities. Results agree very well with experiments

  6. Some recent developments in the recombination model

    International Nuclear Information System (INIS)

    Hwa, R.C.

    1979-01-01

    A critical review of the recombination model for hadron production at low P/sub T/ is first given, emphasizing not so much the successes as unanswered questions that the model faces. A systematic program to answer some of the basic questions is then developed. The theoretical framework is quantum chromodynamics. First, in what may appear as a digression, the possibility of formation of valence quark clusters (called valons) in a nucleon due to gluon bremsstrahlung and quark-pair creation is considered. Evidences are found not only for the valons in neutrino scattering data, but also indications for their momentum distribution in a nucleon. When similar considerations are applied to a meson, the meaning of the recombination function is discussed and its normalization as well as its shape are determined. Next, the problem of quark decay in a hard scattering process (e.g., pion production in e + e - annihilation) is considered. The joint distribution of partons in a quark jet is determined in QCD. The quark decay function for pions in the recombination model is then obtained with excellent fit to the data. Similar investigation is applied to the problem of photoproduction of pions in the fragmentation region; again good agreement with data is achieved. The results indicate the reliability of the recombination model when the two-parton distributions can be calculated in QCD. Finally, hadron initiated reactions are considered. A duality between quark recombination and valon fragmentation is suggested. The picture is consistent with dual Regge model. A possible way to determine the inclusive distribution in the context of QCD is suggested

  7. A molecular recombination map of Antirrhinum majus

    Directory of Open Access Journals (Sweden)

    Hudson Andrew

    2010-12-01

    Full Text Available Abstract Background Genetic recombination maps provide important frameworks for comparative genomics, identifying gene functions, assembling genome sequences and for breeding. The molecular recombination map currently available for the model eudicot Antirrhinum majus is the result of a cross with Antirrhinum molle, limiting its usefulness within A. majus. Results We created a molecular linkage map of A. majus based on segregation of markers in the F2 population of two inbred lab strains of A. majus. The resulting map consisted of over 300 markers in eight linkage groups, which could be aligned with a classical recombination map and the A. majus karyotype. The distribution of recombination frequencies and distorted transmission of parental alleles differed from those of a previous inter-species hybrid. The differences varied in magnitude and direction between chromosomes, suggesting that they had multiple causes. The map, which covered an estimated of 95% of the genome with an average interval of 2 cM, was used to analyze the distribution of a newly discovered family of MITE transposons and tested for its utility in positioning seven mutations that affect aspects of plant size. Conclusions The current map has an estimated interval of 1.28 Mb between markers. It shows a lower level of transmission ratio distortion and a longer length than the previous inter-species map, making it potentially more useful. The molecular recombination map further indicates that the IDLE MITE transposons are distributed throughout the genome and are relatively stable. The map proved effective in mapping classical morphological mutations of A. majus.

  8. Metabolite profiling of recombinant CHO cells: Designing tailored feeding regimes that enhance recombinant antibody production.

    NARCIS (Netherlands)

    Sellick, C.A.; Croxford, A.S.; Maqsood, A.R.; Stephens, G.; Westerhoff, H.V.; Goodacre, R.; Dickson, A.J.

    2011-01-01

    Chinese hamster ovary (CHO) cells are the primary platform for commercial expression of recombinant therapeutic proteins. Obtaining maximum production from the expression platform requires optimal cell culture medium (and associated nutrient feeds). We have used metabolite profiling to define the

  9. Metabolite profiling of recombinant CHO cells: designing tailored feeding regimes that enhance recombinant antibody production.

    NARCIS (Netherlands)

    Sellick, C.A.; Croxford, A.S.; Maqsood, A.R.; Stephens, G.; Westerhoff, H.V.; Goodacre, R.; Dickson, A.J.

    2011-01-01

    Chinese hamster ovary (CHO) cells are the primary platform for commercial expression of recombinant therapeutic proteins. Obtaining maximum production from the expression platform requires optimal cell culture medium (and associated nutrient feeds). We have used metabolite profiling to define the

  10. Sequence and recombination analyses of the geminivirus replication

    Indian Academy of Sciences (India)

    Prakash

    2006-09-18

    Sep 18, 2006 ... Recombination can provide selective advantage in the evolution of viruses .... Program (v 1.08): Recombination Detection Program (RDP). (Martin and Rybicki ..... Sweet potato leaf curl virus - [US:Louisiana:1994]. AF104036.

  11. New rifamycins produced by a recombinant strain of Nocardia mediterranei.

    Science.gov (United States)

    Schupp, T; Traxler, P; Auden, J A

    1981-08-01

    A recombinant strain of Nocardia mediterranei was found to produce a number of new rifamycins which are structurally related to rifamycin S, rifamycin W and rifamycin G. This strain was derived from two Nocardia mediterranei mutants by intraspecific recombination.

  12. High recombination rate in natural populations of Plasmodium falciparum

    NARCIS (Netherlands)

    Conway, D. J.; Roper, C.; Oduola, A. M.; Arnot, D. E.; Kremsner, P. G.; Grobusch, M. P.; Curtis, C. F.; Greenwood, B. M.

    1999-01-01

    Malaria parasites are sexually reproducing protozoa, although the extent of effective meiotic recombination in natural populations has been debated. If meiotic recombination occurs frequently, compared with point mutation and mitotic rearrangement, linkage disequilibrium between polymorphic sites is

  13. Branching innovation, recombinant innovation, and endogenous technological transitions

    NARCIS (Netherlands)

    Frenken, K.; Izquierdo, L.; Zeppini, P.

    2012-01-01

    We propose a model of technological transitions based on two different types of innovations. Branching innovations refer to technological improvements along a particular path, while recombinant innovations represent fusions of multiple paths. Recombinant innovations create "short-cuts" which reduce

  14. Caenorhabditis briggsae recombinant inbred line genotypes reveal inter-strain incompatibility and the evolution of recombination.

    Directory of Open Access Journals (Sweden)

    Joseph A Ross

    2011-07-01

    Full Text Available The nematode Caenorhabditis briggsae is an emerging model organism that allows evolutionary comparisons with C. elegans and exploration of its own unique biological attributes. To produce a high-resolution C. briggsae recombination map, recombinant inbred lines were generated from reciprocal crosses between two strains and genotyped at over 1,000 loci. A second set of recombinant inbred lines involving a third strain was also genotyped at lower resolution. The resulting recombination maps exhibit discrete domains of high and low recombination, as in C. elegans, indicating these are a general feature of Caenorhabditis species. The proportion of a chromosome's physical size occupied by the central, low-recombination domain is highly correlated between species. However, the C. briggsae intra-species comparison reveals striking variation in the distribution of recombination between domains. Hybrid lines made with the more divergent pair of strains also exhibit pervasive marker transmission ratio distortion, evidence of selection acting on hybrid genotypes. The strongest effect, on chromosome III, is explained by a developmental delay phenotype exhibited by some hybrid F2 animals. In addition, on chromosomes IV and V, cross direction-specific biases towards one parental genotype suggest the existence of cytonuclear epistatic interactions. These interactions are discussed in relation to surprising mitochondrial genome polymorphism in C. briggsae, evidence that the two strains diverged in allopatry, the potential for local adaptation, and the evolution of Dobzhansky-Muller incompatibilities. The genetic and genomic resources resulting from this work will support future efforts to understand inter-strain divergence as well as facilitate studies of gene function, natural variation, and the evolution of recombination in Caenorhabditis nematodes.

  15. Evolution of recombination in eutherian mammals: insights into mechanisms that affect recombination rates and crossover interference

    OpenAIRE

    Segura, Joana; Ferretti, Luca; Ramos-Onsins, Sebastián; Capilla, Laia; Farré, Marta; Reis, Fernanda; Oliver-Bonet, Maria; Fernández-Bellón, Hugo; Garcia, Francisca; Garcia-Caldés, Montserrat; Robinson, Terence J.; Ruiz-Herrera, Aurora

    2013-01-01

    Recombination allows faithful chromosomal segregation during meiosis and contributes to the production of new heritable allelic variants that are essential for the maintenance of genetic diversity. Therefore, an appreciation of how this variation is created and maintained is of critical importance to our understanding of biodiversity and evolutionary change. Here, we analysed the recombination features from species representing the major eutherian taxonomic groups Afrotheria, Rodentia, Primat...

  16. Recombinant zoster (shingles) vaccine, RZV - what you need to know

    Science.gov (United States)

    ... year in the United States get shingles. Shingles vaccine (recombinant) Recombinant shingles vaccine was approved by FDA in 2017 for the ... life-threatening allergic reaction after a dose of recombinant shingles vaccine, or has a severe allergy to any component ...

  17. Bimolecular recombination in ambipolar organic field effect transistors

    NARCIS (Netherlands)

    Charrier, D.S.H.; Vries, T. de; Mathijssen, S.G.J.; Geluk, E.-J.; Smits, E.C.P.; Kemerink, M.; Janssen, R.A.J.

    2009-01-01

    In ambipolar organic field effect transistors (OFET) the shape of the channel potential is intimately related to the recombination zone width W, and hence to the electron–hole recombination strength. Experimentally, the recombination profile can be assessed by scanning Kelvin probe microscopy

  18. Intrinsic and experimental quasiparticle recombination times in superconducting films

    International Nuclear Information System (INIS)

    Eisenmenger, W.; Lassmann, K.; Trumpp, H.J.; Krauss, R.

    1977-01-01

    Experimental quasiparticle recombination lifetime data for superconducting Al, Sn, and Pb films are compared with calculations based on a ray acoustic model taking account of the film thickness dependence of the reabsorption of recombination phonons. Information on the true or intrinsic quasiparticle recombination lifetime obtained from these and other data is discussed. (orig.) [de

  19. Bimolecular recombination in ambipolar organic field effect transistors

    NARCIS (Netherlands)

    Charrier, D. S. H.; de Vries, T.; Mathijssen, S. G. J.; Geluk, E. -J.; Smits, E. C. P.; Kemerink, M.; Janssen, R. A. J.

    In ambipolar organic field effect transistors (OFET) the shape of the channel potential is intimately related to the recombination zone width W, and hence to the electron-hole recombination strength. Experimentally, the recombination profile can be assessed by scanning Kelvin probe microscopy

  20. Anti-proliferative activity of recombinant melittin expressed in ...

    African Journals Online (AJOL)

    Recombinant melittin was then successfully expressed in Escherichia coli. The activity of affinity-purified recombinant melittin was determined in human leukemic U937 cells. Results show that the recombinant melittin had the same anti-proliferative activity in human leukemic U937 cells in vitro as natural one. This shows the ...

  1. Recombination and dissociative recombination of H2+ and H3+ ions on surfaces with application to hydrogen negative ion sources

    International Nuclear Information System (INIS)

    Hiskes, J.R.; Karo, A.M.

    1988-12-01

    A four-step model for recombination and dissociative recombination of H 2 + and H 3 + ions on metal surfaces is discussed. Vibrationally excited molecules, H 2 (v''), from H 3 + recombination are produced in a broad spectrum that enhances the excited level distribution. The application of this latter process to hydrogen negative ion discharges is discussed. 5 refs., 3 figs., 1 tab

  2. Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system.

    Science.gov (United States)

    Schwarz, D; Kisselev, P; Honeck, H; Cascorbi, I; Schunck, W H; Roots, I

    2001-06-01

    1. Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (1462V) and CYP1A1.4 (T461N), were co-expressed with human NADPH-P450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus co-infection to elaborate a suitable system for studying the role of CYPA1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. tinder optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethlylase activities (nmol/min(-1) nmol(-1) CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated approximately 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B[a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-co-infection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CY1A1 variants.

  3. Why Do Sex Chromosomes Stop Recombining?

    Science.gov (United States)

    Ponnikas, Suvi; Sigeman, Hanna; Abbott, Jessica K; Hansson, Bengt

    2018-04-28

    It is commonly assumed that sex chromosomes evolve recombination suppression because selection favours linkage between sex-determining and sexually antagonistic genes. However, although the role of sexual antagonism during sex chromosome evolution has attained strong support from theory, experimental and observational evidence is rare or equivocal. Here, we highlight alternative, often neglected, hypotheses for recombination suppression on sex chromosomes, which invoke meiotic drive, heterozygote advantage, and genetic drift, respectively. We contrast the hypotheses, the situations when they are likely to be of importance, and outline why it is surprisingly difficult to test them. Lastly, we discuss future research directions (including modelling, population genomics, comparative approaches, and experiments) to disentangle the different hypotheses of sex chromosome evolution. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. CFD Analysis of Passive Autocatalytic Recombiner

    Directory of Open Access Journals (Sweden)

    B. Gera

    2011-01-01

    Full Text Available In water-cooled nuclear power reactors, significant quantities of hydrogen could be produced following a postulated loss-of-coolant accident (LOCA along with nonavailability of emergency core cooling system (ECCS. Passive autocatalytic recombiners (PAR are implemented in the containment of water-cooled power reactors to mitigate the risk of hydrogen combustion. In the presence of hydrogen with available oxygen, a catalytic reaction occurs spontaneously at the catalyst surfaces below conventional ignition concentration limits and temperature and even in presence of steam. Heat of reaction produces natural convection flow through the enclosure and promotes mixing in the containment. For the assessment of the PAR performance in terms of maximum temperature of catalyst surface and outlet hydrogen concentration an in-house 3D CFD model has been developed. The code has been used to study the mechanism of catalytic recombination and has been tested for two literature-quoted experiments.

  5. Cosmic microwave background bispectrum from recombination.

    Science.gov (United States)

    Huang, Zhiqi; Vernizzi, Filippo

    2013-03-08

    We compute the cosmic microwave background temperature bispectrum generated by nonlinearities at recombination on all scales. We use CosmoLib2nd, a numerical Boltzmann code at second order to compute cosmic microwave background bispectra on the full sky. We consistently include all effects except gravitational lensing, which can be added to our result using standard methods. The bispectrum is peaked on squeezed triangles and agrees with the analytic approximation in the squeezed limit at the few percent level for all the scales where this is applicable. On smaller scales, we recover previous results on perturbed recombination. For cosmic-variance limited data to l(max)=2000, its signal-to-noise ratio is S/N=0.47, corresponding to f(NL)(eff)=-2.79, and will bias a local signal by f(NL)(loc) ~/= 0.82.

  6. Recombinant organisms for production of industrial products

    Science.gov (United States)

    Adrio, Jose-Luis

    2010-01-01

    A revolution in industrial microbiology was sparked by the discoveries of ther double-stranded structure of DNA and the development of recombinant DNA technology. Traditional industrial microbiology was merged with molecular biology to yield improved recombinant processes for the industrial production of primary and secondary metabolites, protein biopharmaceuticals and industrial enzymes. Novel genetic techniques such as metabolic engineering, combinatorial biosynthesis and molecular breeding techniques and their modifications are contributing greatly to the development of improved industrial processes. In addition, functional genomics, proteomics and metabolomics are being exploited for the discovery of novel valuable small molecules for medicine as well as enzymes for catalysis. The sequencing of industrial microbal genomes is being carried out which bodes well for future process improvement and discovery of new industrial products. PMID:21326937

  7. Bacterial Artificial Chromosome Mutagenesis Using Recombineering

    Directory of Open Access Journals (Sweden)

    Kumaran Narayanan

    2011-01-01

    Full Text Available Gene expression from bacterial artificial chromosome (BAC clones has been demonstrated to facilitate physiologically relevant levels compared to viral and nonviral cDNA vectors. BACs are large enough to transfer intact genes in their native chromosomal setting together with flanking regulatory elements to provide all the signals for correct spatiotemporal gene expression. Until recently, the use of BACs for functional studies has been limited because their large size has inherently presented a major obstacle for introducing modifications using conventional genetic engineering strategies. The development of in vivo homologous recombination strategies based on recombineering in E. coli has helped resolve this problem by enabling facile engineering of high molecular weight BAC DNA without dependence on suitably placed restriction enzymes or cloning steps. These techniques have considerably expanded the possibilities for studying functional genetics using BACs in vitro and in vivo.

  8. Recombination clumping factor during cosmic reionization

    International Nuclear Information System (INIS)

    Kaurov, Alexander A.; Gnedin, Nickolay Y.

    2014-01-01

    We discuss the role of recombinations in the intergalactic medium, and the related concept of the clumping factor, during cosmic reionization. The clumping factor is, in general, a local quantity that depends on both the local overdensity and the scale below which the baryon density field can be assumed smooth. That scale, called the filtering scale, depends on over-density and local thermal history. We present a method for building a self-consistent analytical model of inhomogeneous reionization, assuming the linear growth rate of the density fluctuation, which simultaneously accounts for these effects. We show that taking into account the local clumping factor introduces significant corrections to the total recombination rate, compared to the model with a globally uniform clumping factor.

  9. Recombinant organisms for production of industrial products.

    Science.gov (United States)

    Adrio, Jose-Luis; Demain, Arnold L

    2010-01-01

    A revolution in industrial microbiology was sparked by the discoveries of ther double-stranded structure of DNA and the development of recombinant DNA technology. Traditional industrial microbiology was merged with molecular biology to yield improved recombinant processes for the industrial production of primary and secondary metabolites, protein biopharmaceuticals and industrial enzymes. Novel genetic techniques such as metabolic engineering, combinatorial biosynthesis and molecular breeding techniques and their modifications are contributing greatly to the development of improved industrial processes. In addition, functional genomics, proteomics and metabolomics are being exploited for the discovery of novel valuable small molecules for medicine as well as enzymes for catalysis. The sequencing of industrial microbal genomes is being carried out which bodes well for future process improvement and discovery of new industrial products. © 2010 Landes Bioscience

  10. Recombinant cells and organisms having persistent nonstandard amino acid dependence and methods of making them

    Science.gov (United States)

    Church, George M.; Mandell, Daniel J.; Lajoie, Marc J.

    2017-12-05

    Recombinant cells and recombinant organisms persistently expressing nonstandard amino acids (NSAAs) are provided. Methods of making recombinant cells and recombinant organisms dependent on persistently expressing NSAAs for survival are also provided. These methods may be used to make safe recombinant cells and recombinant organisms and/or to provide a selective pressure to maintain one or more reassigned codon functions in recombinant cells and recombinant organisms.

  11. Modelling of procecces in catalytic recombiners

    International Nuclear Information System (INIS)

    Boehm, J.

    2007-01-01

    In order to achieve a high degree of safety in nuclear power plants and prevent possible accident scenarios, their consequences are calculated and analysed with numeric codes. One of the most important part of nuclear safety research of hazardous incidents are development and validation of these numeric models, which are implemented into accident codes. The severe hydrogen release during a core meltdown is one of the considered scenario of performed accident analyses. One of the most important measure for the elimination of the hydrogen is catalytic recombiners. Converting the hydrogen with the atmospheric oxygen to water vapor in an exothermic reaction will prevent possible detonation of the hydrogen/air atmosphere. Within the dissertation the recombiner simulation REKO-DIREKT was developed and validated by an extensive experimental database. The performance of recombiners with regard to the conversion of the hydrogen and the temperature development is modelled. The REKO-DIREKT program is unique and has made significant revolution in research of hydrogen safety. For the first time it has been possible to show the performance of the recombiner so great in detail by using REKO-DIREKT. In the future engineers of nuclear power plants will have opportunity to have precise forecasts about the process of the possible accidents with hydrogen release. Also with presence of water vapor or with oxygen depletion which are included in the model. The major discussion of the hydrogen ignition at hot catalyst steel plates can be evaluated in the future with REKO-DIREKT more reliably than the existing used models. (orig.)

  12. Cultivating Insect Cells To Produce Recombinant Proteins

    Science.gov (United States)

    Spaulding, Glenn; Goodwin, Thomas; Prewett, Tacey; Andrews, Angela; Francis, Karen; O'Connor, Kim

    1996-01-01

    Method of producing recombinant proteins involves growth of insect cells in nutrient solution in cylindrical bioreactor rotating about cylindrical axis, oriented horizontally and infecting cells with viruses into which genes of selected type cloned. Genes in question those encoding production of desired proteins. Horizontal rotating bioreactor preferred for use in method, denoted by acronym "HARV", described in "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662).

  13. Recombinant Amphiphilic Protein Micelles for Drug Delivery

    OpenAIRE

    Kim, Wookhyun; Xiao, Jiantao; Chaikof, Elliot L.

    2011-01-01

    Amphiphilic block polypeptides can self-assemble into a range of nanostructures in solution, including micelles and vesicles. Our group has recently described the capacity of recombinant amphiphilic diblock copolypeptides to form highly stable micelles. In this report, we demonstrate the utility of protein nanoparticles to serve as a vehicle for controlled drug delivery. Drug-loaded micelles were produced by encapsulating dipyridamole as a model hydrophobic drug with anti-inflammatory activit...

  14. Development of Mycoplasma hyopneumoniae Recombinant Vaccines.

    Science.gov (United States)

    Marchioro, Silvana Beutinger; Simionatto, Simone; Dellagostin, Odir

    2016-01-01

    Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia (EP), a disease that affects swine production worldwide. Vaccination is the most cost-effective strategy for the control and prevention of the disease. Research using genome-based approach has the potential to elucidate the biology and pathogenesis of M. hyopneumoniae and contribute to the development of more effective vaccines. Here, we describe the protocol for developing M. hyopneumoniae recombinant vaccines using reverse vaccinology approaches.

  15. Recombinant protein expression in microbial systems

    OpenAIRE

    Rosano, Germán L.; Ceccarelli, Eduardo A.

    2014-01-01

    The emergence of recombinant DNA technology during the early 70's set a revolution in molecular biology. This set of techniques was strengthened even further later on with the introduction of the polymerase chain reaction and allowed scientists to explore and understand essential life processes in an easy and straightforward way. It also marked the birth of the modern biotech industry. At that time, it was shown that eukaryotic DNA could be propagated in Escherichia coli (Morrow et al., 1974)...

  16. Recent advances in DNA repair and recombination.

    Science.gov (United States)

    Iwanejko, L A; Jones, N J

    1998-09-11

    The subjects of the talks at this 1-day DNA Repair Network meeting, held at City University, London on December 15, 1997, encompassed a range of topics and reflected some of the current areas of research in the United Kingdom. Topics included DNA double-strand break repair, V(D)J recombination, DNA ligases, the RecQ family of helicases and Bloom's syndrome, UVB and immunosuppression, the repair of oxidative damage and mismatch repair mechanisms.

  17. Dielectronic recombination of Xe8+ ions

    International Nuclear Information System (INIS)

    Zhang Guoding; Fu Yanbiao; Dong Chenzhong; Zhang Yizhao

    2012-01-01

    Based on the fully relativistic configuration interaction method, theoretical calculations are carried out for the dielectronic recombination (DR) rate coefficients of Xe 8+ ions in the temperature region from 0.1 to 1 650 eV. The comparison of the DR rate coefficients from 4s, 4p and 4d subshell excitations shows that 4d subshell excitation dominates in the whole temperature region. The contribution from 4p subshell excitation is very important at temperature above 10 eV and the contributions from 4s subshell ex- citation is lower than 7.5% in the whole temperature region. Similarly, the comparison of the DR rate coefficients through △n= 0, I and 2 core excitation shows that the contribution from △n= 2 core excitation can not be neglected, the contributions from n'>15 can also not be neglected. The DR rate coefficients of △n=0, 1 and 2 core excitation and the total DR rate coefficients are fitted with some parameters, which are in good agreement with theoretical calculations values (within 1 % difference)The total DR rate coefficients are greater than radiative recombination (RR) and three-body recombination (TBR) rate coefficients at temperature above 1 eV. Therefore, the DR process can strongly influence the ionization balance of laser produced xenon plasmas. (authors)

  18. Dissociative recombination of small molecular ions

    International Nuclear Information System (INIS)

    Mul, P.M.

    1981-01-01

    In this thesis an analysis is given of merged electron-ion beam experiment and work on dissociative recombination of molecular ions and electrons is described. Chapter II covers a brief introduction of the theory of dissociative recombination. In chapter III, a description is given of the merged electron-ion beam experiment and a method is described which allows the determination of the mean angle between the electron and ion trajectories in a merged electron-ion beam experiment. In chapter IV a paper on the three dominant atmospheric diatomic ions NO + , O 2 + and N 2 + is presented and in chapter V the dissociative recombination for N 2 H + and N 2 D + is discussed. In chapter VI two papers on the polyatomic ions of the carbon-containing molecular ions are presented, and in chapter VII a letter with some results of the work presented in more detail in the chapters IV, V and VI is presented. The magnitude and the energy dependence of the cross-section measured by the merged beam technique and by other techniques is compared and discussed. (Auth.)

  19. FASEB Summer Research Conference. Genetic Recombination and Chromosome Rearrangements

    Energy Technology Data Exchange (ETDEWEB)

    Jinks-Robertson, Sue

    2002-02-01

    The 2001 meeting entitled ''Genetic Recombination and Genome Rearrangements'' was held July 21-26 in Snowmass, Colorado. The goal of the meeting was to bring together scientists using diverse approaches to study all aspects of genetic recombination. This goal was achieved by integrating talks covering the genetics, biochemistry and structural biology of homologous recombination, site-specific recombination, and nonhomologous recombination. The format of the meeting consisted of a keynote address on the opening evening, two formal plenary sessions on each of the four full meeting days, a single afternoon workshop consisting of short talks chosen from among submitted abstracts, and afternoon poster sessions on each of the four full meeting days. The eight plenary session were entitled: (1) Recombination Mechanisms, (2) Prokaryotic Recombination, (3) Repair and Recombination, (4) Site-specific Recombination and Transposition, (5) Eukaryotic Recombination I, (6) Genome Rearrangements, (7) Meiosis, and (8) Eukaryotic Recombination II. Each session included a mix of genetic, biochemical and structural talks; talks were limited to 20 minutes, followed by 10 minutes of very lively, general discussion. Much of the data presented in the plenary sessions was unpublished, thus providing attendees with the most up-to-date knowledge of this rapidly-moving field.

  20. Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae, is permissive for AcMNPV infection and produces high levels of recombinant proteins

    Directory of Open Access Journals (Sweden)

    Zhang Sheng

    2010-07-01

    Full Text Available Abstract Background The insect cell line is a critical component in the production of recombinant proteins in the baculovirus expression system and new cell lines hold the promise of increasing both quantity and quality of protein production. Results Seventy cell lines were established by single-cell cloning from a primary culture of cells derived from eggs of the black witch moth (Ascalapha odorata; Lepidoptera, Noctuidae. Among 8 rapidly growing lines, cell line 38 (Ao38 was selected for further analysis, based on susceptibility to AcMNPV infection and production of secreted alkaline phosphatase (SEAP from a baculovirus expression vector. In comparisons with low-passage High Five (BTI-Tn-5B1-4 cells, infected Ao38 cells produced β-galactosidase and SEAP at levels higher (153% and 150%, respectively than those measured from High Five cells. Analysis of N-glycans of SEAP produced in Ao38 cells revealed two N-glycosylation sites and glycosylation patterns similar to those reported for High Five and Sf9 cells. Glycopeptide isoforms consisted of pauci- or oligomannose, with and without fucose on N-acetylglucosamine(s linked to asparagine residues. Estimates of Ao38 cell volume suggest that Ao38 cells are approximately 2.5× larger than Sf9 cells but only approximately 74% of the size of High Five cells. Ao38 cells were highly susceptible to AcMNPV infection, similar to infectivity of Sf9 cells. Production of infectious AcMNPV budded virions from Ao38 cells peaked at approximately 4.5 × 107 IU/ml, exceeding that from High Five cells while lower than that from Sf9 cells. Ao38 cells grew rapidly in stationary culture with a population doubling time of 20.2 hr, and Ao38 cells were readily adapted to serum-free medium (Sf-900III and to a suspension culture system. Analysis of Ao38 and a parental Ascalapha odorata cell line indicated that these lines were free of the alphanodavirus that was recently identified as an adventitious agent in High Five cell

  1. Recombination pattern reanalysis of some HIV-1 circulating recombination forms suggest the necessity and difficulty of revision.

    Directory of Open Access Journals (Sweden)

    Lei Jia

    Full Text Available Recombination is one of the major mechanisms underlying the generation of HIV-1 variability. Currently 61 circulating recombinant forms of HIV-1 have been identified. With the development of recombination detection techniques and accumulation of HIV-1 reference stains, more accurate mosaic structures of circulating recombinant forms (CRFs, like CRF04 and CRF06, have undergone repeated analysis and upgrades. Such revisions may also be necessary for other CRFs. Unlike previous studies, whose results are based primarily on a single recombination detection program, the current study was based on multiple recombination analysis, which may have produced more impartial results.Representative references of 3 categories of intersubtype recombinants were selected, including BC recombinants (CRF07 and CRF08, BG recombinants (CRF23 and CRF24, and BF recombinants (CRF38 and CRF44. They were reanalyzed in detail using both the jumping profile hidden Markov model and RDP3.The results indicate that revisions and upgrades are very necessary and the entire re-analysis suggested 2 types of revision: (i length of inserted fragments; and (ii number of inserted fragments. The reanalysis also indicated that determination of small regions of about 200 bases or fewer should be performed with more caution.Results indicated that the involvement of multiple recombination detection programs is very necessary. Additionally, results suggested two major challenges, one involving the difficulty of accurately determining the locations of breakpoints and the second involving identification of small regions of about 200 bases or fewer with greater caution. Both indicate the complexity of HIV-1 recombination. The resolution would depend critically on development of a recombination analysis algorithm, accumulation of HIV-1 stains, and a higher sequencing quality. With the changes in recombination pattern, phylogenetic relationships of some CRFs may also change. All these results may

  2. Mechanisms and factors that influence high frequency retroviral recombination

    DEFF Research Database (Denmark)

    Delviks-Frankenberry, Krista; Galli, Andrea; Nikolaitchik, Olga

    2011-01-01

    With constantly changing environmental selection pressures, retroviruses rely upon recombination to reassort polymorphisms in their genomes and increase genetic diversity, which improves the chances for the survival of their population. Recombination occurs during DNA synthesis, whereby reverse...... transcriptase undergoes template switching events between the two copackaged RNAs, resulting in a viral recombinant with portions of the genetic information from each parental RNA. This review summarizes our current understanding of the factors and mechanisms influencing retroviral recombination, fidelity...... of the recombination process, and evaluates the subsequent viral diversity and fitness of the progeny recombinant. Specifically, the high mutation rates and high recombination frequencies of HIV-1 will be analyzed for their roles in influencing HIV-1 global diversity, as well as HIV-1 diagnosis, drug treatment...

  3. Mechanisms and Factors that Influence High Frequency Retroviral Recombination

    Science.gov (United States)

    Delviks-Frankenberry, Krista; Galli, Andrea; Nikolaitchik, Olga; Mens, Helene; Pathak, Vinay K.; Hu, Wei-Shau

    2011-01-01

    With constantly changing environmental selection pressures, retroviruses rely upon recombination to reassort polymorphisms in their genomes and increase genetic diversity, which improves the chances for the survival of their population. Recombination occurs during DNA synthesis, whereby reverse transcriptase undergoes template switching events between the two copackaged RNAs, resulting in a viral recombinant with portions of the genetic information from each parental RNA. This review summarizes our current understanding of the factors and mechanisms influencing retroviral recombination, fidelity of the recombination process, and evaluates the subsequent viral diversity and fitness of the progeny recombinant. Specifically, the high mutation rates and high recombination frequencies of HIV-1 will be analyzed for their roles in influencing HIV-1 global diversity, as well as HIV-1 diagnosis, drug treatment, and vaccine development. PMID:21994801

  4. Multiple barriers to recombination between divergent HIV-1 variants revealed by a dual-marker recombination assay

    DEFF Research Database (Denmark)

    Nikolaitchik, Olga A; Galli, Andrea; Moore, Michael D

    2011-01-01

    Recombination is a major force for generating human immunodeficiency virus type 1 (HIV-1) diversity and produces numerous recombinants circulating in the human population. We previously established a cell-based system using green fluorescent protein gene (gfp) as a reporter to study the mechanisms...... of HIV-1 recombination. We now report an improved system capable of detecting recombination using authentic viral sequences. Frameshift mutations were introduced into the gag gene so that parental viruses do not express full-length Gag; however, recombination can generate a progeny virus that expresses...

  5. Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

    Science.gov (United States)

    Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki; Gang, David R.; Sarkanen, Simo; Ford, Joshua D.

    2001-04-03

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  6. Late replicating domains are highly recombining in females but have low male recombination rates: implications for isochore evolution.

    Directory of Open Access Journals (Sweden)

    Catherine J Pink

    Full Text Available In mammals sequences that are either late replicating or highly recombining have high rates of evolution at putatively neutral sites. As early replicating domains and highly recombining domains both tend to be GC rich we a priori expect these two variables to covary. If so, the relative contribution of either of these variables to the local neutral substitution rate might have been wrongly estimated owing to covariance with the other. Against our expectations, we find that sex-averaged recombination rates show little or no correlation with replication timing, suggesting that they are independent determinants of substitution rates. However, this result masks significant sex-specific complexity: late replicating domains tend to have high recombination rates in females but low recombination rates in males. That these trends are antagonistic explains why sex-averaged recombination is not correlated with replication timing. This unexpected result has several important implications. First, although both male and female recombination rates covary significantly with intronic substitution rates, the magnitude of this correlation is moderately underestimated for male recombination and slightly overestimated for female recombination, owing to covariance with replicating timing. Second, the result could explain why male recombination is strongly correlated with GC content but female recombination is not. If to explain the correlation between GC content and replication timing we suppose that late replication forces reduced GC content, then GC promotion by biased gene conversion during female recombination is partly countered by the antagonistic effect of later replicating sequence tending increase AT content. Indeed, the strength of the correlation between female recombination rate and local GC content is more than doubled by control for replication timing. Our results underpin the need to consider sex-specific recombination rates and potential covariates in

  7. Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance

    Science.gov (United States)

    Kempf, Brian J.; Peersen, Olve B.

    2016-01-01

    ABSTRACT RNA recombination is important in the formation of picornavirus species groups and the ongoing evolution of viruses within species groups. In this study, we examined the structure and function of poliovirus polymerase, 3Dpol, as it relates to RNA recombination. Recombination occurs when nascent RNA products exchange one viral RNA template for another during RNA replication. Because recombination is a natural aspect of picornavirus replication, we hypothesized that some features of 3Dpol may exist, in part, to facilitate RNA recombination. Furthermore, we reasoned that alanine substitution mutations that disrupt 3Dpol-RNA interactions within the polymerase elongation complex might increase and/or decrease the magnitudes of recombination. We found that an L420A mutation in 3Dpol decreased the frequency of RNA recombination, whereas alanine substitutions at other sites in 3Dpol increased the frequency of recombination. The 3Dpol Leu420 side chain interacts with a ribose in the nascent RNA product 3 nucleotides from the active site of the polymerase. Notably, the L420A mutation that reduced recombination also rendered the virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin-induced G→A and C→U mutations in viral RNA. We conclude that 3Dpol Leu420 is critically important for RNA recombination and that RNA recombination contributes to ribavirin resistance. IMPORTANCE Recombination contributes to the formation of picornavirus species groups and the emergence of circulating vaccine-derived polioviruses (cVDPVs). The recombinant viruses that arise in nature are occasionally more fit than either parental strain, especially when the two partners in recombination are closely related, i.e., members of characteristic species groups, such as enterovirus species groups A to H or rhinovirus species groups A to C. Our study shows that RNA recombination requires conserved features of the viral polymerase. Furthermore, a

  8. Recombination: the good, the bad and the variable.

    Science.gov (United States)

    Stapley, Jessica; Feulner, Philine G D; Johnston, Susan E; Santure, Anna W; Smadja, Carole M

    2017-12-19

    Recombination, the process by which DNA strands are broken and repaired, producing new combinations of alleles, occurs in nearly all multicellular organisms and has important implications for many evolutionary processes. The effects of recombination can be good , as it can facilitate adaptation, but also bad when it breaks apart beneficial combinations of alleles, and recombination is highly variable between taxa, species, individuals and across the genome. Understanding how and why recombination rate varies is a major challenge in biology. Most theoretical and empirical work has been devoted to understanding the role of recombination in the evolution of sex-comparing between sexual and asexual species or populations. How recombination rate evolves and what impact this has on evolutionary processes within sexually reproducing organisms has received much less attention. This Theme Issue focusses on how and why recombination rate varies in sexual species, and aims to coalesce knowledge of the molecular mechanisms governing recombination with our understanding of the evolutionary processes driving variation in recombination within and between species. By integrating these fields, we can identify important knowledge gaps and areas for future research, and pave the way for a more comprehensive understanding of how and why recombination rate varies. © 2017 The Authors.

  9. Genetic evidence for inducibility of recombination competence in yeast

    International Nuclear Information System (INIS)

    Fabre, F.; Roman, H.

    1977-01-01

    Recombination between unirradiated chromosomes was induced by UV or x-ray irradiation of haploids followed by a mating with heteroallelic diploids of Saccharomyces cerevisiae. The selected event of intragenic recombination did not involve the participation of the irradiated chromosome and apparently was not caused by lesions introduced into the unirradiated chromosomes by some indirect process. The results favor the idea that recombination is repressed in the majority of vegetative cells and that one effect of radiation is the release of some factor(s) necessary for recombination. Consequently, the proportion of competent cells (i.e., cells able to recombine) in the population increases. This competent state seems necessary not only for the recombinational repair of radiation-induced lesions but also, since recombinants are produced in the absence of such lesions, for spontaneous recombination. Photoreactivation of the UV-irradiated haploids led to a decrease in the production of recombinants. Hence, lesions in the DNA appear to be responsible for the induction of the recombinational ability

  10. [Construction and expression of a recombinant adenovirus with LZP3].

    Science.gov (United States)

    Chen, Bang-dang; Zhang, Fu-chun; Sun, Mei-yu; Li, Yi-jie; Ma, Zheng-hai

    2007-08-01

    To explore a new immunocontraceptive vaccine and construct an attenuated recombinant adenoviral vaccine against Lagurus lagurus zona pellucida 3(LZP3). LZP3 gene was subcloned into the shuttle vector pShuttle-CMV, and then a two-step transformation procedure was employed to construct a recombinant adenoviral plasmid with LZP3, which was digested with Pac I and transfected into HEK293 cells to package recombinant adenovirus particles. Finally, HeLa cells were infected by the recombinant adenovirus. LZP3 gene was detected from the recombinant virus by PCR, and its transcription and expression were analyzed by RT-PCR and Western blot. Recombinant adenovirus vector pAd-LZP3 with LZP3 gene was constructed by homologous recombination in E.coli, and a recombinant adenovirus was obtained by transfecting HEK293 cells with pAd-LZP3. PCR test indicated that LZP3 gene was successfully integrated into the adenoviral genome, and the titer of the recombinant adenovirus reached 1.2x10(10) pfu/L. The transcription and expression of LZP3 gene in the infected HeLa cells were confirmed by RT-PCR and Western blot. The recombinant adenovirus RAd-LZP3 can be successfully expressed in the infected HeLa cells, which lays the foundation for further researches into immunizing animals with RAd-LZP3.

  11. V(D)J recombination frequency is affected by the sequence interposed between a pair of recombination signals: sequence comparison reveals a putative recombinational enhancer element

    DEFF Research Database (Denmark)

    Roch, F A; Hobi, R; Berchtold, M W

    1997-01-01

    respectively, can markedly affect the frequency of V(D)J recombination. We report that the entire Emu, the Emu core as well as its flanking 5' and 3' matrix associated regions (5' and 3' MARs) upregulate V(D)J recombination while the downstream section of the 3' MAR of Emu does not. Also, prokaryotic sequences...

  12. Recombination-deficient mutants of Bacillus subtilis

    International Nuclear Information System (INIS)

    Sadaie, Y.; Kada, T.

    1976-01-01

    Two mutant strains of Bacillus subtilis Marburg, NIG43 and NIG45, were isolated. They showed high sensitivities to gamma rays, ultraviolet light (uv), and chemicals. Deficiencies in genetic recombination of these two mutants were shown by the experiments on their capacity in transformation, SPO2 transfection, and PBS1 phage transduction, as well as on their radiation and drug sensitivities and their Hcr + capacity for uv-exposed phage M2. Some of these characteristics were compared with those of the known strains possessing the recA1 or recB2 alleles. Mapping studies revealed that the mutation rec-43 of strain NIG43 lies in the region of chromosome replication origin. The order was purA dna-8132 rec-43. Another mutation, rec-45, of strain NIG45 was found to be tightly linked to recA1. The mutation rec-43 reduced mainly the frequency of PBS1 transduction. On the other hand, the mutation rec-45 reduced the frequency of recombination involved both in transformation and PBS1 tranduction. The mutation rec-43 of strain NIG43 is conditional, but rec-45 of strain NIG45 is not. The uv impairment in cellular survival of strain NIG43 was gradually reverted at higher salt or sucrose concentrations, suggesting cellular possession of a mutated gene product whose function is conditional. In contrast to several other recombination-deficient strains, SPO2 lysogens of strains NIG43 and NIG45 were not inducible, indicating involvement of rec-43 + or rec-45 + gene product in the development of SPO2 prophage to a vegetative form. The uv-induced deoxyribonucleic acid degradation in vegetative cells was higher in rec-43 and rec-45 strains

  13. Recombinational DNA repair and human disease

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Larry H.; Schild, David

    2002-11-30

    We review the genes and proteins related to the homologous recombinational repair (HRR) pathway that are implicated in cancer through either genetic disorders that predispose to cancer through chromosome instability or the occurrence of somatic mutations that contribute to carcinogenesis. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and an ataxia-like disorder (ATLD), are chromosome instability disorders that are defective in the ataxia telangiectasia mutated (ATM), NBS, and Mre11 genes, respectively. These genes are critical in maintaining cellular resistance to ionizing radiation (IR), which kills largely by the production of double-strand breaks (DSBs). Bloom syndrome involves a defect in the BLM helicase, which seems to play a role in restarting DNA replication forks that are blocked at lesions, thereby promoting chromosome stability. The Werner syndrome gene (WRN) helicase, another member of the RecQ family like BLM, has very recently been found to help mediate homologous recombination. Fanconi anemia (FA) is a genetically complex chromosomal instability disorder involving seven or more genes, one of which is BRCA2. FA may be at least partially caused by the aberrant production of reactive oxidative species. The breast cancer-associated BRCA1 and BRCA2 proteins are strongly implicated in HRR; BRCA2 associates with Rad51 and appears to regulate its activity. We discuss in detail the phenotypes of the various mutant cell lines and the signaling pathways mediated by the ATM kinase. ATM's phosphorylation targets can be grouped into oxidative stress-mediated transcriptional changes, cell cycle checkpoints, and recombinational repair. We present the DNA damage response pathways by using the DSB as the prototype lesion, whose incorrect repair can initiate and augment karyotypic abnormalities.

  14. Recombinational DNA repair and human disease

    International Nuclear Information System (INIS)

    Thompson, Larry H.; Schild, David

    2002-01-01

    We review the genes and proteins related to the homologous recombinational repair (HRR) pathway that are implicated in cancer through either genetic disorders that predispose to cancer through chromosome instability or the occurrence of somatic mutations that contribute to carcinogenesis. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and an ataxia-like disorder (ATLD), are chromosome instability disorders that are defective in the ataxia telangiectasia mutated (ATM), NBS, and Mre11 genes, respectively. These genes are critical in maintaining cellular resistance to ionizing radiation (IR), which kills largely by the production of double-strand breaks (DSBs). Bloom syndrome involves a defect in the BLM helicase, which seems to play a role in restarting DNA replication forks that are blocked at lesions, thereby promoting chromosome stability. The Werner syndrome gene (WRN) helicase, another member of the RecQ family like BLM, has very recently been found to help mediate homologous recombination. Fanconi anemia (FA) is a genetically complex chromosomal instability disorder involving seven or more genes, one of which is BRCA2. FA may be at least partially caused by the aberrant production of reactive oxidative species. The breast cancer-associated BRCA1 and BRCA2 proteins are strongly implicated in HRR; BRCA2 associates with Rad51 and appears to regulate its activity. We discuss in detail the phenotypes of the various mutant cell lines and the signaling pathways mediated by the ATM kinase. ATM's phosphorylation targets can be grouped into oxidative stress-mediated transcriptional changes, cell cycle checkpoints, and recombinational repair. We present the DNA damage response pathways by using the DSB as the prototype lesion, whose incorrect repair can initiate and augment karyotypic abnormalities

  15. Creating Porcine Biomedical Models Through Recombineering

    Directory of Open Access Journals (Sweden)

    Lawrence B. Schook

    2006-03-01

    Full Text Available Recent advances in genomics provide genetic information from humans and other mammals (mouse, rat, dog and primates traditionally used as models as well as new candidates (pigs and cattle. In addition, linked enabling technologies, such as transgenesis and animal cloning, provide innovative ways to design and perform experiments to dissect complex biological systems. Exploitation of genomic information overcomes the traditional need to choose naturally occurring models. Thus, investigators can utilize emerging genomic knowledge and tools to create relevant animal models. This approach is referred to as reverse genetics. In contrast to ‘forward genetics’, in which gene(s responsible for a particular phenotype are identified by positional cloning (phenotype to genotype, the ‘reverse genetics’ approach determines the function of a gene and predicts the phenotype of a cell, tissue, or organism (genotype to phenotype. The convergence of classical and reverse genetics, along with genomics, provides a working definition of a ‘genetic model’ organism (3. The recent construction of phenotypic maps defining quantitative trait loci (QTL in various domesticated species provides insights into how allelic variations contribute to phenotypic diversity. Targeted chromosomal regions are characterized by the construction of bacterial artificial chromosome (BAC contigs to isolate and characterize genes contributing towards phenotypic variation. Recombineering provides a powerful methodology to harvest genetic information responsible for phenotype. Linking recombineering with gene-targeted homologous recombination, coupled with nuclear transfer (NT technology can provide ‘clones’ of genetically modified animals.

  16. Functions and structures of eukaryotic recombination proteins

    International Nuclear Information System (INIS)

    Ogawa, Tomoko

    1994-01-01

    We have found that Rad51 and RecA Proteins form strikingly similar structures together with dsDNA and ATP. Their right handed helical nucleoprotein filaments extend the B-form DNA double helixes to 1.5 times in length and wind the helix. The similarity and uniqueness of their structures must reflect functional homologies between these proteins. Therefore, it is highly probable that similar recombination proteins are present in various organisms of different evolutional states. We have succeeded to clone RAD51 genes from human, mouse, chicken and fission yeast genes, and found that the homologues are widely distributed in eukaryotes. The HsRad51 and MmRad51 or ChRad51 proteins consist of 339 amino acids differing only by 4 or 12 amino acids, respectively, and highly homologous to both yeast proteins, but less so to Dmcl. All of these proteins are homologous to the region from residues 33 to 240 of RecA which was named ''homologous core. The homologous core is likely to be responsible for functions common for all of them, such as the formation of helical nucleoprotein filament that is considered to be involved in homologous pairing in the recombination reaction. The mouse gene is transcribed at a high level in thymus, spleen, testis, and ovary, at lower level in brain and at a further lower level in some other tissues. It is transcribed efficiently in recombination active tissues. A clear functional difference of Rad51 homologues from RecA was suggested by the failure of heterologous genes to complement the deficiency of Scrad51 mutants. This failure seems to reflect the absence of a compatible partner, such as ScRad52 protein in the case of ScRad51 protein, between different species. Thus, these discoveries play a role of the starting point to understand the fundamental gene targeting in mammalian cells and in gene therapy. (J.P.N.)

  17. Classification of Recombinant Biologics in the EU

    DEFF Research Database (Denmark)

    Klein, Kevin; De Bruin, Marie L; Broekmans, Andre W

    2015-01-01

    BACKGROUND AND OBJECTIVE: Biological medicinal products (biologics) are subject to specific pharmacovigilance requirements to ensure that biologics are identifiable by brand name and batch number in adverse drug reaction (ADR) reports. Since Member States collect ADR data at the national level...... of biologics by national authorities responsible for ADR reporting. METHODS: A sample list of recombinant biologics from the European Medicines Agency database of European Public Assessment Reports was created to analyze five Member States (Belgium, the Netherlands, Spain, Sweden, and the UK) according...

  18. Hydrogen recombiner catalyst test supporting data

    International Nuclear Information System (INIS)

    Britton, M.D.

    1995-01-01

    This is a data package supporting the Hydrogen Recombiner Catalyst Performance and Carbon Monoxide Sorption Capacity Test Report, WHC-SD-WM-TRP-211, Rev 0. This report contains 10 appendices which consist of the following: Mass spectrometer analysis reports: HRC samples 93-001 through 93-157; Gas spectrometry analysis reports: HRC samples 93-141 through 93-658; Mass spectrometer procedure PNL-MA-299 ALO-284; Alternate analytical method for ammonia and water vapor; Sample log sheets; Job Safety analysis; Certificate of mixture analysis for feed gases; Flow controller calibration check; Westinghouse Standards Laboratory report on Bois flow calibrator; and Sorption capacity test data, tables, and graphs

  19. Nanobodies and recombinant binders in cell biology

    Science.gov (United States)

    Helma, Jonas; Cardoso, M. Cristina; Muyldermans, Serge

    2015-01-01

    Antibodies are key reagents to investigate cellular processes. The development of recombinant antibodies and binders derived from natural protein scaffolds has expanded traditional applications, such as immunofluorescence, binding arrays, and immunoprecipitation. In addition, their small size and high stability in ectopic environments have enabled their use in all areas of cell research, including structural biology, advanced microscopy, and intracellular expression. Understanding these novel reagents as genetic modules that can be integrated into cellular pathways opens up a broad experimental spectrum to monitor and manipulate cellular processes. PMID:26056137

  20. Population inversion in a stationary recombining plasma

    International Nuclear Information System (INIS)

    Otsuka, M.

    1980-01-01

    Population inversion, which occurs in a recombining plasma when a stationary He plasma is brought into contact with a neutral gas, is examined. With hydrogen as a contact gas, noticeable inversion between low-lying levels of H as been found. The overpopulation density is of the order of 10 8 cm -3 , which is much higher then that (approx. =10 5 cm -3 ) obtained previously with He as a contact gas. Relations between these experimental results and the conditions for population inversion are discussed with the CR model

  1. Nanobodies and recombinant binders in cell biology.

    Science.gov (United States)

    Helma, Jonas; Cardoso, M Cristina; Muyldermans, Serge; Leonhardt, Heinrich

    2015-06-08

    Antibodies are key reagents to investigate cellular processes. The development of recombinant antibodies and binders derived from natural protein scaffolds has expanded traditional applications, such as immunofluorescence, binding arrays, and immunoprecipitation. In addition, their small size and high stability in ectopic environments have enabled their use in all areas of cell research, including structural biology, advanced microscopy, and intracellular expression. Understanding these novel reagents as genetic modules that can be integrated into cellular pathways opens up a broad experimental spectrum to monitor and manipulate cellular processes. © 2015 Helma et al.

  2. Dissociative recombination of molecular ions H2+

    International Nuclear Information System (INIS)

    Abarenov, A.V.; Marchenko, V.S.

    1989-01-01

    The total cross sections of dissociation and dissociative recombination of slow electrons and molecular ions H 2 + have been calculated in terms of the quasiclassical and dipole approximations. In the calculations allowance was made for the quantum nature of vibrational motion of heavy particles and presence of autoionization of divergence states of the H 2 (Σ u , nl) molecules. It is shown that the H 2 + ion dissociation cross sections are dominant in increase of the electron energy in the ε >or approx. 2-3 eV region for H 2 + (v) ion distribution over the vibrational levels characteristic for the beam experiments. 15 refs.; 5 figs

  3. Recombinant DNA. Rifkin's regulatory revivalism runs riot.

    Science.gov (United States)

    David, P

    Jeremy Rifkin, activist opponent of genetic engineering, has adopted tactics of litigation, persuasion, and confrontation in his campaign to halt genetic experimentation. The Recombinant DNA Advisory Committee of the National Institutes of Health has often been the target of his criticism, most recently for its failure to prepare an environmental risk assessment for some DNA tests it approved. Rifkin has won support for his position from religious organizations in the United States, and in June 1983 persuaded an ecumenical group of religious leaders to ask Congress to ban genetic experiments that would affect the human germ line.

  4. Guiding recombinant antivenom development by omics technologies

    DEFF Research Database (Denmark)

    Laustsen, Andreas Hougaard

    2017-01-01

    directed towards the different omics technologies (particularly venomics, antivenomics, and toxicovenomics) that are being used to uncover novel animal toxins, shed light on venom complexity, and provide directions for how to determine the medical relevance of individual toxins within whole venoms. Finally......, endogenous animal proteins with toxin-neutralizing capabilities, and recombinant monoclonal antibodies. Harnessing either of these approaches, antivenom development may benefit from an in-depth understanding of venom compositions and the medical importance of individual venom toxins. Focus is thus also......, techniques for assessing antivenom specificity and cross-reactivity are reviewed, with special focus on antivenomics and high-density peptide microarray technology....

  5. How compressible is recombinant battery separator mat?

    Energy Technology Data Exchange (ETDEWEB)

    Pendry, C. [Hollingsworth and Vose, Postlip Mills Winchcombe (United Kingdom)

    1999-03-01

    In the past few years, the recombinant battery separator mat (RBSM) for valve-regulated lead/acid (VRLA) batteries has become the focus of much attention. Compression, and the ability of microglass separators to maintain a level of `springiness` have helped reduce premature capacity loss. As higher compressions are reached, we need to determine what, if any, damage can be caused during the assembly process. This paper reviews the findings when RBSM materials, with different surface areas, are compressed under forces up to 500 kPa in the dry state. (orig.)

  6. Test tube systems with cutting/recombination operations

    Energy Technology Data Exchange (ETDEWEB)

    Freund, R. [Technische Universitaet Wien (Austria); Csuhaj-Varju, E. [Computer and Automation Institute, Budapest (Hungary); Wachtler, F. [Universitaet Wien (Austria)

    1996-12-31

    We introduce test tube systems based on operations that are closely related to the splicing operations, i.e. we consider the operations of cutting a string at a specific site into two pieces with marking them at the cut ends and of recombining two strings with specifically marked endings. Whereas in the splicing of two strings these strings are cut at specific sites and the cut pieces are recombined immediately in a crosswise way, in CR(cutting/recombination)-schemes cutting can happen independently from recombining the cut pieces. Test tube systems based on these operations of cutting and recombination turn out to have maximal generative power even if only very restricted types of input filters for the test tubes are used for the redistribution of the contents of the test tubes after a period of cuttings and recombinations in the test tubes. 10 refs.

  7. Induction of intrachromosomal homologous recombination in whole plants

    International Nuclear Information System (INIS)

    Puchta, H.; Swoboda, P.; Hohn, B.

    1995-01-01

    The influence of different factors on frequencies of intrachromosomal homologous recombination in whole Arabidopsis thaliana and tobacco plants was analyzed using a disrupted β-glucuronidase marker gene. Recombination frequencies were enhanced several fold by DNA damaging agents like UV-light or MMS (methyl methanesulfonate). Applying 3-methoxybenzamide (3-MB), an inhibitor of poly(ADP)ribose polymerase (PARP), an enzyme that is postulated to be involved in DNA repair, enhanced homologous recombination frequencies strongly. These findings indicate that homologous recombination is involved in DNA repair and can (at least partially) compensate for other DNA repair pathways. Indications that recombination in plants can be induced by environmental stress factors that are not likely to be involved in DNA metabolism were also found; Arabidopsis plants growing in a medium containing 0.1 M NaCl exhibited elevated recombination frequencies. The possible general effects of ‘environmental’ challenges on genome flexibility are discussed. (author)

  8. Metal binding proteins, recombinant host cells and methods

    Science.gov (United States)

    Summers, Anne O.; Caguiat, Jonathan J.

    2004-06-15

    The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.

  9. Expression and purification of recombinant hemoglobin in Escherichia coli

    DEFF Research Database (Denmark)

    Natarajan, Chandrasekhar; Jiang, Xiaoben; Fago, Angela

    2011-01-01

    BACKGROUND: Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe...... a protocol for expressing Hbs with low intrinsic solubilities. Since the alpha- and beta-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression......-translational modifications. CONCLUSION/SIGNIFICANCE: Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need...

  10. Bayesian inference of shared recombination hotspots between humans and chimpanzees.

    Science.gov (United States)

    Wang, Ying; Rannala, Bruce

    2014-12-01

    Recombination generates variation and facilitates evolution. Recombination (or lack thereof) also contributes to human genetic disease. Methods for mapping genes influencing complex genetic diseases via association rely on linkage disequilibrium (LD) in human populations, which is influenced by rates of recombination across the genome. Comparative population genomic analyses of recombination using related primate species can identify factors influencing rates of recombination in humans. Such studies can indicate how variable hotspots for recombination may be both among individuals (or populations) and over evolutionary timescales. Previous studies have suggested that locations of recombination hotspots are not conserved between humans and chimpanzees. We made use of the data sets from recent resequencing projects and applied a Bayesian method for identifying hotspots and estimating recombination rates. We also reanalyzed SNP data sets for regions with known hotspots in humans using samples from the human and chimpanzee. The Bayes factors (BF) of shared recombination hotspots between human and chimpanzee across regions were obtained. Based on the analysis of the aligned regions of human chromosome 21, locations where the two species show evidence of shared recombination hotspots (with high BFs) were identified. Interestingly, previous comparative studies of human and chimpanzee that focused on the known human recombination hotspots within the β-globin and HLA regions did not find overlapping of hotspots. Our results show high BFs of shared hotspots at locations within both regions, and the estimated locations of shared hotspots overlap with the locations of human recombination hotspots obtained from sperm-typing studies. Copyright © 2014 by the Genetics Society of America.

  11. Regulation of homologous recombination at telomeres in budding yeast

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine; Lisby, Michael

    2010-01-01

    Homologous recombination is suppressed at normal length telomere sequences. In contrast, telomere recombination is allowed when telomeres erode in the absence of telomerase activity or as a consequence of nucleolytic degradation or incomplete replication. Here, we review the mechanisms that contr...... that contribute to regulating mitotic homologous recombination at telomeres and the role of these mechanisms in signalling short telomeres in the budding yeast Saccharomyces cerevisiae....

  12. Intermediate bands versus levels in non-radiative recombination

    International Nuclear Information System (INIS)

    Luque, Antonio; Marti, Antonio; Antolin, Elisa; Tablero, Cesar

    2006-01-01

    There is a practical interest in developing semiconductors with levels situated within their band gap while preventing the non-radiative recombination that these levels promote. In this paper, the physical causes of this non-radiative recombination are analyzed and the increase in the density of the impurities responsible for the mid-gap levels to the point of forming bands is suggested as the means of suppressing the recombination. Simple models supporting this recommendation and helping in its quantification are presented

  13. In vitro V(D)J recombination: Signal joint formation

    OpenAIRE

    Cortes, Patricia; Weis-Garcia, Frances; Misulovin, Ziva; Nussenzweig, Andre; Lai, Jiann-Shiun; Li, Gloria; Nussenzweig, Michel C.; Baltimore, David

    1996-01-01

    The first step of V(D)J recombination, specific cleavage at the recombination signal sequence (RSS), can be carried out by the recombination activating proteins RAG1 and RAG2. In vivo, the cleaved coding and signal ends must be rejoined to generate functional antigen receptors and maintain chromosomal integrity. We have investigated signal joint formation using deletion and inversion substrates in a cell free system. RAG1 and RAG2 alone or in combination were unabl...

  14. Recombinant protein scaffolds for tissue engineering

    International Nuclear Information System (INIS)

    Werkmeister, Jerome A; Ramshaw, John A M

    2012-01-01

    New biological materials for tissue engineering are now being developed using common genetic engineering capabilities to clone and express a variety of genetic elements that allow cost-effective purification and scaffold fabrication from these recombinant proteins, peptides or from chimeric combinations of these. The field is limitless as long as the gene sequences are known. The utility is dependent on the ease, product yield and adaptability of these protein products to the biomedical field. The development of recombinant proteins as scaffolds, while still an emerging technology with respect to commercial products, is scientifically superior to current use of natural materials or synthetic polymer scaffolds, in terms of designing specific structures with desired degrees of biological complexities and motifs. In the field of tissue engineering, next generation scaffolds will be the key to directing appropriate tissue regeneration. The initial period of biodegradable synthetic scaffolds that provided shape and mechanical integrity, but no biological information, is phasing out. The era of protein scaffolds offers distinct advantages, particularly with the combination of powerful tools of molecular biology. These include, for example, the production of human proteins of uniform quality that are free of infectious agents and the ability to make suitable quantities of proteins that are found in low quantity or are hard to isolate from tissue. For the particular needs of tissue engineering scaffolds, fibrous proteins like collagens, elastin, silks and combinations of these offer further advantages of natural well-defined structural scaffolds as well as endless possibilities of controlling functionality by genetic manipulation. (topical review)

  15. Recombinant antigens for immunodiagnosis of cystic echinococcosis

    Directory of Open Access Journals (Sweden)

    Li Jun

    2004-01-01

    Full Text Available Three cDNAs, termed EpC1, TPxEg and EgG5, were isolated by immunoscreening from an Echinococcus granulosus cDNA library. The recombinant phages exhibited strong reactivity with sera from humans with confirmed cystic echinococcosis (CE and with sera from mice infected with E. granulosus oncospheres. The cDNAs were subcloned into a pET vector, expressed as fusion proteins tagged with GST and affinity purified against the GST tag. Of the three recombinant proteins, EpC1 achieved the highest performance for serodiagnosis of CE in Western blot analysis using a panel of clinically defined human sera to initially address the sensitivity and specificity of the molecules. The protein yielded an overall sensitivity of 92.2% and specificity of 95.6%, levels unprecedented taking into account the large panel of 896 human sera that were tested. The strategy used may also prove suitable for improved immunodiagnosis of other parasitic infections.

  16. Simulation and Optimisation of CLIC's recombination complex

    CERN Document Server

    Costa, Raul; Barroso, Manuel

    In this thesis we present the first Placet2 recombination simulations of the drive beam recombination complex (DBRC) design for the compact linear collider (CLIC). We start by presenting a review of the CLIC project and the DBRC’s role and design within it. We then discuss some of the core principles of beam dynamics and how tracking codes like Placet2 implement them. We follow that by presenting the design issues raised by our simulations and our proposed strategy to address them, key among which is a previously unknown parabolic dependency of the longitudinal position to the momentum (T 566 ), which threat- ens the efficiency of the power extraction structures. Through iterative opti- misation of the design, we eliminated this aberration both in the delay loop and in combiner ring 1. We also found the beam’s horizontal emittance to be significantly over the design budget (150 μm) and attempted to meet that budget, reaching 157 μm. In order to obtain this emittance value, an update to the combiner ring...

  17. Single-crossover recombination in discrete time.

    Science.gov (United States)

    von Wangenheim, Ute; Baake, Ellen; Baake, Michael

    2010-05-01

    Modelling the process of recombination leads to a large coupled nonlinear dynamical system. Here, we consider a particular case of recombination in discrete time, allowing only for single crossovers. While the analogous dynamics in continuous time admits a closed solution (Baake and Baake in Can J Math 55:3-41, 2003), this no longer works for discrete time. A more general model (i.e. without the restriction to single crossovers) has been studied before (Bennett in Ann Hum Genet 18:311-317, 1954; Dawson in Theor Popul Biol 58:1-20, 2000; Linear Algebra Appl 348:115-137, 2002) and was solved algorithmically by means of Haldane linearisation. Using the special formalism introduced by Baake and Baake (Can J Math 55:3-41, 2003), we obtain further insight into the single-crossover dynamics and the particular difficulties that arise in discrete time. We then transform the equations to a solvable system in a two-step procedure: linearisation followed by diagonalisation. Still, the coefficients of the second step must be determined in a recursive manner, but once this is done for a given system, they allow for an explicit solution valid for all times.

  18. Immunoglobulin class-switch recombination deficiencies.

    Science.gov (United States)

    Durandy, Anne; Kracker, Sven

    2012-07-30

    Immunoglobulin class-switch recombination deficiencies (Ig-CSR-Ds) are rare primary immunodeficiencies characterized by defective switched isotype (IgG/IgA/IgE) production. Depending on the molecular defect in question, the Ig-CSR-D may be combined with an impairment in somatic hypermutation (SHM). Some of the mechanisms underlying Ig-CSR and SHM have been described by studying natural mutants in humans. This approach has revealed that T cell-B cell interaction (resulting in CD40-mediated signaling), intrinsic B-cell mechanisms (activation-induced cytidine deaminase-induced DNA damage), and complex DNA repair machineries (including uracil-N-glycosylase and mismatch repair pathways) are all involved in class-switch recombination and SHM. However, several of the mechanisms required for full antibody maturation have yet to be defined. Elucidation of the molecular defects underlying the diverse set of Ig-CSR-Ds is essential for understanding Ig diversification and has prompted better definition of the clinical spectrum of diseases and the development of increasingly accurate diagnostic and therapeutic approaches.

  19. The model of recombination process in TlBr

    International Nuclear Information System (INIS)

    Grigorjeva, L.; Millers, D.

    2002-01-01

    The time-resolved luminescence was used as a tool in the study of recombination process in several undoped TlBr crystals. The spectra and decay kinetics observed under electron beam excitation were investigated. Observation of several luminescence bands with different decay rates shows that more than one recombination center is involved and the recombination process is quite complicated. The band at ∼2.5 eV is dominant under 10 ns excitation pulse (electron beam or nitrogen laser pulses). The results of short-lived absorption and luminescence are used for analysis of possible mechanisms of recombination processes in TlBr

  20. Rogue athletes and recombinant DNA technology: challenges for doping control.

    Science.gov (United States)

    Azzazy, Hassan M E; Mansour, Mai M H

    2007-10-01

    The quest for athletic excellence holds no limit for some athletes, and the advances in recombinant DNA technology have handed these athletes the ultimate doping weapons: recombinant proteins and gene doping. Some detection methods are now available for several recombinant proteins that are commercially available as pharmaceuticals and being abused by dopers. However, researchers are struggling to come up with efficient detection methods in preparation for the imminent threat of gene doping, expected in the 2008 Olympics. This Forum article presents the main detection strategies for recombinant proteins and the forthcoming detection strategies for gene doping as well as the prime analytical challenges facing them.

  1. Experimental evolution across different thermal regimes yields genetic divergence in recombination fraction but no divergence in temperature associated plastic recombination.

    Science.gov (United States)

    Kohl, Kathryn P; Singh, Nadia D

    2018-04-01

    Phenotypic plasticity is pervasive in nature. One mechanism underlying the evolution and maintenance of such plasticity is environmental heterogeneity. Indeed, theory indicates that both spatial and temporal variation in the environment should favor the evolution of phenotypic plasticity under a variety of conditions. Cyclical environmental conditions have also been shown to yield evolved increases in recombination frequency. Here, we use a panel of replicated experimental evolution populations of D. melanogaster to test whether variable environments favor enhanced plasticity in recombination rate and/or increased recombination rate in response to temperature. In contrast to expectation, we find no evidence for either enhanced plasticity in recombination or increased rates of recombination in the variable environment lines. Our data confirm a role of temperature in mediating recombination fraction in D. melanogaster, and indicate that recombination is genetically and plastically depressed under lower temperatures. Our data further suggest that the genetic architectures underlying plastic recombination and population-level variation in recombination rate are likely to be distinct. © 2018 The Author(s). Evolution © 2018 The Society for the Study of Evolution.

  2. Recombination of Globally Circulating Varicella-Zoster Virus

    Science.gov (United States)

    Depledge, Daniel P.; Kundu, Samit; Atkinson, Claire; Brown, Julianne; Haque, Tanzina; Hussaini, Yusuf; MacMahon, Eithne; Molyneaux, Pamela; Papaevangelou, Vassiliki; Sengupta, Nitu; Koay, Evelyn S. C.; Tang, Julian W.; Underhill, Gillian S.; Grahn, Anna; Studahl, Marie; Breuer, Judith; Bergström, Tomas

    2015-01-01

    ABSTRACT Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpesvirus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine in vivo, creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine-wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either in vivo or in vitro during passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade. IMPORTANCE Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the

  3. Evidence of recombination in intrapatient populations of hepatitis C virus.

    Science.gov (United States)

    Sentandreu, Vicente; Jiménez-Hernández, Nuria; Torres-Puente, Manuela; Bracho, María Alma; Valero, Ana; Gosalbes, María José; Ortega, Enrique; Moya, Andrés; González-Candelas, Fernando

    2008-09-18

    Hepatitis C virus (HCV) is a major cause of liver disease worldwide and a potential cause of substantial morbidity and mortality in the future. HCV is characterized by a high level of genetic heterogeneity. Although homologous recombination has been demonstrated in many members of the family Flaviviridae, to which HCV belongs, there are only a few studies reporting recombination on natural populations of HCV, suggesting that these events are rare in vivo. Furthermore, these few studies have focused on recombination between different HCV genotypes/subtypes but there are no reports on the extent of intra-genotype or intra-subtype recombination between viral strains infecting the same patient. Given the important implications of recombination for RNA virus evolution, our aim in this study has been to assess the existence and eventually the frequency of intragenic recombination on HCV. For this, we retrospectively have analyzed two regions of the HCV genome (NS5A and E1-E2) in samples from two different groups: (i) patients infected only with HCV (either treated with interferon plus ribavirin or treatment naïve), and (ii) HCV-HIV co-infected patients (with and without treatment against HIV). The complete data set comprised 17712 sequences from 136 serum samples derived from 111 patients. Recombination analyses were performed using 6 different methods implemented in the program RDP3. Recombination events were considered when detected by at least 3 of the 6 methods used and were identified in 10.7% of the amplified samples, distributed throughout all the groups described and the two genomic regions studied. The resulting recombination events were further verified by detailed phylogenetic analyses. The complete experimental procedure was applied to an artificial mixture of relatively closely viral populations and the ensuing analyses failed to reveal artifactual recombination. From these results we conclude that recombination should be considered as a potentially

  4. A Herpes Simplex Virus Type 1 Mutant Expressing a Baculovirus Inhibitor of Apoptosis Gene in Place of Latency-Associated Transcript Has a Wild-Type Reactivation Phenotype in the Mouse

    Science.gov (United States)

    Jin, Ling; Perng, Guey-Chuen; Mott, Kevin R.; Osorio, Nelson; Naito, Julia; Brick, David J.; Carpenter, Dale; Jones, Clinton; Wechsler, Steven L.

    2005-01-01

    The latency-associated transcript (LAT) is essential for the wild-type herpes simplex virus type 1 (HSV-1) high-reactivation phenotype since LAT− mutants have a low-reactivation phenotype. We previously reported that LAT can decrease apoptosis and proposed that this activity is involved in LAT's ability to enhance the HSV-1 reactivation phenotype. The first 20% of the primary 8.3-kb LAT transcript is sufficient for enhancing the reactivation phenotype and for decreasing apoptosis, supporting this proposal. For this study, we constructed an HSV-1 LAT− mutant that expresses the baculovirus antiapoptosis gene product cpIAP under control of the LAT promoter and in place of the LAT region mentioned above. Mice were ocularly infected with this mutant, designated dLAT-cpIAP, and the reactivation phenotype was determined using the trigeminal ganglion explant model. dLAT-cpIAP had a reactivation phenotype similar to that of wild-type virus and significantly higher than that of (i) the LAT− mutant dLAT2903; (ii) dLAT1.5, a control virus containing the same LAT deletion as dLAT-cpIAP, but with no insertion of foreign DNA, thereby controlling for potential readthrough transcription past the cpIAP insert; and (iii) dLAT-EGFP, a control virus identical to dLAT-cpIAP except that it contained the enhanced green fluorescent protein open reading frame (ORF) in place of the cpIAP ORF, thereby controlling for expression of a random foreign gene instead of the cpIAP gene. These results show that an antiapoptosis gene with no sequence similarity to LAT can efficiently substitute for the LAT function involved in enhancing the in vitro-induced HSV-1 reactivation phenotype in the mouse. PMID:16160155

  5. Toroidal charge exchange recombination spectroscopy on EAST

    Energy Technology Data Exchange (ETDEWEB)

    Ye, Minyou, E-mail: yemy@ustc.edu.cn [School of Nuclear Science and Technology, University of Science and Technology of China, Hefei, Anhui 230026 (China); Institute of Plasma Physics, Chinese Academy of Sciences, Hefei, Anhui 230031 (China); Li, Yingying [Institute of Plasma Physics, Chinese Academy of Sciences, Hefei, Anhui 230031 (China); Yu, Yi [School of Nuclear Science and Technology, University of Science and Technology of China, Hefei, Anhui 230026 (China); Shi, Yuejiang [School of Nuclear Science and Technology, University of Science and Technology of China, Hefei, Anhui 230026 (China); WCI for Fusion Theory, National Fusion Research Institute, 52 Eoeun-Dong, Yusung-Gu, Daejeon 305-333 (Korea, Republic of); Lyu, Bo; Fu, Jia [Institute of Plasma Physics, Chinese Academy of Sciences, Hefei, Anhui 230031 (China); Du, Xuewei; Yin, Xianghui; Zhang, Yi; Wang, Qiuping [School of Nuclear Science and Technology, University of Science and Technology of China, Hefei, Anhui 230026 (China); Wan, Baonian [Institute of Plasma Physics, Chinese Academy of Sciences, Hefei, Anhui 230031 (China); School of Nuclear Science and Technology, University of Science and Technology of China, Hefei, Anhui 230026 (China)

    2015-10-15

    A toroidal charge exchange recombination spectroscopy (CXRS) diagnostic, on the basis of a heating neutral beam injector (NBI), is constructed on EAST tokamak. Simulation of Spectra (SOS) code is used to design and evaluate the diagnostic performance. 30 spatial channels work simultaneously in recent experiment, which covers a radial region from 1.55 m to 2.30 m in the cross section. The CXRS has a radial resolution of 1–3.5 cm from core to edge. The acquisition time is typically 10 ms, limited by the poor photon statistics. The diagnostic can observe not only the normal C{sup 5+} emission line at 529.1 nm but also any interested wavelength in the range of 400–700 nm. In this work, a brief overview on the R&D and the instrument performance for the toroidal CXRS diagnostic is described, together with first results.

  6. Initiation of Meiotic Recombination in Mammals

    Directory of Open Access Journals (Sweden)

    Rajeev Kumar

    2010-12-01

    Full Text Available Meiotic recombination is initiated by the induction of programmed DNA double strand breaks (DSBs. DSB repair promotes homologous interactions and pairing and leads to the formation of crossovers (COs, which are required for the proper reductional segregation at the first meiotic division. In mammals, several hundred DSBs are generated at the beginning of meiotic prophase by the catalytic activity of SPO11. Currently it is not well understood how the frequency and timing of DSB formation and their localization are regulated. Several approaches in humans and mice have provided an extensive description of the localization of initiation events based on CO mapping, leading to the identification and characterization of preferred sites (hotspots of initiation. This review presents the current knowledge about the proteins known to be involved in this process, the sites where initiation takes place, and the factors that control hotspot localization.

  7. Recombinant Brucella abortus gene expressing immunogenic protein

    Energy Technology Data Exchange (ETDEWEB)

    Mayfield, J.E.; Tabatabai, L.B.

    1991-06-11

    This patent describes a synthetic recombinant DNA molecule containing a DNA sequence. It comprises a gene of Brucella abortus encoding an immunogenic protein having a molecular weight of approximately 31,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions, the protein having an isoelectric point around 4.9, and containing a twenty-five amino acid sequence from its amino terminal end consisting of Gln-Ala-Pro-Thr-Phe-Phe-Arg-Ile-Gly-Thr-Gly-Gly-Thr-Ala-Gly-Thr-Tyr-Tyr-Pro-Ile-Gly-Gly-Leu-Ile-Ala, wherein Gln, Ala, Pro, Thr, Phe, Arg, Ile, Gly, Tyr, and Leu, respectively, represent glutamine, alanine, proline, threonine, phenylalanine, arginine, isolecuine, glycine, tyrosine, and leucine.

  8. Nonequilibrium recombination after a curved shock wave

    Science.gov (United States)

    Wen, Chihyung; Hornung, Hans

    2010-02-01

    The effect of nonequilibrium recombination after a curved two-dimensional shock wave in a hypervelocity dissociating flow of an inviscid Lighthill-Freeman gas is considered. An analytical solution is obtained with the effective shock values derived by Hornung (1976) [5] and the assumption that the flow is ‘quasi-frozen’ after a thin dissociating layer near the shock. The solution gives the expression of dissociation fraction as a function of temperature on a streamline. A rule of thumb can then be provided to check the validity of binary scaling for experimental conditions and a tool to determine the limiting streamline that delineates the validity zone of binary scaling. The effects on the nonequilibrium chemical reaction of the large difference in free stream temperature between free-piston shock tunnel and equivalent flight conditions are discussed. Numerical examples are presented and the results are compared with solutions obtained with two-dimensional Euler equations using the code of Candler (1988) [10].

  9. Scavenging and recombination kinetics in radiation chemistry.

    Science.gov (United States)

    Al-Samra, Eyad H; Green, Nicholas J B

    2017-08-02

    This work describes stochastic models developed to study the competition between radical scavenging and recombination for simple model systems typical of radiation chemistry, where the reactive particles are tightly clustered and reactions are assumed fully diffusion limited. Three models are developed: a Monte Carlo random flights model with a periodic boundary condition for scavengers, Monte Carlo simulations in which the scavenging rate is calculated from the Smoluchowski theory for diffusion-limited reactions and a modification of the independent reaction times method where the scavengers close to the spur are explicitly included and the scavengers further away are treated as a continuum. The results indicate that the Smoluchowski theory makes a systematic overestimate of the scavenging rate when such competition is present. A correction for the Smoluchowski rate constant is suggested, an analytical justification is presented and it is tested against the simulations, and shown to be a substantial improvement.

  10. MSD Recombination Method in Statistical Machine Translation

    Science.gov (United States)

    Gros, Jerneja Žganec

    2008-11-01

    Freely available tools and language resources were used to build the VoiceTRAN statistical machine translation (SMT) system. Various configuration variations of the system are presented and evaluated. The VoiceTRAN SMT system outperformed the baseline conventional rule-based MT system in all English-Slovenian in-domain test setups. To further increase the generalization capability of the translation model for lower-coverage out-of-domain test sentences, an "MSD-recombination" approach was proposed. This approach not only allows a better exploitation of conventional translation models, but also performs well in the more demanding translation direction; that is, into a highly inflectional language. Using this approach in the out-of-domain setup of the English-Slovenian JRC-ACQUIS task, we have achieved significant improvements in translation quality.

  11. Guiding recombinant antivenom development by omics technologies

    DEFF Research Database (Denmark)

    Laustsen, Andreas Hougaard

    2017-01-01

    , endogenous animal proteins with toxin-neutralizing capabilities, and recombinant monoclonal antibodies. Harnessing either of these approaches, antivenom development may benefit from an in-depth understanding of venom compositions and the medical importance of individual venom toxins. Focus is thus also...... directed towards the different omics technologies (particularly venomics, antivenomics, and toxicovenomics) that are being used to uncover novel animal toxins, shed light on venom complexity, and provide directions for how to determine the medical relevance of individual toxins within whole venoms. Finally......In this review, the different approaches that have been employed with the aim of developing novel antivenoms against animal envenomings are presented and discussed. Reported efforts have focused on the use of innovative immunization strategies, small molecule inhibitors against enzymatic toxins...

  12. Repair by genetic recombination in bacteria: overview

    International Nuclear Information System (INIS)

    Howard-Flanders, P.

    1975-01-01

    DNA molecules that have been damaged in both strands at the same level are not subject to repair by excision but instead can be repaired through recombination with homologous molecules. Examples of two-strand damage include postreplication gaps opposite pyrimidine dimers, two-strand breaks produced by x-rays, and chemically induced interstrand cross-links. In ultraviolet-irradiated bacteria, and newly synthesized DNA is of length equal to the interdimer spacing. With continued incubation, this low-molecular-weight DNA is joined into high-molecular-weight chains (postreplication repair), a process associated with sister exchanges in bacteria. Recombination is initiated by pyrimidine dimers opposite postreplication gaps and by interstrand cross-links that have been cut by excision enzymes. The free ends at the resulting gaps presumably initiate the exchanges. Postreplication repair in Escherichia coli occurs in recB - and recC - but is greatly slowed in recF - mutants. RecB and recC are the structural genes for exonuclease V, which digests two-stranded DNA by releasing oligonucleotides first from one strand and then from the other. The postreplication sister exchanges in ultraviolet-irradiated bacteria result in the distribution of pyrimidine dimers between parental and daughter strands, indicating that long exchanges involving both strands of each duplex occur. The R1 restriction endonuclease from E. coli has been used to cut the DNA of a bacterial drug-resistance transfer factor with one nuclease-sensitive site, and also DNA from the frog Xenopus enriched for ribosomal 18S and 28S genes. The fragments were annealed with the cut plasmid DNA and ligated, producing a new larger plasmid carrying the eukaryotic rDNA and able to infect and replicate in E. coli

  13. Functional, Responsive Materials Assembled from Recombinant Oleosin

    Science.gov (United States)

    Hammer, Daniel

    Biological cells are surrounded by a plasma membrane made primarily of phospholipids that form a bilayer. This membrane is permselective and compartmentalizes the cell. A simple form of artificial cell is the vesicle, in which a phospholipid bilayer membrane surrounds an aqueous solution. However, there is no a priori reason why a membrane needs to be made of phospholipids. It could be made of any surfactant that forms a bilayer. We have assembled membranes and other structures from the recombinant plant protein oleosin. The ability to assemble from a recombinant protein means that every molecule is identical, we have complete control over the sequence, and hence can build in designer functionality with high fidelity, including adhesion and enzymatic activity. Such incorporation is trivial using the tools of molecular biology. We find that while many variants of oleosin make membranes, others make micelles and sheets. We show how the type of supramolecular structure can be altered by the conditions of solvent, such as ionic strength, and the architecture of the surfactant itself. We show that protease cleavable domains can be incorporated within oleosin, and be engineered to protect other functional domains such as adhesive motifs, to make responsive materials whose activity and shape depend on the action of proteases. We will also present the idea of making ``Franken''-oleosins, where large domains of native oleosin are replaced with domains from other functional proteins, to make hybrids conferred by the donor protein. Thus, we can view oleosin as a template upon which a vast array of designer functionalities can be imparted..

  14. Radio recombination lines from H II regions

    International Nuclear Information System (INIS)

    Silverglate, P.R.

    1978-01-01

    Radio recombination lines have been observed from forty-six H II regions. The Arecibo 1000-foot radio telescope was used to provide high sensitivity and high angular resolution at 1400 MHz (gain approx. 7.7 0 K/Jy, HPBW = 3:2) and 2372 MHZ (gain approx. 6.3 0 K/Jy, HPBW = 2'). Observations were made at 1400 MHz in the frequency switching mode, and at 2372 MHz in the total power mode. Gaussians were fit to be observed lines to derive velocities, line widths, and line temperatures. From the velocities kinematic distances were derived. For eleven sources H I absorption measurements were also made. The absorption spectra enabled the kinematic distance ambiguity to be resolved for some sources. The absorption spectra themselves were found to have extremely sharp, non-gaussian edges. One explanation for these is a model where the interstellar medium contains many H I cloudlets with T/sub s/less than or equal to 100 0 K and turbulent velocities less than or equal to 3 km/s. The H I absorption spectrum is then a superposition of many narrow gaussian profiles. It was also found from a comparison of H I absorption velocities with radio recombination line velocities that peculiar motions exist in the interstellar medium with velocities of up to 10 km/s. Using the measured line temperatures and continuum temperatures, estimates were desired of emission measures, electron temperatures, and electron densities, using a non-LTE analysis. Non-LTE effects were important only for the hottest and densest H II regions. The non-LTE calculations were checked through a comparison derivation of electron temperatures using hydrogen beta lines

  15. Genetic analysis of variation in human meiotic recombination.

    Directory of Open Access Journals (Sweden)

    Reshmi Chowdhury

    2009-09-01

    Full Text Available The number of recombination events per meiosis varies extensively among individuals. This recombination phenotype differs between female and male, and also among individuals of each gender. In this study, we used high-density SNP genotypes of over 2,300 individuals and their offspring in two datasets to characterize recombination landscape and to map the genetic variants that contribute to variation in recombination phenotypes. We found six genetic loci that are associated with recombination phenotypes. Two of these (RNF212 and an inversion on chromosome 17q21.31 were previously reported in the Icelandic population, and this is the first replication in any other population. Of the four newly identified loci (KIAA1462, PDZK1, UGCG, NUB1, results from expression studies provide support for their roles in meiosis. Each of the variants that we identified explains only a small fraction of the individual variation in recombination. Notably, we found different sequence variants associated with female and male recombination phenotypes, suggesting that they are regulated by different genes. Characterization of genetic variants that influence natural variation in meiotic recombination will lead to a better understanding of normal meiotic events as well as of non-disjunction, the primary cause of pregnancy loss.

  16. Collision and recombination driven instabilities in variable charged ...

    Indian Academy of Sciences (India)

    The dust-acoustic instability driven by recombination of electrons and ions on the surface of charged and variably-charged dust grains as well as by collisions in dusty plasmas with significant pressure of background neutrals have been theoretically investigated. The recombination driven instability is shown to be dominant ...

  17. Construction and Characterization of a Recombinant Invertebrate Iridovirus

    NARCIS (Netherlands)

    Ozgen, A.; Muratoglu, H.; Demirbag, Z.; Vlak, J.M.; Oers, van M.M.; Nalcacioglu, R.

    2014-01-01

    Chilo iridescent virus (CIV), officially named Insect iridescent virus 6 (IIV6), is the type species of the genus Iridovirus (family Iridoviridae). In this paper we constructed a recombinant CIV, encoding the green fluorescent protein (GFP). This recombinant can be used to investigate viral

  18. Construction and characterization of a recombinant invertebrate iridovirus.

    Science.gov (United States)

    Ozgen, Arzu; Muratoglu, Hacer; Demirbag, Zihni; Vlak, Just M; van Oers, Monique M; Nalcacioglu, Remziye

    2014-08-30

    Chilo iridescent virus (CIV), officially named Insect iridescent virus 6 (IIV6), is the type species of the genus Iridovirus (family Iridoviridae). In this paper we constructed a recombinant CIV, encoding the green fluorescent protein (GFP). This recombinant can be used to investigate viral replication dynamics. We showed that homologous recombination is a valid method to make CIV gene knockouts and to insert foreign genes. The CIV 157L gene, putatively encoding a non-functional inhibitor of apoptosis (IAP), was chosen as target for foreign gene insertion. The gfp open reading frame preceded by the viral mcp promoter was inserted into the 157L locus by homologous recombination in Anthonomus grandis BRL-AG-3A cells. Recombinant virus (rCIV-Δ157L-gfp) was purified by successive rounds of plaque purification. All plaques produced by the purified recombinant virus emitted green fluorescence due to the presence of GFP. One-step growth curves for recombinant and wild-type CIV were similar and the recombinant was fully infectious in vivo. Hence, CIV157L can be inactivated without altering the replication kinetics of the virus. Consequently, the CIV 157L locus can be used as a site for insertion of foreign DNA, e.g. to modify viral properties for insect biocontrol. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Measurements of EEDF in recombination dominated afterglow plasma

    Science.gov (United States)

    Plasil, R.; Korolov, I.; Kotrik, T.; Varju, J.; Dohnal, P.; Donko, Z.; Bano, G.; Glosik, J.

    2009-11-01

    Electron energy distribution functions (EEDF) have been measured in decaying plasma in Flowing Afterglow Langmuir Probe (FALP) experiment. The measurements have been carried out in diffusion and recombination governed plasmas used for studies of recombination of KrD+ and H3+ ions.

  20. Measurements of EEDF in recombination dominated afterglow plasma

    Energy Technology Data Exchange (ETDEWEB)

    Plasil, R; Korolov, I; Kotrik, T; Varju, J; Dohnal, P; Glosik, J [Charles University, Faculty of Mathematics and Physics, Department of Surface and Plasma Science, Prague (Czech Republic); Donko, Z; Bano, G, E-mail: radek.plasil@mff.cuni.c [Research Institute for Solid State Physics and Optics, Hungarian Academy of Sciences, Budapest (Hungary)

    2009-11-15

    Electron energy distribution functions (EEDF) have been measured in decaying plasma in Flowing Afterglow Langmuir Probe (FALP) experiment. The measurements have been carried out in diffusion and recombination governed plasmas used for studies of recombination of KrD{sup +} and H{sub 3}{sup +} ions.

  1. Measurements of EEDF in recombination dominated afterglow plasma

    International Nuclear Information System (INIS)

    Plasil, R; Korolov, I; Kotrik, T; Varju, J; Dohnal, P; Glosik, J; Donko, Z; Bano, G

    2009-01-01

    Electron energy distribution functions (EEDF) have been measured in decaying plasma in Flowing Afterglow Langmuir Probe (FALP) experiment. The measurements have been carried out in diffusion and recombination governed plasmas used for studies of recombination of KrD + and H 3 + ions.

  2. Co-solute assistance in refolding of recombinant proteins | Gerami ...

    African Journals Online (AJOL)

    Prokaryotic expression system is the most widely used host for the production of recombinant proteins but inclusion body formation is a major bottleneck in the production of recombinant proteins in prokaryotic cells, especially in Escherichia coli. In vitro refolding of inclusion body into the the proteins with native ...

  3. Expression and purification of recombinant Shiga toxin 2B from ...

    African Journals Online (AJOL)

    Expression and purification of recombinant Shiga toxin 2B from Escherichia coli O157:H7. ... (SDS-PAGE) and StxB2 yield was 450 μg ml-1 confirmed by Bradford assay. Recombinant Stx2B protein was produced in highly pure yield using ...

  4. Mitochondrial recombination increases with age in Podospora anserina

    NARCIS (Netherlands)

    van Diepeningen, Anne D; Goedbloed, Daniël J; Slakhorst, S Marijke; Koopmanschap, A Bertha; Maas, Marc F P M; Hoekstra, Rolf F; Debets, Alfons J M

    With uniparental inheritance of mitochondria, there seems little reason for homologous recombination in mitochondria, but the machinery for mitochondrial recombination is quite well-conserved in many eukaryote species. In fungi and yeasts heteroplasmons may be formed when strains fuse and transfer

  5. Three-particle recombination at low temperature: QED approach

    International Nuclear Information System (INIS)

    Bhattacharyya, S.; Roy, A.

    2001-01-01

    A theoretical study of three-body recombination of proton in presence of a spectator electron with electronic beam at near-zero temperature is presented using field theory and invariant Lorentz gauge. Contributions from the Feynman diagrams of different orders give an insight into the physics of the phenomena. Recombination rate coefficient is obtained for low lying principal quantum number n = 1 to 10. At a fixed ion beam temperature (300 K) recombination rate coefficient is found to increase in general with n, having a flat and a sharp peak at quantum states 3 to 5, respectively. In absence of any theoretical and experimental results for low temperature formation of H-atom by three-body recombination at low lying quantum states, we have presented the theoretical results of Stevefelt and group for three-body recombination of deuteron with electron along with the present results. Three-body recombination of antihydrogen in antiproton-positron plasma is expected to yield similar result as that for three-body recombination of hydrogen formation in proton-electron plasma. The necessity for experimental investigation of low temperature three-body recombination at low quantum states is stressed. (author)

  6. Recombination-Driven Genome Evolution and Stability of Bacterial Species.

    Science.gov (United States)

    Dixit, Purushottam D; Pang, Tin Yau; Maslov, Sergei

    2017-09-01

    While bacteria divide clonally, horizontal gene transfer followed by homologous recombination is now recognized as an important contributor to their evolution. However, the details of how the competition between clonality and recombination shapes genome diversity remains poorly understood. Using a computational model, we find two principal regimes in bacterial evolution and identify two composite parameters that dictate the evolutionary fate of bacterial species. In the divergent regime, characterized by either a low recombination frequency or strict barriers to recombination, cohesion due to recombination is not sufficient to overcome the mutational drift. As a consequence, the divergence between pairs of genomes in the population steadily increases in the course of their evolution. The species lacks genetic coherence with sexually isolated clonal subpopulations continuously formed and dissolved. In contrast, in the metastable regime, characterized by a high recombination frequency combined with low barriers to recombination, genomes continuously recombine with the rest of the population. The population remains genetically cohesive and temporally stable. Notably, the transition between these two regimes can be affected by relatively small changes in evolutionary parameters. Using the Multi Locus Sequence Typing (MLST) data, we classify a number of bacterial species to be either the divergent or the metastable type. Generalizations of our framework to include selection, ecologically structured populations, and horizontal gene transfer of nonhomologous regions are discussed as well. Copyright © 2017 by the Genetics Society of America.

  7. Recombination rate variation in mice from an isolated island.

    Science.gov (United States)

    Wang, Richard J; Gray, Melissa M; Parmenter, Michelle D; Broman, Karl W; Payseur, Bret A

    2017-01-01

    Recombination rate is a heritable trait that varies among individuals. Despite the major impact of recombination rate on patterns of genetic diversity and the efficacy of selection, natural variation in this phenotype remains poorly characterized. We present a comparison of genetic maps, sampling 1212 meioses, from a unique population of wild house mice (Mus musculus domesticus) that recently colonized remote Gough Island. Crosses to a mainland reference strain (WSB/EiJ) reveal pervasive variation in recombination rate among Gough Island mice, including subchromosomal intervals spanning up to 28% of the genome. In spite of this high level of polymorphism, the genomewide recombination rate does not significantly vary. In general, we find that recombination rate varies more when measured in smaller genomic intervals. Using the current standard genetic map of the laboratory mouse to polarize intervals with divergent recombination rates, we infer that the majority of evolutionary change occurred in one of the two tested lines of Gough Island mice. Our results confirm that natural populations harbour a high level of recombination rate polymorphism and highlight the disparities in recombination rate evolution across genomic scales. © 2016 John Wiley & Sons Ltd.

  8. Inference of Ancestral Recombination Graphs through Topological Data Analysis

    Science.gov (United States)

    Cámara, Pablo G.; Levine, Arnold J.; Rabadán, Raúl

    2016-01-01

    The recent explosion of genomic data has underscored the need for interpretable and comprehensive analyses that can capture complex phylogenetic relationships within and across species. Recombination, reassortment and horizontal gene transfer constitute examples of pervasive biological phenomena that cannot be captured by tree-like representations. Starting from hundreds of genomes, we are interested in the reconstruction of potential evolutionary histories leading to the observed data. Ancestral recombination graphs represent potential histories that explicitly accommodate recombination and mutation events across orthologous genomes. However, they are computationally costly to reconstruct, usually being infeasible for more than few tens of genomes. Recently, Topological Data Analysis (TDA) methods have been proposed as robust and scalable methods that can capture the genetic scale and frequency of recombination. We build upon previous TDA developments for detecting and quantifying recombination, and present a novel framework that can be applied to hundreds of genomes and can be interpreted in terms of minimal histories of mutation and recombination events, quantifying the scales and identifying the genomic locations of recombinations. We implement this framework in a software package, called TARGet, and apply it to several examples, including small migration between different populations, human recombination, and horizontal evolution in finches inhabiting the Galápagos Islands. PMID:27532298

  9. Engineered mammalian cells for production of recombinant proteins

    DEFF Research Database (Denmark)

    2017-01-01

    The present invention relates to mammalian cells modified to provide for improved expression of a recombinant protein of interest. In particular, the invention relates to CHO cells and other host cells in which the expression of one or more endogenous secreted proteins has been disrupted, as well...... as to the preparation, identification and use of such cells in the production of recombinant proteins....

  10. Improved means and methods for expressing recombinant proteins

    NARCIS (Netherlands)

    Poolman, Berend; Martinez Linares, Daniel; Gul, Nadia

    2014-01-01

    The invention relates to the field of genetic engineering and the production of recombinant proteins in microbial host cells. Provided is a method for enhanced expression of a recombinant protein of interest in a microbial host cell, comprising providing a microbial host cell wherein the function of

  11. Expression and purification of recombinant hemoglobin in Escherichia coli

    DEFF Research Database (Denmark)

    Natarajan, Chandrasekhar; Jiang, Xiaoben; Fago, Angela

    2011-01-01

    BACKGROUND: Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe...

  12. Dielectronic recombination measurements using the Electron Beam Ion Trap

    International Nuclear Information System (INIS)

    Knapp, D.A.

    1991-01-01

    We have used the Electron Beam Ion Trap at LLNL to study dielectronic recombination in highly charged ions. Our technique is unique because we observe the x-rays from dielectronic recombination at the same time we see x-rays from all other electron-ion interactions. We have recently taken high-resolution, state-selective data that resolves individual resonances

  13. Recombination in Avian Gamma-Coronavirus Infectious Bronchitis Virus

    Directory of Open Access Journals (Sweden)

    Mark W. Jackwood

    2011-09-01

    Full Text Available Recombination in the family Coronaviridae has been well documented and is thought to be a contributing factor in the emergence and evolution of different coronaviral genotypes as well as different species of coronavirus. However, there are limited data available on the frequency and extent of recombination in coronaviruses in nature and particularly for the avian gamma-coronaviruses where only recently the emergence of a turkey coronavirus has been attributed solely to recombination. In this study, the full-length genomes of eight avian gamma-coronavirus infectious bronchitis virus (IBV isolates were sequenced and along with other full-length IBV genomes available from GenBank were analyzed for recombination. Evidence of recombination was found in every sequence analyzed and was distributed throughout the entire genome. Areas that have the highest occurrence of recombination are located in regions of the genome that code for nonstructural proteins 2, 3 and 16, and the structural spike glycoprotein. The extent of the recombination observed, suggests that this may be one of the principal mechanisms for generating genetic and antigenic diversity within IBV. These data indicate that reticulate evolutionary change due to recombination in IBV, likely plays a major role in the origin and adaptation of the virus leading to new genetic types and strains of the virus.

  14. Ensuring an exit strategy: RTEL1 restricts rogue recombination.

    Science.gov (United States)

    Villeneuve, Anne M

    2008-10-17

    Success of homologous recombination-based DNA repair depends not only on recombinases, which promote invasion of the homologous DNA duplex that serves as a template for repair, but also on antirecombinases, which dismantle recombination intermediates to allow completion of repair. In this issue, Barber et al. (2008) identify a previously elusive antirecombinase activity important for maintaining genome stability in animals.

  15. The Λ0 polarization and the recombination mechanism

    International Nuclear Information System (INIS)

    Herrera, G.; Montano, L.M.

    1997-01-01

    We use the recombination and the Thomas Precession Model to obtain a prediction for the Λ 0 polarization in the p+p → Λ 0 + X reaction. We study the effect of the recombination function on the Λ 0 polarization. (author)

  16. A study on the hydrogen recombination rates of catalytic recombiners and deliberate ignition

    International Nuclear Information System (INIS)

    Fineschi, F.; Bazzichi, M.; Carcassi, M.

    1994-01-01

    A study is being carried out by the Department of Nuclear and Mechanical Constructions (DCMN) at the University of Pisa on catalytic recombiners and on deliberately induced weak deflagration. The recombination rates of different types of catalytic devices were obtained from a thorough analysis of published experimental data. The main parameter that affects the effectiveness of these devices seems to be the molar density of the deficiency reactant rather than its volumetric concentration. The recombination rate of weak deflagrations in vented compartments has been assessed with experimental tests carried out in a small scale glass vessel. Through a computerized system of analysis of video recordings of the deflagrations, the flame surface and the burned gas volume were obtained as functions of time. Although approximations are inevitable, the method adopted to identify the position of the flame during propagation is more reliable than other non-visual methods (thermocouples and ion-probes). It can only easily be applied to vented weak deflagrations, i.e. when the hydrogen concentration is far from stoichiometric conditions and near to flammability limits, because the pressurization has to be limited due to the low mechanical resistance of the glass. The values of flame surface and burned gas volume were used as inputs for a computer code to calculate the recombining rate, the burning velocity and the pressure transient in the experimental test. The code is being validated with a methodology principally based on a comparison of the measurements of pressure with the calculated values. The research gave some very interesting results on a small scale which should in the future be compared with large scale data

  17. Recombinant expression systems: the obstacle to helminth vaccines?

    Science.gov (United States)

    Geldhof, Peter; De Maere, Veerle; Vercruysse, Jozef; Claerebout, Edwin

    2007-11-01

    The need for alternative ways to control helminth parasites has in recent years led to a boost in vaccination experiments with recombinant antigens. Despite the use of different expression systems, only a few recombinants induced high levels of protection against helminths. This is often attributed to the limitations of the current expression systems. Therefore, the need for new systems that can modify and glycosylate the expressed antigens has been advocated. However, analysis of over 100 published vaccine trials with recombinant helminth antigens indicates that it is often not known whether the native parasite antigen itself can induce protection or, if it does, which epitopes are important. This information is vital for a well-thought-out strategy for recombinant production. So, in addition to testing more expression systems, it should be considered that prior evaluation and characterization of the native antigens might help the development of recombinant vaccines against helminths in the long term.

  18. Recombinant human bone morphogenetic protein induces bone formation

    International Nuclear Information System (INIS)

    Wang, E.A.; Rosen, V.; D'Alessandro, J.S.; Bauduy, M.; Cordes, P.; Harada, T.; Israel, D.I.; Hewick, R.M.; Kerns, K.M.; LaPan, P.; Luxenberg, D.P.; McQuaid, D.; Moutsatsos, I.K.; Nove, J.; Wozney, J.M.

    1990-01-01

    The authors have purified and characterized active recombinant human bone morphogenetic protein (BMP) 2A. Implantation of the recombinant protein in rats showed that a single BMP can induce bone formation in vivo. A dose-response and time-course study using the rat ectopic bone formation assay revealed that implantation of 0.5-115 μg of partially purified recombinant human BMP-2A resulted in cartilage by day 7 and bone formation by day 14. The time at which bone formation occurred was dependent on the amount of BMP-2A implanted; at high doses bone formation could be observed at 5 days. The cartilage- and bone-inductive activity of the recombinant BMP-2A is histologically indistinguishable from that of bone extracts. Thus, recombinant BMP-2A has therapeutic potential to promote de novo bone formation in humans

  19. Low-temperature radiative recombination of electrons with bare nuclei

    International Nuclear Information System (INIS)

    Omidvar, K.

    1993-01-01

    Aside from empirical formulas, the radiative-recombination cross section and coefficient are usually given in tabulated forms instead of analytic formulas. Here, we give analytic expressions in the form of expansions for the recombination cross section as a function of the electron energy E for low E, and for the recombination coefficient as a function of the temperature T for low T. The expansion coefficients are combinations of confluent hypergeometric functions, and are tabulated for a large number of the final principal and angular-momentum quantum numbers n and l. It is shown that the recombination cross section for arbitrary nuclear charge number Z is independent of Z, while the recombination coefficient for T/Z 2 much-lt 1.58x10 5 K increases as Z 2 . Excellent agreement is found with the published tabulated values

  20. Three-body recombination of cold fermionic atoms

    International Nuclear Information System (INIS)

    Suno, H; Esry, B D; Greene, Chris H

    2003-01-01

    Recombination of identical, spin-polarized fermions in cold three-body collisions is investigated. We parametrize the mechanisms for recombination in terms of the 'scattering volume' V p and another length scale r 0 . Model two-body interactions were used within the framework of the adiabatic hyperspherical representation. We examine the recombination rate K 3 as a function of the collision energy E for various values of V p . Not only do we consider the dominant J Π = 1 + case, but also the next-leading order contributions from J Π = 1 - and 3 - . We discuss the behaviour near a two-body resonance and the expected universality of fermionic recombination. Comparisons with boson recombination are considered in detail

  1. Total and partial recombination cross sections for F6+

    International Nuclear Information System (INIS)

    Mitnik, D.M.; Pindzola, M.S.; Badnell, N.R.

    1999-01-01

    Total and partial recombination cross sections for F 6+ are calculated using close-coupling and distorted-wave theory. For total cross sections, close-coupling and distorted-wave results, which include interference between the radiative and dielectronic pathways, are found to be in good agreement with distorted-wave results based on a sum of independent processes. Total cross sections near zero energy are dominated by contributions from low-energy dielectronic recombination resonances. For partial cross sections, the close-coupling and distorted-wave theories predict strong interference for recombination into the final recombined ground state 1s 2 2s 21 S 0 of F 5+ , but only weak interference for recombination into the levels of the 1s 2 2s2p configuration. copyright 1999 The American Physical Society

  2. Novel canine circovirus strains from Thailand: Evidence for genetic recombination.

    Science.gov (United States)

    Piewbang, Chutchai; Jo, Wendy K; Puff, Christina; van der Vries, Erhard; Kesdangsakonwut, Sawang; Rungsipipat, Anudep; Kruppa, Jochen; Jung, Klaus; Baumgärtner, Wolfgang; Techangamsuwan, Somporn; Ludlow, Martin; Osterhaus, Albert D M E

    2018-05-14

    Canine circoviruses (CanineCV's), belonging to the genus Circovirus of the Circoviridae family, were detected by next generation sequencing in samples from Thai dogs with respiratory symptoms. Genetic characterization and phylogenetic analysis of nearly complete CanineCV genomes suggested that natural recombination had occurred among different lineages of CanineCV's. Similarity plot and bootscaning analyses indicated that American and Chinese viruses had served as major and minor parental viruses, respectively. Positions of recombination breakpoints were estimated using maximum-likelihood frameworks with statistical significant testing. The putative recombination event was located in the Replicase gene, intersecting with open reading frame-3. Analysis of nucleotide changes confirmed the origin of the recombination event. This is the first description of naturally occurring recombinant CanineCV's that have resulted in the circulation of newly emerging CanineCV lineages.

  3. Looking for the optimal rate of recombination for evolutionary dynamics

    Science.gov (United States)

    Saakian, David B.

    2018-01-01

    We consider many-site mutation-recombination models of evolution with selection. We are looking for situations where the recombination increases the mean fitness of the population, and there is an optimal recombination rate. We found two fitness landscapes supporting such nonmonotonic behavior of the mean fitness versus the recombination rate. The first case is related to the evolution near the error threshold on a neutral-network-like fitness landscape, for moderate genome lengths and large population. The more realistic case is the second one, in which we consider the evolutionary dynamics of a finite population on a rugged fitness landscape (the smooth fitness landscape plus some random contributions to the fitness). We also give the solution to the horizontal gene transfer model in the case of asymmetric mutations. To obtain nonmonotonic behavior for both mutation and recombination, we need a specially designed (ideal) fitness landscape.

  4. Charge carrier recombination dynamics in perovskite and polymer solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Paulke, Andreas; Kniepert, Juliane; Kurpiers, Jona; Wolff, Christian M.; Schön, Natalie; Brenner, Thomas J. K.; Neher, Dieter [Institute of Physics and Astronomy, University of Potsdam, Karl-Liebknecht-Str. 24–25, 14476, Potsdam (Germany); Stranks, Samuel D. [Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, Oxford OX1 3PU (United Kingdom); Research Laboratory of Electronics, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139 (United States); Cavendish Laboratory, JJ Thomson Avenue, Cambridge CB3 0HE (United Kingdom); Snaith, Henry J. [Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, Oxford OX1 3PU (United Kingdom)

    2016-03-14

    Time-delayed collection field experiments are applied to planar organometal halide perovskite (CH{sub 3}NH{sub 3}PbI{sub 3}) based solar cells to investigate charge carrier recombination in a fully working solar cell at the nanosecond to microsecond time scale. Recombination of mobile (extractable) charges is shown to follow second-order recombination dynamics for all fluences and time scales tested. Most importantly, the bimolecular recombination coefficient is found to be time-dependent, with an initial value of ca. 10{sup −9} cm{sup 3}/s and a progressive reduction within the first tens of nanoseconds. Comparison to the prototypical organic bulk heterojunction device PTB7:PC{sub 71}BM yields important differences with regard to the mechanism and time scale of free carrier recombination.

  5. Sex recombination, and reproductive fitness: an experimental study using Paramecium

    Energy Technology Data Exchange (ETDEWEB)

    Nyberg, D.

    1982-08-01

    The effect of sex and recombination on reproductive fitness are measured using five wild stocks of Paramecium primaurelia. Among the wild stocks there were highly significant differences in growth rates. No hybrid had as low a fitness as the least fit parental stock. Recombination produced genotypes of higher fitness than those of either parent only in the cross between the two stocks of lowest fitness. The increase in variance of fitness as a result of recombination was almost exclusively attributable to the generation lines with low fitness. The fitness consequences of sexuality and mate choice were stock specific; some individuals leaving the most descendants by inbreeding, others by outcrossing. For most crosses the short-term advantage of sex, if any, accrue from the fusion of different gametes (hybrid vigor) and not from recombination. Since the homozygous genotype with the highest fitnes left the most progeny by inbreeding (no recombination), the persistence of conjugation in P. primaurelia is paradoxical. (JMT)

  6. In vitro V(D)J recombination: signal joint formation.

    Science.gov (United States)

    Cortes, P; Weis-Garcia, F; Misulovin, Z; Nussenzweig, A; Lai, J S; Li, G; Nussenzweig, M C; Baltimore, D

    1996-11-26

    The first step of V(D)J recombination, specific cleavage at the recombination signal sequence (RSS), can be carried out by the recombination activating proteins RAG1 and RAG2. In vivo, the cleaved coding and signal ends must be rejoined to generate functional antigen receptors and maintain chromosomal integrity. We have investigated signal joint formation using deletion and inversion substrates in a cell free system. RAG1 and RAG2 alone or in combination were unable to generate signal joints. However, RAG1 and RAG2 complemented with nuclear extracts were able to recombine an extrachromosomal substrate and form precise signal joints. The in vitro reaction resembled authentic V(D)J recombination in being Ku-antigen-dependent.

  7. Correlation between pairing initiation sites, recombination nodules and meiotic recombination in Sordaria macrospora.

    Science.gov (United States)

    Zickler, D; Moreau, P J; Huynh, A D; Slezec, A M

    1992-09-01

    The decrease of meiotic exchanges (crossing over and conversion) in two mutants of Sordaria macrospora correlated strongly with a reduction of chiasmata and of both types of "recombination nodules." Serial section reconstruction electron microscopy was used to compare the synapsis pattern of meiotic prophase I in wild type and mutants. First, synapsis occurred but the number of synaptonemal complex initiation sites was reduced in both mutants. Second, this reduction was accompanied by, or resulted in, modifications of the pattern of synapsis. Genetic and synaptonemal complex maps were compared in three regions along one chromosome arm divided into well marked intervals. Reciprocal exchange frequencies and number of recombination nodules correlated in wild type in the three analyzed intervals, but disparity was found between the location of recombination nodules and exchanges in the mutants. Despite the twofold exchange decrease, sections of the genome such as the short arm of chromosome 2 and telomere regions were sheltered from nodule decrease and from pairing modifications. This indicated a certain amount of diversity in the control of these features and suggested that exchange frequency was dependent not only on the amount of effective pairing but also on the localization of the pairing sites, as revealed by the synaptonemal complex progression in the mutants.

  8. Expression and purification of recombinant hemoglobin in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Chandrasekhar Natarajan

    Full Text Available Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species.As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus, a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications.Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis.

  9. Expression and purification of recombinant hemoglobin in Escherichia coli.

    Science.gov (United States)

    Natarajan, Chandrasekhar; Jiang, Xiaoben; Fago, Angela; Weber, Roy E; Moriyama, Hideaki; Storz, Jay F

    2011-01-01

    Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species. As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus), a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications. Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis.

  10. Effect of sex, age, and breed on genetic recombination features in cattle

    Science.gov (United States)

    Meiotic recombination is a fundamental biological process which generates genetic diversity, affects fertility, and influences evolvability. Here we investigate the roles of sex, age, and breed in cattle recombination features, including recombination rate, location and crossover interference. Usin...

  11. Cold Spring Harbor symposia on quantitative biology: Volume 49, Recombination at the DNA level

    International Nuclear Information System (INIS)

    1984-01-01

    This volume contains full papers prepared by the participants to the 1984 Cold Springs Harbor Symposia on Quantitative Biology. This year's theme is entitled Recombination at the DNA level. The volume consists of 93 articles grouped into subject areas entitled chromosome mechanics, yeast systems, mammalian homologous recombination, transposons, mu, plant transposons/T4 recombination, topoisomerase, resolvase and gyrase, Escherichia coli general recombination, RecA, repair, leukaryotic enzymes, integration and excision of bacteriophage, site-specific recombination, and recombination in vitro

  12. Retroviral Vectors for Analysis of Viral Mutagenesis and Recombination

    Directory of Open Access Journals (Sweden)

    Jonathan M.O. Rawson

    2014-09-01

    Full Text Available Retrovirus population diversity within infected hosts is commonly high due in part to elevated rates of replication, mutation, and recombination. This high genetic diversity often complicates the development of effective diagnostics, vaccines, and antiviral drugs. This review highlights the diverse vectors and approaches that have been used to examine mutation and recombination in retroviruses. Retroviral vectors for these purposes can broadly be divided into two categories: those that utilize reporter genes as mutation or recombination targets and those that utilize viral genes as targets of mutation or recombination. Reporter gene vectors greatly facilitate the detection, quantification, and characterization of mutants and/or recombinants, but may not fully recapitulate the patterns of mutagenesis or recombination observed in native viral gene sequences. In contrast, the detection of mutations or recombination events directly in viral genes is more biologically relevant but also typically more challenging and inefficient. We will highlight the advantages and disadvantages of the various vectors and approaches used as well as propose ways in which they could be improved.

  13. Experimental study of para- and ortho-H3+ recombination

    International Nuclear Information System (INIS)

    Plasil, R; Varju, J; Hejduk, M; Dohnal, P; KotrIk, T; Glosik, J

    2011-01-01

    Recombination of H 3 + with electrons is a key process for many plasmatic environments. Recent experiments on storage ring devices used ion sources producing H 3 + with enhanced populations of H 3 + ions in the para nuclear spin configuration to shed light on the theoretically predicted faster recombination of para states. Although increased recombination rates were observed, no in situ characterization of recombining ions was performed. We present a state selective recombination study of para- and ortho-H 3 + ions with electrons at 77 K in afterglow plasma in a He/Ar/H 2 gas-mixture. Both spin configurations of H 3 + have been observed in situ with a near infrared cavity ring down spectrometer (NIR-CRDS) using the two lowest energy levels of H 3 + . Using hydrogen with an enhanced population of H 2 molecules in para states allowed us to influence the [para-H 3 + ]/[ortho-H 3 + ] ratio in the discharge and in the afterglow. We observed an increase in the measured effective recombination rate coefficients with the increase of the fraction of para-H 3 + . Measurements with different fractions of para-H 3 + at otherwise identical conditions allowed us to determine the binary recombination rate coefficients for pure para-H 3 + p α bin (77 K) = (2.0±0.4)x10 -7 cm 3 s -1 and pure ortho-H 3 + o α bin (77 K) = (4±3)x10 -8 cm 3 s -1 .

  14. Triplet formation in the ion recombination in irradiated liquids

    International Nuclear Information System (INIS)

    Bartczak, W.M.; Tachiya, M.; Hummel, A.

    1990-01-01

    The formation of singlet and triplet excited stages in the ion recombination in groups of oppositely charged ions (or positive ions and electrons) in nonpolar liquids, as occurs in the tracks of high energy electrons, is considered. Theoretical studies on triplet formation in groups of ion pairs have thus far concentrated on the case where recombination of the negative ions with any of the positive ions in the group is equally probable (random recombination). In this paper the probability for geminate recombination (electron and parent positive ion) vs cross-recombination (an electron with a positive ion other than its parent ion) in multiple ion pair groups is calculated by computer simulation and the effect of the initial spatial configuration of the charged species is investigated. It is also shown explicitly that the probability for singlet formation as a result of cross recombination is equal to 1/4, when spin relaxation by magnetic interaction with the medium and by exchange interaction can be neglected. The effect of the preferential recombination on the singlet formation probability is illustrated and recent experimental results on singlet to triplet ratios are discussed. (author)

  15. A Glance at Recombination Hotspots in the Domestic Cat.

    Directory of Open Access Journals (Sweden)

    Hasan Alhaddad

    Full Text Available Recombination has essential roles in increasing genetic variability within a population and in ensuring successful meiotic events. The objective of this study is to (i infer the population-scaled recombination rate (ρ, and (ii identify and characterize regions of increased recombination rate for the domestic cat, Felis silvestris catus. SNPs (n = 701 were genotyped in twenty-two East Asian feral cats (random bred. The SNPs covered ten different chromosomal regions (A1, A2, B3, C2, D1, D2, D4, E2, F2, X with an average region size of 850 Kb and an average SNP density of 70 SNPs/region. The Bayesian method in the program inferRho was used to infer regional population recombination rates and hotspots localities. The regions exhibited variable population recombination rates and four decisive recombination hotspots were identified on cat chromosome A2, D1, and E2 regions. As a description of the identified hotspots, no correlation was detected between the GC content and the locality of recombination spots, and the hotspots enclosed L2 LINE elements and MIR and tRNA-Lys SINE elements.

  16. Recombination via point defects and their complexes in solar silicon

    Energy Technology Data Exchange (ETDEWEB)

    Peaker, A.R.; Markevich, V.P.; Hamilton, B. [Photon Science Institute, University of Manchester, Manchester M13 9PL (United Kingdom); Parada, G.; Dudas, A.; Pap, A. [Semilab, 2 Prielle Kornelia Str, 1117 Budapest (Hungary); Don, E. [Semimetrics, PO Box 36, Kings Langley, Herts WD4 9WB (United Kingdom); Lim, B.; Schmidt, J. [Institute for Solar Energy Research (ISFH) Hamlen, 31860 Emmerthal (Germany); Yu, L.; Yoon, Y.; Rozgonyi, G. [Department of Materials Science and Engineering, North Carolina State University, Raleigh, NC 27695-7907 (United States)

    2012-10-15

    Electronic grade Czochralski and float zone silicon in the as grown state have a very low concentration of recombination generation centers (typically <10{sup 10} cm{sup -3}). Consequently, in integrated circuit technologies using such material, electrically active inadvertent impurities and structural defects are rarely detectable. The quest for cheap photovoltaic cells has led to the use of less pure silicon, multi-crystalline material, and low cost processing for solar applications. Cells made in this way have significant extrinsic recombination mechanisms. In this paper we review recombination involving defects and impurities in single crystal and in multi-crystalline solar silicon. Our main techniques for this work are recombination lifetime mapping measurements using microwave detected photoconductivity decay and variants of deep level transient spectroscopy (DLTS). In particular, we use Laplace DLTS to distinguish between isolated point defects, small precipitate complexes and decorated extended defects. We compare the behavior of some common metallic contaminants in solar silicon in relation to their effect on carrier lifetime and cell efficiency. Finally, we consider the role of hydrogen passivation in relation to transition metal contaminants, grain boundaries and dislocations. We conclude that recombination via point defects can be significant but in most multi-crystalline material the dominant recombination path is via decorated dislocation clusters within grains with little contribution to the overall recombination from grain boundaries. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  17. Damage-induced ectopic recombination in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Kupiec, M; Steinlauf, R

    1997-06-09

    Mitotic recombination in the yeast Saccharomyces cerevisiae is induced when cells are irradiated with UV or X-rays, reflecting the efficient repair of damage by recombinational repair mechanisms. We have used multiply marked haploid strains that allow the simultaneous detection of several types of ectopic recombination events. We show that inter-chromosomal ectopic conversion of lys2 heteroalleles and, to a lesser extent, direct repeat recombination (DRR) between non-tandem repeats, are increased by DNA-damaging agents; in contrast, ectopic recombination of the naturally occurring Ty element is not induced. We have tested several hypotheses that could explain the preferential lack of induction of Ty recombination by DNA-damaging agents. We have found that the lack of induction cannot be explained by a cell cycle control or by an effect of the mating-type genes. We also found no role for the flanking long terminal repeats (LTRs) of the Ty in preventing the induction. Ectopic conversion, DRR, and forward mutation of artificial repeats show different kinetics of induction at various positions of the cell cycle, reflecting different mechanisms of recombination. We discuss the mechanistic and evolutionary aspects of these results.

  18. The influence of recombination on human genetic diversity.

    Directory of Open Access Journals (Sweden)

    Chris C A Spencer

    2006-09-01

    Full Text Available In humans, the rate of recombination, as measured on the megabase scale, is positively associated with the level of genetic variation, as measured at the genic scale. Despite considerable debate, it is not clear whether these factors are causally linked or, if they are, whether this is driven by the repeated action of adaptive evolution or molecular processes such as double-strand break formation and mismatch repair. We introduce three innovations to the analysis of recombination and diversity: fine-scale genetic maps estimated from genotype experiments that identify recombination hotspots at the kilobase scale, analysis of an entire human chromosome, and the use of wavelet techniques to identify correlations acting at different scales. We show that recombination influences genetic diversity only at the level of recombination hotspots. Hotspots are also associated with local increases in GC content and the relative frequency of GC-increasing mutations but have no effect on substitution rates. Broad-scale association between recombination and diversity is explained through covariance of both factors with base composition. To our knowledge, these results are the first evidence of a direct and local influence of recombination hotspots on genetic variation and the fate of individual mutations. However, that hotspots have no influence on substitution rates suggests that they are too ephemeral on an evolutionary time scale to have a strong influence on broader scale patterns of base composition and long-term molecular evolution.

  19. Recombination properties of dislocations in GaN

    Science.gov (United States)

    Yakimov, Eugene B.; Polyakov, Alexander Y.; Lee, In-Hwan; Pearton, Stephen J.

    2018-04-01

    The recombination activity of threading dislocations in n-GaN with different dislocation densities and different doping levels was studied using electron beam induced current (EBIC). The recombination velocity on a dislocation, also known as the dislocation recombination strength, was calculated. The results suggest that dislocations in n-GaN giving contrast in EBIC are charged and surrounded by a space charge region, as evidenced by the observed dependence of dislocation recombination strength on dopant concentration. For moderate (below ˜108 cm-2) dislocation densities, these defects do not primarily determine the average diffusion length of nonequilibrium charge carriers, although locally, dislocations are efficient recombination sites. In general, it is observed that the effect of the growth method [standard metalorganic chemical vapor deposition (MOCVD), epitaxial lateral overgrowth versions of MOCVD, and hydride vapor phase epitaxy] on the recombination activity of dislocations is not very pronounced, although the average diffusion lengths can widely differ for various samples. The glide of basal plane dislocations at room temperature promoted by low energy electron irradiation does not significantly change the recombination properties of dislocations.

  20. Phylogenetic and recombination analysis of tomato spotted wilt virus.

    Directory of Open Access Journals (Sweden)

    Sen Lian

    Full Text Available Tomato spotted wilt virus (TSWV severely damages and reduces the yield of many economically important plants worldwide. In this study, we determined the whole-genome sequences of 10 TSWV isolates recently identified from various regions and hosts in Korea. Phylogenetic analysis of these 10 isolates as well as the three previously sequenced isolates indicated that the 13 Korean TSWV isolates could be divided into two groups reflecting either two different origins or divergences of Korean TSWV isolates. In addition, the complete nucleotide sequences for the 13 Korean TSWV isolates along with previously sequenced TSWV RNA segments from Korea and other countries were subjected to phylogenetic and recombination analysis. The phylogenetic analysis indicated that both the RNA L and RNA M segments of most Korean isolates might have originated in Western Europe and North America but that the RNA S segments for all Korean isolates might have originated in China and Japan. Recombination analysis identified a total of 12 recombination events among all isolates and segments and five recombination events among the 13 Korea isolates; among the five recombinants from Korea, three contained the whole RNA L segment, suggesting reassortment rather than recombination. Our analyses provide evidence that both recombination and reassortment have contributed to the molecular diversity of TSWV.

  1. Analysis of intermolecular RNA-RNA recombination by rubella virus

    International Nuclear Information System (INIS)

    Adams, Sandra D.; Tzeng, W.-P.; Chen, M.-H.; Frey, Teryl K.

    2003-01-01

    To investigate whether rubella virus (RUB) undergoes intermolecular RNA-RNA recombination, cells were cotransfected with pairs of in vitro transcripts from genomic cDNA plasmid vectors engineered to contain nonoverlapping deletions: the replicative transcript maintained the 5'-proximal nonstructural (NS) ORF (which contained the replicase, making it RNA replication competent), had a deletion in the 3'-proximal structural protein (SP) ORF, and maintained the 3' end of the genome, including the putative 3' cis-acting elements (CSE), while the nonreplicative transcript consisted of the 3' half of the genome including the SP-ORF and 3' CSE. Cotransfection yielded plaque-forming virus that synthesized the standard genomic and subgenomic RNAs and thus was generated by RNA-RNA recombination. Using transcripts tagged with a 3'-terminal deletion, it was found that recombinants contained the 3' end derived from the replicative strand, indicating a cis-preference for initiation of negative-strand synthesis. In cotransfections in which the replicative transcript lacked the 3' CSE, recombination occurred, albeit at lower efficiency, indicating that initiation in trans from the NS-ORF can occur. The 3' CSE was sufficient as a nonreplicative transcript, showing that it can serve as a promoter for negative-strand RNA synthesis. While deletion mutagenesis showed that the presence of the junction untranslated region (J-UTR) between the ORFs appeared to be necessary on both transcripts for recombination in this region of the genome, analysis with transcripts tagged with restriction sites showed that the J-UTR was not a hot spot for recombination compared to neighboring regions in both ORFs. Sequence analysis of recombinants revealed that both precise (homologous) and imprecise recombination (aberrant, homologous resulting in duplications) occurred; however, imprecise recombination only involved the J-UTR or the 3' end of the NS-ORF and the J-UTR (maintaining the NS-ORF), indicating

  2. Recombination among multiple mitochondrial pseudogenes from a passerine genus

    DEFF Research Database (Denmark)

    Nielsen, Kirstine Klitgaard; Arctander, P.

    2001-01-01

    to the observed differences in substitution patterns 58% of the cloned sequences were identified as pseudogenes. Recombination could be traced in 19% of the inferred nuclear pseudogenes, but this figure probably represents a Significant underestimation of the factual recombination events. The nonrecombined...... pseudogenes consisted of multiple haplotypes found to diverge from 1 to 16% from the mitochondrial gene. The number of mitochondrial nuclear copies and their apparent frequent recombination suggest that pseudogenes constitute a serious potential risk in confounding phylogenetic studies and population genetic...

  3. Recombinative generalization of subword units using matching to sample.

    LENUS (Irish Health Repository)

    Mahon, Catherine

    2010-01-01

    The purpose of the current study was to develop and test a computerized matching-to-sample (MTS) protocol to facilitate recombinative generalization of subword units (onsets and rimes) and recognition of novel onset-rime and onset-rime-rime words. In addition, we sought to isolate the key training components necessary for recombinative generalization. Twenty-five literate adults participated. Conditional discrimination training emerged as a crucial training component. These findings support the effectiveness of MTS in facilitating recombinative generalization, particularly when conditional discrimination training with subword units is used.

  4. Plasmodium knowlesi Sporozoite Antigen: Expression by Infectious Recombinant Vaccinia Virus

    Science.gov (United States)

    Smith, Geoffrey L.; Godson, G. Nigel; Nussenzweig, Victor; Nussenzweig, Ruth S.; Barnwell, John; Moss, Bernard

    1984-04-01

    The gene coding for the circumsporozoite antigen of the malaria parasite Plasmodium knowlesi was inserted into the vaccinia virus genome under the control of a defined vaccinia virus promoter. Cells infected with the recombinant virus synthesized polypeptides of 53,000 to 56,000 daltons that reacted with monoclonal antibody against the repeating epitope of the malaria protein. Furthermore, rabbits vaccinated with the recombinant virus produced antibodies that bound specifically to sporozoites. These data provide evidence for expression of a cloned malaria gene in mammalian cells and illustrate the potential of vaccinia virus recombinants as live malaria vaccines.

  5. Dielectronic Recombination of Al-Like Ions

    Science.gov (United States)

    Abdel-Naby, Shahin; Nikolic, Dragan; Gorczyca, Thomas W.; Badnell, Nigel R.; Savin, Daniel W.

    2008-05-01

    Accurate dielectronic recombination (DR) data are important for cosmic and laboratory plasma modeling. Over the past few years, our group has computed reliable DR data for all isoelectronic sequences up through Mg-like ions. Recently, we have focused our work on the complex third-row M-shell isoelectronic sequences, especially Al-like. Previous calculations for the DR rate coefficient for S^3+ were performed only within a non-relativistic LS-coupling approximation. Fe^13+ DR calculations, including semi-relativistic effects, have been completed and tested against the Heidelberg heavy-ion Test Storage Ring facility measurements. Here we present semi-relativistic DR rate coefficient calculations for a wide range of Al-like ions using AUTOSTRUCTURE, a level-resolved distorted-wave program package. The important effect of fine structure splitting in the Al-like ground state will be discussed. Finally, our results are fitted into a simple formula for use by astrophysical plasma modelers.This work was funded in part by NASA (APRA), NASA (SHP) SR&T, and UK PPARC grants.

  6. Recombinant viruses as vaccines against viral diseases

    Directory of Open Access Journals (Sweden)

    A.P.D. Souza

    2005-04-01

    Full Text Available Vaccine approaches to infectious diseases are widely applied and appreciated. Amongst them, vectors based on recombinant viruses have shown great promise and play an important role in the development of new vaccines. Many viruses have been investigated for their ability to express proteins from foreign pathogens and induce specific immunological responses against these antigens in vivo. Generally, gene-based vaccines can stimulate potent humoral and cellular immune responses and viral vectors might be an effective strategy for both the delivery of antigen-encoding genes and the facilitation and enhancement of antigen presentation. In order to be utilized as a vaccine carrier, the ideal viral vector should be safe and enable efficient presentation of required pathogen-specific antigens to the immune system. It should also exhibit low intrinsic immunogenicity to allow for its re-administration in order to boost relevant specific immune responses. Furthermore, the vector system must meet criteria that enable its production on a large-scale basis. Several viral vaccine vectors have thus emerged to date, all of them having relative advantages and limits depending on the proposed application, and thus far none of them have proven to be ideal vaccine carriers. In this review we describe the potential, as well as some of the foreseeable obstacles associated with viral vaccine vectors and their use in preventive medicine.

  7. Applications of recombinant antibodies in plant pathology.

    Science.gov (United States)

    Ziegler, Angelika; Torrance, Lesley

    2002-09-01

    Summary Advances in molecular biology have made it possible to produce antibody fragments comprising the binding domains of antibody molecules in diverse heterologous systems, such as Escherichia coli, insect cells, or plants. Antibody fragments specific for a wide range of antigens, including plant pathogens, have been obtained by cloning V-genes from lymphoid tissue, or by selection from large naive phage display libraries, thus avoiding the need for immunization. The antibody fragments have been expressed as fusion proteins to create different functional molecules, and fully recombinant assays have been devised to detect plant viruses. The defined binding properties and unlimited cheap supply of antibody fusion proteins make them useful components of standardized immunoassays. The expression of antibody fragments in plants was shown to confer resistance to several plant pathogens. However, the antibodies usually only slowed the progress of infection and durable 'plantibody' resistance has yet to be demonstrated. In future, it is anticipated that antibody fragments from large libraries will be essential tools in high-throughput approaches to post-genomics research, such as the assignment of gene function, characterization of spatio-temporal patterns of protein expression, and elucidation of protein-protein interactions.

  8. Recombinant albumin monolayers on latex particles.

    Science.gov (United States)

    Sofińska, Kamila; Adamczyk, Zbigniew; Kujda, Marta; Nattich-Rak, Małgorzata

    2014-01-14

    The adsorption of recombinant human serum albumin (rHSA) on negatively charged polystyrene latex micro-particles was studied at pH 3.5 and the NaCl concentration range of 10(-3) to 0.15 M. The electrophoretic mobility of latex monotonically increased with the albumin concentration in the suspension. The coverage of adsorbed albumin was quantitatively determined using the depletion method, where the residual protein concentration was determined by electrokinetic measurements and AFM imaging. It was shown that albumin adsorption was irreversible. Its maximum coverage on latex varied between 0.7 mg m(-2) for 10(-3) M NaCl to 1.3 mg m(-2) for 0.15 M NaCl. The latter value matches the maximum coverage previously determined for human serum albumin on mica using the streaming potential method. The increase in the maximum coverage was interpreted in terms of reduced electrostatic repulsion among adsorbed molecules. These facts confirm that albumin adsorption at pH 3.5 is governed by electrostatic interactions and proceeds analogously to colloid particle deposition. The stability of albumin monolayers was measured in additional experiments where changes in the latex electrophoretic mobility and the concentration of free albumin in solutions were monitored over prolonged time periods. Based on these experimental data, a robust procedure of preparing albumin monolayers on latex particles of well-controlled coverage and molecule distribution was proposed.

  9. Overview of the purification of recombinant proteins.

    Science.gov (United States)

    Wingfield, Paul T

    2015-04-01

    When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches, many of which were described and mentioned throughout Current Protocols in Protein Science. In the interim, there has been a shift toward an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein, and whether to engineer a self-cleavage system or simply leave them on. We will briefly address some of these issues. Also, although this overview focuses on E.coli, protein expression and purification, other commonly used expression systems are mentioned and, apart from cell-breakage methods, protein purification methods and strategies are essentially the same. Copyright © 2015 John Wiley & Sons, Inc.

  10. Therapeutic Use of Native and Recombinant Enteroviruses

    Directory of Open Access Journals (Sweden)

    Jani Ylä-Pelto

    2016-02-01

    Full Text Available Research on human enteroviruses has resulted in the identification of more than 100 enterovirus types, which use more than 10 protein receptors and/or attachment factors required in cell binding and initiation of the replication cycle. Many of these “viral” receptors are overexpressed in cancer cells. Receptor binding and the ability to replicate in specific target cells define the tropism and pathogenesis of enterovirus types, because cellular infection often results in cytolytic response, i.e., disruption of the cells. Viral tropism and cytolytic properties thus make native enteroviruses prime candidates for oncolytic virotherapy. Copy DNA cloning and modification of enterovirus genomes have resulted in the generation of enterovirus vectors with properties that are useful in therapy or in vaccine trials where foreign antigenic epitopes are expressed from or on the surface of the vector virus. The small genome size and compact particle structure, however, set limits to enterovirus genome modifications. This review focuses on the therapeutic use of native and recombinant enteroviruses and the methods that have been applied to modify enterovirus genomes for therapy.

  11. Epigenetic codes programming class switch recombination

    Directory of Open Access Journals (Sweden)

    Bharat eVaidyanathan

    2015-09-01

    Full Text Available Class switch recombination imparts B cells with a fitness-associated adaptive advantage during a humoral immune response by using a precision-tailored DNA excision and ligation process to swap the default constant region gene of the antibody with a new one that has unique effector functions. This secondary diversification of the antibody repertoire is a hallmark of the adaptability of B cells when confronted with environmental and pathogenic challenges. Given that the nucleotide sequence of genes during class switching remains unchanged (genetic constraints, it is logical and necessary therefore, to integrate the adaptability of B cells to an epigenetic state, which is dynamic and can be heritably modulated before, after or even during an antibody-dependent immune response. Epigenetic regulation encompasses heritable changes that affect function (phenotype without altering the sequence information embedded in a gene, and include histone, DNA and RNA modifications. Here, we review current literature on how B cells use an epigenetic code language as a means to ensure antibody plasticity in light of pathogenic insults.

  12. Recombination-assisted megaprimer (RAM) cloning

    Science.gov (United States)

    Mathieu, Jacques; Alvarez, Emilia; Alvarez, Pedro J.J.

    2014-01-01

    No molecular cloning technique is considered universally reliable, and many suffer from being too laborious, complex, or expensive. Restriction-free cloning is among the simplest, most rapid, and cost-effective methods, but does not always provide successful results. We modified this method to enhance its success rate through the use of exponential amplification coupled with homologous end-joining. This new method, recombination-assisted megaprimer (RAM) cloning, significantly extends the application of restriction-free cloning, and allows efficient vector construction with much less time and effort when restriction-free cloning fails to provide satisfactory results. The following modifications were made to the protocol:•Limited number of PCR cycles for both megaprimer synthesis and the cloning reaction to reduce error propagation.•Elimination of phosphorylation and ligation steps previously reported for cloning methods that used exponential amplification, through the inclusion of a reverse primer in the cloning reaction with a 20 base pair region of homology to the forward primer.•The inclusion of 1 M betaine to enhance both reaction specificity and yield. PMID:26150930

  13. Recombinant phage probes for Listeria monocytogenes

    Energy Technology Data Exchange (ETDEWEB)

    Carnazza, S; Gioffre, G; Felici, F; Guglielmino, S [Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Messina (Italy)

    2007-10-03

    Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 10{sup 4} cells ml{sup -1}. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

  14. Recombinant phage probes for Listeria monocytogenes

    Science.gov (United States)

    Carnazza, S.; Gioffrè, G.; Felici, F.; Guglielmino, S.

    2007-10-01

    Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 104 cells ml-1. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

  15. Fluctuation characteristics in detached recombining plasmas

    International Nuclear Information System (INIS)

    Ohno, Noriyasu; Tanaka, Naoyuki; Takamura, Shuichi; Budaev, Viatcheslav

    2002-01-01

    Fluctuation in detached recombining plasmas has been investigated experimentally in the linear divertor plasma simulator, NAGDIS-II. As increasing neutral gas pressure, floating potential fluctuation of the target plate installed at the end of the NADIS-II device becomes larger and bursty negative spikes are observed in the signal associated with a transition from attached to detached a plasmas. The fluctuation property has been analyzed by using Fast Fourier Transform (FFT), probability distribution function (PDF) and wavelet transform. The PDF of the floating potential fluctuation in the attached plasma condition obeys the Gaussian distribution function, on the other hand, the PDF in detached plasma shows a strong deviation from the Gaussian distribution function, which can be characterized by flatness and skewness. Comparison of the fluctuation properties between the floating potential and the optical emission from the detached plasma has been done based on the wavelet transform to show that a strong correlation between them, which could indicate bursty transport of energetic electrons from upstream to downstream region along the magnetic field. (author)

  16. Expression of feline immunodeficiency virus gag and env precursor proteins in Spodoptera frugiperda cells and their use in immunodiagnosis

    NARCIS (Netherlands)

    Horzinek, M.C.; Verschoor, E.J.; Vliet, A.L.W. van; Egberink, H.F.; Hesselink, W.; Ronde, A. de

    1993-01-01

    The gag and env genes of the feline immunodeficiency virus strain UT113 were cloned into a baculovirus transfer vector. The recombinant plasmids were used to create recombinant baculoviruses that expressed either the gag or the env precursor protein in insect cells (Sf9 cells). Leader sequence

  17. Two novel porcine epidemic diarrhea virus (PEDV) recombinants from a natural recombinant and distinct subtypes of PEDV variants.

    Science.gov (United States)

    Chen, Nanhua; Li, Shuangjie; Zhou, Rongyun; Zhu, Meiqin; He, Shan; Ye, Mengxue; Huang, Yucheng; Li, Shuai; Zhu, Cong; Xia, Pengpeng; Zhu, Jianzhong

    2017-10-15

    Porcine epidemic diarrhea virus (PEDV) causes devastating impact on global pig-breeding industry and current vaccines have become not effective against the circulating PEDV variants since 2011. During the up-to-date investigation of PEDV prevalence in Fujian China 2016, PEDV was identified in vaccinated pig farms suffering severe diarrhea while other common diarrhea-associated pathogens were not detected. Complete genomes of two PEDV representatives (XM1-2 and XM2-4) were determined. Genomic comparison showed that these two viruses share the highest nucleotide identities (99.10% and 98.79%) with the 2011 ZMDZY strain, but only 96.65% and 96.50% nucleotide identities with the attenuated CV777 strain. Amino acid alignment of spike (S) proteins indicated that they have the similar mutation, insertion and deletion pattern as other Chinese PEDV variants but also contain several unique substitutions. Phylogenetic analysis showed that 2016 PEDV variants belong to the cluster of recombination strains but form a new branch. Recombination detection suggested that both XM1-2 and XM2-4 are inter-subgroup recombinants with breakpoints within ORF1b. Remarkably, the natural recombinant HNQX-3 isolate serves as a parental virus for both natural recombinants identified in this study. This up-to-date investigation provides the direct evidence that natural recombinants may serve as parental viruses to generate recombined PEDV progenies that are probably associated with the vaccination failure. Copyright © 2017. Published by Elsevier B.V.

  18. Rapid generation of markerless recombinant MVA vaccines by en passant recombineering of a self-excising bacterial artificial chromosome.

    Science.gov (United States)

    Cottingham, Matthew G; Gilbert, Sarah C

    2010-09-01

    The non-replicating poxviral vector modified vaccinia virus Ankara (MVA) is currently a leading candidate for development of novel recombinant vaccines against globally important diseases. The 1980s technology for making recombinant MVA (and other poxviruses) is powerful and robust, but relies on rare recombination events in poxviral-infected cells. In the 21st century, it has become possible to apply bacterial artificial chromosome (BAC) technology to poxviruses, as first demonstrated by B. Moss' lab in 2002 for vaccinia virus. A similar BAC clone of MVA was subsequently derived, but while recombination-mediated genetic engineering for rapid production was used of deletion mutants, an alternative method was required for efficient insertion of transgenes. Furthermore "markerless" viruses, which carry no trace of the selectable marker used for their isolation, are increasingly required for clinical trials, and the viruses derived via the new method contained the BAC sequence in their genomic DNA. Two methods are adapted to MVA-BAC to provide more rapid generation of markerless recombinants in weeks rather than months. "En passant" recombineering is applied to the insertion of a transgene expression cassette and the removal of the selectable marker in bacteria; and a self-excising variant of MVA-BAC is constructed, in which the BAC cassette region is rapidly and efficiently lost from the viral genome following rescue of the BAC into infectious virus. These methods greatly facilitate and accelerate production of recombinant MVA, including markerless constructs. Copyright 2010 Elsevier B.V. All rights reserved.

  19. Producción y purificación de la proteína core del virus de la hepatitis C en el sistema de expresión del baculovirus para ensayos biológicos.

    Directory of Open Access Journals (Sweden)

    Ivonne Rubio

    2005-03-01

    Full Text Available Introducción. La infección por el virus de la hepatitis C (VHC se caracteriza por la alta frecuencia de infección persistente. La capacidad del VHC para inducir alteraciones en la función de las células del sistema inmune, específicamente en las células dendríticas, parece ser una de las estrategias virales implicada en el establecimiento de la infección persistente. Esta estrategia parece estar mediada en gran parte por una de las proteínas estructurales codificada por el genoma del VHC, la proteína Core, unidad estructural de la cápside viral. Objetivo. Una de las limitantes para la evaluación de las propiedades de Core es la obtención y la purificación de la proteína nativa (191 aa. El objetivo de este estudio fue la producción y la purificación de la proteína Core completa en un sistema eucariote para evaluar las propiedades de la proteína en cultivos de células dendríticas humanas. Resultados. En este estudio se logró la producción de la proteína Core completa en el sistema de expresión de baculovirus y su purificación por la técnica de separación por punto isoeléctrico y electroelución. La pureza de la proteína obtenida se confirmó por Western blot y tinción con plata. Estos análisis mostraron dos bandas únicas correspondientes a las isoformas p23 y p21 de la proteína Core, previamente descritas en la literatura. Conclusiones. La proteína obtenida posee varias de las condiciones de la proteína Core nativa, como peso molecular, isoformas y localización subcelular. Los procedimientos descritos en este artículo son aplicables a proteínas asociadas a membranas producidas en sistemas de expresión eucarióticos.

  20. Bio-equivalent doses of recombinent HCG and recombinent LH during ovarian stimulation result in similar oestradiol output

    DEFF Research Database (Denmark)

    Alsbjerg, Birgit; Elbaek, Helle Olesen; Laursen, Rita Jakubcionyte

    2017-01-01

    In nature, HCG is secreted by the implanting embryo from peri-implantation and onwards. In contrast, LH is mandatory for steroidogenesis and follicular development during the follicular phase, working in synergy with FSH. Moreover, LH is mandatory for the function of the corpus luteum. Although LH...... and HCG bind to the same receptor, significant molecular, structural and functional differences exist, inducing differences in bioactivity. This randomized controlled study compared the effect of recombinant FSH stimulation combined with daily either micro-dose recombinant HCG or recombinant LH...