WorldWideScience
 
 
1

Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes.  

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Replication protein A (RPA) is a DNA single-strand binding protein essential for DNA replication, recombination and repair. In human cells treated with the topoisomerase inhibitors camptothecin or etoposide (VP-16), we find that RPA2, the middle-sized subunit of RPA, becomes rapidly phosphorylated. This response appears to be due to DNA-dependent protein kinase (DNA-PK) and to be independent of p53 or the ataxia telangiectasia mutated (ATM) protein. RPA2 phosphorylation in response to camptot...

1999-01-01

2

RPA antagonizes microhomology-mediated repair of DNA double-strand breaks.  

Science.gov (United States)

Microhomology-mediated end joining (MMEJ) is a Ku- and ligase IV-independent mechanism for the repair of DNA double-strand breaks that contributes to chromosome rearrangements. Here we used a chromosomal end-joining assay to determine the genetic requirements for MMEJ in Saccharomyces cerevisiae. We found that end resection influences the ability to expose microhomologies; however, it is not rate limiting for MMEJ in wild-type cells. The frequency of MMEJ increased by up to 350-fold in rfa1 hypomorphic mutants, suggesting that replication protein A (RPA) bound to the single-stranded DNA (ssDNA) overhangs formed by resection prevents spontaneous annealing between microhomologies. In vitro, the mutant RPA complexes were unable to fully extend ssDNA and were compromised in their ability to prevent spontaneous annealing. We propose that the helix-destabilizing activity of RPA channels ssDNA intermediates from mutagenic MMEJ to error-free homologous recombination, thus preserving genome integrity. PMID:24608368

Deng, Sarah K; Gibb, Bryan; de Almeida, Mariana Justino; Greene, Eric C; Symington, Lorraine S

2014-04-01

3

DNA-PK, ATM and ATR collaboratively regulate p53-RPA interaction to facilitate homologous recombination DNA repair.  

Science.gov (United States)

Homologous recombination (HR) and nonhomologous end joining (NHEJ) are two distinct DNA double-stranded break (DSB) repair pathways. Here, we report that DNA-dependent protein kinase (DNA-PK), the core component of NHEJ, partnering with DNA-damage checkpoint kinases ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), regulates HR repair of DSBs. The regulation was accomplished through modulation of the p53 and replication protein A (RPA) interaction. We show that upon DNA damage, p53 and RPA were freed from a p53-RPA complex by simultaneous phosphorylations of RPA at the N-terminus of RPA32 subunit by DNA-PK and of p53 at Ser37 and Ser46 in a Chk1/Chk2-independent manner by ATR and ATM, respectively. Neither the phosphorylation of RPA nor of p53 alone could dissociate p53 and RPA. Furthermore, disruption of the release significantly compromised HR repair of DSBs. Our results reveal a mechanism for the crosstalk between HR repair and NHEJ through the co-regulation of p53-RPA interaction by DNA-PK, ATM and ATR. PMID:22797063

Serrano, M A; Li, Z; Dangeti, M; Musich, P R; Patrick, S; Roginskaya, M; Cartwright, B; Zou, Y

2013-05-01

4

FRET-based assays to monitor DNA binding and annealing by Rad52 recombination mediator protein.  

Science.gov (United States)

During homologous recombination and homology-directed repair of broken chromosomes, proteins that mediate and oppose recombination form dynamic complexes on damaged DNA. Quantitative analysis of these nucleoprotein assemblies requires a robust signal, which reports on the association of a recombination mediator with its substrate and on the state of substrate DNA within the complex. Eukaryotic Rad52 protein mediates recombination, repair, and restart of collapsed replication forks by facilitating replacement of ssDNA binding protein replication protein A (RPA) with Rad51 recombinase and by mediating annealing of two complementary DNA strands protected by RPA. The characteristic binding mode whereby ssDNA is wrapped around the Rad52 ring allowed us to develop robust and sensitive FRET-based assays for monitoring Rad52 interactions with protein-free DNA and ssDNA-RPA complexes. By reporting on the configuration of ssDNA dually labeled with Cy3 and Cy5 fluorescent dyes, solution-based FRET is used to analyze Rad52-RPA-DNA interactions under equilibrium binding conditions. Finally, FRET between Cy3 and Cy5 dyes incorporated into two homologous ssDNA molecules can be used to analyze interplay between Rad52-mediated DNA strand annealing and duplex DNA destabilization by RPA. PMID:21660711

Grimme, Jill M; Spies, Maria

2011-01-01

5

Phase I Study of Safety and Immunogenicity of an Escherichia coli-Derived Recombinant Protective Antigen (rPA) Vaccine to Prevent Anthrax in Adults  

Science.gov (United States)

Background The fatal disease caused by Bacillus anthracis is preventable with a prophylactic vaccine. The currently available anthrax vaccine requires a lengthy immunization schedule, and simpler and more immunogenic options for protection against anthrax are a priority for development. In this report we describe a phase I clinical trial testing the safety and immunogenicity of an anthrax vaccine using recombinant Escherichia coli-derived, B. anthracis protective antigen (rPA). Methodology/Principal Findings A total of 73 healthy adults ages 18–40 were enrolled and 67 received 2 injections separated by 4 weeks of either buffered saline placebo, or rPA formulated with or without 704 µg/ml Alhydrogel® adjuvant in increasing doses (5, 25, 50, 100 µg) of rPA. Participants were followed for one year and safety and immunologic data were assessed. Tenderness and warmth were the most common post-injection site reactions. No serious adverse events related to the vaccine were observed. The most robust humoral immune responses were observed in subjects receiving 50 µg of rPA formulated with Alhydrogel® with a geometric mean concentration of anti-rPA IgG antibodies of 283 µg/ml and a toxin neutralizing geometric 50% reciprocal geometric mean titer of 1061. The highest lymphoproliferative peak cellular response (median Lymphocyte Stimulation Index of 29) was observed in the group receiving 25 µg Alhydrogel®-formulated rPA. Conclusions/Significance The vaccine was safe, well tolerated and stimulated a robust humoral and cellular response after two doses. Trial Registration ClinicalTrials.gov NCT00057525

Brown, Bruce K.; Cox, Josephine; Gillis, Anita; VanCott, Thomas C.; Marovich, Mary; Milazzo, Mark; Antonille, Tanya Santelli; Wieczorek, Lindsay; McKee, Kelly T.; Metcalfe, Karen; Mallory, Raburn M.; Birx, Deborah; Polonis, Victoria R.; Robb, Merlin L.

2010-01-01

6

Purified human BRCA2 stimulates RAD51-mediated recombination  

Science.gov (United States)

Mutation of the breast cancer susceptibility gene, BRCA2, leads to breast and ovarian cancers. Mechanistic insight into the functions of human BRCA2 has been limited by the difficulty of isolating this large protein (3,418 amino acids). Here we report purification of full length BRCA2 and show that it both binds RAD51 and potentiates recombinational DNA repair by promoting assembly of RAD51 onto single-stranded DNA (ssDNA). BRCA2 acts by: targeting RAD51 to ssDNA over double-stranded DNA; enabling RAD51 to displace Replication protein-A (RPA) from ssDNA; and stabilizing RAD51-ssDNA filaments by blocking ATP hydrolysis. BRCA2 does not anneal ssDNA complexed with RPA, implying it does not directly function in repair processes that involve ssDNA annealing. Our findings show that BRCA2 is a key mediator of homologous recombination, and they provide a molecular basis for understanding how this DNA repair process is disrupted by BRCA2 mutations, which lead to chromosomal instability and cancer.

Jensen, Ryan B.; Carreira, Aura; Kowalczykowski, Stephen C.

2010-01-01

7

Purified human BRCA2 stimulates RAD51-mediated recombination.  

Science.gov (United States)

Mutation of the breast cancer susceptibility gene, BRCA2, leads to breast and ovarian cancers. Mechanistic insight into the functions of human BRCA2 has been limited by the difficulty of isolating this large protein (3,418 amino acids). Here we report the purification of full-length BRCA2 and show that it both binds RAD51 and potentiates recombinational DNA repair by promoting assembly of RAD51 onto single-stranded DNA (ssDNA). BRCA2 acts by targeting RAD51 to ssDNA over double-stranded DNA, enabling RAD51 to displace replication protein-A (RPA) from ssDNA and stabilizing RAD51-ssDNA filaments by blocking ATP hydrolysis. BRCA2 does not anneal ssDNA complexed with RPA, implying it does not directly function in repair processes that involve ssDNA annealing. Our findings show that BRCA2 is a key mediator of homologous recombination, and they provide a molecular basis for understanding how this DNA repair process is disrupted by BRCA2 mutations, which lead to chromosomal instability and cancer. PMID:20729832

Jensen, Ryan B; Carreira, Aura; Kowalczykowski, Stephen C

2010-10-01

8

Molecular anatomy of the recombination mediator function of Saccharomyces cerevisiae Rad52  

DEFF Research Database (Denmark)

A helical filament of Rad51 on single-strand DNA (ssDNA), called the presynaptic filament, catalyzes DNA joint formation during homologous recombination. Rad52 facilitates presynaptic filament assembly, and this recombination mediator activity is thought to rely on the interactions of Rad52 with Rad51, the ssDNA-binding protein RPA, and ssDNA. The N-terminal region of Rad52, which has DNA binding activity and an oligomeric structure, is thought to be crucial for mediator activity and recombination. Unexpectedly, we find that the C-terminal region of Rad52 also harbors a DNA binding function. Importantly, the Rad52 C-terminal portion alone can promote Rad51 presynaptic filament assembly. The middle portion of Rad52 associates with DNA-bound RPA and contributes to the recombination mediator activity. Accordingly, expression of a protein species that harbors the middle and C-terminal regions of Rad52 in the rad52 Delta 327 background enhances the association of Rad51 protein with a HO-made DNA double-strand break and partially complements the methyl-methane sulfonate sensitivity of the mutant cells. Our results provide a mechanistic framework for rationalizing the multi-faceted role of Rad52 in recombination and DNA repair.

Seong, C.; Sehorn, M.G.

2008-01-01

9

Role of Saccharomyces Single-Stranded DNA-Binding Protein RPA in the Strand Invasion Step of Double-Strand Break Repair  

Directory of Open Access Journals (Sweden)

Full Text Available The single-stranded DNA (ssDNA-binding protein replication protein A (RPA is essential for both DNA replication and recombination. Chromatin immunoprecipitation techniques were used to visualize the kinetics and extent of RPA binding following induction of a double-strand break (DSB and during its repair by homologous recombination in yeast. RPA assembles at the HO endonuclease-cut MAT locus simultaneously with the appearance of the DSB, and binding spreads away from the DSB as 5' to 3' exonuclease activity creates more ssDNA. RPA binding precedes binding of the Rad51 recombination protein. The extent of RPA binding is greater when Rad51 is absent, supporting the idea that Rad51 displaces RPA from ssDNA. RPA plays an important role during RAD51-mediated strand invasion of the MAT ssDNA into the donor sequence HML. The replication-proficient but recombination-defective rfa1-t11 (K45E mutation in the large subunit of RPA is normal in facilitating Rad51 filament formation on ssDNA, but is unable to achieve synapsis between MAT and HML. Thus, RPA appears to play a role in strand invasion as well as in facilitating Rad51 binding to ssDNA, possibly by stabilizing the displaced ssDNA.

Wang Xuan

2004-01-01

10

The Karyopherin Kap95 and the C-Termini of Rfa1, Rfa2, and Rfa3 Are Necessary for Efficient Nuclear Import of Functional RPA Complex Proteins in Saccharomyces cerevisiae  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Nuclear protein import in eukaryotic cells is mediated by karyopherin proteins, which bind to specific nuclear localization signals on substrate proteins and transport them across the nuclear envelope and into the nucleus. Replication protein A (RPA) is a nuclear protein comprised of three subunits (termed Rfa1, Rfa2, and Rfa3 in Saccharomyces cerevisiae) that binds single-stranded DNA and is essential for DNA replication, recombination, and repair. RPA associates with two different karyopher...

Belanger, Kenneth D.; Griffith, Amanda L.; Baker, Heather L.; Hansen, Jeanne N.; Simmons Kovacs, Laura A.; Seconi, Justin S.; Strine, Andrew C.

2011-01-01

11

PRP19 transforms into a sensor of RPA-ssDNA after DNA damage and drives ATR activation via a ubiquitin-mediated circuitry.  

Science.gov (United States)

PRP19 is a ubiquitin ligase involved in pre-mRNA splicing and the DNA damage response (DDR). Although the role for PRP19 in splicing is well characterized, its role in the DDR remains elusive. Through a proteomic screen for proteins that interact with RPA-coated single-stranded DNA (RPA-ssDNA), we identified PRP19 as a sensor of DNA damage. PRP19 directly binds RPA and localizes to DNA damage sites via RPA, promoting RPA ubiquitylation in a DNA-damage-induced manner. PRP19 facilitates the accumulation of ATRIP, the regulatory partner of the ataxia telangiectasia mutated and Rad3-related (ATR) kinase, at DNA damage sites. Depletion of PRP19 compromised the phosphorylation of ATR substrates, recovery of stalled replication forks, and progression of replication forks on damaged DNA. Importantly, PRP19 mutants that cannot bind RPA or function as an E3 ligase failed to support the ATR response, revealing that PRP19 drives ATR activation by acting as an RPA-ssDNA-sensing ubiquitin ligase during the DDR. PMID:24332808

Maréchal, Alexandre; Li, Ju-Mei; Ji, Xiao Ye; Wu, Ching-Shyi; Yazinski, Stephanie A; Nguyen, Hai Dang; Liu, Shizhou; Jiménez, Amanda E; Jin, Jianping; Zou, Lee

2014-01-23

12

The karyopherin Kap95 and the C-termini of Rfa1, Rfa2, and Rfa3 are necessary for efficient nuclear import of functional RPA complex proteins in Saccharomyces cerevisiae.  

Science.gov (United States)

Nuclear protein import in eukaryotic cells is mediated by karyopherin proteins, which bind to specific nuclear localization signals on substrate proteins and transport them across the nuclear envelope and into the nucleus. Replication protein A (RPA) is a nuclear protein comprised of three subunits (termed Rfa1, Rfa2, and Rfa3 in Saccharomyces cerevisiae) that binds single-stranded DNA and is essential for DNA replication, recombination, and repair. RPA associates with two different karyopherins in yeast, Kap95, and Msn5/Kap142. However, it is unclear which of these karyopherins is responsible for RPA nuclear import. We have generated GFP fusion proteins with each of the RPA subunits and demonstrate that these Rfa-GFP chimeras are functional in yeast cells. The intracellular localization of the RPA proteins in live cells is similar in wild-type and msn5? deletion strains but becomes primarily cytoplasmic in cells lacking functional Kap95. Truncating the C-terminus of any of the RPA subunits results in mislocalization of the proteins to the cytoplasm and a loss of protein-protein interactions between the subunits. Our data indicate that Kap95 is likely the primary karyopherin responsible for RPA nuclear import in yeast and that the C-terminal regions of Rfa1, Rfa2, and Rfa3 are essential for efficient nucleocytoplasmic transport of each RPA subunit. PMID:21332387

Belanger, Kenneth D; Griffith, Amanda L; Baker, Heather L; Hansen, Jeanne N; Kovacs, Laura A Simmons; Seconi, Justin S; Strine, Andrew C

2011-09-01

13

Stn1?Ten1 is an Rpa2?Rpa3-like complex at telomeres  

Energy Technology Data Exchange (ETDEWEB)

In budding yeast, Cdc13, Stn1, and Ten1 form a heterotrimeric complex (CST) that is essential for telomere protection and maintenance. Previous bioinformatics analysis revealed a putative oligonucleotide/oligosaccharide-binding (OB) fold at the N terminus of Stn1 (Stn1N) that shows limited sequence similarity to the OB fold of Rpa2, a subunit of the eukaryotic ssDNA-binding protein complex replication protein A (RPA). Here we present functional and structural analyses of Stn1 and Ten1 from multiple budding and fission yeast. The crystal structure of the Candida tropicalis Stn1N complexed with Ten1 demonstrates an Rpa2N-Rpa3-like complex. In both structures, the OB folds of the two components pack against each other through interactions between two C-terminal helices. The structure of the C-terminal domain of Saccharomyces cerevisiae Stn1 (Stn1C) was found to comprise two related winged helix-turn-helix (WH) motifs, one of which is most similar to the WH motif at the C terminus of Rpa2, again supporting the notion that Stn1 resembles Rpa2. The crystal structure of the fission yeast Schizosaccharomyces pombe Stn1N-Ten1 complex exhibits a virtually identical architecture as the C. tropicalis Stn1N-Ten1. Functional analyses of the Candida albicans Stn1 and Ten1 proteins revealed critical roles for these proteins in suppressing aberrant telomerase and recombination activities at telomeres. Mutations that disrupt the Stn1-Ten1 interaction induce telomere uncapping and abolish the telomere localization of Ten1. Collectively, our structural and functional studies illustrate that, instead of being confined to budding yeast telomeres, the CST complex may represent an evolutionarily conserved RPA-like telomeric complex at the 3' overhangs that works in parallel with or instead of the well-characterized POT1-TPP1/TEBP{alpha}-{beta} complex.

Sun, Jia; Yu, Eun Young; Yang, Yuting; Confer, Laura A.; Sun, Steven H.; Wan, Ke; Lue, Neal F.; Lei, Ming; (Weill); (Michigan-Med)

2010-09-02

14

Relativistic RPA in axial symmetry  

CERN Document Server

Covariant density functional theory, in the framework of self-consistent Relativistic Mean Field (RMF) and Relativistic Random Phase approximation (RPA), is for the first time applied to axially deformed nuclei. The fully self-consistent RMF+RRPA equations are posed for the case of axial symmetry and non-linear energy functionals, and solved with the help of a new parallel code. Formal properties of RPA theory are studied and special care is taken in order to validate the proper decoupling of spurious modes and their influence on the physical response. Sample applications to the magnetic and electric dipole transitions in $^{20}$Ne are presented and analyzed.

Arteaga, D Pena; 10.1103/PhysRevC.77.034317

2009-01-01

15

RPA theory in wave turbulence  

Energy Technology Data Exchange (ETDEWEB)

We develop a generalized RPA(Random Phase and Amplitude) formalism in Wave Turbulence(WT). RPA theory employ the Hamiliton mechanics description, derived directly from the 3-D inviscid and incompressible Navier-Stokes equation through the Clebsch transformation. We expand randomness even to amplitudes besides phases of wave-like motions. The newly derived equation is also a Wave Kinetic Equation(WKE). We exploit the PDF analysis to analyze the WKE and obtain some new good results. Those are based on PDF analysis from the analysis of WT spectrum, the exact solution of the Hamilton equation.

Choi, Yeon Taek; Lee, Chang Hoon [Yonsei Univ., Seoul (Korea, Republic of); Jo, Sang Gyu [Kyungpook National Univ., Daegu (Korea, Republic of)

2005-07-01

16

RPA Systems Opportunities Final Report  

available before the CAP2013 changes are upon us and in an environment .... \\2013 and before then RPA must change two of its key contracts. ... the systems \\and services become increasingly an enabler of the business. ... rules engines \\and a settlement function, it will be possible to improve current performance while\\.

17

Requirement for PCNA and RPA in interstrand crosslink-induced DNA synthesis  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Proliferating nuclear cell antigen (PCNA) and replication protein A (RPA) have proven to be essential elements in many aspects of DNA metabolism including replication, repair and recombination. We have developed an in vitro assay in which the presence of an interstrand crosslink stimulates the incorporation of radiolabeled nucleotides into both damaged and undamaged plasmid DNAs. Using this assay we have investigated the roles of PCNA and RPA in crosslink-induced DNA synthesis. p21, a potent ...

2000-01-01

18

GENERATION OF RECOMBINANT BACULOVIRUS VIA LIPOSOME MEDIATED TRANSFECTION  

Science.gov (United States)

Baculovirus expression vectors have become a popular method of producing recombinant proteins. Production of recombinant virus requires the transfection of both the native viral DNA and a transfer plasmid into insect cells where recombination takes place. While several methods of...

19

RecTE(Psy)-mediated recombineering in Pseudomonas syringae.  

Science.gov (United States)

A recently developed Pseudomonas syringae recombineering system simplifies the procedure for installing specific mutations at a chosen genomic locus. The procedure involves transforming P. syringae cells expressing recombineering functions with a PCR product that contains desired changes flanked by sequences homologous to a target location. Cells transformed with the substrate undergo homologous recombination between the genomic DNA and the recombineering substrate. The recombinants are found by selection for traits carried by the recombineering substrate, usually antibiotic resistance. PMID:24557893

Swingle, Bryan

2014-01-01

20

Effect of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Restriction enzyme-mediated events (REM events; integration of transforming DNA catalyzed by in vivo action of a restriction enzyme) and illegitimate recombination events (IR events; integration of transforming DNA that shares no homology with the host genomic sequences) have been previously characterized in Saccharomyces cerevisiae. This study determines the effect of mutations in genes that are involved in homologous recombination and/or in the repair of double-stranded DNA breaks on these ...

Schiestl, R. H.; Zhu, J.; Petes, T. D.

1994-01-01

 
 
 
 
21

Recombinant adenoviral mediated gene transfer in ischemic impaired wound healing.  

Science.gov (United States)

Chronic nonhealing wounds represent a large clinical problem resulting in severe disabilities and large healthcare expenditures. Despite the scope of this problem, effective new therapies are lacking. The deficiency of growth factors in chronic wounds has brought attention to the topical application of growth factors, but initial clinical trials have resulted in only modest improvements in healing despite large, repetitive doses. The modest improvement in healing observed in these trials show that growth factors can improve chronic wound healing, but a better means of growth factor delivery is needed. We hypothesized that gene therapy using a recombinant adenoviral vector could be used to induce transgene production directly by cells in the wound. An adenovirus containing the beta-galactosidase reporter transgene (Ad-LacZ) was used in the ischemic rabbit ear model to test this hypothesis. Ad-LacZ resulted in efficient transgene delivery to cells participating in the wound healing response, with expression up to 2 weeks. However, wound reepithelialization was impaired in Ad-LacZ treated wounds compared to vehicle control wounds. Adenoviral mediated gene transfer is a promising efficient means of growth factor delivery to chronic wounds. However, selection of the proper transgene with appropriate biologic activity in wound healing may be essential to overcome the potential adverse effects of adenoviral infection. PMID:10417750

Liechty, K W; Sablich, T J; Adzick, N S; Crombleholme, T M

1999-01-01

22

Stimulation and suppression of PCR-mediated recombination.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Recombination, or chimera formation, is known to occur between related template sequences present in a single PCR amplification. To characterize the conditions under which such recombinant amplification products form we monitored the exchange of sequence between two homologous templates carrying different restriction sites separated by 282 bp. Using a typical cycling program the rates of recombination between the two restriction sites were 1 and 7% using Taq and Vent polymerases respectively ...

Judo, M. S.; Wedel, A. B.; Wilson, C.

1998-01-01

23

Triapine disrupts CtIP-mediated homologous recombination repair and sensitizes ovarian cancer cells to PARP and topoisomerase inhibitors.  

Science.gov (United States)

PARP inhibitors exploit synthetic lethality to target epithelial ovarian cancer (EOC) with hereditary BRCA mutations and defects in homologous recombination repair (HRR). However, such an approach is limited to a small subset of EOC patients and compromised by restored HRR due to secondary mutations in BRCA genes. Here, it was demonstrated that triapine, a small-molecule inhibitor of ribonucleotide reductase, enhances the sensitivity of BRCA wild-type EOC cells to the PARP inhibitor olaparib and the topoisomerase II inhibitor etoposide. Triapine abolishes olaparib-induced BRCA1 and Rad51 foci, and disrupts the BRCA1 interaction with the Mre11-Rad50-Nbs1 (MRN) complex in BRCA1 wild-type EOC cells. It has been shown that phosphorylation of CtIP (RBBP8) is required for the interaction with BRCA1 and with MRN to promote DNA double-strand break (DSB) resection during S and G(2) phases of the cell cycle. Mechanistic studies within reveal that triapine inhibits cyclin-dependent kinase (CDK) activity and blocks olaparib-induced CtIP phosphorylation through Chk1 activation. Furthermore, triapine abrogates etoposide-induced CtIP phosphorylation and DSB resection as evidenced by marked attenuation of RPA32 phosphorylation. Concurrently, triapine obliterates etoposide-induced BRCA1 foci and sensitizes BRCA1 wild-type EOC cells to etoposide. Using a GFP-based HRR assay, it was determined that triapine suppresses HRR activity induced by an I-SceI-generated DSB. These results suggest that triapine augments the sensitivity of BRCA wild-type EOC cells to drug-induced DSBs by disrupting CtIP-mediated HRR. Implications: These findings provide a strong rationale for combining triapine with PARP or topoisomerase inhibitors to target HRR-proficient EOC cells. PMID:24413181

Lin, Z Ping; Ratner, Elena S; Whicker, Margaret E; Lee, Yashang; Sartorelli, Alan C

2014-03-01

24

Functional analysis of the four DNA binding domains of Replication Protein A: the role of RPA2 in ssDNA binding*  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Replication Protein A (RPA), the heterotrimeric SSB of eukaryotes, contains four ssDNA binding domains (DBDs) within its two largest subunits, RPA1 and RPA2. We analyzed the contribution of the four DBDs to ssDNA binding affinity by assaying recombinant heterotrimeric RPA in which a single DBD (A, B, C or D) was inactive. Inactivation was accomplished by mutating the two conserved aromatic stacking residues present in each DBD. Using a short substrate, such as (dT)12, no stable interaction co...

Bastin-shanower, Suzanne A.; Brill, Steven J.

2001-01-01

25

Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A procedure of reversible immortalization of primary cells was devised by retrovirus-mediated transfer of an oncogene that could be subsequently excised by site-specific recombination. This study focused on the early stages of immortalization: global induction of proliferation and life span extension of cell populations. Comparative analysis of Cre/LoxP and FLP/FRT recombination in this system indicated that only Cre/LoxP operates efficiently in primary cells. Pure populations of cells in whi...

1996-01-01

26

Potential role of recombinant secretory leucoprotease inhibitor in the prevention of neutrophil mediated matrix degradation.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

BACKGROUND--Neutrophil elastase is able to degrade connective tissue matrices and is thought to be involved in the pathogenesis of destructive lung diseases. METHODS--The ability of recombinant secretory leucoprotease inhibitor (rSLPI) to inhibit neutrophil mediated degradation of fibronectin in vitro is demonstrated and its efficacy compared with native alpha-1-proteinase inhibitor (n alpha 1-PI), recombinant alpha-1-proteinase inhibitor (r alpha 1-PI), and the chemical elastase inhibitor IC...

Llewellyn-jones, C. G.; Lomas, D. A.; Stockley, R. A.

1994-01-01

27

Finite amplitude method for the RPA solution  

CERN Multimedia

We propose a practical method to solve the random-phase approximation (RPA) in the self-consistent Hartree-Fock (HF) and density-functional theory. The method is based on numerical evaluation of the residual interactions utilizing finite amplitude of single-particle wave functions. The method only requires calculations of the single-particle Hamiltonian constructed with independent bra and ket states. Using the present method, the RPA calculation becomes possible with a little extension of a numerical code of the static HF calculation. We demonstrate usefulness and accuracy of the present method performing test calculations for isoscalar responses in deformed 20Ne.

Nakatsukasa, Takashi; Yabana, Kazuhiro

2007-01-01

28

Purified human BRCA2 stimulates RAD51-mediated recombination  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Mutation of the breast cancer susceptibility gene, BRCA2, leads to breast and ovarian cancers. Mechanistic insight into the functions of human BRCA2 has been limited by the difficulty of isolating this large protein (3,418 amino acids). Here we report purification of full length BRCA2 and show that it both binds RAD51 and potentiates recombinational DNA repair by promoting assembly of RAD51 onto single-stranded DNA (ssDNA). BRCA2 acts by: targeting RAD51 to ssDNA over double-stranded DNA; ena...

Jensen, Ryan B.; Carreira, Aura; Kowalczykowski, Stephen C.

2010-01-01

29

Plasmin-mediated fibrinolysis by variant recombinant tissue plasminogen activators.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A rapid and quantitative fibrinolytic assay has been used to measure the overall activity of a recombinant tissue plasminogen activator (rTPA) preparation for dissolution of a fibrin clot by its ability to activate [Glu1]plasminogen (containing glutamic acid at position 1) to plasmin. A standard curve constructed for wild-type two-chain rTPA that contains, from the amino terminus, the finger (F)-growth factor (E)-kringle 1 (K1)-kringle 2 (K2)-serine protease (P) domains was used to assess the...

Urano, S.; Metzger, A. R.; Castellino, F. J.

1989-01-01

30

Extended GIPQ description of the RPA excitations  

International Nuclear Information System (INIS)

The work presents an application of the GIPQ quantization method, extended to include the wave function quantization. By explicit calculation on a particular proton-neutron system it is shown that the extended quantization gives the same excitation operator and energy as the RPA. (authors)

1989-01-01

31

Entropy and Effective temperature in second RPA dynamics  

International Nuclear Information System (INIS)

The temporal behavior of the entropy and effective temperature of the system of collective RPA phonons is studied by deriving closed formulas of these quantities from the formulation based on the second RPA dynamics at finite temperature

1989-05-01

32

RPA facilitates telomerase activity at chromosome ends in budding and fission yeasts  

Science.gov (United States)

In Saccharomyces cerevisiae, the telomerase complex binds to chromosome ends and is activated in late S-phase through a process coupled to the progression of the replication fork. Here, we show that the single-stranded DNA-binding protein RPA (replication protein A) binds to the two daughter telomeres during telomere replication but only its binding to the leading-strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex. We further demonstrate that RPA specifically co-precipitates with yKu, Cdc13 and telomerase. The interaction of RPA with telomerase appears to be mediated by both yKu and the telomerase subunit Est1. Moreover, a mutation in Rfa1 that affects both the interaction with yKu and telomerase reduces the dramatic increase in telomere length of a rif1?, rif2? double mutant. Finally, we show that the RPA/telomerase association and function are conserved in Schizosaccharomyces pombe. Our results indicate that in both yeasts, RPA directly facilitates telomerase activity at chromosome ends.

Luciano, Pierre; Coulon, Stephane; Faure, Virginie; Corda, Yves; Bos, Julia; Brill, Steven J; Gilson, Eric; Simon, Marie-Noelle; Geli, Vincent

2012-01-01

33

RPA facilitates telomerase activity at chromosome ends in budding and fission yeasts.  

Science.gov (United States)

In Saccharomyces cerevisiae, the telomerase complex binds to chromosome ends and is activated in late S-phase through a process coupled to the progression of the replication fork. Here, we show that the single-stranded DNA-binding protein RPA (replication protein A) binds to the two daughter telomeres during telomere replication but only its binding to the leading-strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex. We further demonstrate that RPA specifically co-precipitates with yKu, Cdc13 and telomerase. The interaction of RPA with telomerase appears to be mediated by both yKu and the telomerase subunit Est1. Moreover, a mutation in Rfa1 that affects both the interaction with yKu and telomerase reduces the dramatic increase in telomere length of a rif1?, rif2? double mutant. Finally, we show that the RPA/telomerase association and function are conserved in Schizosaccharomyces pombe. Our results indicate that in both yeasts, RPA directly facilitates telomerase activity at chromosome ends. PMID:22354040

Luciano, Pierre; Coulon, Stéphane; Faure, Virginie; Corda, Yves; Bos, Julia; Brill, Steven J; Gilson, Eric; Simon, Marie-Noelle; Géli, Vincent

2012-04-18

34

Tunnelling-mediated recombination-induced luminescence in polyethylene naphthalate  

Science.gov (United States)

Electroluminescence-EL- is excited in Poly(ethylene 2,6- naphthalate)-PEN-films submitted to a DC field of the order of 3 MV/cm. The decay of the light emitted after field removal, so-called delayed luminescence, is analyzed and it is shown that light emission can be interpreted in terms of tunnelling recombination of deeply trapped electrons to the luminescent centers forme d by the ionized molecules. The EL decay obeys an equation of the form I varies direct as (1 + (alpha) .t)-m and appears temperature independent. A similar behavior was obtained after ambipolar charging of the film surface by a cold plasma discharge. Wavelength resolved emission spectra obtained in the different situations are compared to the photoluminescence spectrum of the polymer. The predominance of phosphorescence in the electroluminescence spectrum of PEN is explained by a direct coupling between the trapping center and the triplet state of the molecules.

Teyssedre, G.; Laurent, C.

1999-12-01

35

Transposon-mediated mutagenesis and recombination in Vibrio cholerae.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

An efficient method for introducing transposons into the Vibrio cholerae chromosome or the P plasmid was developed by using F'tslac+ plasmids from Escherichia coli as suicide delivery vehicles. Hybrid P::Tn1, Tn5 and P::Tn1, Tn10 plasmids containing Tn5 or Tn10 in either of the two possible orientations were constructed. During conjugation, these hybrid plasmids mediated oriented transfer of markers from the sites of insertion of homologous transposons in the donor chromosome. The collection ...

Newland, J. W.; Green, B. A.; Holmes, R. K.

1984-01-01

36

Human replication protein A: Global fold of the N-terminal RPA-70 domain reveals a basic cleft and flexible C-terminal linker+  

International Nuclear Information System (INIS)

Human Replication Protein A (hsRPA) is required for multiple cellular processes in DNA metabolism including DNA repair, replication and recombination. It binds single-stranded DNA with high affinity and interacts specifically with multiple proteins. hsRPA forms a heterotrimeric complex composed of 70-, 32- and 14-kDa subunits (henceforth RPA70, RPA32, and RPA14). The N-terminal 168 residues of RPA70 form a structurally distinct domain that stimulates DNA polymerase ? activity, interacts with several transcriptional activators including tumor suppressor p53, and during the cell cycle it signals escape from the DNA damage induced G2/M checkpoint. We have solved the global fold of the fragment corresponding to this domain (RPA70?169) and we find residues 8-108 of the N-terminal domain are structured. The remaining C-terminal residues are unstructured and may form a flexible linker to the DNA-binding domain of RPA70. The globular region forms a five-stranded anti-parallel ?-barrel. The ends of the barrel are capped by short helices. Two loops on one side of the barrel form a large basic cleft which is a likely site for binding the acidic motifs of transcriptional activators. Many lethal or conditional lethal yeast point mutants map to this cleft, whereas no mutations with severe phenotype have been found in the linker region

1999-08-01

37

Self-consistent medium polarization in RPA  

International Nuclear Information System (INIS)

The standard random-phase approximation for finite systems is extended by including the effect of the exchange of the RPA phonons in the residual interaction selfconsistently. It is shown that this particle-hole interaction is strongly energy dependent due to the presence of poles corresponding to 2p2h (and more complex) excitations. The RPA eigenvalue problem with this energy-dependent residual interaction also provides solutions for these predominantly 2p2h-like states. In addition a modified normalization condition is obtained. This new scheme is applied to "5"6Ni("5"6Co) in a large (up to 7(h/2?)?) configuration space using a residual interaction of G-matrix type. It is shown that the lowest 2"+ eigenvalue, which in the standard RPA becomes imaginary, is stabilized when the selfconsistent screening is taken into account. Another feature observed is the splitting of the M1 strength as an example of 1p1h and 2p2h mixing. (orig.)

1986-03-24

38

Caffeine suppresses homologous recombination through interference with RAD51-mediated joint molecule formation  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Caffeine is a widely used inhibitor of the protein kinases that play a central role in the DNA damage response. We used chemical inhibitors and genetically deficient mouse embryonic stem cell lines to study the role of DNA damage response in stable integration of the transfected DNA and found that caffeine rapidly, efficiently and reversibly inhibited homologous integration of the transfected DNA as measured by several homologous recombination-mediated gene-targeting assays. Biochemical and s...

Zelensky, Alex N.; Sanchez, Humberto; Ristic, Dejan; Vidic, Iztok; Rossum-fikkert, Sari E.; Essers, Jeroen; Wyman, Claire; Kanaar, Roland

2013-01-01

39

Tissue-specific and inducible Cre-mediated recombination in the gut epithelium.  

Science.gov (United States)

We generated two complementary systems for Cre-mediated recombination of target genes in the mouse digestive epithelium and tested them with a Cre-reporter mouse strain. Cre was expressed under the control of a 9 kb regulatory region of the murine villin gene (vil-Cre). Genetic recombination was initiated at embryonic day (E) 9 in the visceral endoderm, and by E12.5 in the entire intestinal epithelium, but not in other tissues. Cre expression was maintained throughout adulthood. Furthermore, transgenic mice bearing a tamoxifen-dependent Cre recombinase (vil-Cre-ERT2) expressed under the control of the villin promoter were created to perform targeted spatiotemporally controlled somatic recombination. After tamoxifen treatment, recombination was detectable throughout the digestive epithelium. The recombined locus persisted for 60 days after tamoxifen administration, despite rapid intestinal cell renewal, indicating that epithelial progenitor cells had been targeted. The villin-Cre and villin-Cre-ERT2 mice provide valuable tools for studies of cell lineage allocation and gene function in the developing and adult intestine. PMID:15282745

el Marjou, Fatima; Janssen, Klaus-Peter; Chang, Benny Hung-Junn; Li, Mei; Hindie, Valérie; Chan, Lawrence; Louvard, Daniel; Chambon, Pierre; Metzger, Daniel; Robine, Sylvie

2004-07-01

40

A stabilized respiratory syncytial virus reverse genetics system amenable to recombination-mediated mutagenesis.  

Science.gov (United States)

We describe the first example of combining bacterial artificial chromosome (BAC) recombination-mediated mutagenesis with reverse genetics for a negative strand RNA virus. A BAC-based respiratory syncytial virus (RSV) rescue system was established. An important advantage of this system is that RSV antigenomic cDNA was stabilized in the BAC vector. The RSV genotype chosen was A2-line19F, a chimeric strain previously shown to recapitulate in mice key features of RSV pathogenesis. We recovered two RSV reporter viruses, one expressing the red fluorescent protein monomeric Katushka 2 (A2-K-line19F) and one expressing Renilla luciferase (A2-RL-line19F). As proof of principle, we efficiently generated a RSV gene deletion mutant (A2-line19F?NS1/NS2) and a point mutant (A2-K-line19F-I557V) by recombination-mediated BAC mutagenesis. Together with sequence-optimized helper expression plasmids, BAC-RSV is a stable, versatile, and efficient reverse genetics platform for generation of a recombinant Pneumovirus. PMID:23062737

Hotard, Anne L; Shaikh, Fyza Y; Lee, Sujin; Yan, Dan; Teng, Michael N; Plemper, Richard K; Crowe, James E; Moore, Martin L

2012-12-01

 
 
 
 
41

Mechanism of RecA-mediated homologous recombination revisited by single molecule nanomanipulation  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The mechanisms of RecA-mediated three-strand homologous recombination are investigated at the single-molecule level, using magnetic tweezers. Probing the mechanical response of DNA molecules and nucleoprotein filaments in tension and in torsion allows a monitoring of the progression of the exchange in real time, both from the point of view of the RecA-bound single-stranded DNA and from that of the naked double-stranded DNA (dsDNA). We show that strand exchange is able to generate torsion even...

Fulconis, Renaud; Mine, Judith; Bancaud, Aure?lien; Dutreix, Marie; Viovy, Jean-louis

2006-01-01

42

Porcine hepatocyte isolation and reversible immortalization mediated by retroviral transfer and site-specific recombination  

Directory of Open Access Journals (Sweden)

Full Text Available AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40Tag.METHODS: We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subsequently excised by Cre/LoxP site-specific recombination.RESULTS: The resultant hepatocytes with high viability (97% were successfully immortalized with retroviral vector SSR#69. One of the immortalized clones showed the typical morphological appearance, TJPH-1, and was selected by clone rings and expanded in culture. After excision of the SV40T gene with Cre-recombinase, cells stopped growing. The population of reverted cells exhibited the characteristics of differentiated hepatocytes.CONCLUSION: In conclusion, we herein describe a modified method of hepatocyte isolation and subsequently established a porcine hepatocyte cell line mediated by retroviral transfer and site-specific recombination.

Fan-Ying Meng, Zhi-Shui Chen, Meng Han, Xin-Peng Hu, Xing-Xing He, Yong Liu, Wen-Tao He, Wei Huang, Hui Guo, Ping Zhou

2010-04-01

43

Optimized axolotl (Ambystoma mexicanum) husbandry, breeding, metamorphosis, transgenesis and tamoxifen-mediated recombination.  

Science.gov (United States)

The axolotl (Mexican salamander, Ambystoma mexicanum) has become a very useful model organism for studying limb and spinal cord regeneration because of its high regenerative capacity. Here we present a protocol for successfully mating and breeding axolotls in the laboratory throughout the year, for metamorphosing axolotls by a single i.p. injection and for axolotl transgenesis using I-SceI meganuclease and the mini Tol2 transposon system. Tol2-mediated transgenesis provides different features and advantages compared with I-SceI-mediated transgenesis, and it can result in more than 30% of animals expressing the transgene throughout their bodies so that they can be directly used for experimentation. By using Tol2-mediated transgenesis, experiments can be performed within weeks (e.g., 5-6 weeks for obtaining 2-3-cm-long larvae) without the need to establish germline transgenic lines (which take 12-18 months). In addition, we describe here tamoxifen-induced Cre-mediated recombination in transgenic axolotls. PMID:24504478

Khattak, Shahryar; Murawala, Prayag; Andreas, Heino; Kappert, Verena; Schuez, Maritta; Sandoval-Guzmán, Tatiana; Crawford, Karen; Tanaka, Elly M

2014-03-01

44

Angular momentum coupling in the RPA-equations  

International Nuclear Information System (INIS)

A formal and tensor algebraic reduction of the state vectors of a many-fermion system, which are described by the Random Phase Approximation (RPA), to eigenvectors of the square and the z-component of the angular momentum operator, will be outlined. It is shown how the angular momentum coupled RPA equations can be obtained by the reduction of the uncoupled equations to a set of independent equations for each irreducible component of the RPA state vector. These equations are also written in a form which is symmetric with regard to the treatment of particle and hole states

1984-07-09

45

Imaginary eigenvalue solution in RPA and phase transition  

International Nuclear Information System (INIS)

The phase transition (PT) of a many-particle system with a close-shell configuration, the stability of the Hartree-Fock (HF) solution and the random phase approximation (RPA) are studied by means of a generalized three-level solvable model. The question whether the occurrence of an imaginary eigenvalue solution in RPA (OISA) may be considered as a signature of PT is explored in some detail. It is found that there is no close relation between OISA and PT. Generally, OISA shows that RPA becomes poor

1993-08-01

46

Cre recombination-mediated cassette exchange for building versatile transgenic human embryonic stem cells lines.  

Science.gov (United States)

To circumvent the silencing effect of transgene expression in human embryonic stem cells (hESCs), we employed the Cre recombination-mediated cassette exchange strategy to target the silencing-resistant site in the genome. We have identified new loci that sustain transgene expression during stem cell expansion and differentiation to cells representing the three germ layers in vitro and in vivo. The built-in double loxP cassette in the established master hESC lines was specifically replaced by a targeting vector containing the same loxP sites, using the cell-permeable Cre protein transduction method, resulting in successful generation of new hESC lines with constitutive functional gene expression, inducible transgene expression, and lineage-specific reporter gene expression. This strategy and the master cell lines allow for rapid production of transgenic hESC lines in ordinary laboratories. PMID:19415769

Du, Zhong-Wei; Hu, Bao-Yang; Ayala, Melvin; Sauer, Brian; Zhang, Su-Chun

2009-05-01

47

PCR-mediated recombination in development of microsatellite markers: mechanism and implications  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english Protocols for microsatellite-enrichment libraries have been widely applied to several species in order to supply the most informative molecular markers for population and inbreeding studies. One drawback of these protocols is the ratio of designed primer pairs that fail to amplify the expected fragm [...] ent, even after exhaustive optimization attempts. A possible cause of unsuccessful microsatellite primers may be that such loci are artifacts resulting from chimeric PCR products, instead of real genomic sequences. The microsatellite-enriched library constructed for Aegla longirostri (Crustacea, Decapoda, Anomura) showed that 29% of sequenced clones were chimeric products because these sequences shared one of the flanking regions around the same repeat motif but not the other. PCR-mediated recombination is a well-known event described for several procedures in which related sequences are used as a template. We have associated this phenomenon with microsatellite marker development. This study explained the high ratio of recombinant sequences generated in the A. longirostri microsatellite-enriched library. We discuss the mechanism and implications of PCR chimeric-product formation during microsatellite isolation.

Roratto, Paula A.; Buchmann, Darine; Santos, Sandro; Bartholomei-Santos, Marlise L..

48

DNA polymerases are error-prone at RecA-mediated recombination intermediates.  

Science.gov (United States)

Genetic studies have suggested that Y-family translesion DNA polymerase IV (DinB) performs error-prone recombination-directed replication (RDR) under conditions of stress due to its ability to promote mutations during double-strand break (DSB) repair in growth-limited E. coli cells. In recent studies we have demonstrated that pol IV is preferentially recruited to D-loop recombination intermediates at stress-induced concentrations and is highly mutagenic during RDR in vitro. These findings verify longstanding genetic data that have implicated pol IV in promoting stress-induced mutagenesis at D-loops. In this Extra View, we demonstrate the surprising finding that A-family pol I, which normally exhibits high-fidelity DNA synthesis, is highly error-prone at D-loops like pol IV. These findings indicate that DNA polymerases are intrinsically error-prone at RecA-mediated D-loops and suggest that auxiliary factors are necessary for suppressing mutations during RDR in non-stressed proliferating cells. PMID:23907132

Pomerantz, Richard T; Goodman, Myron F; O'Donnell, Michael E

2013-08-15

49

Hydrophobin signal sequence mediates efficient secretion of recombinant proteins in Pichia pastoris.  

Science.gov (United States)

Pichia pastoris is an important eukaryotic organism for the expression, processing, and secretion of recombinant proteins. Here, the secretion of enhanced green fluorescent protein (EGFP) in P. pastoris by using three novel secretion signals originating from the HFBI and HFBII class 2 hydrophobins of Trichoderma reesei was investigated. EGFP was fused to the carboxyl terminus of hydrophobin secretion signals and expressed under the control of the constitutive GAP promoter. In every case, recombinant EGFP entered the secretory pathway of P. pastoris. SDS-polyacrylamide gel electrophoresis, Western blot analysis of the cells' supernatant, and fluorescence measurements on single-cell level via flow cytometry confirmed the efficient secretion of EGFP mediated by the novel secretion sequences. In conclusion, the data clearly show that the secretion sequences derived from HFBI and HFBII of T. reesei have the potential to achieve an efficient secretion of heterologous proteins in P. pastoris. Due to the small size of the hydrophobin-derived secretion signals, their coding sequence can be easily introduced to the gene of interest by PCR. PMID:21484207

Kottmeier, Kirsten; Ostermann, Kai; Bley, Thomas; Rödel, Gerhard

2011-07-01

50

Recombinant-antibody-mediated resistance against Tomato yellow leaf curl virus in Nicotiana benthamiana.  

Science.gov (United States)

Tomato yellow leaf curl virus (TYLCV) is a geminivirus species whose members cause severe crop losses in the tropics and subtropics. We report the expression of a single-chain variable fragment (scFv) antibody that protected Nicotiana benthamiana plants from a prevalent Iranian isolate of the virus (TYLCV-Ir). Two recombinant antibodies (scFv-ScRep1 and scFv-ScRep2) interacting with the multifunctional replication initiator protein (Rep) were obtained from phage display libraries and expressed in plants, both as stand-alone proteins and as N-terminal GFP fusions. Initial results indicated that both scFvs and both fusions accumulated to a detectable level in the cytosol and nucleus of plant cells. Transgenic plants challenged with TYLCV-Ir showed that the scFv-ScRep1, but more so the fusion proteins, were able to suppress TYLCV-Ir replication. These results show that expression of a scFv-ScRep1-GFP fusion protein can attenuate viral DNA replication and prevent the development of disease symptoms. The present article describes the first successful application of a recombinant antibody-mediated resistance approach against a plant DNA virus. PMID:19234665

Safarnejad, Mohammad Reza; Fischer, Rainer; Commandeur, Ulrich

2009-01-01

51

Radiosensitization effect of recombinant adenoviral-mediated PUMA gene on pancreatic carcinoma cells  

International Nuclear Information System (INIS)

Objective: To study the effect of PUMA gene mediated by recombinant adenovirus vector combined with radiation on the pancreatic carcinoma. Methods: The PANC-1 cells were infected with Ad- PUMA (MOI=10, 50 and 100, respectively) for 48 h. The expression of PUMA mRNA and protein was detected by RT-PCR and Western blot, respectively. PANC-1 cells were divided into 4 groups: control group, transfection group, irradiation group and combined treatment group. The cell growth inhibition rate and apoptotic rate of PANC-1 cells were assessed by MTT assay and flow cytometry. Human pancreatic carcinomas were transplanted subcutaneously in nude mice, which were randomized into 4 groups: control group, transfection group, irradiation group and combined treatment group. Tumor growth rate and apoptotic index at different time points were recorded in 35 days. Results: The expression of PUMA mRNA and protein was increased with the increase of MOI of Ad-PUMA, which was does-dependant (MOI=10, mRNA=0.46± 0.02, protein=0.75± 0.09; MOI=50, mRNA=1.12±0.09, protein=1.01±0.18; MOI=100, mRNA=1.50±0.08, protein= 1.80±0.15; P3, (39.5±9.23)mm3, (33.6±10.3)mm3 and (52.0±11.43)mm3, respectively, P<0.05]. And the apoptotic index was increased in the same manner (AI=0.43±0.05, 0.29±0.10, 0.24±0.05 and 0.00±0.00, respectively, P<0.05). Conclusions: Recombinant adenoviral-mediated PUMA gene combined with irradiation could increase the cell-killing effect on pancreatic carcinoma. It is better than that of either one kind of therapy. (authors)

2009-02-01

52

BLM and RMI1 Alleviate RPA Inhibition of TopoIIIa Decatenase Activity  

DEFF Research Database (Denmark)

RPA is a single-stranded DNA binding protein that physically associates with the BLM complex. RPA stimulates BLM helicase activity as well as the double Holliday junction dissolution activity of the BLM-topoisomerase IIIa complex. We investigated the effect of RPA on the ssDNA decatenase activity of topoisomerase IIIa. We found that RPA and other ssDNA binding proteins inhibit decatenation by topoisomerase IIIa. Complex formation between BLM, TopoIIIa, and RMI1 ablates inhibition of decatenation by ssDNA binding proteins. Together, these data indicate that inhibition by RPA does not involve species-specific interactions between RPA and BLM-TopoIIIa-RMI1, which contrasts with RPA modulation of double Holliday junction dissolution. We propose that topoisomerase IIIa and RPA compete to bind to single-stranded regions of catenanes. Interactions with BLM and RMI1 enhance toposiomerase IIIa activity, promoting decatenation in the presence of RPA.

Yang, Jay; Bachrati, Csanad Z

2012-01-01

53

Mechanism of RecA-mediated homologous recombination revisited by single molecule nanomanipulation.  

Science.gov (United States)

The mechanisms of RecA-mediated three-strand homologous recombination are investigated at the single-molecule level, using magnetic tweezers. Probing the mechanical response of DNA molecules and nucleoprotein filaments in tension and in torsion allows a monitoring of the progression of the exchange in real time, both from the point of view of the RecA-bound single-stranded DNA and from that of the naked double-stranded DNA (dsDNA). We show that strand exchange is able to generate torsion even along a molecule with freely rotating ends. RecA readily depolymerizes during the reaction, a process presenting numerous advantages for the cell's 'protein economy' and for the management of topological constraints. Invasion of an untwisted dsDNA by a nucleoprotein filament leads to an exchanged duplex that remains topologically linked to the exchanged single strand, suggesting multiple initiations of strand exchange on the same molecule. Overall, our results seem to support several important assumptions of the monomer redistribution model. PMID:16946710

Fulconis, Renaud; Mine, Judith; Bancaud, Aurélien; Dutreix, Marie; Viovy, Jean-Louis

2006-09-20

54

RPA-like calculations within limited particle-hole spaces  

International Nuclear Information System (INIS)

Working in a boson formalism, we define a new class of phonon operators for RPA-like calculations. These operators remind the standard RPA ones, namely they have 'upward' and 'backward' amplitudes and an overall one-particle one-hole nature. However, their structure is more complex. As a basic feature, the vacuum of these phonon operators is characterized by only a limited (and variable at will) set of particle-hole excitations. In addition, multiphonon states constructed by repeated actions of these phonon operators on the vacuum state have a particle-hole structure never exceeding in complexity that of the vacuum itself. We discuss advantages and disadvantages deriving from the use of this new class of phonon operators and show an application of the formalism within the Lipkin model. Diagonalizations in multiphonon spaces built in terms of new and standard RPA phonon operators exhibit a faster convergence toward the exact results in the former case

2003-02-24

55

A method for the solution of the RPA eigenvalue  

International Nuclear Information System (INIS)

The RPA eigenvalue problem requires the diagonalization of a 2nx2n matrix. In practical calculations, n (the number of particle-hole basis states) can be a few hundred and the diagonalization of such a large non-symmetric matrix may take quite a long time. In this report we firstly discuss sufficient conditions for real and non-zero RPA eigenvalues. The presence of zero or imaginary eigenvalues is related to the relative importance of the groundstate correlations to the total interaction energy. We then rewrite the RPA eigenvalue problem for the cases where these conditions are fulfilled in a form which only requires the diagonalization of two symmetric nxn matrices. The extend to which this method can be applied when zero eigenvalues occur, is also discussed

1985-07-09

56

Mutually exclusive recombination of wild-type and mutant loxP sites in vivo facilitates transposon-mediated deletions from both ends of genomic DNA in PACs  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Recombination of wild-type and mutant loxP sites mediated by wild-type Cre protein was analyzed in vivo using a sensitive phage P1 transduction assay. Contrary to some earlier reports, recombination between loxP sites was found to be highly specific: a loxP site recombined in vivo only with another of identical sequence, with no crossover recombination either between a wild-type and mutant site; or between two different mutant sites tested. Mutant loxP sites of identical sequence recombined a...

Chatterjee, Pradeep K.; Shakes, Leighcraft A.; Srivastava, Deepak K.; Garland, Douglas M.; Harewood, Ken R.; Moore, Kyle J.; Coren, Jonathon S.

2004-01-01

57

Delivery of Recombinant Adeno-Associated Virus-Mediated Human Tissue Kallikrein for Therapy of Chronic Renal Failure in Rats  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The tissue kallikrein–kinin system is important in regulating cardiovascular and renal function, and dysregulation of the system has been implicated in heart and kidney pathologies. These findings suggest that if balance can be restored to the kallikrein-kinin axis, then associated disease progression may be attenuated. To test this hypothesis, recombinant adeno-associated virus (rAAV)-mediated human tissue kallikrein (HK) expression was induced in a rodent model of chronic renal failure in...

2008-01-01

58

Recombinant antibody mediated delivery of organelle-specific DNA pH sensors along endocytic pathways  

Science.gov (United States)

DNA has been used to build nanomachines with potential in cellulo and in vivo applications. However their different in cellulo applications are limited by the lack of generalizable strategies to deliver them to precise intracellular locations. Here we describe a new molecular design of DNA pH sensors with response times that are nearly 20 fold faster. Further, by changing the sequence of the pH sensitive domain of the DNA sensor, we have been able to tune their pH sensitive regimes and create a family of DNA sensors spanning ranges from pH 4 to 7.6. To enable a generalizable targeting methodology, this new sensor design also incorporates a `handle' domain. We have identified, using a phage display screen, a set of three recombinant antibodies (scFv) that bind sequence specifically to the handle domain. Sequence analysis of these antibodies revealed several conserved residues that mediate specific interactions with the cognate DNA duplex. We also found that all three scFvs clustered into different branches indicating that their specificity arises from mutations in key residues. When one of these scFvs is fused to a membrane protein (furin) that traffics via the cell surface, the scFv-furin chimera binds the `handle' and ferries a family of DNA pH sensors along the furin endocytic pathway. Post endocytosis, all DNA nanodevices retain their functionality in cellulo and provide spatiotemporal pH maps of retrogradely trafficking furin inside living cells. This new molecular technology of DNA-scFv-protein chimeras can be used to site-specifically complex DNA nanostructures for bioanalytical applications.DNA has been used to build nanomachines with potential in cellulo and in vivo applications. However their different in cellulo applications are limited by the lack of generalizable strategies to deliver them to precise intracellular locations. Here we describe a new molecular design of DNA pH sensors with response times that are nearly 20 fold faster. Further, by changing the sequence of the pH sensitive domain of the DNA sensor, we have been able to tune their pH sensitive regimes and create a family of DNA sensors spanning ranges from pH 4 to 7.6. To enable a generalizable targeting methodology, this new sensor design also incorporates a `handle' domain. We have identified, using a phage display screen, a set of three recombinant antibodies (scFv) that bind sequence specifically to the handle domain. Sequence analysis of these antibodies revealed several conserved residues that mediate specific interactions with the cognate DNA duplex. We also found that all three scFvs clustered into different branches indicating that their specificity arises from mutations in key residues. When one of these scFvs is fused to a membrane protein (furin) that traffics via the cell surface, the scFv-furin chimera binds the `handle' and ferries a family of DNA pH sensors along the furin endocytic pathway. Post endocytosis, all DNA nanodevices retain their functionality in cellulo and provide spatiotemporal pH maps of retrogradely trafficking furin inside living cells. This new molecular technology of DNA-scFv-protein chimeras can be used to site-specifically complex DNA nanostructures for bioanalytical applications. Electronic supplementary information (ESI) available: Detailed description of all oligonucleotide sequences used in this study; list of figures that support claims from the main text. Mainly these show sensor sequences, phage display results, scFv purification and binding data, cell images clamped at different pH and co-localization studies with endocytic tracers. See DOI: 10.1039/c3nr03769j

Modi, Souvik; Halder, Saheli; Nizak, Clément; Krishnan, Yamuna

2013-12-01

59

Generation of Cell Lines to Complement Adenovirus Vectors using Recombination-Mediated Cassette Exchange  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Adenovirus serotype 5 (Ad5 has many favourable characteristics for development as a gene therapy vector. However, the utility of current Ad5 vectors is limited by transient transgene expression, toxicity and immunogenicity. The most promising form of vector is the high capacity type, which is deleted for all viral genes. However, these vectors can only be produced to relatively low titres and with the aid of helper virus. Therefore a continuing challenge is the generation of more effective Ad5 vectors that can still be grown to high titres. Our approach is to generate complementing cell lines to support the growth of Ad5 vectors with novel late gene deficiencies. Results We have used LoxP/Cre recombination mediated cassette exchange (RMCE to generate cell lines expressing Ad5 proteins encoded by the L4 region of the genome, the products of which play a pivotal role in the expression of Ad5 structural proteins. A panel of LoxP parent 293 cell lines was generated, each containing a GFP expression cassette under the control of a tetracycline-regulated promoter inserted at a random genome location; the cassette also contained a LoxP site between the promoter and GFP sequence. Clones displayed a variety of patterns of regulation, stability and level of GFP expression. Clone A1 was identified as a suitable parent for creation of inducible cell lines because of the tight inducibility and stability of its GFP expression. Using LoxP-targeted, Cre recombinase-mediated insertion of an L4 cassette to displace GFP from the regulated promoter in this parent clone, cell line A1-L4 was generated. This cell line expressed L4 100K, 22K and 33K proteins at levels sufficient to complement L4-33K mutant and L4-deleted viruses. Conclusions RMCE provides a method for rapid generation of Ad5 complementing cell lines from a pre-selected parental cell line, chosen for its desirable transgene expression characteristics. Parent cell lines can be selected for high or low gene expression, and for tight regulation, allowing viral protein expression to mirror that found during infection. Cell lines derived from a single parent will allow the growth of different vectors to be assessed without the complication of varying complementing protein expression.

Farley Daniel C

2010-12-01

60

BLM and RMI1 Alleviate RPA Inhibition of TopoIII? Decatenase Activity  

Digital Repository Infrastructure Vision for European Research (DRIVER)

RPA is a single-stranded DNA binding protein that physically associates with the BLM complex. RPA stimulates BLM helicase activity as well as the double Holliday junction dissolution activity of the BLM-topoisomerase III? complex. We investigated the effect of RPA on the ssDNA decatenase activity of topoisomerase III?. We found that RPA and other ssDNA binding proteins inhibit decatenation by topoisomerase III?. Complex formation between BLM, TopoIII?, and RMI1 ablates inhibition of decat...

Yang, Jay; Bachrati, Csanad Z.; Hickson, Ian D.; Brown, Grant W.

2012-01-01

 
 
 
 
61

Recombinant NFAT1 (NFATp) is regulated by calcineurin in T cells and mediates transcription of several cytokine genes.  

Science.gov (United States)

Transcription factors of the NFAT family play a key role in the transcription of cytokine genes and other genes during the immune response. We have identified two new isoforms of the transcription factor NFAT1 (previously termed NFATp) that are the predominant isoforms expressed in murine and human T cells. When expressed in Jurkat T cells, recombinant NFAT1 is regulated, as expected, by the calmodulin-dependent phosphatase calcineurin, and its function is inhibited by the immunosuppressive agent cyclosporin A (CsA). Transactivation by recombinant NFAT1 in Jurkat T cells requires dual stimulation with ionomycin and phorbol 12-myristate 13-acetate; this activity is potentiated by coexpression of constitutively active calcineurin and is inhibited by CsA. Immunocytochemical analysis indicates that recombinant NFAT1 localizes in the cytoplasm of transiently transfected T cells and translocates into the nucleus in a CsA-sensitive manner following ionomycin stimulation. When expressed in COS cells, however, NFAT1 is capable of transactivation, but it is not regulated correctly: its subcellular localization and transcriptional function are not affected by stimulation of the COS cells with ionomycin and phorbol 12-myristate 13-acetate. Recombinant NFAT1 can mediate transcription of the interleukin-2, interleukin-4, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor promoters in T cells, suggesting that NFAT1 contributes to the CsA-sensitive transcription of these genes during the immune response. PMID:8668213

Luo, C; Burgeon, E; Carew, J A; McCaffrey, P G; Badalian, T M; Lane, W S; Hogan, P G; Rao, A

1996-07-01

62

Fusion protein bilayer fabrication composed of recombinant azurin/cytochrome P450 by the sortase-mediated ligation method.  

Science.gov (United States)

Recently, the fabrication of protein bilayer has been required for the development of protein or enzyme complex formation. In the present study, we fabricated a fusion protein bilayer composed of recombinant azurin-cytochrome P450, which was synthesized by a site-specific sortase-mediated ligation method. The Pseudomonas aeruginosa azurin was modified by DNA recombinant technique, for enzymatic ligation and immobilization. The Pseudomonas putida cytochrome P450 was also modified for enzymatic ligation. The recombinant metalloproteins were conjugated via the sortase A. The conjugation was confirmed by SDS-PAGE and UV-vis. Then, the prepared fusion protein was immobilized on Au substrate, by the self-assembly method. The Azu-P450 (recombinant azurin-cytochrome P450) fusion protein layer was confirmed by AFM (Atomic Force Microscopy) and SERS (Surface-enhanced Raman Spectroscopy), to confirm the fusion protein bilayer orientation. Moreover, the electrochemical property of Azu-P450 was observed by cyclic voltammetry (CV). As a result, the Azu-P450 fusion protein bilayer shows good orientation on the Au substrate. Also, the original redox property of this fusion protein bilayer has been well maintained. The proposed fusion protein bilayer can. PMID:24924834

Lee, Taek; Min, Junhong; Hirakawa, Hidehiko; Nagamune, Teruyuki; Choi, Jeong-Woo

2014-08-01

63

Cell cycle-specific UNG2 phosphorylations regulate protein turnover, activity and association with RPA  

DEFF Research Database (Denmark)

Human UNG2 is a multifunctional glycosylase that removes uracil near replication forks and in non-replicating DNA, and is important for affinity maturation of antibodies in B cells. How these diverse functions are regulated remains obscure. Here, we report three new phosphoforms of the non-catalytic domain that confer distinct functional properties to UNG2. These are apparently generated by cyclin-dependent kinases through stepwise phosphorylation of S23, T60 and S64 in the cell cycle. Phosphorylation of S23 in late G1/early S confers increased association with replication protein A (RPA) and replicating chromatin and markedly increases the catalytic turnover of UNG2. Conversely, progressive phosphorylation of T60 and S64 throughout S phase mediates reduced binding to RPA and flag UNG2 for breakdown in G2 by forming a cyclin E/c-myc-like phosphodegron. The enhanced catalytic turnover of UNG2 p-S23 likely optimises the protein to excise uracil along with rapidly moving replication forks. Our findings may aid further studies of how UNG2 initiates mutagenic rather than repair processing of activation-induced deaminase-generated uracil at Ig loci in B cells.

Hagen, Lars; Kavli, Bodil

2008-01-01

64

Elevated recombination in immortal human cells is mediated by HsRAD51 recombinase.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Normal diploid cells have a limited replicative potential in culture, with progressively increasing interdivision time. Rarely, cell lines arise which can divide indefinitely; like tumor cells, such "immortal" lines display frequent chromosomal aberrations which may reflect high rates of recombination. Recombination frequencies within a plasmid substrate were 3.5-fold higher in nine immortal human cell lines than in six untransformed cell strains. Expression of HsRAD51, a human homolog of the...

Xia, S. J.; Shammas, M. A.; Shmookler Reis, R. J.

1997-01-01

65

Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Gene targeting by homologous recombination in embryonic stem cells is extensively used to generate specific mouse mutants. However, most mammalian species lack tools for targeted gene manipulation. Since double-strand breaks strongly increase the rate of homologous recombination at genomic loci, we explored whether gene targeting can be directly performed in zygotes by the use of zinc-finger nucleases. Here we report that gene targeting is achieved in 1.7–4.5% of murine one-cell embryos upo...

2010-01-01

66

Agrobacterium tumefaciens-Mediated Transformation for Investigation of Somatic Recombination in the Fungal Pathogen Armillaria mellea?  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Armillaria root disease is one of the most damaging timber and fruit tree diseases in the world. Despite its economic importance, many basic questions about the biology of the causal fungi, Armillaria spp., are unanswered. For example, Armillaria undergoes matings between diploid and haploid mycelia, which can result in a recombinant diploid without meiosis. Evidence of such somatic recombination in natural populations suggests that this reproductive mode may affect the pathogen's ecology. In...

Baumgartner, Kendra; Fujiyoshi, Phillip; Foster, Gary D.; Bailey, Andy M.

2010-01-01

67

CTCF Binding Elements Mediate Control of V(D)J Recombination  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Immunoglobulin heavy chain (IgH) variable region exons are assembled from VH, D and JH gene segments in developing B lymphocytes. Within the 2.7 megabase (Mb) mouse IgH locus (IgH), V(D)J recombination is regulated to ensure specific and diverse antibody repertoires. Herein, we report a key IgH V(D)J recombination regulatory region, termed InterGenic Control Region-1 (IGCR1), that lies between the VH and D clusters. Functionally, IGCR1 employs CTCF looping/insulator factor binding elements an...

Guo, Chunguang; Yoon, Hye Suk; Franklin, Andrew; Jain, Suvi; Ebert, Anja; Cheng, Hwei-ling; Hansen, Erica; Despo, Orion; Bossen, Claudia; Vettermann, Christian; Bates, Jamie G.; Richards, Nicholas; Myers, Darienne; Patel, Harin; Gallagher, Michael

2011-01-01

68

A set of vectors for introduction of antibiotic resistance genes by in vitro Cre-mediated recombination  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Introduction of new antibiotic resistance genes in the plasmids of interest is a frequent task in molecular cloning practice. Classical approaches involving digestion with restriction endonucleases and ligation are time-consuming. Findings We have created a set of insertion vectors (pINS carrying genes that provide resistance to various antibiotics (puromycin, blasticidin and G418 and containing a loxP site. Each vector (pINS-Puro, pINS-Blast or pINS-Neo contains either a chloramphenicol or a kanamycin resistance gene and is unable to replicate in most E. coli strains as it contains a conditional R6K? replication origin. Introduction of the antibiotic resistance genes into the vector of interest is achieved by Cre-mediated recombination between the replication-incompetent pINS and a replication-competent target vector. The recombination mix is then transformed into E. coli and selected by the resistance marker (kanamycin or chloramphenicol present in pINS, which allows to recover the recombinant plasmids with 100% efficiency. Conclusion Here we propose a simple strategy that allows to introduce various antibiotic-resistance genes into any plasmid containing a replication origin, an ampicillin resistance gene and a loxP site.

Vassetzky Yegor S

2008-12-01

69

Expression of Active Recombinant Human Tissue-Type Plasminogen Activator by Using In Vivo Polyhydroxybutyrate Granule Display?  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Recombinant human tissue plasminogen activator (rPA) is a truncated version of tissue plasminogen activator (tPA), which contains nine disulfide bonds and is prone to forming inactive inclusion bodies when expressed in bacteria. To obtain functional rPA expression, we displayed the rPA on the surface of polyhydroxybutyrate (PHB) granules using phasin as the affinity tag. rPA was fused to the N terminus of the phasin protein with a thrombin cleavage site as the linker. Sodium dodecyl sulfate-p...

Geng, Yanping; Wang, Shengjun; Qi, Qingsheng

2010-01-01

70

The Interaction of cos with Chi Is Separable from DNA Packaging in recA- recBC-Mediated Recombination of Bacteriophage Lambda  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Chi (5'-GCTGGTGG) is a recombinator in RecA- RecBC-mediated recombination in Escherichia coli. In bacteriophage ? vegetative recombination, Chi is fully active only when it is correctly oriented with respect to cos, the site that defines the ends of the packaged chromosome. Here we demonstrate that packaging from cos is not necessary for this cos-Chi interaction. Our evidence suggests that correctly oriented cos is an activator of Chi. cos, as an activator, is (1) dominant over cos-, (2) ac...

Kobayashi, Ichizo; Stahl, Mary M.; Leach, David; Stahl, Franklin W.

1983-01-01

71

PCR-mediated recombination between Cryptosporidium spp. of lizards and snakes.  

Science.gov (United States)

The presence or absence of genetic recombination has often been used as one of the criteria for Cryptosporidium species designation and population structure delineation. During a recent study of cryptosporidiosis in reptiles that were housed in the same room, 4 lizards were found to have concurrent infections of C. serpentis (a gastric parasite) and C. saurophilum (an intestinal parasite), and 6 snakes were concurrently infected with C. serpentis, C. saurophilum and a new Cryptosporidium as indicated by PCR-RFLP analysis of the SSU rRNA gene. DNA sequence analysis of cloned PCR products confirmed the diagnosis of mixed infections. Surprisingly, it appeared that 11 of the 22 clones (8 and 14 clones from a lizard and a snake, respectively) had chimeric sequences of two Cryptosporidium spp. BootScan analysis indicated the existence of recombinants among the cloned sequences and detection of the informative sites confirmed the BootScan results. Because the probability for genetic recombination between gastric and intestinal parasites is small, these hybrid sequences were likely results of PCR artifacts due to the presence of multiple templates. This was confirmed by PCR-sequencing analysis of single-copy templates using diluted DNA samples. Direct sequencing of 69 PCR products from 100- to 1,000-fold diluted DNAs from the same snake and lizard produced only sequences of C. serpentis, C. saurophilum and the unnamed Cryptosporidium sp. Thus, care should be taken to eliminate PCR artifacts when determining the presence of genetic recombination or interpreting results of population genetic studies. PMID:14736163

Zhou, Ling; Yang, Chunfu; Xiao, Lihua

2003-01-01

72

Transgenic or Plant Expression Vector-Mediated Recombination of Plum Pox Virus  

Science.gov (United States)

Different mutants of an infectious full-length clone (p35PPV-NAT) of Plum pox virus (PPV) were constructed: three mutants with mutations of the assembly motifs RQ and DF in the coat protein gene (CP) and two CP chimeras with exchanges in the CP core region of Zucchini yellow mosaic virus and Potato virus Y. The assembly mutants were restricted to single infected cells, whereas the PPV chimeras were able to produce systemic infections in Nicotiana benthamiana plants. After passages in different transgenic N. benthamiana plants expressing the PPV CP gene with a complete (plant line 4.30.45.) or partially deleted 3?-nontranslated region (3?-NTR) (plant line 17.27.4.), characterization of the viral progeny of all mutants revealed restoration of wild-type virus by recombination with the transgenic CP RNA only in the presence of the complete 3?-NTR (4.30.45.). Reconstitution of wild-type virus was also observed following cobombardment of different assembly-defective p35PPV-NAT together with a movement-defective plant expression vector of Potato virus X expressing the intact PPV-NAT CP gene transiently in nontransgenic N. benthamiana plants. Finally, a chimeric recombinant virus was detected after cobombardment of defective p35PPV-NAT with a plant expression vector-derived CP gene from the sour cherry isolate of PPV (PPV-SoC). This chimeric virus has been established by a double recombination event between the CP-defective PPV mutant and the intact PPV-SoC CP gene. These results demonstrate that viral sequences can be tested for recombination events without the necessity for producing transgenic plants.

Varrelmann, Mark; Palkovics, Laszlo; Maiss, Edgar

2000-01-01

73

Long Telomeres are Preferentially Extended During Recombination-Mediated Telomere Maintenance  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Most human somatic cells do not express telomerase. Consequently, with each cell division their telomeres progressively shorten until replicative senescence is induced. Approximately 15% of human cancers maintain their telomeres using telomerase-independent, recombination-based mechanisms collectively termed Alternative Lengthening of Telomeres (ALT). In the yeast Saccharomyces cerevisiae, ALT cells are referred to as “survivors”. One type of survivor (type II) resembles human ALT cells i...

Chang, Michael; Dittmar, John C.; Rothstein, Rodney

2011-01-01

74

RPA sum rules for giant resonances at finite temperature  

International Nuclear Information System (INIS)

Finite-temperature random-phase-approximation (RPA) sum-rule expressions have been derived for the moments of the strength function. It has been found that the same expressions as in the T=0 case hold, the only change being the necessary thermal average on the statistical ensemble. Numerical results are presented for L=0-4 isoscalar giant resonances obtained with finite-temperature Hartree-Fock (HF) and Thomas-Fermi (TF) methods. A careful discussion of the Thomas-Fermi approach is presented. (orig.)

1985-11-04

75

Generation of TALEN-Mediated GRdim Knock-In Rats by Homologous Recombination  

Science.gov (United States)

Transcription Activator-Like Effector Nucleases (TALEN) are potential tools for precise genome engineering of laboratory animals. We report the first targeted genomic integration in the rat using TALENs (Transcription Activator-Like Effector Nucleases) by homology-derived recombination (HDR). We assembled TALENs and designed a linear donor insert targeting a pA476T mutation in the rat Glucocorticoid Receptor (Nr3c1) namely GRdim, that prevents receptor homodimerization in the mouse. TALEN mRNA and linear double-stranded donor were microinjected into rat one-cell embryos. Overall, we observed targeted genomic modifications in 17% of the offspring, indicating high TALEN cutting efficiency in rat zygotes.

Ponce de Leon, Veronica; Merillat, Anne-Marie; Tesson, Laurent; Anegon, Ignacio; Hummler, Edith

2014-01-01

76

Microhomology-mediated end joining in fission yeast is repressed by pku70 and relies on genes involved in homologous recombination.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Two DNA repair pathways are known to mediate DNA double-strand-break (DSB) repair: homologous recombination (HR) and nonhomologous end joining (NHEJ). In addition, a nonconservative backup pathway showing extensive nucleotide loss and relying on microhomologies at repair junctions was identified in NHEJ-deficient cells from a variety of organisms and found to be involved in chromosomal translocations. Here, an extrachromosomal assay was used to characterize this microhomology-mediated end-joi...

Decottignies, Anabelle

2007-01-01

77

P-glycoprotein-mediated resistance to chemotherapy in cancer cells: using recombinant cytosolic domains to establish structure-function relationships  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english Resistance to chemotherapy in cancer cells is mainly mediated by overexpression of P-glycoprotein (Pgp), a plasma membrane ATP-binding cassette (ABC) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. Pgp consists of two homologous halves each containing a transmembrane dom [...] ain and a cytosolic nucleotide-binding domain (NBD) which contains two consensus Walker motifs, A and B, involved in ATP binding and hydrolysis. The protein also contains an S signature characteristic of ABC transporters. The molecular mechanism of Pgp-mediated drug transport is not known. Since the transporter has an extraordinarily broad substrate specificity, its cellular function has been described as a "hydrophobic vacuum cleaner". The limited knowledge about the mechanism of Pgp, partly due to the lack of a high-resolution structure, is well reflected in the failure to efficiently inhibit its activity in cancer cells and thus to reverse multidrug resistance (MDR). In contrast to the difficulties encountered when studying the full-length Pgp, the recombinant NBDs can be obtained in large amounts as soluble proteins. The biochemical and biophysical characterization of recombinant NBDs is shown here to provide a suitable alternative route to establish structure-function relationships. NBDs were shown to bind ATP and analogues as well as potent modulators of MDR, such as hydrophobic steroids, at a region close to the ATP site. Interestingly, flavonoids also bind to NBDs with high affinity. Their binding site partly overlaps both the ATP-binding site and the steroid-interacting region. Therefore flavonoids constitute a new promising class of bifunctional modulators of Pgp.

A., Di Pietro; G., Dayan; G., Conseil; E., Steinfels; T., Krell; D., Trompier; H., Baubichon-Cortay; J.-M., Jault.

78

Comparative Vaccine Efficacy of Different Isoforms of Recombinant Protective Antigen Against Bacillus anthracis Spore Challenge in Rabbits.  

Science.gov (United States)

The next-generation human anthrax vaccine developed by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) is based upon purified Bacillus anthracis recombinant protective antigen (rPA) adsorbed to aluminum hydroxide adjuva...

W. J. Ribot B. S. Powell B. E. Ivins S. F. Little W. M. Johnson

2006-01-01

79

MODEST: a web-based design tool for oligonucleotide-mediated genome engineering and recombineering  

DEFF Research Database (Denmark)

Recombineering and multiplex automated genome engineering (MAGE) offer the possibility to rapidly modify multiple genomic or plasmid sites at high efficiencies. This enables efficient creation of genetic variants including both single mutants with specifically targeted modifications as well as combinatorial cell libraries. Manual design of oligonucleotides for these approaches can be tedious, time-consuming, and may not be practical for larger projects targeting many genomic sites. At present, the change from a desired phenotype (e.g. altered expression of a specific protein) to a designed MAGE oligo, which confers the corresponding genetic change, is performed manually. To address these challenges, we have developed the MAGE Oligo Design Tool (MODEST). This web-based tool allows designing of MAGE oligos for (i) tuning translation rates by modifying the ribosomal binding site, (ii) generating translational gene knockouts and (iii) introducing other coding or non-coding mutations, including amino acid substitutions, insertions, deletions and point mutations. The tool automatically designs oligos based on desired genotypic or phenotypic changes defined by the user, which can be used for high efficiency recombineering and MAGE. MODEST is available for free and is open to all users at http://modest.biosustain.dtu.dk.

Bonde, Mads; Anderson, Mads Valdemar

2014-01-01

80

MODEST: a web-based design tool for oligonucleotide-mediated genome engineering and recombineering.  

Science.gov (United States)

Recombineering and multiplex automated genome engineering (MAGE) offer the possibility to rapidly modify multiple genomic or plasmid sites at high efficiencies. This enables efficient creation of genetic variants including both single mutants with specifically targeted modifications as well as combinatorial cell libraries. Manual design of oligonucleotides for these approaches can be tedious, time-consuming, and may not be practical for larger projects targeting many genomic sites. At present, the change from a desired phenotype (e.g. altered expression of a specific protein) to a designed MAGE oligo, which confers the corresponding genetic change, is performed manually. To address these challenges, we have developed the MAGE Oligo Design Tool (MODEST). This web-based tool allows designing of MAGE oligos for (i) tuning translation rates by modifying the ribosomal binding site, (ii) generating translational gene knockouts and (iii) introducing other coding or non-coding mutations, including amino acid substitutions, insertions, deletions and point mutations. The tool automatically designs oligos based on desired genotypic or phenotypic changes defined by the user, which can be used for high efficiency recombineering and MAGE. MODEST is available for free and is open to all users at http://modest.biosustain.dtu.dk. PMID:24838561

Bonde, Mads T; Klausen, Michael S; Anderson, Mads V; Wallin, Annika I N; Wang, Harris H; Sommer, Morten O A

2014-07-01

 
 
 
 
81

DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2.  

Science.gov (United States)

The repair of DNA double-strand breaks (DSBs) by homologous recombination requires processing of broken ends. For repair to start, the DSB must first be resected to generate a 3'-single-stranded DNA (ssDNA) overhang, which becomes a substrate for the DNA strand exchange protein, Rad51 (ref. 1). Genetic studies have implicated a multitude of proteins in the process, including helicases, nucleases and topoisomerases. Here we biochemically reconstitute elements of the resection process and reveal that it requires the nuclease Dna2, the RecQ-family helicase Sgs1 and the ssDNA-binding protein replication protein-A (RPA). We establish that Dna2, Sgs1 and RPA constitute a minimal protein complex capable of DNA resection in vitro. Sgs1 helicase unwinds the DNA to produce an intermediate that is digested by Dna2, and RPA stimulates DNA unwinding by Sgs1 in a species-specific manner. Interestingly, RPA is also required both to direct Dna2 nucleolytic activity to the 5'-terminated strand of the DNA break and to inhibit 3' to 5' degradation by Dna2, actions that generate and protect the 3'-ssDNA overhang, respectively. In addition to this core machinery, we establish that both the topoisomerase 3 (Top3) and Rmi1 complex and the Mre11-Rad50-Xrs2 complex (MRX) have important roles as stimulatory components. Stimulation of end resection by the Top3-Rmi1 heterodimer and the MRX proteins is by complex formation with Sgs1 (refs 5, 6), which unexpectedly stimulates DNA unwinding. We suggest that Top3-Rmi1 and MRX are important for recruitment of the Sgs1-Dna2 complex to DSBs. Our experiments provide a mechanistic framework for understanding the initial steps of recombinational DNA repair in eukaryotes. PMID:20811461

Cejka, Petr; Cannavo, Elda; Polaczek, Piotr; Masuda-Sasa, Taro; Pokharel, Subhash; Campbell, Judith L; Kowalczykowski, Stephen C

2010-09-01

82

Direct interferon-?-mediated protection caused by a recombinant coxsackievirus B3  

International Nuclear Information System (INIS)

Coxsackievirus B3 (CVB3) is one of the most important causes of viral myocarditis. Cytokines are involved in the control of CVB3 replication and pathogenesis. Local expression of specific cytokines by recombinant CVB3 confers prevention of virus-caused myocarditis. Expression of IFN-? by CVB3(IFN-?) protected BALB/c and C57BL/6 mice when the lethal infection with the highly pathogenic CVB3H3 variant was given directly after or prior to CVB3(IFN-?) inoculation by decreasing the viral load and spread as well as tissue destruction. This direct effect was not restricted to the homologous virus. In vitro, cocultivation of CVB3(IFN-?)-infected cells induced a reduction of CVB3H3 replication and virus-induced cytopathogenicity

2003-10-25

83

The Epistatic Relationship between BRCA2 and the Other RAD51 Mediators in Homologous Recombination  

Digital Repository Infrastructure Vision for European Research (DRIVER)

RAD51 recombinase polymerizes at the site of double-strand breaks (DSBs) where it performs DSB repair. The loss of RAD51 causes extensive chromosomal breaks, leading to apoptosis. The polymerization of RAD51 is regulated by a number of RAD51 mediators, such as BRCA1, BRCA2, RAD52, SFR1, SWS1, and the five RAD51 paralogs, including XRCC3. We here show that brca2-null mutant cells were able to proliferate, indicating that RAD51 can perform DSB repair in the absence of BRCA2. We disrupted the BR...

Qing, Yong; Yamazoe, Mitsuyoshi; Hirota, Kouji; Dejsuphong, Donniphat; Sakai, Wataru; Yamamoto, Kimiyo N.; Bishop, Douglas K.; Wu, Xiaohua; Takeda, Shunichi

2011-01-01

84

Microscopic nuclear-dissipation mechanism as damping of collective motion in the second RPA  

International Nuclear Information System (INIS)

A microscopic model for the damping of the one-phonon RPA collective state, absolute value c > = Q/sub c/ 0 > /sub S//sub R/, has been previously described. This one-phonon RPA collective state is defined within a restricted subspace, S/sub R/, of the discrete 1p-1h structure. Its damping is described within an extended subspace, S = S/sub R/ + S/sub A/, by the time evolution of a wave packet according to the RPA and the Second RPA approximations of the complete Schroedinger equation when initialized with the one-phonon state. The one-phonon state, however, is unable to describe time-varying oscillations of the mean field. Such oscillations require wave packets formed by linear superposition of the RPA many-phonon eigenstates. Coherent time-varying oscillations of the mean field (multi-phonon initial states) are discussed

1982-01-23

85

Thermal nuclear pairing within the self-consistent quasiparticle RPA  

International Nuclear Information System (INIS)

The self-consistent quasiparticle RPA (SCQRPA) is constructed to study the effects of fluctuations on pairing properties in nuclei at finite temperature and z-projection M of angular momentum. Particle-number projection (PNP) is taken into account within the Lipkin-Nogami method. Several issues such as the smoothing of superfluid-normal phase transition, thermally assisted pairing in hot rotating nuclei, extraction of the nuclear pairing gap using an improved odd-even mass difference are discussed. A novel approach of embedding the PNP SCQRPA eigenvalues in the canonical and microcanonical ensembles is proposed and applied to describe the recent empirical thermodynamic quantities for iron, molybdenum, dysprosium, and ytterbium isotopes.

2011-01-01

86

Thermal nuclear pairing within the self-consistent quasiparticle RPA  

CERN Document Server

The self-consistent quasiparticle RPA (SCQRPA) is constructed to study the effects of fluctuations on pairing properties in nuclei at finite temperature and z-projection M of angular momentum. Particle-number projection (PNP) is taken into account within the Lipkin-Nogami method. Several issues such as the smoothing of superfluid-normal phase transition, thermally assisted pairing in hot rotating nuclei, extraction of the nuclear pairing gap using an improved odd-even mass difference are discussed. A novel approach of embedding the PNP SCQRPA eigenvalues in the canonical and microcanonical ensembles is proposed and applied to describe the recent empirical thermodynamic quantities for iron, molybdenum, dysprosium, and ytterbium isotopes.

Dang, N Dinh

2010-01-01

87

Soluble recombinant neprilysin induces aggrecanase-mediated cleavage of aggrecan in cartilage explant cultures.  

Science.gov (United States)

Neprilysin (neutral endopeptidase, enkephalinase, CALLA, CD10, NEP) is a regulatory Zn metallopeptidase expressed in the brush border membranes of the kidney and has been found in porcine chondrocytes and rat articular cartilage as well as other cell types and tissues. Although its function in cartilage is not currently known, previous observations of high levels of NEP enzymatic activity in the synovial fluid of arthritic patients and on the chondrocyte membranes of human osteoarthritic cartilage have led to the hypothesis that NEP is involved in the inflammation or degradation pathways in articular cartilage. Our study localized endogenous NEP to the membranes of mature bovine articular chondrocytes in a tissue explant model and demonstrated that the addition of soluble recombinant NEP (sNEP) to the culture medium of bovine cartilage explants leads to the degradation of aggrecan through the action of aggrecanase. A 6-day exposure to sNEP was necessary to initiate the degradation, suggesting that the chondrocytes were responding in a delayed manner to an altered composition of regulatory peptides. This NEP-induced degradation was completely inhibited by the NEP inhibitors thiorphan and phosphoramidon. These results suggest that NEP is present as a transmembrane enzyme on articular chondrocytes where it can cleave regulatory peptides and lead to the induction of aggrecanase. PMID:11747295

Chevrier, A; Mort, J S; Crine, P; Hoemann, C D; Buschmann, M D

2001-12-15

88

Suppression of mutagenesis by Rad51D-mediated homologous recombination  

Energy Technology Data Exchange (ETDEWEB)

Homologous recombinational repair (HRR) restores chromatid breaks arising during DNA replication and prevents chromosomal rearrangements that can occur from the misrepair of such breaks. In vertebrates, five Rad51 paralogs are identified that contribute in a nonessential but critical manner to HRR efficiency. We constructed and characterized a Rad51D knockout cell line in widely studied CHO cells. The rad51d mutant (51D1) displays sensitivity to a wide spectrum of induced DNA damage, indicating the broad relevance of HRR to genotoxicity. Untreated 51D1 cells exhibit {approx}5-fold elevated chromosomal breaks, a 12-fold increased rate of hprt mutation, and 4- to 10-fold increased rates of gene amplification at the dhfr and CAD loci, respectively. These results explicitly show the quantitative importance of HHR in preventing these types genetic alterations, which are associated with carcinogenesis. Thus, HRR copes in an error-free manner with spontaneous DNA damage encountered during DNA replication, and Rad51D is essential for this fidelity.

Hinz, J M; Tebbs, R S; Wilson, P F; Nham, P B; Salazar, E P; Nagasawa, H; Urbin, S S; Thompson, L H

2005-11-15

89

Current Challenges and Future Directions in Recombinant AAV-Mediated Gene Therapy of Duchenne Muscular Dystrophy  

Directory of Open Access Journals (Sweden)

Full Text Available Various characteristics of adeno-associated virus (AAV-based vectors with long-term safe expression have made it an exciting transduction tool for clinical gene therapy of Duchenne muscular dystrophy (DMD. Although host immune reactions against the vector as well as transgene products were detected in some instances of the clinical studies, there have been promising observations. Methods of producing AAV vectors for considerable in vivo experimentation and clinical investigations have been developed and a number of studies with AAV vector-mediated muscle transduction were attempted. Notably, an intravenous limb perfusion transduction technique enables extensive transgene expression in the skeletal muscles without noticeable adverse events. Furthermore, cardiac transduction by the rAAV9-microdystrophin would be promising to prevent development of cardiac dysfunction. Recent achievements in transduction technology suggest that long-term transgene expression with therapeutic benefits in DMD treatment would be achieved by the rAAV-mediated transduction strategy with an adequate regimen to regulate host immune response.

Shin'ichi Takeda

2013-06-01

90

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Inducible conditional knockout animals are widely used to get insight in the function of genes and the pathogenesis of human diseases. These models frequently rely on Cre-mediated recombination of sequences flanked by Lox-P sites. To understand the consequences of gene disruption, it is essential to know the efficiency of the recombination process. Results Here, we describe a modification of the multiplex ligation-dependent probe amplification (MLPA, called extension-MLPA (eMLPA, which enables quantification of relatively small differences in DNA that are a consequence of Cre-mediated recombination. eMLPA, here applied on an inducible Pkd1 conditional deletion mouse model, simultaneously measures both the reduction of the floxed allele and the increase of the deletion allele in a single reaction thereby minimizing any type of experimental variation. Interestingly, with this method we were also able to observe the presence of the excised DNA fragment. This extra-chromosomal deletion-circle was detectable up to 5 months after activation of Cre. Conclusion eMLPA is a novel strategy which easily can be applied to measure the Cre-mediated recombination efficiency in each experimental case with high accuracy. In addition the fate of the deletion-circle can be followed simultaneously.

Breuning Martijn H

2008-02-01

91

Peptide nucleic acid-mediated recombination for targeted genomic repair and modification.  

Science.gov (United States)

The ability to directly manipulate the human genome to correct a disease-related mutation, introduce a sequence change that would lead to site-specific gene knockout, or increase gene expression is a very powerful tool with tremendous clinical value. Triplex formation by synthetic DNA-binding molecules such as peptide nucleic acids (PNAs) has been studied for over 20 years and much of the work in the last 10 years has shown its great promise in its use to direct site-specific gene modification for the use in gene therapy. In this chapter, detailed protocols are described for the design and use of triplex-forming PNAs to bind and mediate gene modification at specific chromosomal targets. Target site identification, PNA and donor oligonucleotide design, in vitro characterization of binding, optimization with reporter systems, as well as various methods to assess gene modification and isolate modified cells are described. PMID:24297362

Schleifman, Erica B; Glazer, Peter M

2014-01-01

92

Imaging of human sodium-iodide symporter gene expression mediated by recombinant adenovirus in skeletal muscle of living rats  

International Nuclear Information System (INIS)

We evaluated the feasibility of non-invasive imaging of recombinant adenovirus-mediated human sodium-iodide symporter (hNIS) gene expression by 99mTcO4- scintigraphy in skeletal muscle of rats. Replication-defective recombinant adenovirus encoding hNIS gene [Rad-CMV-hNIS 5 x 107, 2 x 108 or 1 x 109 plaque forming units (pfu)] or ?-galactosidase gene (Rad-CMV-LacZ 1 x 109 pfu) was injected into the right biceps femoris muscle of rats (n=5-6 for each group). Three days after gene transfer, scintigraphy was performed using a gamma camera 30 min after injection of 99mTcO4- (1.85 MBq). An additional two rats injected with 1 x 109 pfu of Rad-CMV-hNIS underwent 99mTcO4- scintigraphy with sodium perchlorate. After the imaging studies, rats were sacrificed for assessment of the biodistribution of 99mTcO4- and measurement of hNIS mRNA expression. In all the rats injected with 1 x 109 pfu of Rad-CMV-hNIS, hNIS expression was successfully imaged by 99mTcO4- scintigraphy, while rats injected with Rad-CMV-LacZ or lower doses of Rad-CMV-hNIS failed to show uptake. The biodistribution studies indicated that a significantly different amount of 99mTcO4- was retained in the liver (p9 pfu of Rad-CMV-hNIS. The muscular hNIS mRNA level quantified by real-time reverse transcription-polymerase chain reaction was significantly higher in rats injected with 1 x 109 pfu of Rad-CMV-hNIS (p9 pfu of Rad-CMV-hNIS were specifically inhibited by sodium perchlorate. This study illustrated that 99mTcO4- scintigraphy can monitor Rad-CMV-hNIS-mediated gene expression in skeletal muscle of rats, non-invasively and quantitatively. (orig.)

2004-09-01

93

Crystallization and Preliminary X-ray Analysis of Bacteriophasge T4 UvsY Recombination Mediator Protein  

International Nuclear Information System (INIS)

Bacteriophage T4 UvsY protein is considered to be the prototype of recombination mediator proteins, a class of proteins which assist in the loading of recombinases onto DNA. Wild-type and Se-substituted UvsY protein have been expressed and purified and crystallized by hanging-drop vapor diffusion. The crystals diffract to 2.4 (angstrom) using in-house facilities and to 2.2 (angstrom) at NSLS, Brookhaven National Laboratory. The crystals belong to space group P422, P4222, P4212 or P42212, the ambiguity arising from pseudo-centering, with unit-cell parameters a = b = 76.93, c = 269.8 (angstrom). Previous biophysical characterization of UvsY indicates that it exists primarily as a hexamer in solution. Along with the absence of a crystallographic threefold, this suggests that the asymmetric unit of these crystals is likely to contain either three monomers, giving a solvent content of 71%, or six monomers, giving a solvent content of 41%

2006-01-01

94

RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia.  

Science.gov (United States)

The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL) cases, is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near breakpoints, incorporation of non-templated sequence at junctions, ?30-fold enrichment at promoters and enhancers of genes actively transcribed in B cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single-cell tracking shows that this mechanism is active throughout leukemic evolution, with evidence of localized clustering and reiterated deletions. Integration of data on point mutations and rearrangements identifies ATF7IP and MGA as two new tumor-suppressor genes in ALL. Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1-positive lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B cell differentiation. PMID:24413735

Papaemmanuil, Elli; Rapado, Inmaculada; Li, Yilong; Potter, Nicola E; Wedge, David C; Tubio, Jose; Alexandrov, Ludmil B; Van Loo, Peter; Cooke, Susanna L; Marshall, John; Martincorena, Inigo; Hinton, Jonathan; Gundem, Gunes; van Delft, Frederik W; Nik-Zainal, Serena; Jones, David R; Ramakrishna, Manasa; Titley, Ian; Stebbings, Lucy; Leroy, Catherine; Menzies, Andrew; Gamble, John; Robinson, Ben; Mudie, Laura; Raine, Keiran; O'Meara, Sarah; Teague, Jon W; Butler, Adam P; Cazzaniga, Giovanni; Biondi, Andrea; Zuna, Jan; Kempski, Helena; Muschen, Markus; Ford, Anthony M; Stratton, Michael R; Greaves, Mel; Campbell, Peter J

2014-02-01

95

Radiation protection authorized persons (RPA) in Germany; Der Strahlenschutzbevollmaechtigte in Deutschland  

Energy Technology Data Exchange (ETDEWEB)

The radiation protection authorized person (RPA) is playing an important role in the fields of organization, realization and checking the radiation protection in Germany, first of all in big institutions like research centers, facilities and medical centers. The paper deals with the legal status of the RPA especially the clear dividing line between his tasks and the tasks of the radiation protection supervisor and the radiation protection commissioner. The paper shows that the embodiment of the RPA in the radiation protection law has advantages also in coordinating the tasks of radiation protection officer and radiation protection expert recommended by the European Union. (orig.)

Sahre, P.; Beutmann, A.; Lorenz, J. [Verein fuer Kernverfahrenstechnik und Analytik Rossendorf e.V., Dresden (Germany); Leder, F. [Saechsisches Staatsministerium fuer Umwelt und Landwirtschaft, Dresden (Germany); Philipp, T. [Saechsisches Landesamt fuer Umwelt, Landwirtschaft und Geologie, Dresden (Germany)

2013-07-01

96

A point mutation in the AF-2 domain of thyroid hormone receptor alpha1 expressed after CRE mediated recombination partially recapitulates hypothyroidism.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Thyroid hormones act directly on transcription by binding to TR?1, TR?1, TR?2 nuclear receptors, regulating many aspects of post-natal development and homeostasis. To precisely analyze the implication of the widely expressed TR?1 isoform in this pleiotropic action, we have generated transgenic mice with a point mutation in the TR?1 coding sequence, which is expressed only after CRE/loxP mediated DNA recombination. The amino-acid change prevents interaction between TR?1 and histone acety...

Quignodon, Laure; Vincent, Se?verine; Winter, Harald; Samarut, Jacques; Flamant, Fre?de?ric

2007-01-01

97

Formation of lipoxins and leukotrienes during receptor-mediated interactions of human platelets and recombinant human granulocyte/macrophage colony-stimulating factor-primed neutrophils  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The generation of lipoxygenase products of arachidonic acid is considered an important event in inflammation. This study demonstrates the levels of both lipoxins and leukotrienes (LTC4, LTD4, LTB4, and omega-oxidized LTB4) generated from endogenous sources of arachidonate by PMN primed with recombinant human granulocyte/macrophage colony- stimulating factor and in coincubations with platelets (1:1 to 1:100 ratio). Upon exposure to receptor-mediated stimuli (FMLP and thrombin), the levels of l...

1990-01-01

98

The V(D)J recombinational and transcriptional activities of the immunoglobulin heavy-chain intronic enhancer can be mediated through distinct protein-binding sites in a transgenic substrate.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Immunoglobulin and T-cell receptor gene transcriptional enhancers encompass sequences which stimulate V(D)J recombination of associated variable gene segments. To address the question of whether enhancer-mediated transcriptional activation and recombinational activation depend on the same cis-regulatory sequences, we have produced transgenic mice by using recombination substrates containing various mutations in the immunoglobulin heavy-chain intronic enhancer (E mu). Analysis of substrate rea...

Fernex, C.; Capone, M.; Ferrier, P.

1995-01-01

99

Scattering in particle-hole space: simple approximations to nuclear RPA calculations in the continuum  

International Nuclear Information System (INIS)

The Random Phase Approximation (RPA) treatment of nuclear small amplitude vibrations including particle-hole continua is handled in terms of previously developed techniques to treat single-particle resonances in a reaction theoretical framework. A hierarchy of interpretable approximations is derived and a simple working approximation is proposed which involves a numerical effort no larger than that involved in standard, discrete RPA calculations. (Author)

1987-01-01

100

Validation of the RTOG recursive partitioning analysis (RPA) classification for brain metastases  

International Nuclear Information System (INIS)

Purpose: The Radiation Therapy Oncology Group (RTOG) previously developed three prognostic classes for brain metastases using recursive partitioning analysis (RPA) of a large database. These classes were based on Karnofsky performance status (KPS), primary tumor status, presence of extracranial system metastases, and age. An analysis of RTOG 91-04, a randomized study comparing two dose-fractionation schemes with a comparison to the established RTOG database, was considered important to validate the RPA classes. Methods and Materials: A total of 445 patients were randomized on RTOG 91-04, a Phase III study of accelerated hyperfractionation versus accelerated fractionation. No difference was observed between the two treatment arms with respect to survival. Four hundred thirty-two patients were included in this analysis. The majority of the patients were under age 65, had KPS 70-80, primary tumor controlled, and brain-only metastases. The initial RPA had three classes, but only patients in RPA Classes I and II were eligible for RTOG 91-04. Results: For RPA Class I, the median survival time was 6.2 months and 7.1 months for 91-04 and the database, respectively. The 1-year survival was 29% for 91-04 versus 32% for the database. There was no significant difference in the two survival distributions (p = 0.72). For RPA Class II, the median survival time was 3.8 months for 91-04 versus 4.2 months for the database. The 1-year survival was 12% and 16% for 91-04 and the database, respectively (p = 0.22). Conclusion: This analysis indicates that the RPA classes are valid and reliable for historical comparisons. Both the RTOG and other clinical trial organizers should currently utilize this RPA classification as a stratification factor for clinical trials

2000-07-01

 
 
 
 
101

Single-step purification of recombinant proteins using elastin-like peptide-mediated inverse transition cycling and self-processing module from Neisseria meningitides FrpC.  

Science.gov (United States)

Purification of recombinant proteins is a major task and challenge in biotechnology and medicine. In this paper we report a novel single-step recombinant protein purification system which was based on elastin-like peptide (ELP)-mediated reversible phase transition and FrpC self-processing module (SPM)-mediated cleavage. After construction of a SPM-ELP fusion expression vector, we cloned the coding sequence for green fluorescent protein (GFP), the Fc portion of porcine IgG (pFc) or human ? defensin 3 (HBD3) into the vector, transformed the construct into Escherichia coli, and induced the fusion protein expression with IPTG. The target-SPM-ELP fusion proteins GFP-SPM-ELP, Fc-SPM-ELP and HBD3-SPM-ELP were expressed in a soluble form and efficiently purified from the clarified cell extracts by two rounds of inverse transition cycling (ITC). Under the optimized conditions, the SPM-mediated cleavage efficiencies for the three fusion proteins ranged from 92% to 93%. After an additional round of ITC, the target proteins GFP, pFc and HBD3 were recovered with purities ranging from 90% to 100% and yields ranging from 1.1 to 36mg/L in shake flasks. The endotoxin levels in all of the three target proteins were <0.03EU/mg. The three target proteins were functionally active with the expected molecular weights. These experimental results confirmed the high specificity and efficiency of SPM-mediated cleavage, and suggested the applicability of SPM-ELP fusion system for purification of recombinant proteins. PMID:24607361

Liu, Wen-Jun; Wu, Qian; Xu, Bi; Zhang, Xin-Yu; Xia, Xiao-Li; Sun, Huai-Chang

2014-06-01

102

Smc5-Smc6 mediate DNA double-strand-break repair by promoting sister-chromatid recombination.  

Science.gov (United States)

DNA double-strand breaks (DSB) can arise during DNA replication, or after exposure to DNA-damaging agents, and their correct repair is fundamental for cell survival and genomic stability. Here, we show that the Smc5-Smc6 complex is recruited to DSBs de novo to support their repair by homologous recombination between sister chromatids. In addition, we demonstrate that Smc5-Smc6 is necessary to suppress gross chromosomal rearrangements. Our findings show that the Smc5-Smc6 complex is essential for genome stability as it promotes repair of DSBs by error-free sister-chromatid recombination (SCR), thereby suppressing inappropriate non-sister recombination events. PMID:16892052

De Piccoli, Giacomo; Cortes-Ledesma, Felipe; Ira, Gregory; Torres-Rosell, Jordi; Uhle, Stefan; Farmer, Sarah; Hwang, Ji-Young; Machin, Felix; Ceschia, Audrey; McAleenan, Alexandra; Cordon-Preciado, Violeta; Clemente-Blanco, Andrés; Vilella-Mitjana, Felip; Ullal, Pranav; Jarmuz, Adam; Leitao, Beatriz; Bressan, Debra; Dotiwala, Farokh; Papusha, Alma; Zhao, Xiaolan; Myung, Kyungjae; Haber, James E; Aguilera, Andrés; Aragón, Luis

2006-09-01

103

Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained inside E. coli allows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination syste...

Dulal, Kalpana; Silver, Benjamin; Zhu, Hua

2012-01-01

104

Frequent and sex-biased deletion of SLX4IP by illegitimate V(D)J-mediated recombination in childhood acute lymphoblastic leukemia.  

Science.gov (United States)

Acute lymphoblastic leukemia (ALL) accounts for ?25% of pediatric malignancies. Of interest, the incidence of ALL is observed ?20% higher in males relative to females. The mechanism behind the phenomenon of sex-specific differences is presently not understood. Employing genome-wide genetic aberration screening in 19 ALL samples, one of the most recurrent lesions identified was monoallelic deletion of the 5' region of SLX4IP. We characterized this deletion by conventional molecular genetic techniques and analyzed its interrelationships with biological and clinical characteristics using specimens and data from 993 pediatric patients enrolled into trial AIEOP-BFM ALL 2000. Deletion of SLX4IP was detected in ?30% of patients. Breakpoints within SLX4IP were defined to recurrent positions and revealed junctions with typical characteristics of illegitimate V(D)J-mediated recombination. In initial and validation analyses, SLX4IP deletions were significantly associated with male gender and ETV6/RUNX1-rearranged ALL (both overall P < 0.0001). For mechanistic validation, a second recurrent deletion affecting TAL1 and caused by the same molecular mechanism was analyzed in 1149 T-cell ALL patients. Validating a differential role by sex of illegitimate V(D)J-mediated recombination at the TAL1 locus, 128 out of 1149 T-cell ALL samples bore a deletion and males were significantly more often affected (P = 0.002). The repeatedly detected association of SLX4IP deletion with male sex and the extension of the sex bias to deletion of the TAL1 locus suggest that differential illegitimate V(D)J-mediated recombination events at specific loci may contribute to the consistent observation of higher incidence rates of childhood ALL in boys compared with girls. PMID:24045615

Meissner, Barbara; Bartram, Thies; Eckert, Cornelia; Trka, Jan; Panzer-Grümayer, Renate; Hermanova, Ivana; Ellinghaus, Eva; Franke, Andre; Möricke, Anja; Schrauder, André; Teigler-Schlegel, Andrea; Dörge, Petra; von Stackelberg, Arend; Basso, Giuseppe; Bartram, Claus R; Kirschner-Schwabe, Renate; Bornhäuser, Beat; Bourquin, Jean-Pierre; Cazzaniga, Giovanni; Hauer, Julia; Attarbaschi, Andishe; Izraeli, Shai; Zaliova, Marketa; Cario, Gunnar; Zimmermann, Martin; Avigad, Smadar; Sokalska-Duhme, Magdalena; Metzler, Markus; Schrappe, Martin; Koehler, Rolf; Te Kronnie, Geertruy; Stanulla, Martin

2014-02-01

105

Recombinant Lz-8 from Ganoderma lucidum induces endoplasmic reticulum stress-mediated autophagic cell death in SGC-7901 human gastric cancer cells  

Digital Repository Infrastructure Vision for European Research (DRIVER)

In Asia, the mushroom of the fungus Ganoderma lucidum has been widely used as a traditional medicine for the past two millennia. The aim of this study was to investigate the anticancer activity of recombinant Lz-8 (rLz-8), a protein belonging to a family of fungal immunomodulatory proteins. We report that rLz-8 induces endoplasmic reticulum (ER) stress-mediated autophagic cell death in the human gastric cancer cell line SGC-7901. Our results show that rLz-8 induces autophagic cell death by ag...

Liang, Chongyang; Li, Hongrui; Zhou, Hui; Zhang, Shuqin; Liu, Zhiyi; Zhou, Qiuli; Sun, Fei

2012-01-01

106

Subtraction of the spurious translational mode from the RPA response function  

CERN Document Server

It is well known that within self-consistent Random Phase Approximation (RPA) on top of Hartree-Fock (HF), the translational symmetry should be restored. Due to approximations at the level of the practical implementation, this restoration may be only partial. As a result, one has spurious contributions in the physical quantities that are extracted from RPA. While there are several recipes in the literature to overcome this drawback in order to produce transition densites or strength functions that are free from spurious contamination, there is no formalism associated with the full RPA response function. We present such formalism in this paper. Our goal is to avoid spurious contamination when the response function is used in many-body frameworks like the particle-vibration coupling theory.

Mizuyama, Kazuhito

2011-01-01

107

RPA classification has prognostic significance for surgically resected single brain metastasis  

International Nuclear Information System (INIS)

Purpose: To retrospectively evaluate prognostic factors that correlate with overall survival among patients with a surgically resected single brain metastasis. Methods and Materials: An Institutional Review Board-approved database of Cleveland Clinic Brain Tumor Institute was queried for patients with a single brain metastasis treated by surgical resection between February 1984 and January 2004. The primary endpoint was overall survival from the date of surgery by the Kaplan-Meier method. Results: A total of 271 patients were included. Statistically significant variables for improved survival on multivariate analysis included age <65 years, lack of extracranial metastases, control of primary tumor, histology (non-small-cell lung carcinoma), and use of stereotactic radiosurgery. The median survival for all patients was 10.2 months. Survival of patients in recursive partitioning analysis (RPA) class 1 was better (21.4 months) than those in RPA class 2 (9.0 months, p < 0.001), RPA class 3 (8.9 months, p = 0.15), or the combined group of RPA classes 2 and 3 (9.0 months, p < 0.001). Patients had a median survival of 10.6 months after documented gross total resection and 8.7 months after subtotal resection, which approached statistical significance (p 0.07). Those who were treated with stereotactic radiosurgery had a median survival of 17.1 months, which was greater than patients who were not treated with stereotactic radiosurgery (8.9 months, p = 0.006). Conclusions: This analysis supports the prognostic significance of the RPA classification in patients with a single brain metastasis who undergo surgical resection and adjuvant therapy. RPA class 1 patients have a very favorable prognosis with a median survival of 21.4 months

2006-11-01

108

The resolution and regeneration of a cointegrate plasmid reveals a model for plasmid evolution mediated by conjugation and oriT site-specific recombination.  

Science.gov (United States)

Cointegrate plasmids are useful models for the study of plasmid evolution if their evolutionary processes can be replicated under laboratory conditions. pBMB0228, a 17?706?bp native plasmid originally isolated from Bacillus thuringiensis strain YBT-1518, carries two nematicidal crystal protein genes, cry6Aa and cry55Aa. In this study, we show that pBMB0228 is in fact a cointegrate of two plasmids and contains two functional replication regions and two functional mobilization regions. Upon introduction into B.?thuringiensis strain BMB171, pBMB0228 spontaneously resolves into two constituent plasmids via recombination at its oriT1 and oriT2 sites. The resolution does not require conjugation but can be promoted by conjugation. We further confirm that the resolution is mediated by oriT site-specific recombination requiring Mob02281 or Mob02282. Additionally, the two constituent plasmids of pBMB0228 are mobilizable, and can fuse back via oriT site-specific integration after entering into the same cell by conjugation. Our study confirms that native plasmid can reversibly interconvert between a cointegrate structure and its constituent plasmids. This study provides insight into the evolution of cointegrate plasmids, linking plasmid evolution with conjugation and the oriT site-specific recombination function of relaxase. PMID:23826996

Wang, Pengxia; Zhang, Chunyi; Zhu, Yiguang; Deng, Yun; Guo, Suxia; Peng, Donghai; Ruan, Lifang; Sun, Ming

2013-12-01

109

I-SceI-Mediated Double-Strand Break Does Not Increase the Frequency of Homologous Recombination at the Dct Locus in Mouse Embryonic Stem Cells  

Science.gov (United States)

Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells.

Fenina, Myriam; Simon-Chazottes, Dominique; Vandormael-Pournin, Sandrine; Soueid, Jihane; Langa, Francina; Cohen-Tannoudji, Michel; Bernard, Bruno A.; Panthier, Jean-Jacques

2012-01-01

110

Intermolecular recombination between DNAs introduced into mouse L cells is mediated by a nonconservative pathway that leads to crossover products.  

Science.gov (United States)

We describe experiments designed to measure the efficiency of intermolecular recombination between mutant herpesvirus thymidine kinase (tk) genes introduced into mouse L cells. Recombinants were scored as stable transformants containing a functional tk gene. The two recombination substrates used were ptkB8, a pBR322-based plasmid containing a mutant tk gene, with a BamHI linker in an SphI restriction site that is centrally located within the gene, and mp10tk delta 3' delta 5', an mp10 vector with a tk gene deleted at both the 3' and 5' ends. The only homology shared by the two DNAs is 885 base pairs within the tk gene. To determine whether the double-strand break repair model that has been used to explain recombination in yeast cells (J. W. Szostak, T. L. Orr-Weaver, R. J. Rothstein, and F. W. Stahl, Cell 33:25-35, 1983) can account for recombination during the introduction of these DNAs into mammalian cells, we transformed cells with BamHI-linearized ptkB8 and supercoiled mp10tk delta 3' delta 5' replicative-form DNA. These two DNAs should recombine efficiently according to that model and should generate gene conversion products. In this reaction, the supercoiled DNA acts as the donor of information to repair the cleaved tk gene. Our results indicated that the efficiency of this reaction was very low (less than 10 transformants were obtained per 0.1 microgram of each DNA used in the reaction per 10(6) cells). In contrast, if BamHI-cleaved ptkB8 DNA was cotransformed into cells along with a circular DNA molecule containing a tk gene deleted only at its 3' end or only at its 5' end (mp10tk delta 3' or mp10tk delta 5'), then the efficiency of recombination could be more than 4 orders of magnitude higher than it was with circular mp10tk delta 3' delta 5' DNA. Recombination frequencies were highest when the tk delta 3' or tk delta 5' DNA used was cleaved at the tk deletion junction. Southern analyses of DNA from TK+ transformants generated with BamHI-cleaved ptkB8 and BamHI-cleaved mp10tk delta 3' DNAs indicated that recombination was almost always associated with the reassortment of markers flanking the reconstructed tk DNA. Together, these results are more consistent with the nonconservative single-strand annealing model for recombination that we proposed several years ago (F.-L. Lin, K. Sperle, and N. Sternberg, Mol. Cell. Biol. 4:1020-1034, 1984) than they are with the double-strand break repair model. PMID:2294396

Lin, F L; Sperle, K; Sternberg, N

1990-01-01

111

Recombination mediates production of an extrachromosomal circular DNA containing a transposon-like human element, THE-1  

Energy Technology Data Exchange (ETDEWEB)

An abundant class of HeLa extrachromosomal circular DNA containing the transposon-like element, THE-1, is shown to arise via site specific recombination. The chromosomal locus from which these circles are derived, however, is single-copy. Northern blot analysis detects homology to two polyadenylated RNAs in HeLa cells. The possible presence of an origin of replication and its role in generating these small polydisperse circles is discussed.

Misra, R.; Rush, M.G. (New York Univ. School of Medicine, NY (USA)); Matera, A.G.; Schmid, C.W. (Univ. of California, Davis (USA))

1989-10-25

112

Recombinant NFAT1 (NFATp) is regulated by calcineurin in T cells and mediates transcription of several cytokine genes.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Transcription factors of the NFAT family play a key role in the transcription of cytokine genes and other genes during the immune response. We have identified two new isoforms of the transcription factor NFAT1 (previously termed NFATp) that are the predominant isoforms expressed in murine and human T cells. When expressed in Jurkat T cells, recombinant NFAT1 is regulated, as expected, by the calmodulin-dependent phosphatase calcineurin, and its function is inhibited by the immunosuppressive a...

1996-01-01

113

Engineering neonatal Fc receptor-mediated recycling and transcytosis in recombinant proteins by short terminal peptide extensions  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The importance of therapeutic recombinant proteins in medicine has led to a variety of tactics to increase their circulation time or to enable routes of administration other than injection. One clinically successful tactic to improve both protein circulation and delivery is to fuse the Fc domain of IgG to therapeutic proteins so that the resulting fusion proteins interact with the human neonatal Fc receptor (FcRn). As an alternative to grafting the high molecular weight Fc domain to therapeut...

Sockolosky, Jonathan T.; Tiffany, Matthew R.; Szoka, Francis C.

2012-01-01

114

TRESK gene recombinant adenovirus vector inhibits capsaicin-mediated substance P release from cultured rat dorsal root ganglion neurons  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The present study was conducted to determine whether the activation of TRESK in the dorsal root ganglion (DRG) by the TRESK gene recombinant adenovirus vector inhibits the capsaicin-evoked substance P (SP) release using a radioimmunoassay. TRESK is an outwardly rectifying K+ current channel that contributes to the resting potential and is the most important background potassium channel in DRG. Previous studies have shown that neuropathic pain (NP) is closely related to the regulation of certa...

Zhou, Jun; Yao, Shang-long; Yang, Cheng-xiang; Zhong, Ji-ying; Wang, Han-bing; Zhang, Yan

2012-01-01

115

A dual role of BRCA1 in two distinct homologous recombination mediated repair in response to replication arrest  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Homologous recombination (HR) is a major mechanism utilized to repair blockage of DNA replication forks. Here, we report that a sister chromatid exchange (SCE) generated by crossover-associated HR efficiently occurs in response to replication fork stalling before any measurable DNA double-strand breaks (DSBs). Interestingly, SCE produced by replication fork collapse following DNA DSBs creation is specifically suppressed by ATR, a central regulator of the replication checkpoint. BRCA1 depletio...

Feng, Zhihui; Zhang, Junran

2012-01-01

116

Lyapunov stability and Poisson structure of the thermal TDHF and RPA equations  

International Nuclear Information System (INIS)

The thermal TDHF equation is analyzed in the Liouville representation of quantum mechanics, where the matrix elements of the single-particle (s.p.) density ? behave as classical dynamical variables. By introducing the Lie-Poisson bracket associated with the unitary group of the s.p. Hilbert space, we show that TDHF has a hamiltonian, but non-canonical, classical form. Within this Poisson structure, either the s.p. energy or the s.p. grand potential ?(?) act as a Hamilton function. The Lyapunov stability of both the TDHF and RPA equations around a HF state then follows, since the HF approximation for thermal equilibrium is determined by minimizing ?(?). The RPA matrix in the Liouville space is expressed as the product of the Poisson tensor with the HF stability matrix, interpreted as a metric tensor generated by the entropy. This factorization displays the roles of the energy and entropy terms arising from ?(?) in the RPA dynamics, and it helps to construct the RPA modes. Several extensions are considered

1989-01-01

117

Spin waves in the 2D Hubbard model beyond the RPA  

International Nuclear Information System (INIS)

The spin-wave velocity c for the repulsive Hubbard model on a square lattice at half-filling is calculated as the square root of spin stiffness over perpendicular susceptibility using the variational Monte Carlo method with Gutzwiller wave function. For 3?U/t?12, c increases by 34-15% compared to the RPA result. ((orig.))

1994-04-01

118

RPA response to a static point charge near a metal surface  

Energy Technology Data Exchange (ETDEWEB)

In the present letter the RPA treatment is developed of the response to an electric point charge q embedded in the metal which fills the z<0 half-space at a distance a from the limiting surface represented as a finite barrier.

Vecchio, G.L.; Magnaterra, A. (Ferrara Univ. (Italy). Ist. di Fisica)

1981-08-01

119

Rapid construction of Drosophila RNAi transgenes using pRISE, a P-element-mediated transformation vector exploiting an in vitro recombination system.  

Science.gov (United States)

RNAi is a gene-silencing phenomenon mediated by double-stranded RNA (dsRNA) and has become a powerful tool to elucidate gene function. To accomplish rapid construction of transgenes expressing dsRNA in Drosophila, we developed a novel transformation vector, pRISE, which contains an inverted repeat of the attR1-ccdB-attR2 cassette for in vitro recombination and a pentameric GAL4 binding site for conditional expression. These features enabled us to construct RNAi transgenes without a complicated cloning scheme. In cultured cells and transgenic flies, pRISE constructs carrying dsRNA transgenes induced effective RNAi against an EGFP transgene and the endogenous white gene, respectively. These results indicate that pRISE is a convenient transformation vector for studies of multiple Drosophila genes for which functional information is lacking. PMID:16755136

Kondo, Takefumi; Inagaki, Sachi; Yasuda, Kunio; Kageyama, Yuji

2006-04-01

120

Human DNA topoisomerase I-mediated cleavage and recombination of duck hepatitis B virus DNA in vitro.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

In this study, we report that eukaryotic topoisomerase I (top1) can linearize the open circular DNA of duck hepatitis B virus (DHBV). Using synthetic oligonucleotides mimicking the three-strand flap DR1 region of the DHBV genome, we found that top1 cleaves the DNA plus strand in a suicidal manner, which mimics the linearization of the virion DNA. We also report that top1 can cleave the DNA minus strand at specific sites and can linearize the minus strand via a non-homologous recombination rea...

Pourquier, P.; Jensen, A. D.; Gong, S. S.; Pommier, Y.; Rogler, C. E.

1999-01-01

 
 
 
 
121

Recombinant adenovirus vector-mediated human MDA-7 gene transfection suppresses hepatocellular carcinoma growth in a mouse xenograft model?  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Hepatocellular carcinoma is one of the most common tumors in the world. The purpose of the present study was to investigate the inhibitory effects of adenoviral transduction of human melanoma differentiation-associated gene-7 (MDA-7) gene on hepatocellular carcinoma, so as to provide a theoretical basis for gene therapy of the disease. The human MDA-7 gene was cloned into replication-defective adenovirus specific to HepG2 cells using recombinant virus technology. RT-PCR and Western blotting a...

Pan, Xinting; Wu, Liqun; Cao, Jingyu; Guo, Weidong; Wang, Zusen; Han, Bing; Hu, Weiyu

2012-01-01

122

Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells  

Science.gov (United States)

Microhomology-mediated end joining (MMEJ) is a major pathway for Ku-independent alternative nonhomologous end joining, which contributes to chromosomal translocations and telomere fusions, but the underlying mechanism of MMEJ in mammalian cells is not well understood. In this study, we demonstrated that, distinct from Ku-dependent classical nonhomologous end joining, MMEJ—even with very limited end resection—requires cyclin-dependent kinase activities and increases significantly when cells enter S phase. We also showed that MMEJ shares the initial end resection step with homologous recombination (HR) by requiring meiotic recombination 11 homolog A (Mre11) nuclease activity, which is needed for subsequent recruitment of Bloom syndrome protein (BLM) and exonuclease 1 (Exo1) to DNA double-strand breaks (DSBs) to promote extended end resection and HR. MMEJ does not require S139-phosphorylated histone H2AX (?-H2AX), suggesting that initial end resection likely occurs at DSB ends. Using a MMEJ and HR competition repair substrate, we demonstrated that MMEJ with short end resection is used in mammalian cells at the level of 10–20% of HR when both HR and nonhomologous end joining are available. Furthermore, MMEJ is used to repair DSBs generated at collapsed replication forks. These studies suggest that MMEJ not only is a backup repair pathway in mammalian cells, but also has important physiological roles in repairing DSBs to maintain cell viability, especially under genomic stress.

Truong, Lan N.; Li, Yongjiang; Shi, Linda Z.; Hwang, Patty Yi-Hwa; He, Jing; Wang, Hailong; Razavian, Niema; Berns, Michael W.; Wu, Xiaohua

2013-01-01

123

Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells.  

Science.gov (United States)

Microhomology-mediated end joining (MMEJ) is a major pathway for Ku-independent alternative nonhomologous end joining, which contributes to chromosomal translocations and telomere fusions, but the underlying mechanism of MMEJ in mammalian cells is not well understood. In this study, we demonstrated that, distinct from Ku-dependent classical nonhomologous end joining, MMEJ--even with very limited end resection--requires cyclin-dependent kinase activities and increases significantly when cells enter S phase. We also showed that MMEJ shares the initial end resection step with homologous recombination (HR) by requiring meiotic recombination 11 homolog A (Mre11) nuclease activity, which is needed for subsequent recruitment of Bloom syndrome protein (BLM) and exonuclease 1 (Exo1) to DNA double-strand breaks (DSBs) to promote extended end resection and HR. MMEJ does not require S139-phosphorylated histone H2AX (?-H2AX), suggesting that initial end resection likely occurs at DSB ends. Using a MMEJ and HR competition repair substrate, we demonstrated that MMEJ with short end resection is used in mammalian cells at the level of 10-20% of HR when both HR and nonhomologous end joining are available. Furthermore, MMEJ is used to repair DSBs generated at collapsed replication forks. These studies suggest that MMEJ not only is a backup repair pathway in mammalian cells, but also has important physiological roles in repairing DSBs to maintain cell viability, especially under genomic stress. PMID:23610439

Truong, Lan N; Li, Yongjiang; Shi, Linda Z; Hwang, Patty Yi-Hwa; He, Jing; Wang, Hailong; Razavian, Niema; Berns, Michael W; Wu, Xiaohua

2013-05-01

124

Characterization of transferrin receptor-mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice (Oryza sativa L.  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Transferrin (TF plays a critical physiological role in cellular iron delivery via the transferrin receptor (TFR-mediated endocytosis pathway in nearly all eukaryotic organisms. Human serum TF (hTF is extensively used as an iron-delivery vehicle in various mammalian cell cultures for production of therapeutic proteins, and is also being explored for use as a drug carrier to treat a number of diseases by employing its unique TFR-mediated endocytosis pathway. With the increasing concerns over the risk of transmission of infectious pathogenic agents of human plasma-derived TF, recombinant hTF is preferred to use for these applications. Here, we carry out comparative studies of the TFR binding, TFR-mediated endocytosis and cellular iron delivery of recombinant hTF from rice (rhTF, and evaluate its suitability for biopharmaceutical applications. Result Through a TFR competition binding affinity assay with HeLa human cervic carcinoma cells (CCL-2 and Caco-2 human colon carcinoma cells (HTB-37, we show that rhTF competes similarly as hTF to bind TFR, and both the TFR binding capacity and dissociation constant of rhTF are comparable to that of hTF. The endocytosis assay confirms that rhTF behaves similarly as hTF in the slow accumulation in enterocyte-like Caco-2 cells and the rapid recycling pathway in HeLa cells. The pulse-chase assay of rhTF in Caco-2 and HeLa cells further illustrates that rice-derived rhTF possesses the similar endocytosis and intracellular processing compared to hTF. The cell culture assays show that rhTF is functionally similar to hTF in the delivery of iron to two diverse mammalian cell lines, HL-60 human promyelocytic leukemia cells (CCL-240 and murine hybridoma cells derived from a Sp2/0-Ag14 myeloma fusion partner (HB-72, for supporting their proliferation, differentiation, and physiological function of antibody production. Conclusion The functional similarity between rice derived rhTF and native hTF in their cellular iron delivery, TFR binding, and TFR-mediated endocytosis and intracellular processing support that rice-derived rhTF can be used as a safe and animal-free alternative to serum hTF for bioprocessing and biopharmaceutical applications.

Zhang Deshui

2012-11-01

125

Experimental study on the effects of recombinant adenoviral-mediated mI?B? gene combined with irradiation on the treatment of hepatocarcinoma  

International Nuclear Information System (INIS)

Objective: To explore the effect of recombinant adenovirus vector mediated mutant I?B? (mI?B?) combined with radiation on the hepatocarcinoma. Methods: Limited dilution method was used to test the virus titer in 293 cells. The HCC9204 cells were infected with MOI 10,20,30 and 50 for 48 h, respectively. The expression of p65 and mI?B? protein was analyzed by Western blot. Transfected HCC9204 cells and controls were treated with 4 Gy ? rays. The inhibition rate of HCC9204 cells was examined by MTT. Rat models of HCC9204 was constructed. AdmI?B? plasmids were injected into tumor tissue and the tumors were administered with 6 Gy ? irradiation 48 hours later. Tumor growth at different time points was recorded during 28 days. Results: The titer of AdmI?B? is 1.252 x 109 pfu/ml. The expression of mI?B? protein was increased with titer of AdmI?B?, and p65 protein began to decrease when MOI was 10, and reached the lowest when MOI was 50, they were all dose-dependent. The proliferation of HCC9204 cell lines were suppressed, as was more significant combined with radiation, and the effect was in a viral dose-dependent manner. From days 7 to 28 after AdmI?B? gene and radiotherapy, the tumor growth was significantly slower than after irradiation or gene therapy alone. Conclusions: Recombinant adenoviral-mediated mI?B? gene, combined with irradiation, can increase the cell-killing effect. It is better than that of either one alone. (authors)

2007-10-01

126

BLM–DNA2–RPA–MRN and EXO1–BLM–RPA–MRN constitute two DNA end resection machineries for human DNA break repair  

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Repair of dsDNA breaks requires processing to produce 3?-terminated ssDNA. We biochemically reconstituted DNA end resection using purified human proteins: Bloom helicase (BLM); DNA2 helicase/nuclease; Exonuclease 1 (EXO1); the complex comprising MRE11, RAD50, and NBS1 (MRN); and Replication protein A (RPA). Resection occurs via two routes. In one, BLM and DNA2 physically and specifically interact to resect DNA in a process that is ATP-dependent and requires BLM helicase and DNA2 nuclease fu...

Nimonkar, Amitabh V.; Genschel, Jochen; Kinoshita, Eri; Polaczek, Piotr; Campbell, Judith L.; Wyman, Claire; Modrich, Paul; Kowalczykowski, Stephen C.

2011-01-01

127

Recombinant Listeria monocytogenes as a Live Vaccine Vehicle for the Induction of Protective Anti-Viral Cell-Mediated Immunity  

Science.gov (United States)

Listeria monocytogenes (LM) is a Gram-positive bacterium that is able to enter host cells, escape from the endocytic vesicle, multiply within the cytoplasm, and spread directly from cell to cell without encountering the extracellular milieu. The ability of LM to gain access to the host cell cytosol allows proteins secreted by the bacterium to efficiently enter the pathway for major histocompatibility complex class I antigen processing and presentation. We have established a genetic system for expression and secretion of foreign antigens by recombinant strains, based on stable site-specific integration of expression cassettes into the LM genome. The ability of LM recombinants to induce protective immunity against a heterologous pathogen was demonstrated with lymphocytic choriomeningitis virus (LCMV). LM strains expressing the entire LCMV nucleoprotein or an H-2L^d-restricted nucleoprotein epitope (aa 118-126) were constructed. Immunization of mice with LM vaccine strains conferred protection against challenge with virulent strains of LCMV that otherwise establish chronic infection in naive adult mice. In vivo depletion of CD8^+ T cells from vaccinated mice abrogated their ability to clear viral infection, showing that protective anti-viral immunity was due to CD8^+ T cells.

Shen, Hao; Slifka, Mark K.; Matloubian, Mehrdad; Jensen, Eric R.; Ahmed, Rafi; Miller, Jeff F.

1995-04-01

128

Comparison of humoral and cell-mediated immune responses to cationic PLGA microspheres containing recombinant hepatitis B antigen.  

Science.gov (United States)

Presently available marketed alum adsorbed hepatitis B vaccine used for prophylactic immunization, can effectively elicit humoral immunity but is poor inducer of cell-mediated immunity (CMI). Besides, conventional alum-adjuvant vaccines require multiple injections to achieve long-lasting protective immune responses. Therefore, as a result of insufficient immunization, infections are still the leading killer among diseases. The present investigation was therefore, aimed at developing "single-shot" HBsAg adsorbed microspheres of poly (DL)-lactide-co-glycolide (PLGA) (L/G 50:50 and 75:25) and their capability to stimulate the cell mediated immune response against hepatitis B surface antigen. These microspheres were characterized in vitro for their size, shape polydispersity index, percentage HBsAg adsorption efficiency and in vitro release profile. The immune-stimulating activities were also studied following subcutaneous injection of HBsAg adsorbed PLGA microspheres (single-dose on day 0) and compared with alum adsorbed vaccines (two-doses on 0 and 28 days) in Balb/c mice. Specific cell-mediated immune responses such as lymphocyte transformation assay (stimulation-index) including release of interferon-gamma (IFN-?), interleukin-2 (IL-2) and nitric-oxide were determined. Cellular responses in case of alum adsorb HBsAg vaccine was very low. These studies demonstrate the potential of cationic polymeric microspheres based vaccine in stimulating cell mediated immune response along with humoral response against hepatitis B. PMID:21291968

Saini, Vinay; Jain, Vikas; Sudheesh, M S; Jaganathan, K S; Murthy, P K; Kohli, D V

2011-04-15

129

Collective excitations and pairing effects in drip-line nuclei. Continuum RPA in coordinate-space HFB  

Energy Technology Data Exchange (ETDEWEB)

We discuss novel features of a new continuum RPA formulated in the coordinate-space Hartree-Fock-Bogoliubov framework. This continuum quasiparticle RPA takes into account both the one- and two-particle escaping channels. The theory is tested with numerical calculations for monopole, dipole and quadrupole excitations in neutron-rich oxygen isotopes near the drip-line. Effects of the particle-particle RPA correlation caused by the pairing interaction are discussed in detail, and importance of the selfconsistent treatment is emphasized. (author)

Matsuo, Masayuki [Niigata University, Graduate School of Science and Technology, Niigata (Japan)

2002-12-01

130

Particle-particle and hole-hole RPA correlations at finite temperature and the temperature dependence of the level density parameter  

International Nuclear Information System (INIS)

The pp-hh RPA equations obtained by summing the infinite series of ladder, upwards and backwards going diagrams in the temperature two particle Green's functions are derived at finite temperature. The contribution to the thermodynamic grand potential due to pp-hh RPA correlations is calculated simultaneously to that of ph RPA correlations. A schematic model is constructed which shows that, as for ph RPA states, the energies of pp and hh RPA states have no temperature dependence at not too high temperature. Within the same model, the temperature dependence of the level density parameter is discussed

1987-01-01

131

Recombinant Adeno-associated virus (rAAV)-mediated transduction and optogenetic manipulation of cortical neurons in vitro  

Science.gov (United States)

Genetically encoded light-sensitive proteins can be used to manipulate and observe cellular functions. According to different modes of action, these proteins are divided into actuators like the blue-light gated cation channel Channelrhodopsin-2 (ChR2) and detectors like the calcium sensor GCaMP. In order to optogenetically control and study the activity of rat primary cortical neurons, we established a transduction procedure using recombinant Adeno-associated viruses (rAAVs) as gene-ferries. Thereby, we achieved high transduction rates of these neurons with ChR2. In ChR2 expressing neurons, action potentials could be repeatedly and precisely elicited with laser pulses and measured via patch clamp recording.

Lange, Wienke; Jin, Lei; Maybeck, Vanessa; Meisenberg, Annika; Baumann, Arnd; Offenhäusser, Andreas

2014-03-01

132

Microhomology-mediated end joining in fission yeast is repressed by pku70 and relies on genes involved in homologous recombination.  

Science.gov (United States)

Two DNA repair pathways are known to mediate DNA double-strand-break (DSB) repair: homologous recombination (HR) and nonhomologous end joining (NHEJ). In addition, a nonconservative backup pathway showing extensive nucleotide loss and relying on microhomologies at repair junctions was identified in NHEJ-deficient cells from a variety of organisms and found to be involved in chromosomal translocations. Here, an extrachromosomal assay was used to characterize this microhomology-mediated end-joining (MMEJ) mechanism in fission yeast. MMEJ was found to require at least five homologous nucleotides and its efficiency was decreased by the presence of nonhomologous nucleotides either within the overlapping sequences or at DSB ends. Exo1 exonuclease and Rad22, a Rad52 homolog, were required for repair, suggesting that MMEJ is related to the single-strand-annealing (SSA) pathway of HR. In addition, MMEJ-dependent repair of DSBs with discontinuous microhomologies was strictly dependent on Pol4, a PolX DNA polymerase. Although not strictly required, Msh2 and Pms1 mismatch repair proteins affected the pattern of MMEJ repair. Strikingly, Pku70 inhibited MMEJ and increased the minimal homology length required for efficient MMEJ. Overall, this study strongly suggests that MMEJ does not define a distinct DSB repair mechanism but reflects "micro-SSA." PMID:17483423

Decottignies, Anabelle

2007-07-01

133

P-glycoprotein-mediated resistance to chemotherapy in cancer cells: using recombinant cytosolic domains to establish structure-function relationships  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Resistance to chemotherapy in cancer cells is mainly mediated by overexpression of P-glycoprotein (Pgp), a plasma membrane ATP-binding cassette (ABC) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. Pgp consists of two homologous halves each containing a transmembrane domain and a cytosolic nucleotide-binding domain (NBD) which contains two consensus Walker motifs, A and B, involved in ATP binding and hydrolysis. The protein also contains an S signature characteris...

Di Pietro A; Dayan G.; Conseil G.; Steinfels E.; Krell T.; Trompier D.; Baubichon-Cortay H; -m, Jault J.

1999-01-01

134

Proton-neutron quasiparticle RPA with separable Gamow-Teller forces  

International Nuclear Information System (INIS)

A comprehensive representation is presented of a generalized form of the proton-neutron quasiparticle RPA model, originally introduced by Halbleib and Sorensen almost thirty years ago. The model uses separable Gamow-Teller forces, including, in addition to the particle-hole force of the former model, the particle-particle force, which is of decisive importance for ?+ decay and ?? decay. The above model has further been extended to the treatment of odd-odd nuclei. An extension is also made to transitions from nuclear excited states. This is essential for calculations of nuclear weak transition rates in the high-temperature interior of massive stars. Complementing the discussion of Halbleib and Sorensen on the particle-hole force, the structure of the RPA dispersion relation is discussed with emphasis on effects of the particle-particle force. (orig.)

1992-01-01

135

Giant Resonances using Correlated Realistic Interactions: The Case for Second RPA  

CERN Document Server

Lately we have been tackling the problem of describing nuclear collective excitations starting from correlated realistic nucleon-nucleon (NN) interactions. The latter are constructed within the Unitary Correlation Operator Method (UCOM), starting from realistic NN potentials. It has been concluded that first-order RPA with a two-body UCOM interaction is not capable, in general, of reproducing quantitatively the properties of giant resonances (GRs), due to missing higher-order configurations and long-range correlations as well as neglected three-body terms in the Hamiltonian. Here we report results on GRs obtained by employing a UCOM interaction based on the Argonne V18 potential in Second RPA (SRPA) calculations. The same interaction is used to describe the Hartree-Fock (HF) ground state and the residual interactions. We find that the inclusion of second-order configurations -- which effectively dress the underlying HF single-particle states with self-energy insertions -- produces sizable corrections. The eff...

Papakonstantinou, P

2007-01-01

136

Self-Consistent Separable Rpa Approach for Skyrme Forces: Axial Nuclei  

CERN Document Server

The self-consistent separable RPA (random phase approximation) method is formulated for Skyrme forces with pairing. The method is based on a general self-consistent procedure for factorization of the two-body interaction. It is relevant for various density- and current-dependent functionals. The contributions of the time-even and time-odd Skyrme terms as well as of the Coulomb and pairing terms to the residual interaction are taken self-consistently into account. Most of the expression have a transparent analytical form, which makes the method convenient for the treatment and analysis. The separable character of the residual interaction allows to avoid diagonalization of high-rank RPA matrices and thus to minimize the calculation effort. The previous studies have demonstrated high numerical accuracy and efficiency of the method for spherical nuclei. In this contribution, the method is specified for axial nuclei. We provide systematic and detailed presentation of formalism and discuss different aspects of the ...

Nesterenko, V O; Kleinig, W; Kvasil, J; Reinhard, P G

2005-01-01

137

Self-consistent quasi-particle RPA for the description of superfluid Fermi systems  

CERN Document Server

Self-Consistent Quasi-Particle RPA (SCQRPA) is for the first time applied to a more level pairing case. Various filling situation and values for the coupling constant are considered. Very encouraging results in comparison with the exact solution of the model are obtaining. The nature of the low lying mode in SCQRPA is identified. The strong reduction of the number fluctuation in SCQRPA vs BCS is pointed out. The transition from superfluidity to the normal fluid case is carefully investigated.

Rahbi, A; Chanfray, G; Schuck, P

2002-01-01

138

Self-Consistent Quasi-Particle RPA for the Description of Superfluid Fermi Systems  

CERN Multimedia

Self-Consistent Quasi-Particle RPA (SCQRPA) is for the first time applied to a more level pairing case. Various filling situations and values for the coupling constant are considered. Very encouraging results in comparison with the exact solution of the model are obtained. The nature of the low lying mode in SCQRPA is identified. The strong reduction of the number fluctuation in SCQRPA vs BCS is pointed out. The transition from superfluidity to the normal fluid case is carefully investigated.

Rabhi, A; Chanfray, G; Schuck, P

2002-01-01

139

Fully self-consistent RPA description of the many level pairing model  

International Nuclear Information System (INIS)

The self-consistent RPA (SCRPA) equations in the particle-particle channel are solved without any approximation for the picket fence model. The results are in excellent agreement with the exact solutions found with the Richardson method. Particularly interesting features are that screening corrections reverse the sign of the interaction and that SCRPA yields the exact energies in the case of two levels with two particles

2002-03-15

140

The unrestricted Gutzwiller+RPA approach and its application to stripes in cuprates  

International Nuclear Information System (INIS)

We use the time-dependent Gutzwiller approximation for the Hubbard model in order to compute random-phase approximation-like (RPA) fluctuations on top of the Gutzwiller approximation (GA). No restrictions are imposed on the charge and spin configurations which makes the method suitable for the calculation of linear excitations around symmetry-broken solutions. Within this formalism, we investigate static and dynamical properties in the charge and spin channel of stripe textures in cuprates

2005-04-30

 
 
 
 
141

The unrestricted Gutzwiller+RPA approach and its application to stripes in cuprates  

Energy Technology Data Exchange (ETDEWEB)

We use the time-dependent Gutzwiller approximation for the Hubbard model in order to compute random-phase approximation-like (RPA) fluctuations on top of the Gutzwiller approximation (GA). No restrictions are imposed on the charge and spin configurations which makes the method suitable for the calculation of linear excitations around symmetry-broken solutions. Within this formalism, we investigate static and dynamical properties in the charge and spin channel of stripe textures in cuprates.

Seibold, G. [Institut fuer Physik, BTU Cottbus, P. Box 101344, 03013 Cottbus (Germany)]. E-mail: goetz@physik.tu-cottbus.de; Lorenzana, J. [SMC-INFM, Dipartimento di Fisica, Universita di Roma La Sapienza, P. Aldo Moro 2, 00185 Rome (Italy)

2005-04-30

142

Test of the pnRPA method within an extended Lipkin-type model  

International Nuclear Information System (INIS)

An extended Lipkin-Meshkov-Glick (LMG) model for testing the proton-neutron Random Phase Approximation (pnRPA) method is developed taking into account explicitly proton and neutron degrees of freedom. Besides the proton and neutron single particle terms two types of residual proton-neutron interactions, one simulating a particle-particle and the other a particle-hole interaction, are included in the model Hamiltonian so that the model is exactly solvable in an isospin SU(2) x SU(2) basis. The behavior of the first excited (collective) state obtained by i) exact diagonalization of the Hamiltonian matrix and ii) with the pnRPA, is studied as function of the model parameters and the two results compared with each other. Furthermore, charge-changing operators simulating nuclear beta decay and their action on eigenfunctions of the model Hamiltonian are defined and transition amplitudes of them are calculated using exact, and the Tamm-Dancoff and pnRPA eigenfunctions. (authors)

2000-01-01

143

Microneedle mediated intradermal delivery of adjuvanted recombinant HIV-1 CN54gp140 effectively primes mucosal boost inoculations  

Science.gov (United States)

Dissolving polymeric microneedle arrays formulated to contain recombinant CN54 HIVgp140 and the TLR4 agonist adjuvant MPLA were assessed for their ability to elicit antigen-specific immunity. Using this novel microneedle system we successfully primed antigen-specific responses that were further boosted by an intranasal mucosal inoculation to elicit significant antigen-specific immunity. This prime-boost modality generated similar serum and mucosal gp140-specific IgG levels to the adjuvanted and systemic subcutaneous inoculations. While the microneedle primed groups demonstrated a balanced Th1/Th2 profile, strong Th2 polarization was observed in the subcutaneous inoculation group, likely due to the high level of IL-5 secretion from cells in this group. Significantly, the animals that received a microneedle prime and intranasal boost regimen elicited a high level IgA response in both the serum and mucosa, which was greatly enhanced over the subcutaneous group. The splenocytes from this inoculation group secreted moderate levels of IL-5 and IL-10 as well as high amounts of IL-2, cytokines known to act in synergy to induce IgA. This work opens up the possibility for microneedle-based HIV vaccination strategies that, once fully developed, will greatly reduce risk for vaccinators and patients, with those in the developing world set to benefit most.

Pattani, Aditya; McKay, Paul F.; Garland, Martin J.; Curran, Rhonda M.; Migalska, Katarzyna; Cassidy, Corona M.; Malcolm, R. Karl; Shattock, Robin J.; McCarthy, Helen O.; Donnelly, Ryan F.

2012-01-01

144

Use of Aspirin in normalization of recombinant human erythropoietin-mediated hyper-reactivity of platelets in rats  

Science.gov (United States)

Objectives: The cytokine erythropoietin is the primary stimulator of erythropoiesis and recombinant human erythropoietin (rHuEPO), which is widely used in the treatment of anemia associated with advanced chronic kidney disease (CKD). Adverse cardiovascular outcomes have been observed during clinical trials of anemia correction with rHuEPO in CKD patients. We investigated the effects of short-term, high-dose treatment with rHuEPO on platelet reactivity and effects of aspirin on platelet reactivity in healthy rats. Materials and Methods: Animals received three daily dose of rHuEPO (25 ?g/kg s.c.). Platelets were isolated after 48 h of last dose of rHuEPO to study the arachidonic acid-induced platelet aggregation. Aspirin (75 mg/kg p.o.) was given to animals just before 1 h of isolation of platelets. Results: In rats, treatment with rHuEPO increased platelet reactivity and platelet count. The increased platelet reactivity was paralleled by decreased time-to-occlusion (TTO) in arterial thrombosis model, and decreased bleeding time after tail transection in rats. Treatment with rHuEPO followed by single dose of aspirin showed significant reduction in TTO and bleeding time as compared with aspirin-treated group. Conclusions: These findings suggest that rHuEPO increases platelet reactivity and aspirin normalizes the hyper-reactive platelet and may reduce the cardiovascular events associated with rHuEPO in CKD patients.

Soni, Hitesh M.; Vekaria, Amit M.; Rath, Akshyaya C.; Belemkar, Sateesh; Jain, Mukul R.

2014-01-01

145

Correlation between Lethal Toxin-Neutralizing Antibody Titers and Protection from Intranasal Challenge with Bacillus anthracis Ames Strain Spores in Mice after Transcutaneous Immunization with Recombinant Anthrax Protective Antigen  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Transcutaneous immunization of mice with recombinant protective antigen (rPA) of Bacillus anthracis resulted in significantly higher lethal toxin-neutralizing antibody titers than did intramuscular injection of alum-adsorbed rPA. Immunized mice were partially protected against intranasal challenge with 235,000 (10 50% lethal doses) Ames strain B. anthracis spores. A highly significant correlation was observed between toxin-neutralizing antibody titer and survival after challenge. Future exper...

Peachman, Kristina K.; Rao, Mangala; Alving, Carl R.; Burge, Robert; Leppla, Stephen H.; Rao, Venigalla B.; Matyas, Gary R.

2006-01-01

146

Recombinant TLR5 Agonist CBLB502 Promotes NK Cell-Mediated Anti-CMV Immunity in Mice  

Science.gov (United States)

Prior work using allogeneic bone marrow transplantation (allo-BMT) models showed that peritransplant administration of flagellin, a toll-like receptor 5 (TLR5) agonist protected murine allo-BMT recipients from CMV infection while limiting graft-vs-host disease (GvHD). However, the mechanism by which flagellin-TLR5 interaction promotes anti-CMV immunity was not defined. Here, we investigated the anti-CMV immunity of NK cells in C57BL/6 (B6) mice treated with a highly purified cGMP grade recombinant flagellin variant CBLB502 (rflagellin) followed by murine CMV (mCMV) infection. A single dose of rflagellin administered to mice between 48 to 72 hours prior to MCMV infection resulted in optimal protection from mCMV lethality. Anti-mCMV immunity in rflagellin-treated mice correlated with a significantly reduced liver viral load and increased numbers of Ly49H+ and Ly49D+ activated cytotoxic NK cells. Additionally, the increased anti-mCMV immunity of NK cells was directly correlated with increased numbers of IFN-?, granzyme B- and CD107a producing NK cells following mCMV infection. rFlagellin-induced anti-mCMV immunity was TLR5-dependent as rflagellin-treated TLR5 KO mice had ?10-fold increased liver viral load compared with rflagellin-treated WT B6 mice. However, the increased anti-mCMV immunity of NK cells in rflagellin-treated mice is regulated indirectly as mouse NK cells do not express TLR5. Collectively, these data suggest that rflagellin treatment indirectly leads to activation of NK cells, which may be an important adjunct benefit of administering rflagellin in allo-BMT recipients.

Hossain, Mohammad S.; Ramachandiran, Sampath; Gewirtz, Andrew T.; Waller, Edmund K.

2014-01-01

147

Recombinant AAV-mediated BEST1 transfer to the retinal pigment epithelium: analysis of serotype-dependent retinal effects.  

Science.gov (United States)

Mutations in the BEST1 gene constitute an underlying cause of juvenile macular dystrophies, a group of retinal disorders commonly referred to as bestrophinopathies and usually diagnosed in early childhood or adolescence. The disease primarily affects macular and paramacular regions of the eye leading to major declines in central vision later in life. Currently, there is no cure or surgical management for BEST1-associated disorders. The recently characterized human disease counterpart, canine multifocal retinopathy (cmr), recapitulates a full spectrum of clinical and molecular features observed in human bestrophinopathies and offers a valuable model system for development and testing of therapeutic strategies. In this study, the specificity, efficiency and safety of rAAV-mediated transgene expression driven by the human VMD2 promoter were assessed in wild-type canine retinae. While the subretinal delivery of rAAV2/1 vector serotype was associated with cone damage in the retina when BEST1 and GFP were co-expressed, the rAAV2/2 vector serotype carrying either GFP reporter or BEST1 transgene under control of human VMD2 promoter was safe, and enabled specific transduction of the RPE cell monolayer that was stable for up to 6 months post injection. These encouraging studies with the rAAV2/2 vector lay the groundwork for development of gene augmentation therapy for human bestrophinopathies. PMID:24143172

Guziewicz, Karina E; Zangerl, Barbara; Komáromy, András M; Iwabe, Simone; Chiodo, Vincent A; Boye, Sanford L; Hauswirth, William W; Beltran, William A; Aguirre, Gustavo D

2013-01-01

148

Immunological ignorance allows long-term gene expression after perinatal recombinant adeno-associated virus-mediated gene transfer to murine airways.  

Science.gov (United States)

Abstract Gene therapy of the lung has the potential to treat life-threatening diseases such as cystic fibrosis and ?1-antitrypsin or surfactant deficiencies. A major hurdle for successful gene therapy is the development of an immune response against the transgene and/or viral vector. We hypothesized that by targeting the airways in the perinatal period, induction of an immune response against the vector particle could be prevented because of immaturity of the immune system, in turn allowing repeated gene transfer later in adult life to ensure long-term gene expression. Therefore, we readministered recombinant adeno-associated viral vector serotype 5 (rAAV2/5) to mouse airways 3 and 6 months after initial perinatal gene transfer. Our findings demonstrate that perinatal rAAV2/5-mediated gene transfer to the airways avoids a strong immune response. This immunological ignorance allows the readministration of an autologous vector later in adult life, resulting in efficient and stable gene transfer up to 7 months, without evidence of a decrease in transgene expression. Together, these data provide a basis to further explore perinatal gene therapy for pulmonary conditions with adequate gene expression up to 7 months. PMID:24548076

Carlon, Marianne S; Vidovi?, Dragana; Dooley, James; da Cunha, Marina Mori; Maris, Michael; Lampi, Youlia; Toelen, Jaan; Van den Haute, Chris; Baekelandt, Veerle; Deprest, Jan; Verbeken, Erik; Liston, Adrian; Gijsbers, Rik; Debyser, Zeger

2014-06-01

149

Recombinant Lz-8 from Ganoderma lucidum induces endoplasmic reticulum stress-mediated autophagic cell death in SGC-7901 human gastric cancer cells.  

Science.gov (United States)

In Asia, the mushroom of the fungus Ganoderma lucidum has been widely used as a traditional medicine for the past two millennia. The aim of this study was to investigate the anticancer activity of recombinant Lz-8 (rLz-8), a protein belonging to a family of fungal immunomodulatory proteins. We report that rLz-8 induces endoplasmic reticulum (ER) stress-mediated autophagic cell death in the human gastric cancer cell line SGC-7901. Our results show that rLz-8 induces autophagic cell death by aggregating in the ER, triggering ER stress and the ATF4-CHOP pathway. A foreign protein, in the ER rLz-8 causes the activation of the ubiquitine/proteasome ER-associated degradation (ERAD) system. The autophagic arm of this system is then overstimulated by an excessive abundance of rLz-8 and causes the cell's death through an over-autophagic response. We also found that caspase inhibitors do not prevent rLz-8-induced cell death, and therefore the autophagic response induced by rLz-8 is independent of caspase activation. PMID:22179718

Liang, Chongyang; Li, Hongrui; Zhou, Hui; Zhang, Shuqin; Liu, Zhiyi; Zhou, Qiuli; Sun, Fei

2012-04-01

150

Recombinant adeno-associated virus-mediated microRNA delivery into the postnatal mouse brain reveals a role for miR-134 in dendritogenesis in vivo  

Directory of Open Access Journals (Sweden)

Full Text Available Recent studies using primary neuronal cultures have revealed important roles of the microRNA pathway in the regulation of neuronal development and morphology. For example, miR-134 is involved in dendritogenesis and spine development in hippocampal neurons by regulating local mRNA translation in dendrites. The in vivo roles of microRNAs in these processes are still uninvestigated, partly due to the lack of tools enabling stable in vivo delivery of microRNAs or microRNA inhibitors into neurons of the mammalian brain. Here we describe the construction and validation of a vector-based tool for stable delivery of microRNAs in vivo by use of recombinant adeno-associated virus (rAAV. rAAV-mediated overexpression of miR-134 in neurons of the postnatal mouse brain provided evidence for a negative role of miR-134 in dendritic arborization of cortical layer V pyramidal neurons in vivo, thereby confirming previous findings obtained with cultured neurons. Our system provides researchers with a unique tool to study the role of any candidate microRNA in vivo and can easily be adapted to microRNA loss-of-function studies. This platform should therefore greatly facilitate investigations on the role of microRNAs in synapse development, plasticity and behavior in vivo.

MetteChristensen

2010-01-01

151

RPA Review  

for the constructive way in which we and the report have been received. ...... will \\be integrated into the core processing work and will be carried out by case \\workers. ... problems with Remote Sensing, principally related to cloud cover \\rendering.

152

Application of the Remotely Piloted Aircraft (RPA) 'MASC' in Atmospheric Boundary Layer Research  

Science.gov (United States)

The remotely piloted aircraft (RPA) MASC (Multipurpose Airborne Sensor Carrier) was developed at the University of Tübingen in cooperation with the University of Stuttgart, University of Applied Sciences Ostwestfalen-Lippe and 'ROKE-Modelle'. Its purpose is the investigation of thermodynamic processes in the atmospheric boundary layer (ABL), including observations of temperature, humidity and wind profiles, as well as the measurement of turbulent heat, moisture and momentum fluxes. The aircraft is electrically powered, has a maximum wingspan of 3.40 m and a total weight of 5-8 kg, depending on battery- and payload. The standard meteorological payload consists of temperature sensors, a humidity sensor, a flow probe, an inertial measurement unit and a GNSS. In normal operation, the aircraft is automatically controlled by the ROCS (Research Onboard Computer System) autopilot to be able to fly predefined paths at constant altitude and airspeed. Since 2010 the system has been tested and improved intensively. In September 2012 first comparative tests could successfully be performed at the Lindenberg observatory of Germany's National Meteorological Service (DWD). In 2013, several campaigns were done with the system, including fundamental boundary layer research, wind energy meteorology and assistive measurements to aerosol investigations. The results of a series of morning transition experiments in summer 2013 will be presented to demonstrate the capabilities of the measurement system. On several convective days between May and September, vertical soundings were done to record the evolution of the ABL in the early morning, from about one hour after sunrise, until noon. In between the soundings, flight legs of up to 1 km length were performed to measure turbulent statistics and fluxes at a constant altitude. With the help of surface flux measurements of a sonic anemometer, methods of similarity theory could be applied to the RPA flux measurements to compare them to literature. The results show prospects and limitations of boundary layer research with a single RPA at the present state of the art.

Wildmann, Norman; Bange, Jens

2014-05-01

153

Generalized Brueckner-Hartree-Fock theory and self-consistent RPA  

International Nuclear Information System (INIS)

Self-consistent RPA (SCRPA) theory is developed in the particle-particle (pp) channel. It is pointed out that in this way vertex and self-energy corrections are taken into account on an equal footing whereas in Brueckner-Hartree-Fock (BHF) this is not the case. We discuss in detail the interconnection between both theories and apply them to a model case. Excellent agreement with the exact solution is found for SCRPA where as BHF gives somewhat poorer results. In an appendix it is demonstrated how SCRPA connects to a variational principle and how, for the particle-hole case, sum rules and conservation laws are fulfilled. (orig.)

1998-01-05

154

Superallowed Fermi transitions in RPA with a relativistic point-coupling energy functional  

CERN Document Server

The self-consistent random phase approximation (RPA) approach with the residual interaction derived from a relativistic point-coupling energy functional is applied to evaluate the isospin symmetry-breaking corrections {\\delta}c for the 0+\\to0+ superallowed Fermi transitions. With these {\\delta}c values, together with the available experimental ft values and the improved radiative corrections, the unitarity of the Cabibbo-Kobayashi-Maskawa (CKM) matrix is examined. Even with the consideration of uncertainty, the sum of squared top-row elements has been shown to deviate from the unitarity condition by 0.1% for all the employed relativistic energy functionals.

Li, Z X; Chen, H; 10.1007/s11433-011-4320-2

2011-01-01

155

A Remotely Piloted Aircraft (RPA) as a Measurement Tool for Wind-Energy Research  

Science.gov (United States)

In wind energy meteorology, RPA have the clear advantage compared to manned aircraft that they allow to fly very close to the ground and even in between individual wind turbines in a wind farm. Compared to meteorological towers and lidar systems, the advantage is the flexibility of the system, which makes it possible to measure at the desired site on short notice and not only in main wind direction. At the Center of Applied Geoscience at the University of Tübingen, the research RPA MASC (Multi-purpose Airborne Sensor Carrier) was developed. RPA of type MASC have a wingspan of about 3 m and a maximum take-off weight of 7.5 kg, including payload. The standard meteorological payload includes instruments for temperature, humidity, barometric pressure and wind measurement. It is possible to resolve turbulence fluctuations of wind and temperature up to 20 Hz. The autopilot ROCS (Research Onboard Computer System), which is developed at the Institute of Flight Mechanics and Control, University of Stuttgart, makes it possible to automatically follow predefined waypoints at constant altitude and airspeed. At a cruising speed of 24 m/s and a battery life of approx. one hour, a range of 80 km is feasible. The project 'Lidar Complex', funded by the German Federal Ministry for the Environment, Nature Conservation and Nuclear Safety, is part of the research network 'WindForS', based in Southern Germany. The goal of the project is to establish lidar technology for wind energy plant site evaluation in complex terrain. Additional goals are the comparison of different measurement techniques and the validation of wind-field models in not IEC 61400 conform terrain. It is planned to design a turbulent wind-field generator, fed by real measurement data, which can be used to analyse WEC behaviour. Two test sites were defined for the 'Lidar Complex' project, one in IEC-conform terrain about 15 km from the Baltic Sea, the other in the Swabian Alb, only 2 km downstream of a 100 m steep escarpment. At both sites, flight measurements were performed in 2013 with the RPA MASC. The data that was collected allows to investigate the influence of thermal stability of the atmosphere at the test site and turbulence intensity around individual wind energy converters (WECs). Several measurement flights were done to investigate the wake structure downstream a running WEC. Preliminary results will be presented as well as an outlook for future research with the instrument.

Wildmann, Norman; Bange, Jens

2014-05-01

156

An effective interaction derived from HJ potential for use in TDA and RPA calculations  

International Nuclear Information System (INIS)

An effective interaction is derived by fitting the matrix elements of a sum of Yukawa terms to the G-matrix elements of a sum of Yukawa terms to the G-matrix elements derived from the Hamada-Johnston potential. Central, spin-orbit and tensor components are taken into account. Numerical results are given and compared with those obtained from the Paris and Reid potentials. As an application, the excitation spectra in "1"6O are investigated in the framework of the TDA and RPA. (author). 12 refs, 3 tabs

1990-01-01

157

Superallowed Fermi transitions in RPA with a relativistic point-coupling energy functional  

Science.gov (United States)

The self-consistent random phase approximation (RPA) approach with the residual interaction derived from a relativistic point-coupling energy functional is applied to evaluate the isospin symmetry-breaking corrections ? c for the 0+ ? 0+ superallowed Fermi transitions. With these ? c values, together with the available experimental f t values and the improved radiative corrections, the unitarity of the Cabibbo-Kobayashi-Maskawa (CKM) matrix is examined. Even with the consideration of uncertainty, the sum of squared top-row elements has been shown to deviate from the unitarity condition by 0.1% for all the employed relativistic energy functionals.

Li, Zhaoxi; Yao, Jiangming; Chen, Hong

2011-06-01

158

Human recombinant interleukin-1 beta- and tumor necrosis factor alpha-mediated suppression of heparin-like compounds on cultured porcine aortic endothelial cells  

International Nuclear Information System (INIS)

Cytokines are known to tip the balance of the coagulant-anticoagulant molecules on the endothelial cell surface toward intravascular coagulation. Their effects on endothelial cell surface-associated heparin-like compounds have not been examined yet. Incorporation of [35S]sulfate into heparan sulfate on cultured porcine aortic endothelial cells was suppressed by human recombinant interleukin-1 beta (rIL-1 beta) or tumor necrosis factor alpha (rTNF alpha) in a dose- and time-dependent manner with little effect on cell number, protein content, and [3H]leucine incorporation of cells. Maximal inhibition was achieved by incubation of cells with 100 ng/ml of rIL-1 beta or 5 ng/ml of rTNF alpha for 12-24 hours, resulting in a reduction of the synthesis of heparan sulfate on the cell surface by approximately 50%. The dose dependency was consistent with that seen in the stimulation of endothelial cell procoagulant activity by each cytokine. The suppression of heparan sulfate synthesis was sustained for at least 48 hours after pretreatment of cells with cytokines and was unchanged after the addition of indomethacin or polymyxin B. The rate of degradation of prelabeled 35S-heparan sulfate on the cell surface was not altered by cytokine treatments. Neither the size, the net negative charge, nor the proportion of the molecule with high affinity for antithrombin III of endothelial cell heparan sulfate was changed by cytokines. Furthermore, specific binding of 125I-labeled antithrombin III to the endothelial cell surface was reduced to 40-60% of control by cytokines. In parallel with reduction in binding, antithrombin III cofactor activity was partially diminished in cytokine-treated endothelial cells. Thus, cytokine-mediated suppression of heparin-like substance on endothelial cells appears to be another cytokine-inducible endothelial effects affecting coagulation

1990-01-01

159

Recombinant adeno-associated virus 2-mediated transfer of the human superoxide-dismutase gene does not confer radioresistance on HeLa cervical carcinoma cells  

International Nuclear Information System (INIS)

Background and purpose: The success rate of any therapeutic approach depends on the therapeutic window, which can be increased by either raising the resistance of the normal tissue without protecting the tumor cells or by sensitizing the tumor cells but not the normal cells. Two promising candidate genes for normal tissue protection against radiation-induced damage may be the copper-zinc (CuZnSOD) and manganese superoxide-dismutase genes (MnSOD). The recombinant adeno-associated virus 2 (rAAV-2) offers attractive advantages over other vector systems: low immunogenicity, ability to infect dividing and non-dividing tissues and a low chance of insertional mutagenesis, due to extra-chromosomal localization. We report the production of novel rAAV-2-SOD vectors and the investigation of their modulating effects on HeLa-RC cells after irradiation. Material and methods: rAAV-2 vectors were cloned containing the human CuZnSOD or MnSOD as transgene and vector stocks were produced. In the initial experiments human cervix carcinoma (HeLa-RC) cells were chosen for their susceptibility to rAAV-2. On day 0, cells were seeded and transduced with the rAAV-2-SOD vectors. On day 3, cells were harvested, irradiated (0.5-8 Gy) and reseeded in different assays (FACS, SOD, MTT and colony assays). Results: Although >70% of all cells expressed SOD and significant amounts of functional SOD protein were detected, no radioprotective effect of SOD was observed after transduction of HeLa-RC cells. Conclusions: Novel rAAV-2-SOD vectors that could be produced at high titer, were able to efficiently infect cells and express the SOD genes. The absence of a radioprotective effect in HeLa-RC cancer cells indicates an additional safety feature and suggests that rAAV-mediated MnSOD overexpression might contribute to increasing the therapeutic index when applied for normal tissue protection

2004-09-01

160

Human recombinant interleukin-1 beta- and tumor necrosis factor alpha-mediated suppression of heparin-like compounds on cultured porcine aortic endothelial cells  

Energy Technology Data Exchange (ETDEWEB)

Cytokines are known to tip the balance of the coagulant-anticoagulant molecules on the endothelial cell surface toward intravascular coagulation. Their effects on endothelial cell surface-associated heparin-like compounds have not been examined yet. Incorporation of (35S)sulfate into heparan sulfate on cultured porcine aortic endothelial cells was suppressed by human recombinant interleukin-1 beta (rIL-1 beta) or tumor necrosis factor alpha (rTNF alpha) in a dose- and time-dependent manner with little effect on cell number, protein content, and (3H)leucine incorporation of cells. Maximal inhibition was achieved by incubation of cells with 100 ng/ml of rIL-1 beta or 5 ng/ml of rTNF alpha for 12-24 hours, resulting in a reduction of the synthesis of heparan sulfate on the cell surface by approximately 50%. The dose dependency was consistent with that seen in the stimulation of endothelial cell procoagulant activity by each cytokine. The suppression of heparan sulfate synthesis was sustained for at least 48 hours after pretreatment of cells with cytokines and was unchanged after the addition of indomethacin or polymyxin B. The rate of degradation of prelabeled 35S-heparan sulfate on the cell surface was not altered by cytokine treatments. Neither the size, the net negative charge, nor the proportion of the molecule with high affinity for antithrombin III of endothelial cell heparan sulfate was changed by cytokines. Furthermore, specific binding of 125I-labeled antithrombin III to the endothelial cell surface was reduced to 40-60% of control by cytokines. In parallel with reduction in binding, antithrombin III cofactor activity was partially diminished in cytokine-treated endothelial cells. Thus, cytokine-mediated suppression of heparin-like substance on endothelial cells appears to be another cytokine-inducible endothelial effects affecting coagulation.

Kobayashi, M.; Shimada, K.; Ozawa, T. (Kochi Medical School (Japan))

1990-09-01

 
 
 
 
161

SANS [small-angle neutron scattering] evaluation of the RPA [random phase approximation] theory for binary homopolymer mixtures  

International Nuclear Information System (INIS)

A well characterized binary mixture of normal (protonated) and perdeuterated monodisperse 1,2 polybutenes has been studied by small-angle neutron scattering (SANS). For scattering wavevectors q greater than the inverse radius-of-gyration R/sub g/-1, the SANS intensity is quantitatively predicted by the random phase approximation (RPA) theory of deGennes over all measured values of the segment-segment interaction parameter Chi. In the region (Chi s-Chi)Chi s-1 > 0.5 the interaction parameter determined using the RPA theory for q > R/sub g/-1 is greater than that calculated from the zero-angle intensity based on an Ornstein-Zernike plot, where Chi s represents the limit of single phase stability. These findings indicate a correlation between the critical fluctuation length ? and R/sub g/ which is not accounted for by the RPA theory

1986-12-01

162

Validation of the RTOG recursive partitioning analysis (RPA) classification for small-cell lung cancer-only brain metastases  

International Nuclear Information System (INIS)

Purpose: Radiation Therapy Oncology Group (RTOG) developed a prognostic classification based on a recursive partitioning analysis (RPA) of patient pretreatment characteristics from three completed brain metastases randomized trials. Clinical trials for patients with brain metastases generally exclude small-cell lung cancer (SCLC) cases. We hypothesize that the RPA classes are valid in the setting of SCLC brain metastases. Methods and Materials: A retrospective review of 154 SCLC patients with brain metastases treated between April 1983 and May 2005 was performed. RPA criteria used for class assignment were Karnofsky performance status (KPS), primary tumor status (PT), presence of extracranial metastases (ED), and age. Results: Median survival was 4.9 months, with 4 patients (2.6%) alive at analysis. Median follow-up was 4.7 months (range, 0.3-40.3 months). Median age was 65 (range, 42-85 years). Median KPS was 70 (range, 40-100). Number of patients with controlled PT and no ED was 20 (13%) and with ED, 27 (18%); without controlled PT and ED, 34 (22%) and with ED, 73 (47%). RPA class distribution was: Class I: 8 (5%); Class II: 96 (62%); Class III: 51 (33%). Median survivals (in months) by RPA class were: Class I: 8.6; Class II: 4.2; Class III: 2.3 (p = 0.0023). Conclusions: Survivals for SCLC-only brain metastases replicate the results from the RTOG RPA classification. These classes are therefore valid for brain metastases from SCLC, support the inclusion of SCLC patients in future brain metastases trials, and may also serve as a basis for historical comparisons

2007-01-01

163

Study of the RPA pair-correlation function in GaAs-AlGaAs parabolic quantum well wires  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english The ground state intrasubband pair-correlation function for a quasi-one-dimensional electron gas confined in a GaAs-Al xGa1-xAs parabolic quantum well wire within the Random-Phase Approximation (RPA) is calculated. We have considered two wires with subband energies separation homega = 2:0 meV and ho [...] mega = 2:5 meV. The dependence of the pair-correlation function on the electronic density was studied and the regions where the RPA approach cannot be used were precisely determined.

J. B. B. da, Cunha; J. F. R. da, Cunha; P. C. M., Machado; F. A. P., Osório; A. N., Borges.

164

A mitotic recombination system for mouse chromosome 17  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Mitotic recombination between homologous chromosomes is a genetic technique for mosaic analysis in model organisms. The general application of this technique in the mouse depends on establishment of effective recombination systems for individual chromosomes and reliable and sensitive methods for detection of recombination events. Here, we established a Cre/LoxP-mediated recombination system in mice for mosaic analysis of full-length chromosome 17. Cre-mediated germ-line recombination between ...

Sun, Lei; Wu, Xiaohui; Han, Min; Xu, Tian; Zhuang, Yuan

2008-01-01

165

Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination.  

Science.gov (United States)

DNA double-strand breaks (DSBs) are highly cytotoxic lesions and pose a major threat to genome stability if not properly repaired. We and others have previously shown that a class of DSB-induced small RNAs (diRNAs) is produced from sequences around DSB sites. DiRNAs are associated with Argonaute (Ago) proteins and play an important role in DSB repair, though the mechanism through which they act remains unclear. Here, we report that the role of diRNAs in DSB repair is restricted to repair by homologous recombination (HR) and that it specifically relies on the effector protein Ago2 in mammalian cells. Interestingly, we show that Ago2 forms a complex with Rad51 and that the interaction is enhanced in cells treated with ionizing radiation. We demonstrate that Rad51 accumulation at DSB sites and HR repair depend on catalytic activity and small RNA-binding capability of Ago2. In contrast, DSB resection as well as RPA and Mre11 loading is unaffected by Ago2 or Dicer depletion, suggesting that Ago2 very likely functions directly in mediating Rad51 accumulation at DSBs. Taken together, our findings suggest that guided by diRNAs, Ago2 can promote Rad51 recruitment and/or retention at DSBs to facilitate repair by HR. PMID:24662483

Gao, Min; Wei, Wei; Li, Ming-Ming; Wu, Yong-Sheng; Ba, Zhaoqing; Jin, Kang-Xuan; Li, Miao-Miao; Liao, You-Qi; Adhikari, Samir; Chong, Zechen; Zhang, Ting; Guo, Cai-Xia; Tang, Tie-Shan; Zhu, Bing-Tao; Xu, Xing-Zhi; Mailand, Niels; Yang, Yun-Gui; Qi, Yijun; Rendtlew Danielsen, Jannie M

2014-05-01

166

Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination  

DEFF Research Database (Denmark)

DNA double-strand breaks (DSBs) are highly cytotoxic lesions and pose a major threat to genome stability if not properly repaired. We and others have previously shown that a class of DSB-induced small RNAs (diRNAs) is produced from sequences around DSB sites. DiRNAs are associated with Argonaute (Ago) proteins and play an important role in DSB repair, though the mechanism through which they act remains unclear. Here, we report that the role of diRNAs in DSB repair is restricted to repair by homologous recombination (HR) and that it specifically relies on the effector protein Ago2 in mammalian cells. Interestingly, we show that Ago2 forms a complex with Rad51 and that the interaction is enhanced in cells treated with ionizing radiation. We demonstrate that Rad51 accumulation at DSB sites and HR repair depend on catalytic activity and small RNA-binding capability of Ago2. In contrast, DSB resection as well as RPA and Mre11 loading is unaffected by Ago2 or Dicer depletion, suggesting that Ago2 very likely functions directly in mediating Rad51 accumulation at DSBs. Taken together, our findings suggest that guided by diRNAs, Ago2 can promote Rad51 recruitment and/or retention at DSBs to facilitate repair by HR.

Gao, Min; Wei, Wei

2014-01-01

167

Phase 1 study of a recombinant mutant protective antigen of Bacillus anthracis.  

Science.gov (United States)

A phase 1 study of a recombinant mutant protective antigen (rPA) vaccine was conducted in 186 healthy adults aged 18 to 45 years. Volunteers were randomized to receive one of three formulations of rPA (formalin treated, alum adsorbed, or both), in 10- or 20-?g dosages each, or the licensed vaccine, AVA. Three injections were given at 2-month intervals and a 4th 1 year after the 3rd. Vaccinees were examined at the clinic once following each injection, at 48 to 72 h postinjection. Adverse reactions were recorded in diaries for 7 days. Sera were collected before each injection and 1 week after the 1st, 2 weeks after the 3rd and 4th, and 1 year after the 4th. Serum anti-PA IgG was assayed by enzyme-linked immunosorbent assay (ELISA) and toxin neutralization assay (TNA). All formulations at both dosages were safe and immunogenic, inducing booster responses, with the highest antibody levels following the 4th injection (354 to 732 ?g/ml). The lowest levels were induced by the formalin-only-treated rPA; there was no statistical difference between levels induced by alum-adsorbed and formalin-treated/alum-adsorbed rPA or by the two dosages. The antibody levels declined in all groups during the 1-year intervals after the 3rd and 4th injections but less so during the 2nd year, after the 4th injection (fold decreases were 10 to 25 versus 3.4 to 7.0, P < 0.001). There were too few AVA recipients for statistical comparisons, but their antibody levels followed those of rPA. Anti-rPA measured by ELISA correlated with TNA titers (r = 0.97). These data support studying alum-adsorbed rPA in children. PMID:22190398

Bellanti, Joseph A; Lin, Feng-Ying C; Chu, Chiayung; Shiloach, Joseph; Leppla, Stephen H; Benavides, German A; Karpas, Arthur; Moayeri, Mahtab; Guo, Chunyan; Robbins, John B; Schneerson, Rachel

2012-02-01

168

Nuclear vorticity in isoscalar E1 modes: Skyrme-RPA analysis  

CERN Document Server

Two basic concepts of nuclear vorticity, hydrodynamical (HD) and Rawenthall-Wambach (RW), are critically inspected. As a test case, we consider the interplay of irrotational and vortical motion in isoscalar electric dipole E1(T=0) modes in $^{208}$Pb, namely the toroidal and compression modes. The modes are described in a self-consistent random-phase-approximation (RPA) with the Skyrme force SLy6. They are examined in terms of strength functions, transition densities, current fields, and formfactors. It is shown that the RW conception (suggesting the upper component of the nuclear current as the vorticity indicator) is not robust. The HD vorticity is not easily applicable either because the definition of a velocity field is too involved in nuclear systems. Instead, the vorticity is better characterized by the toroidal strength which closely corresponds to HD treatment and is approximately decoupled from the continuity equation.

Reinhard, P -G; Repko, A; Kvasil, J

2013-01-01

169

Effect of isospin mixing on superallowed Fermi beta decay in self-consistent relativistic RPA approaches  

CERN Document Server

Self-consistent Random Phase Approximation (RPA) approaches in the relativistic framework are applied to calculate the isospin symmetry-breaking corrections $\\delta_c$ for the $0^+\\to0^+$ superallowed transitions. It is found that the corrections $\\delta_c$ are sensitive to the proper treatments of the Coulomb mean field, but not so much to specific effective interactions. With these corrections $\\delta_c$, the nucleus-independent $\\mathcal{F}t$ values are obtained in combination the experimental $ft$ values in the most recent survey and the improved radiative corrections. It is found that the constancy of the $\\mathcal{F}t$ values is satisfied for all effective interactions employed. Furthermore, the element $V_{ud}$ and unitarity of the Cabibbo-Kobayashi-Maskawa matrix are discussed.

Liang, Haozhao; Meng, Jie

2009-01-01

170

Disruption of the p53-mediated G{sub 1}/S cell cycle checkpoint results in elevated rates of spontaneous genetic recombination in human fibroblasts  

Energy Technology Data Exchange (ETDEWEB)

A key feature of the cancer-prone inherited disease ataxia-telangiectasia (A-T) is genetic instability. We recently demonstrated that one aspect of genetic instability in A-T is a marked elevation in the spontaneous rates of intrachromosomal mitotic recombination. We have proposed a model for A-T that attributes these high recombination rates to a lack of DNA damage-sensitive cell cycle checkpoints. One prediction of this model is that disrupting p53 function in normal cells should increase their spontaneous rates of recombination by interfering with their p53-dependent G{sub 1}/S cell cycle checkpoint. To test this prediction, we transfected control and A-T fibroblast lines that each harbor a single integrated copy of lacZ-based recombination vector (pLrec) with derivatives of a eukaryotic expression vector (pRep5) that contain either a dominant-negative p53 mutant (143{sup val{yields}ala}) or a human papilloma virus E6 gene (HPV18 E6). Expression of either of these genes results in loss of p53 function and abolition of the G{sub 1}/S cell cycle checkpoint. Four independent p53{sup 143ala} transformants of the control line showed 25-80 fold elevations in spontaneous recombination rates when compared to their parent cell line. Elevations in spontaneous recombination rates were also detected following transfection with the HPV18 E6 gene. In contrast, four independent p53{sup 143ala} transformants of the A-T cell line showed no significant changes in their already high spontaneous recombination rates. We are now extending these observations to additional normal human fibroblast lines and carrying out molecular analyses of the products of these recombinational events. Our results support our hypothesis that the lack of a p53-dependent G{sub 1}/S cell cycle checkpoint contributes to the hyperrecombination seen in A-T.

Strasfeld, L.; Brainerd, E.; Meyn, M.S. [Yale Univ. School of Medicine, New Haven, CT (United States)

1994-09-01

171

Mutant Telomeric Repeats in Yeast Can Disrupt the Negative Regulation of Recombination-Mediated Telomere Maintenance and Create an Alternative Lengthening of Telomeres-Like Phenotype?  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Some human cancers maintain telomeres using alternative lengthening of telomeres (ALT), a process thought to be due to recombination. In Kluyveromyces lactis mutants lacking telomerase, recombinational telomere elongation (RTE) is induced at short telomeres but is suppressed once telomeres are moderately elongated by RTE. Recent work has shown that certain telomere capping defects can trigger a different type of RTE that results in much more extensive telomere elongation that is reminiscent o...

Bechard, Laura H.; Butuner, Bilge D.; Peterson, George J.; Mcrae, Will; Topcu, Zeki; Mceachern, Michael J.

2009-01-01

172

Pairing and recombination features during meiosis in Cebus paraguayanus (Primates: Platyrrhini)  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Abstract Background Among neotropical Primates, the Cai monkey Cebus paraguayanus (CPA) presents long, conserved chromosome syntenies with the human karyotype (HSA) as well as numerous C+ blocks in different chromosome pairs. In this study, immunofluorescence (IF) against two proteins of the Synaptonemal Complex (SC), namely REC8 and SYCP1, two recombination protein markers (RPA and MLH1), and one protein involved in the pachytene checkpoint machinery (BRCA1) ...

Garcia-Cruz Raquel; Robles Pedro; Steinberg Eliana R; Camats Nuria; Brieño Miguel A; Garcia-Caldés Montserrat; Mudry Marta D

2009-01-01

173

Mph1p promotes gross chromosomal rearrangement through partial inhibition of homologous recombination.  

Science.gov (United States)

Gross chromosomal rearrangement (GCR) is a type of genomic instability associated with many cancers. In yeast, multiple pathways cooperate to suppress GCR. In a screen for genes that promote GCR, we identified MPH1, which encodes a 3'-5' DNA helicase. Overexpression of Mph1p in yeast results in decreased efficiency of homologous recombination (HR) as well as delayed Rad51p recruitment to double-strand breaks (DSBs), which suggests that Mph1p promotes GCR by partially suppressing HR. A function for Mph1p in suppression of HR is further supported by the observation that deletion of both mph1 and srs2 synergistically sensitize cells to methyl methanesulfonate-induced DNA damage. The GCR-promoting activity of Mph1p appears to depend on its interaction with replication protein A (RPA). Consistent with this observation, excess Mph1p stabilizes RPA at DSBs. Furthermore, spontaneous RPA foci at DSBs are destabilized by the mph1Delta mutation. Therefore, Mph1p promotes GCR formation by partially suppressing HR, likely through its interaction with RPA. PMID:18591428

Banerjee, Soma; Smith, Stephanie; Oum, Ji-Hyun; Liaw, Hung-Jiun; Hwang, Ji-Young; Sikdar, Nilabja; Motegi, Akira; Lee, Sang Eun; Myung, Kyungjae

2008-06-30

174

The RecF pathway of homologous recombination can mediate the initiation of DNA damage-inducible replication of the Escherichia coli chromosome.  

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DNA damage-inducible DNA replication in SOS-induced Escherichia coli cells, termed inducible stable DNA replication (iSDR), has previously been shown to require either the RecBCD or the RecE pathway of homologous recombination for initiation. Here, we demonstrate that recB recC sbcC quadruple mutant cells are capable of iSDR induction and that a mutation in the recJ gene abolishes the inducibility. These results indicate that the RecF pathway of homologous recombination can also catalyze iSDR...

Asai, T.; Kogoma, T.

1994-01-01

175

Ice recrystallization inhibition mediated by a nuclear-expressed and -secreted recombinant ice-binding protein in the microalga Chlamydomonas reinhardtii.  

Science.gov (United States)

A Lolium perenne ice-binding protein (LpIBP) demonstrates superior ice recrystallization inhibition (IRI) activity and has proposed applications in cryopreservation, food texturing, as well as in being a "green" gas hydrate inhibitor. Recombinant production of LpIBP has been previously conducted in bacterial and yeast systems for studies of protein characterization, but large-scale applications have been hitherto limited due to high production costs. In this work, a codon-optimized LpIBP was recombinantly expressed and secreted in a novel one-step vector system from the nuclear genome of the green microalga Chlamydomonas reinhardtii. Both mixotrophic and photoautotrophic growth regimes supported LpIBP expression, indicating the feasibility of low-cost production using minimal medium, carbon dioxide, and light energy as input. In addition, multiple growth and bioproduct extraction cycles were performed by repetitive batch cultivation trials, demonstrating the potential for semi-continuous production and biomass harvesting. Concentrations of recombinant protein reached in this proof of concept approach were sufficient to demonstrate IRI activity in culture media without additional purification or concentration, with activity further verified by thermal hysteresis and morphology assays. The incorporation of the recombinant LpIBP into a model gas hydrate offers the promise that algal production may eventually find application as a "green" hydrate inhibitor. PMID:24037309

Lauersen, Kyle J; Vanderveer, Tara L; Berger, Hanna; Kaluza, Isabell; Mussgnug, Jan H; Walker, Virginia K; Kruse, Olaf

2013-11-01

176

Effective on-site Coulomb interaction in transition metals from constrained RPA  

Energy Technology Data Exchange (ETDEWEB)

Effective on-site Coulomb interaction (Hubbard U) between localized d electrons in 3d, 4d, and 5d transition metals are calculated employing the recently developed constrained random-phase approximation (cRPA) within the full-potential linearized augmented plane-wave (FLAPW) method using Wannier functions. The obtained Hubbard U parameters lie between 1 and 5 eV and show a non-monotonic behavior across the transition-metal series. We find that the U depends on the crystal structure, spin polarization, d-electron number and filling of the d orbitals rather than d-character of the elements. For most of the isovalent transition metals, U assumes similar values. The obtained U parameters for the 3d series are in good agreement with previous studies as well as available experimental data. Using calculated U parameters we discuss the strength of the electronic correlations and instability of the paramagnetic state towards the ferromagnetic one for 3d transition metals.

Sasioglu, Ersoy; Friedrich, Christoph; Bluegel, Stefan [Peter Gruenberg Institut and Institute for Advanced Simulation, Forschungszentrum Juelich and JARA, 52425 Juelich (Germany)

2011-07-01

177

RPA-CPA theory for magnetism in disordered Heisenberg binary systems with long range exchange integrals  

CERN Multimedia

We present a theory based on Green's function formalism to study magnetism in disordered Heisenberg systems with long range exchange integrals. Disordered Green's function are decoupled within Tyablicov scheme and solved with a CPA method. The CPA method is the extension of Blackmann-Esterling-Beck approach to system with environmental disorder term which uses cumulant summation of the single-site non crossing diagrams. The crucial point is that we are able to treat simultaneously and self-consistently the RPA and CPA loops. It is shown that the summation of s-scattering contribution can always be performed analytically. While the p,d,f .. contributions are difficult to handle in the case of long-range coupling. To overcome this difficulty we propose and provide a test of a simplified treatment of these terms. In the case of 3D disordered nearest-neighbor Heisenberg system, a good agreement between the simplified treatment and the full calculation is achieved. Our theory allows in particular to calculate the ...

Bouzerar, G

2002-01-01

178

Self-Consistent Separable Rpa for Skyrme Forces: Giant Resonances in Axial Nuclei  

CERN Document Server

We formulate the self-consistent separable random-phase-approximation (SRPA) method and specify it for Skyrme forces with pairing for the case of axially symmetric deformed nuclei. The factorization of the residual interaction allows to avoid diagonalization of high-rank RPA matrices, which dramatically reduces the computational expense. This advantage is crucial for the systems with a huge configuration space, first of all for deformed nuclei. SRPA takes self-consistently into account the contributions of both time-even and time-odd Skyrme terms as well as of the Coulomb force and pairing. The method is implemented to description of isovector E1 and isoscalar E2 giant resonances in a representative set of deformed nuclei: $^{154}$Sm, $^{238}$U, and $^{254}$No. Four different Skyrme parameterizations (SkT6, SkM*, SLy6, and SkI3) are employed to explore dependence of the strength distributions on some basic characteristics of the Skyrme functional and nuclear matter. In particular, we discuss the role of isosc...

Nesterenko, V O; Kleinig, W; Kvasil, J; Reinhard, P G; Vesely, P

2006-01-01

179

Skyrme-Rpa Description of Dipole Giant Resonance in Heavy and Superheavy Nuclei  

CERN Document Server

The E1(T=1) isovector dipole giant resonance (GDR) in heavy and super-heavy deformed nuclei is analyzed over a sample of 18 rare-earth nuclei, 4 actinides and three chains of super-heavy elements (Z=102, 114 and 120). Basis of the description is self-consistent separable RPA (SRPA) using the Skyrme force SLy6. The self-consistent model well reproduces the experimental data (energies and widths) in the rare-earth and actinide region. The trend of the resonance peak energies follows the estimates from collective models, showing a bias to the volume mode for the rare-earths isotopes and a mix of volume and surface modes for actinides and super-heavy elements. The widths of the GDR are mainly determined by the Landau fragmentation which in turn is found to be strongly influenced by deformation. A deformation splitting of the GDR can contribute about one third to the width and about 1 MeV further broadening can be associated to mechanism beyond the mean-field description (escape, coupling with complex configuratio...

Kleinig, W; Kvasil, J; Reinhard, P -G; Vesely, P

2008-01-01

180

Local density and the RPA corrections in charge current quasielastic neutrino on Oxygen, Argon and Iron scattering  

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Numerical computations of cross sections for quasielastic charge current scattering of neutrino on Oxygen, Argon and Iron in Local Density Approximation (LDA) are presented. We consider processes for a few GeV neutrino energy. We include corrections from nucleon re-interaction in nucleus described by relativistic Random Phase Approximation (RPA). We adopt the relativistic Fermi gas model of nucleus with and without taking into account the effective mass of nucleons.

Graczyk, Krzysztof M.

2004-01-01

 
 
 
 
181

Dividing patients with brain metastases into classes derived from the RTOG recursive partitioning analysis (RPA) with emphasis on prognostic poorer patient groups  

International Nuclear Information System (INIS)

Background. The aim of our study was to investigate whether selecting the patients with brain metastases by classifying them into three classes according to the results of the recursive partitioning analysis (RPA) of the Radiation Therapy Oncology Group (RTOG) is useful or not for further decision concerning altered treatment schedules in patients. Patients and methods. The investigated group included 57 male and 48 female patients having received whole brain radiotherapy in a total dose of 30 Gy / 3 Gy daily / 5 days a week. Patients who had surgical excision of brain metastases or had radiosurgical intervention were excluded. All patients were stratified according to the findings of RPA (Class I: Karnofsky Performance Status (KPS) =70, age 65) had an impact on survival according to multivariate analysis. Conclusions. Selecting the patients by dividing them into the three RPA classes seems to be useful. Considering the short survival time in RPA Class III, those patients might be well treated with a shorter treatment course. (author)

2001-01-01

182

Photon - A Fortran programme for the calculation of photoreaction cross-sections and polarizations in RPA theory with a Skyrme interaction  

International Nuclear Information System (INIS)

Description is given for the Photon programme written for the Ibm 370/168 computer in Fortran 4. language. The programme calculates the photoreaction cross-sections, polarization and asymmetries for closed shell nuclei in RPA theory

1984-01-01

183

Detection of a Rickettsia Closely Related to Rickettsia aeschlimannii, “Rickettsia heilongjiangensis,” Rickettsia sp. Strain RpA4, and Ehrlichia muris in Ticks Collected in Russia and Kazakhstan  

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Using PCR, we screened 411 ticks from four genera collected in Russia and Kazakhstan for the presence of rickettsiae and ehrlichiae. In Russia, we detected “Rickettsia heilongjiangensis,” Rickettsia sp. strain RpA4, and Ehrlichia muris. In Kazakhstan, we detected Rickettsia sp. strain RpA4 and a rickettsia closely related to Rickettsia aeschlimannii. These agents should be considered in a differential diagnosis of tick-borne infections in these areas.

Shpynov, Stanislav; Fournier, Pierre-edouard; Rudakov, Nikolay; Tankibaev, Marat; Tarasevich, Irina; Raoult, Didier

2004-01-01

184

Functional redundancy between repair factor XLF and damage response mediator 53BP1 in V(D)J recombination and DNA repair  

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The classical nonhomologous DNA end-joining (C-NHEJ) double-strand break (DSB) repair pathway in mammalian cells maintains genome stability and is required for V(D)J recombination and lymphocyte development. Mutations in the XLF C-NHEJ factor or ataxia telangiectasia-mutated (ATM) DSB response protein cause radiosensitivity and immunodeficiency in humans. Although potential roles for XLF in C-NHEJ are unknown, ATM activates a general DSB response by phosphorylating substrates, including histo...

Oksenych, Valentyn; Alt, Frederick W.; Kumar, Vipul; Schwer, Bjoern; Wesemann, Duane R.; Hansen, Erica; Patel, Harin; Su, Arthur; Guo, Chunguang

2012-01-01

185

Class switch recombination and somatic hypermutation in early mouse B cells are mediated by B cell- and Toll-like receptors.  

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Activation-induced cytidine deaminase (AID) is required for immunoglobulin (Ig) gene class switch recombination (CSR), somatic hypermutation (SHM) and somatic hyperconversion. In general high levels of AID expression are found in mature B cells responding to antigens. However, AID expression and SHM have also been detected in developing B cells from transgenic mice that have a limited Ig repertoire. Here we demonstrate that AID expression and active CSR/SHM occur in developing B cells from wi...

2007-01-01

186

Enhanced T cell-mediated protection against malaria in human challenges by using the recombinant poxviruses FP9 and modified vaccinia virus Ankara  

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Malaria is a major global health problem for which an effective vaccine is required urgently. Prime-boost vaccination regimes involving plasmid DNA and recombinant modified vaccinia virus Ankara-encoding liver-stage malaria antigens have been shown to be powerfully immunogenic for T cells and capable of inducing partial protection against experimental malaria challenge in humans, manifested as a delay in time to patent parasitemia. Here, we report that substitution of plasmid DNA as the primi...

Webster, Daniel P.; Dunachie, Susanna; Vuola, Jenni M.; Berthoud, Tamara; Keating, Sheila; Laidlaw, Stephen M.; Mcconkey, Samuel J.; Poulton, Ian; Andrews, Laura; Andersen, Rikke F.; Bejon, Philip; Butcher, Geoff; Sinden, Robert; Skinner, Michael A.; Gilbert, Sarah C.

2005-01-01

187

I-SceI-Mediated Double-Strand Break Does Not Increase the Frequency of Homologous Recombination at the Dct Locus in Mouse Embryonic Stem Cells  

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Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embed...

Fenina, Myriam; Simon-chazottes, Dominique; Vandormael-pournin, Sandrine; Soueid, Jihane; Langa, Francina; Cohen-tannoudji, Michel; Bernard, Bruno A.; Panthier, Jean-jacques

2012-01-01

188

Humoral and cell-mediated immune-responses after administration of a single-shot recombinant hepatitis B surface antigen vaccine formulated with cationic poly(l-lactide) microspheres.  

Science.gov (United States)

The present investigations were aimed to compare the humoral and cell-mediated immune responses between recombinant hepatitis B surface antigens (HBsAg) adsorbed L-PLA microspheres (Ms) vaccine (single-shot) and marketed alum-HBsAg vaccine (two-doses). The blank cationic (cetyltrimethyammoniumbromide) microspheres were prepared by the double emulsion (w/o/w) solvent evaporation technique. The HBsAg was adsorbed onto the surface of blank cationic microspheres. These microspheres were characterized in vitro for their size, shape, adsorption-efficiency, in-process stability, and HBsAg release studies. Specific humoral immune responses (IgM and IgG) and cell-mediated immune responses (cellular-proliferation) assay including release of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and nitric oxide (NO) from host's cells stimulated with HBsAg or lipopolysaccharide (LPS)/ concanavalin A (con A) in-vitro were determined. Based on these findings, it was concluded that the single injection (using subcutaneous-route) of the polymeric microspheres produced better immune response (both humoral and cell-mediated) than two injections of a conventional alum-HBsAg vaccine. These data demonstrate high potential of polymeric microspheres for their use as a carrier adjuvant for hepatitis B vaccine. PMID:19883203

Saini, Vinay; Jain, Vikas; Sudheesh, M S; Dixit, Saurabh; Gaur, R L; Sahoo, M K; Joseph, S K; Verma, S K; Jaganathan, K S; Murthy, P K; Kohli, Dharmveer

2010-04-01

189

Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria  

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Novel recombinant adeno-associated virus vectors pseudo-typed with serotype 8 capsid (rAAV2/8) have recently shown exciting promise as effective liver-directed gene transfer reagents. We have produced a novel liver-specific rAAV2/8 vector expressing the mouse phenylalanine hydroxylase (Pah) cDNA and have administered this vector to hyperphenylalaninemic PAH-deficient Pahenu2 mice, a model of human phenylketonuria (PKU). Our hypothesis was that this vector would produce sufficient hepatocyte t...

Harding, Co; Gillingham, Mb; Hamman, K.; Clark, H.; Goebel-daghighi, E.; Bird, A.; Koeberl, Dd

2006-01-01

190

Anti-Tumor Therapy Mediated by 5-Fluorocytosine and a Recombinant Fusion Protein Containing TSG-6 Hyaluronan Binding Domain and Yeast Cytosine Deaminase  

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Matrix Attachment Therapy (MAT) is an enzyme prodrug strategy that targets hyaluronan in the tumor extracellular matrix to deliver a prodrug converting enzyme near the tumor cells. A recombinant fusion protein containing the hyaluronan binding domain of TSG-6 (Link) and yeast cytosine deaminase (CD) with an N-terminal His(×6) tag was constructed to test MAT on the C26 colon adenocarcinoma in Balb/c mice that were given 5-fluorocytosine (5-FC) in the drinking water. LinkCD was expressed in E....

Park, Joshua I.; Cao, Limin; Platt, Virginia M.; Huang, Zhaohua; Stull, Robert A.; Dy, Edward E.; Sperinde, Jeffrey J.; Yokoyama, Jennifer S.; Szoka, Francis C.

2009-01-01

191

Recombinant Human Erythropoietin Antagonizes Trastuzumab Treatment of Breast Cancer Cells via Jak2-Mediated Activation of Src and Inactivation of PTEN  

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We found that the receptor for erythropoietin (EpoR) is coexpressed with human epidermal growth factor receptor-2 (HER2) in a significant percentage of human breast tumor specimens and breast cancer cell lines. Exposure of HER2 and EpoR dual-positive breast cancer cells to recombinant human erythropoietin (rHuEPO) activated cell signaling. Concurrent treatment of the cells with rHuEPO and trastuzumab reduced the cells’ response to trastuzumab both in vitro and in vivo. We identified Jak2-me...

Liang, Ke; Esteva, Francisco J.; Albarracin, Constance; Stemke-hale, Katherine; Lu, Yang; Bianchini, Giampaolo; Yang, Ching-yi; Li, Yong; Li, Xinqun; Chen, Chun-te; Mills, Gordon B.; Hortobagyi, Gabriel N.; Mendelsohn, John; Hung, Mien-chie; Fan, Zhen

2010-01-01

192

5' untranslated sequences modulate rapid mRNA degradation mediated by 3' AU-rich element in v-/c-fos recombinants.  

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One major determinant of rapid mRNA decay is the presence of AU-rich sequences located in 3' untranslated regions (UTR). To assess for the contribution of upstream sequences on the activity of the 3' AU-rich destabilizing element, we have determined the decay-rates of v-/c-fos hybrid transcripts by quantitative RNA protection analysis. In a transient expression assay, v-/c-fos recombinants generated two mRNA populations via alternative splicing and removal of an optional intron entirely locat...

Roy, N.; Laflamme, G.; Raymond, V.

1992-01-01

193

Orientation of cohesive end site cos determines the active orientation of chi sequence in stimulating recA . recBC-mediated recombination in phage lambda lytic infections.  

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Sequence chi, 5'G-C-T-G-G-T-G-G, locally enhances homologous recombination by recA and recBC proteins of Escherichia coli. Previous work showed that, in phage lambda, chi is more active in one orientation (leftward) than in the other (rightward). Inverting cos, the sequence for the mature DNA ends of lambda, reverses this orientation dependence: the rightward chi becomes fully active, and the leftward chi becomes relatively inactive. We surmise that chi action in phage lambda is coupled with ...

Kobayashi, I.; Murialdo, H.; Crasemann, J. M.; Stahl, M. M.; Stahl, F. W.

1982-01-01

194

Recombinant adenovirus expressing F and H fusion proteins of peste des petits ruminants virus induces both humoral and cell-mediated immune responses in goats.  

Science.gov (United States)

Peste des petits ruminants (PPR) is an acute and contagious disease of some small ruminants caused by peste des petits ruminants virus (PPRV). Fusion (F) protein and hemagglutinin (H) protein are two glycoproteins of PPRV that might induce a protective immune response. In this study, three replication-defective recombinant adenoviruses were constructed and the immunogenicity was evaluated in goats (the natural host). The recombinant adenoviruses (rAds) expressing F, H, and F-H fusion protein were named rAd-F, rAd-H, and rAd-F-H, respectively. In vitro, the proteins expressed in AAV-293 cells infected with different rAds were identified by Western blotting and immunofluorescence. The results showed that the proteins could be expressed in vitro. Three groups of goats (6 goats per group) were inoculated subcutaneously twice at 3-week intervals with the rAds. As negative controls, two additional groups were inoculated with wild-type adenovirus (wtAd) or PBS. In vivo, goats immunized with the rAds developed PPRV-specific virus neutralizing antibody (VNA) by 3 weeks after primary immunization. Moreover, the seroconversions were maintained for approximately 21 weeks after primary immunization. Stronger lymphocyte proliferation responses were induced in goats immunized with the three rAds than in the negative controls (PDIVA) vaccines for preventing PPRV infection. Notably, the rAd-F-H expressing F-H fusion protein is likely the most potent candidate of the rAds. PMID:23707075

Wang, Yong; Liu, Guangqing; Chen, Zongyan; Li, Chuanfeng; Shi, Lijun; Li, Wenchao; Huang, Huaxin; Tao, Chunai; Cheng, Chaofei; Xu, Binrui; Li, Gang

2013-07-15

195

Recombinant translation initiation factor-1 of Wolbachia is an immunogenic excretory secretory protein that elicits Th2 mediated immune protection against Brugia malayi.  

Science.gov (United States)

Wolbachia, the intracellular alpha-proteobacteria are required for the development, fertility and survival of filarial parasites. Wolbachia Translation initiation factor-1 (Wol Tl IF-1) is one of the factors required for Wolbachia growth and viability. In the present study, we cloned, over expressed and purified Wol Tl IF-1 that exhibited strong immuno-reactivity with various categories of bancroftian sera. Immunization with the recombinant protein resulted into significant reduction in microfilarial density (70-72%) and adult worm establishment (61-63%) in susceptible Mastomys coucha. Protection offered by Wol Tl IF-1 was found associated with humoral immune arm as observed by an increased antibody level with preponderance of IgE, IgM, IgG1 and IgG2a isotypes. The anti-Wol Tl IF-1 antibodies promoted profound adherence of peritoneal exudates cells to the surface of microfilariae and infective larvae causing cytotoxicity and their death. The present study indicates potential of recombinant Wol Tl IF-1 as a promising vaccine candidate against human lymphatic filarial infection. PMID:23079772

Nag, Jeetendra Kumar; Shrivastava, Nidhi; Gupta, Jyoti; Misra-Bhattacharya, Shailja

2013-01-01

196

Circulating Anti-Wild-Type Adeno-Associated Virus Type 2 (AAV2) Antibodies Inhibit Recombinant AAV2 (rAAV2)-Mediated, but Not rAAV5-Mediated, Gene Transfer in the Brain  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Epidemiological studies report that 80% of the population maintains antibodies (Ab) to wild-type (wt) adeno-associated virus type 2 (AAV2), with 30% expressing neutralizing Ab (NAb). The blood-brain barrier (BBB) provides limited immune privilege to brain parenchyma, and the immune response to recombinant AAV (rAAV) administration in the brain of a naive animal is minimal. However, central nervous system transduction in preimmunized animals remains unstudied. Vector administration may disrupt...

Peden, Carmen S.; Burger, Corinna; Muzyczka, Nicholas; Mandel, Ronald J.

2004-01-01

197

Recombining WMAP: Beyond standard recombination  

CERN Multimedia

We place new constraints on sources of ionizing and resonance radiation at the epoch of the recombination process using the recent CMB temperature and polarization spectra coming from WMAP. We find that non-standard recombination scenarios are still consistent with the current data. In light of this we study the impact that such models can have on the determination of several cosmological parameters. In particular, the constraints on curvature and baryon density appear to be weakly affected by a modified recombination scheme. However, it may affect the current WMAP constraints on inflationary parameters like the spectral index and its running. Physically motivated models, like those based on primordial black hole or super heavy dark matter decay, are able to provide a good fit to the current data. Future observations in both temperature and polarization will be needed to more stringently test these models.

Bean, R; Silk, J; Bean, Rachel; Melchiorri, Alessandro; Silk, Joe

2003-01-01

198

VDJ recombination.  

Science.gov (United States)

The ability of lymphocyte receptor V, D and J gene segments to rearrange generates much of the receptor diversity that is the hallmark of the immune system. Naturally, the mechanisms of immunoglobulin and T-cell receptor gene recombination are of enormous interest. Here, Fred Alt and colleagues review current understanding of the process and speculate on future findings. PMID:1510813

Alt, F W; Oltz, E M; Young, F; Gorman, J; Taccioli, G; Chen, J

1992-08-01

199

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)

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 insulin unresponsiveness in human SHSY-5Y and C3A cells in culture due to the activation of central protein kinases of the insulin signalling pathway. This phenomenon can be avoided when studying insulin signalling by using recombinant baculovirus as a heterologous viral expression system. In addition, our data may contribute to an understanding of the molecular mechanisms underlying baculovirus infection of human cells.

Selander Martin

2007-02-01

200

Recursive partitioning analysis (RPA) class does not predict survival in patients with four or more brain metastases  

International Nuclear Information System (INIS)

Background: We evaluated prognostic factors for survival in patients with four or more brain metastases in order to determine whether intense local treatment might be justified for some of them. If up to three brain metastases are present, surgical resection or radiosurgery are currently being considered in case of favorable prognostic factors. Patients and Methods: Retrospective intention-to-treat analysis of 113 patients who underwent whole-brain radiotherapy without surgical resection or radiosurgery at a single institution. Standard treatment was given with ten fractions of 3 Gy. Higher total doses were administered in 13% of patients. Recursive partitioning analysis (RPA) prognostic classes have been described by the radiation therapy oncology group (RTOG) in 1997 (class I: Karnofsky performance status [KPS] ? 70%, age ? 65 years, no extracranial metastases, controlled primary tumor; class III: KPS 50 years, p = 0.05). Strong trends were found for KPS, extracranial metastases, control of the primary tumor, and breast primary tumor. Number of brain metastases, RPA class, and treatment-related factors such as total dose or remission of brain metastases had no appreciable influence on survival (Figure 1). Multivariate analysis failed to identify any significant prognostic factor. Conclusions: Patients with four or more brain metastases seem to represent a group with unfavorable prognosis where remission of brain metastases or administration of more than 30 Gy were not associated with increased survival. The number of patients in RPA class I was too small to draw final conclusions. However, there was absolutely no survival difference between patients in class II (median survival 3.6 months) and III (median 4.2 months). (orig.)

2003-01-01

 
 
 
 
201

Approximated calculation of the glueball mass in the 2 + 1-D SU(2) LGT with the RPA method  

International Nuclear Information System (INIS)

The coupled cluster method is improved with the random phase approximation (RPA) to calculate the glueball mass in the 2 + 1-D SU(2) lattice gauge theory. In this calculating, the trial wave function consists of single-hollow graphs, the Feymann-Hellman theorem is used to obtain a set of closed equations. When the calculation proceeds up to the sixth order, the calculated results of glueball mass show scaling window and a sign of converges at weak coupling region 1/g2>1.0. The results of this paper indicate that the seventh order glueball mass will show good scaling behavior and the converges

2004-06-01

202

Protective Effect of Recombinant Adeno-Associated Virus 2/8-Mediated Gene Therapy from the Maternal Hyperphenylalaninemia in Offsprings of a Mouse Model of Phenylketonuria  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Phenylketonuria (PKU) is an autosomal recessively inherited metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH). The accumulation of phenylalanine leads to severe mental and psychomotor retardation, and the fetus of an uncontrolled pregnant female patient presents with maternal PKU syndrome. We have reported previously on the cognitive outcome of biochemical and phenotypic reversal of PKU in a mouse model, Pahenu2, by the AAV serotype 2-mediated gene delivery of a hum...

Jung, Sung-chul; Park, Joo-won; Oh, Hyun-jeong; Choi, Jin-ok; Seo, Kyung-in; Park, Eun-sook; Park, Hae-young

2008-01-01

203

The PSO4 protein complex associates with replication protein A (RPA) and modulates the activation of ataxia telangiectasia-mutated and Rad3-related (ATR).  

Science.gov (United States)

The PSO4 core complex is composed of PSO4/PRP19/SNEV, CDC5L, PLRG1, and BCAS2/SPF27. Besides its well defined functions in pre-mRNA splicing, the PSO4 complex has been shown recently to participate in the DNA damage response. However, the specific role for the PSO4 complex in the DNA damage response pathways is still not clear. Here we show that both the BCAS2 and PSO4 subunits of the PSO4 complex directly interact and colocalize with replication protein A (RPA). Depletion of BCAS2 or PSO4 impairs the recruitment of ATR-interacting protein (ATRIP) to DNA damage sites and compromises CHK1 activation and RPA2 phosphorylation. Moreover, we demonstrate that both the RPA1-binding ability of BCAS2 and the E3 ligase activity of PSO4 are required for efficient accumulation of ATRIP at DNA damage sites and the subsequent CHK1 activation and RPA2 phosphorylation. Our results suggest that the PSO4 complex functionally interacts with RPA and plays an important role in the DNA damage response. PMID:24443570

Wan, Li; Huang, Jun

2014-03-01

204

The Structure of the yFACT Pob3-M Domain, Its Interaction with the DNA Replication Factor RPA, and a Potential Role in Nucleosome Deposition  

Energy Technology Data Exchange (ETDEWEB)

We report the crystal structure of the middle domain of the Pob3 subunit (Pob3-M) of S. cerevisiae FACT (yFACT, facilitates chromatin transcription), which unexpectedly adopts an unusual double pleckstrin homology (PH) architecture. A mutation within a conserved surface cluster in this domain causes a defect in DNA replication that is suppressed by mutation of replication protein A (RPA). The nucleosome reorganizer yFACT therefore interacts in a physiologically important way with the central single-strand DNA (ssDNA) binding factor RPA to promote a step in DNA replication. Purified yFACT and RPA display a weak direct physical interaction, although the genetic suppression is not explained by simple changes in affinity between the purified proteins. Further genetic analysis suggests that coordinated function by yFACT and RPA is important during nucleosome deposition. These results support the model that the FACT family has an essential role in constructing nucleosomes during DNA replication, and suggest that RPA contributes to this process.

VanDemark,A.; Blanksma, M.; Ferris, E.; Heroux, A.; Hill, C.; Formosa, T.

2006-01-01

205

Exotic Recombination  

Energy Technology Data Exchange (ETDEWEB)

I review few examples of the numerous physical processes that might change the standard model of recombination. The high precision of current and future CMB data may allow the detection of these processes, that leave recognizable imprints on the angular power spectra. I review some of the results obtained in constraining i) a variation of the gravitational constant G ii) the presence of extra-sources of energetic photons in the primeval plasma and iii) annihilation of dark matter particles.

Galli, Silvia [Physics Department, Universita di Roma ' La Sapienza' , Ple Aldo Moro 2, 00185, Rome (Italy); Laboratoire Astroparticule et Cosmologie (APC), Universite Paris Diderot - 75205 PARIS cedex 13 (Italy)

2009-10-15

206

Synthesis, characterization and immunological properties of LPS-based conjugate vaccine composed of O-polysaccharide from pseudomonas aeruginosa IATS 10 bound to recombinant exoprotein A  

International Nuclear Information System (INIS)

Pseudomonas aeruginosa is an improtant opportunistic pathogen that can cause infection in immunocompromised patient. Lipopolysaccharide (LPS), the major surface antigen of P. aeruginosa, is immunogenic and elieits protective antibodies in animals. The O-polysaccharids (O-PS) from international Antigenic typing Scheme (IATS) 10, the antigenic determinant of LPS, was coupled to recombinant exoprotein A (rPA) through adipic acid dihydrazide (ADH) mediated by carbodiimide condensation reaction. Mice were immunized with the conjugate emulsifield with monophosphoryl lipid A-trehalose dicorynomycolate (MPL-T) and freund's adjuvants. The conjiugate emulsified with MPL-T adjuvant elicited the highest level of IgG and IgM followed by freuns's adjuvant. IgG titers using both MPL-T and freund's adjuvants were recorded to be higher than IgM titers after the second post of the immunization. Immunization of mice with the prepared conjugates emulsified with MPL-T and freund's adjvaided provide high level of protection (100%) against ten times the LD50 of homologous strain of P. aeruginsoa. the elicited high IgG level and the in vivo protection test results provided good evidences for the possible protection of the conjugate aginst subsequent infection with the pathogen. These findings will enable us to use it as protective vaccine candidate (authors).

2008-01-01

207

BLM helicase measures DNA unwound before switching strands and hRPA promotes unwinding reinitiation  

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Bloom syndrome (BS) is a rare genetic disorder characterized by genomic instability and a high predisposition to cancer. The gene defective in BS, BLM, encodes a member of the RecQ family of 3'-5' DNA helicases, and is proposed to function in recombinational repair during DNA replication. Here, we have utilized single-molecule fluorescence resonance energy transfer microscopy to examine the behaviour of BLM on forked DNA substrates. Strikingly, BLM unwound individual DNA molecules in a repeti...

Yodh, J. G.; Stevens, B. C.; Kanagaraj, R.; Janscak, P.; Ha, T.

2009-01-01

208

BLM helicase measures DNA unwound before switching strands and hRPA promotes unwinding reinitiation  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Bloom syndrome (BS) is a rare genetic disorder characterized by genomic instability and a high predisposition to cancer. The gene defective in BS, BLM, encodes a member of the RecQ family of 3?–5? DNA helicases, and is proposed to function in recombinational repair during DNA replication. Here, we have utilized single-molecule fluorescence resonance energy transfer microscopy to examine the behaviour of BLM on forked DNA substrates. Strikingly, BLM unwound individual DNA molecules in a ...

Yodh, Jaya G.; Stevens, Benjamin C.; Kanagaraj, Radhakrishnan; Janscak, Pavel; Ha, Taekjip

2009-01-01

209

Gimeracil, an inhibitor of dihydropyrimidine dehydrogenase, inhibits the early step in homologous recombination  

International Nuclear Information System (INIS)

Gimeracil (5-chloro-2, 4-dihydroxypyridine) is an inhibitor of dihydropyrimidine dehydrogenase (DPYD), which degrades pyrimidine including 5-fluorouracil in the blood. Gimeracil was originally added to an oral fluoropyrimidine derivative S-1 to yield prolonged 5-fluorouracil concentrations in serum and tumor tissues. We have already reported that gimeracil had radiosensitizing effects by partially inhibiting homologous recombination (HR) in the repair of DNA double strand breaks. We investigated the mechanisms of gimeracil radiosensitization. Comet assay and radiation-induced focus formation of various kinds of proteins involved in HR was carried out. Small interfering RNA (siRNA) for DPYD were transfected to HeLa cells to investigate the target protein for radiosensitization with gimeracil. SCneo assay was carried out to examine whether DPYD depletion by siRNA inhibited HR repair of DNA double strand breaks. Tail moments in neutral comet assay increased in gimeracil-treated cells. Gimeracil restrained the formation of foci of Rad51 and replication protein A (RPA), whereas it increased the number of foci of Nbs1, Mre11, Rad50, and FancD2. When HeLa cells were transfected with the DPYD siRNA before irradiation, the cells became more radiosensitive. The degree of radiosensitization by transfection of DPYD siRNA was similar to that of gimeracil. Gimeracil did not sensitize DPYD-depleted cells. Depletion of DPYD by siRNA significantly reduced the frequency of neopositive clones in SCneo assay. Gimeracil partially inhibits the early step in HR. It was found that DPYD is the target protein for radiosensitization by gimeracil. The inhibitors of DPYD, such as gimeracil, could enhance the efficacy of radiotherapy through partial suppression of HR-mediated DNA repair. (author)

2011-09-01

210

Recombinant adeno-associated virus serotype 6 (rAAV2/6-mediated gene transfer to nociceptive neurons through different routes of delivery  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV. Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6 to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice. Results Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependant manner. More than 90% of transduced cells were small and medium sized neurons (2, predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (? 60% and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell types. Conclusion We have found that rAAV2/6 is an efficient vector to deliver transgenes to nociceptive neurons in mice. Furthermore, the characterization of the transduction profile may facilitate gene transfer studies to dissect mechanisms behind neuropathic pain.

Beggah Ahmed T

2009-09-01

211

A Preliminary and Comparative Evaluation of a Novel Ad5 [E1-, Eb2-] Recombinant Based Vaccine Used to Induce Cell Mediated Immune Responses  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Adenovirus vectors have been shown to be highly effective as vaccine platforms capable of inducing both humoral and cell mediated immune responses. An Ad serotype 5 vector containing unique deletions in the E2b region (Ad5 [E1-, E2b-]) has been reported to have several advantages over conventional Ad5 vectors deleted in only the E1 region (Ad5 [E1-]), including increased carrying capacity and diminished viral late gene expression. Here, we evaluated a novel Ad5 [E1-, E2b-] vector utilizing th...

2009-01-01

212

Nuclear dissipation as damping of collective motion in the time-dependent RPA and extensions of it  

International Nuclear Information System (INIS)

We have formulated a nonperturbative, microscopic dissipative process in the limit of an infinite mean free path which does not require any statistical assumptions. It attributes the damping of the collective motion to real transitions from the collective state to degenerate, more complicated nucelar states. The dissipation is described through wave packets which solve an approximate Schroedinger equation within extended subspaces, larger than the original subspace of the undamped motion. When the simple RPA is used, this process associates the dissipation with the escape width for direct particle emission. When the Second RPA is used, it associates the dissipation with the spreading width for transitions to the 2p-2h components of the nuclear compound states. The energy loss rate for sharp n-phonon initial states is proportional to the total collective energy. The classical dissipation, however, is obtained for coherent, multiphonon, initial packets which describe the damping of the mean field oscillations, and allow a theoretical connection with the Vibrating Potential Model, and thereby with models of one-body dissipation. The present model contrasts with linear response theories. Canonical coordinates for the collective degree of freedom are explicitly introduced. This allows the construction of a nonlinear frictional Hamiltonian which provides a connection with quantal friction. The dissipation process developed here is properly reversible rather than irreversible, in the sense that it is described by an approximate Schroedinger equation which honors time reversibility, rather than by a coarse grained master equation which violates it. Thus, the present theory contrasts with transport theories

1982-01-01

213

Nuclear dissipation as damping of collective motion in the time-dependent RPA and extensions of it  

Energy Technology Data Exchange (ETDEWEB)

We have formulated a nonperturbative, microscopic dissipative process in the limit of an infinite mean free path which does not require any statistical assumptions. It attributes the damping of the collective motion to real transitions from the collective state to degenerate, more complicated nucelar states. The dissipation is described through wave packets which solve an approximate Schroedinger equation within extended subspaces, larger than the original subspace of the undamped motion. When the simple RPA is used, this process associates the dissipation with the escape width for direct particle emission. When the Second RPA is used, it associates the dissipation with the spreading width for transitions to the 2p-2h components of the nuclear compound states. The energy loss rate for sharp n-phonon initial states is proportional to the total collective energy. The classical dissipation, however, is obtained for coherent, multiphonon, initial packets which describe the damping of the mean field oscillations, and allow a theoretical connection with the Vibrating Potential Model, and thereby with models of one-body dissipation. The present model contrasts with linear response theories. Canonical coordinates for the collective degree of freedom are explicitly introduced. This allows the construction of a nonlinear frictional Hamiltonian which provides a connection with quantal friction. The dissipation process developed here is properly reversible rather than irreversible, in the sense that it is described by an approximate Schroedinger equation which honors time reversibility, rather than by a coarse grained master equation which violates it. Thus, the present theory contrasts with transport theories.

Yannouleas, C.P.

1982-07-01

214

Two fast temperature sensors for probing of the atmospheric boundary layer using small remotely piloted aircraft (RPA  

Directory of Open Access Journals (Sweden)

Full Text Available Two types of temperature sensors are designed and tested: a thermocouple and a fine wire resistance thermometer. The intention of this study is to figure out which kind of measurement principle is in general more suited for atmospheric boundary layer meteorology with small remotely piloted aircraft (RPA. The sensors are calibrated in a NIST traceable climate chamber and validated in flight against tower measurements, radiosondes and remote sensing. The sensors have a measurement range of at least ?10–50 °C, an absolute RMS error of less than ±0.2 K which is stable over the lifetime of the sensors, and a resolution of about 0.01 K. Both devices are tested for typical errors like radiation error and adiabatic heating, as well as for their dynamic response. Spectral resolutions of up to approximately 10 Hz can be obtained with both sensors, which makes them suitable for turbulence measurement. Their low cost of less than 100 EUR in pure hardware is a major advantage for research with small RPA.

N. Wildmann

2013-08-01

215

Two fast temperature sensors for probing of the Atmospheric Boundary Layer using small Remotely Piloted Aircraft (RPA  

Directory of Open Access Journals (Sweden)

Full Text Available Two types of temperature sensors are designed and tested, a thermocouple and a fine wire resistance thermometer. The intention of this study is to figure out which kind of measurement principle is in general more suited for atmospheric boundary layer meteorology with small RPA. The sensors are calibrated in a NIST traceable climate chamber and validated in flight against tower measurements, radiosondes and remote sensing. The sensors have a measurement range of at least ?10...50° C, an absolute RMS error of less than ±0.2 K which is stable over the lifetime of the sensors, and a resolution of about 0.01 K. Both devices are tested for typical errors like radiation error and adiabatic heating, as well as for their dynamic response. Spectral resolutions of up to approximately 10 Hz can be obtained with both sensors, which makes them suitable for turbulence measurements. Their low cost of less than 100 EUR in pure hardware is a major advantage for research with small RPA.

N. Wildmann

2013-03-01

216

Chromosome fusions following telomere loss are mediated by single-strand annealing.  

Science.gov (United States)

Progressive telomere shortening eventually results in chromosome fusions and genome instability as the cell's ability to distinguish chromosome ends from DNA double-strand breaks is compromised. In fission yeast, such events frequently produce stable survivors with all circular chromosomes. To shed light on the repair pathways that mediate chromosome end fusions and generate circular chromosomes, we have examined a diverse array of DNA repair factors. We show that telomere attrition-induced chromosome fusions are dependent on the fission yeast homologs of Rad52, the ERCC1/XPF endonuclease, the single-stranded DNA-binding protein RPA, and the Srs2 and Werner/Bloom helicases, but not Ku and ligase 4. Consistent with a recombinational mechanism of single-strand annealing, cloned junctions map to four of five homology regions in subtelomeric DNA. A comparison with telomere uncapping caused by the absence of the double-stranded telomere-binding protein Taz1 demonstrates that the circumstances and cause of telomere dysfunction profoundly affect which DNA repair pathway is engaged. PMID:18722173

Wang, Xiaorong; Baumann, Peter

2008-08-22

217

A functional recT gene for recombineering of Clostridium.  

Science.gov (United States)

Recombineering is an efficient genetic manipulation method employing the mechanism of phagenic RecT-mediated homologous recombination. To develop a recombineering method for Clostridium, a putative recT gene (CPF0939) from Clostridium perfringens genome was functionally verified in a clostridial host Clostridium acetobutylicum. We show that a short synthetic oligonucleotide can be introduced into the target site for specific point mutation. This functional recT gene would therefore contribute to development of recombineering tools for Clostridium. PMID:24384234

Dong, Hongjun; Tao, Wenwen; Gong, Fuyu; Li, Yin; Zhang, Yanping

2014-03-10

218

Cell-Mediated and Humoral Immune Responses after Immunization of Calves with a Recombinant Multiantigenic Mycobacterium avium subsp. paratuberculosis Subunit Vaccine at Different Ages  

DEFF Research Database (Denmark)

Neonates and juvenile ruminants are very susceptible to paratuberculosis infection. This is likely due to a high degree of exposure from their dams and an immature immune system. To test the influence of age on vaccine-induced responses, a cocktail of recombinant Mycobacterium avium subsp. paratuberculosis proteins (MAP0217, MAP1508, MAP3701c, MAP3783, and MAP1609c/Ag85B) was formulated in a cationic liposome adjuvant (CAF01) and used to vaccinate animals of different ages. Male jersey calves were divided into three groups that were vaccinated at 2, 8, or 16 weeks of age and boosted twice at weeks 4 and 12 relative to the first vaccination. Vaccine-induced immune responses, the gamma interferon (IFN-γ) cytokine secretion and antibody responses, were followed for 20 weeks. In general, the specific responses were significantly elevated in all three vaccination groups after the first booster vaccination with no or only a minor effect from the second booster. However, significant differences were observed in the immunogenicity levels of the different proteins, and it appears that the older age group produced a more consistent IFN-γ response. In contrast, the humoral immune response is seemingly independent of vaccination age as we found no difference in the IgG1 responses when we compared the three vaccination groups. Combined, our results suggest that an appropriate age of vaccination should be considered in vaccination protocols and that there is a possible interference of vaccine-induced immune responses with weaning (week 8).

Thakur, Aneesh; Aagaard, Claus

2013-01-01

219

Recombinations in Staphylococcal Cassette Chromosome mec Elements Compromise the Molecular Detection of Methicillin Resistance in Staphylococcus aureus  

Science.gov (United States)

Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (?4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates.

Hill-Cawthorne, Grant A.; Hudson, Lyndsey O.; El Ghany, Moataz Fouad Abd; Piepenburg, Olaf; Nair, Mridul; Dodgson, Andrew; Forrest, Matthew S.

2014-01-01

220

Adeno-associated virus-mediated delivery of a recombinant single-chain antibody against misfolded superoxide dismutase for treatment of amyotrophic lateral sclerosis.  

Science.gov (United States)

There is emerging evidence that the misfolding of superoxide dismutase 1 (SOD1) may represent a common pathogenic event in both familial and sporadic amyotrophic lateral sclerosis (ALS). To reduce the burden of misfolded SOD1 species in the nervous system, we have tested a novel therapeutic approach based on adeno-associated virus (AAV)-mediated tonic expression of a DNA construct encoding a secretable single-chain fragment variable (scFv) antibody composed of the variable heavy and light chain regions of a monoclonal antibody (D3H5) binding specifically to misfolded SOD1. A single intrathecal injection of the AAV encoding the single-chain antibody in SOD1(G93A) mice at 45 days of age resulted in sustained expression of single-chain antibodies in the spinal cord, and it delayed disease onset and extension of life span by up to 28%, in direct correlation with scFv titers in the spinal cord. The treatment caused attenuation of neuronal stress signals and reduction in levels of misfolded SOD1 in the spinal cord of SOD1(G93A) mice. From these results, we propose that an immunotherapy based on intrathecal inoculation of AAV encoding a secretable scFv against misfolded SOD1 should be considered as potential treatment for ALS, especially for individuals carrying SOD1 mutations. PMID:24394188

Patel, Priyanka; Kriz, Jasna; Gravel, Mathieu; Soucy, Geneviève; Bareil, Christine; Gravel, Claude; Julien, Jean-Pierre

2014-03-01

 
 
 
 
221

Production, recovery and immunogenicity of the protective antigen from a recombinant strain of Bacillus anthracis.  

Science.gov (United States)

The protective antigen (PA) is one of the three components of the anthrax toxin. It is a secreted nontoxic protein with a molecular weight of 83 kDa and is the major component of the currently licensed human vaccine for anthrax. Due to limitations found in the existing vaccine formulation, it has been proposed that genetically modified PA may be more effective as a vaccine. The expression and the stability of two recombinant PA (rPA) variants, PA-SNKE-deltaFF-E308D and PA-N657A, were studied. These proteins were expressed in the nonsporogenic avirulent strain BH445. Initial results indicated that PA-SNKE-deltaFF-E308D, which lacks two proteolysis-sensitive sites, is more stable than PA-N657A. Process development was conducted to establish an efficient production and purification process for PA-SNKE-deltaFF-E308D. pH, media composition, growth strategy and protease inhibitors composition were analyzed. The production process chosen was based on batch growth of B. anthracis using tryptone and yeast extract as the only source of carbon, pH control at 7.5, and antifoam 289. Optimal harvest time was 14-18 h after inoculation, and EDTA (5 mM) was added upon harvest for proteolysis control. Recovery of the rPA was performed by expanded-bed adsorption (EBA) on a hydrophobic interaction chromatography (HIC) resin, eliminating the need for centrifugation, microfiltration and diafiltration. The EBA step was followed by ion exchange and gel filtration. rPA yields before and after purification were 130 and 90 mg/l, respectively. The purified rPA, without further treatment, treated with small amounts of formalin or adsorbed on alum, induced, high levels of IgG anti-PA with neutralization activities. PMID:11986925

Ramirez, D M; Leppla, S H; Schneerson, R; Shiloach, J

2002-04-01

222

Unwinding of synthetic replication and recombination substrates by Srs2.  

Science.gov (United States)

The budding yeast Srs2 protein possesses 3' to 5' DNA helicase activity and channels untimely recombination to post-replication repair by removing Rad51 from ssDNA. However, it also promotes recombination via a synthesis-dependent strand-annealing pathway (SDSA). Furthermore, at the replication fork, Srs2 is required for fork progression and prevents the instability of trinucleotide repeats. To better understand the multiple roles of the Srs2 helicase during these processes, we analysed the ability of Srs2 to bind and unwind various DNA substrates that mimic structures present during DNA replication and recombination. While leading or lagging strands were efficiently unwound, the presence of ssDNA binding protein RPA presented an obstacle for Srs2 translocation. We also tested the preferred directionality of unwinding of various substrates and studied the effect of Rad51 and Mre11 proteins on Srs2 helicase activity. These biochemical results help us understand the possible role of Srs2 in the processing of stalled or blocked replication forks as a part of post-replication repair as well as homologous recombination (HR). PMID:22921573

Marini, Victoria; Krejci, Lumir

2012-10-01

223

M1 strength and (e,e') form factors of 46,48Ti within the RPA  

International Nuclear Information System (INIS)

The abundant experimental information on low- and high-energy M1 excitations in 46,48Ti, obtained recently through (e, e'), (p, p') and (?, ?') experiments, is well described within a quasiparticle RPA approach, based on a deformed Woods-Saxon potential plus spin-spin and quadrupole-quadrupole interactions. It includes a procedure which restores the rotational invariance of the hamiltonian. The low-lying strong M1 states overlap up to 36% with the scissors state of the two rotor model. These states, as well as some medium-energy 1+ excitations, represent a weakly collective scissors motion, more pronounced in 46Ti than in 48Ti. The titanium isotopes offer a better approximation to the scissors state than do the heavy nuclei because in lighter nuclei the scissors state is less collective. (orig.)

1991-10-28

224

Recursive partitioning analysis (RPA) of prognostic factors in three Radiation Therapy Oncology Group (RTOG) brain metastases trials  

International Nuclear Information System (INIS)

Purpose: Promising results from new approaches such as radiosurgery or stereotactic surgery of brain metastases have recently been reported. Are these results due to the therapy alone or can the results be attributed in part to patient selection? An analysis of tumor/patient characteristics and treatment variables in previous Radiation Therapy Oncology Group (RTOG) brain metastases studies was considered necessary to fully evaluate the benefit of these new interventions. Methods and Materials: The database included 1200 patients from three consecutive RTOG trials conducted between 1979 and 1993, which tested several different dose fractionation schemes and radiation sensitizers. Using recursive partitioning analysis (RPA), a statistical methodology which creates a regression tree according to prognostic significance, eighteen pretreatment characteristics and three treatment-related variables were analyzed. Results: According to the RPA tree the best survival (median: 7.1 months) was observed in patients < 65 years of age with a Karnofsky Performance Status (KPS) of at least 70, and a controlled primary tumor with the brain the only site of metastases. The worst survival (median: 2.3 months) was seen in patients with a KPS less than 70. All other patients had relatively minor differences in observed survival, with a median of 4.2 months. Conclusions: Based on this analysis, we suggest the following three classes: Class 1: patients with KPS ? 70, < 65 years of age with controlled primary and no extracranial metastases; Class 3: KPS < 70; Class 2- all others. Using these classes or stages, new treatment techniques can be tested on homogeneous patient groups

1997-03-01

225

Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Recombinase encoded by the R gene of pSR1 of Zygosaccharomyces rouxii mediates reciprocal recombination between two specific recombination sites (RSs) to induce excision or inversion of the DNA segment that is flanked by the RSs. We report here that site-specific recombination mediated by this system takes place effeciently in tobacco cells. To monitor the recombination events in tobacco cells, we have constructed two types of cryptic beta-glucuronidase reporter gene in such a way that recomb...

Onouchi, H.; Yokoi, K.; Machida, C.; Matsuzaki, H.; Oshima, Y.; Matsuoka, K.; Nakamura, K.; Machida, Y.

1991-01-01

226

Mixtures of recombinant growth factors inhibit the production of pro-inflammatory mediators and cytokines in LPS-stimulated RAW 264.7 cells by inactivating the ERK and NF-?B pathways.  

Science.gov (United States)

Growth factors are important for regulating a variety of cellular processes and typically act as signaling molecules between cells. In the present study, we examined the mechanisms underlying the inhibitory effects of mixtures of recombinant growth factors (MRGFs) on nitric oxide (NO) and pro-inflammatory cytokine production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. We also examined whether these effects are mediated through the mitogen?activated protein kinase (MAPK) and nuclear factor-?B (NF-?B) signal transduction pathways. NO production was assessed by measuring nitrite acucmulation using the Greiss reaction. Cytokine concentrations were measured using respective ELISA kits for each cytokine. Our results revealed that the MRGFs significantly attenuated the LPS-induced production of pro-inflammatory cytokines and NO in a dose-dependent manner. To elucidate the mechanisms underlying the inhibitory effects of MRGFs, we examined the effects of the LPS-induced phosphorylation of MAPKs and the activation of the NF-?B signaling pathway on the stabilization of NF-?B nuclear translocation and inhibitory factor-?B (I?B) degradation. Western blot analysis was performed to determine the total and phosphorylated levels of ERK, as well as the nuclear translocation of NF-?B, and I?B phosphorylation and degradation. Our results demonstrated that treatment with MRGFs resulted in a reduction in the phosphorylation of the ERK and NF-?B signaling pathways, whereas the phosphorylation of JNK and p38 was not affected. Taken together, our results suggest that MRGFs inhibit the production of pro-inflammatory cytokines and NO by downregulating inducible NO synthase gene expression and blocking the phosphorylation of the ERK and NF-?B signaling pathways. These findings may provide direct evidence of the potential application of MRGFs in the prevention and treatment of inflammatory diseases. PMID:24888317

Lee, Yonghee; Lee, Dohyun; Koo, Kyotan; Lee, Jay; Song, Yi Seop; Yoon, Ho Sang; Choi, Yoo Mi; Kim, Beom Joon

2014-08-01

227

Recombinant Technology and Probiotics  

Directory of Open Access Journals (Sweden)

Full Text Available Recombinant technology has led the way to monumental advances in the development of useful molecules, including the development of safe probiotics. The development of novel approaches using recombinant technology and probiotics that allow accurate targeting of therapeutics to the mucosa is an interesting area of research. The creation and use of recombinant probiotics expressing recombinantovalbumin, recombinant ovalbumin mutants and yet-to-be-designed recombinant hypo/non-allergenic molecules offer the opportunity to further investigate their effects for food, nutrition, environment andhealth. This review highlights advances in native probiotics and recombinant probiotics expressing native and recombinant molecules for food, nutrition, environment and health.

Icy D’Silva

2011-09-01

228

Chromatin remodeling directly activates V(D)J recombination  

Digital Repository Infrastructure Vision for European Research (DRIVER)

V(D)J recombination substrate choice is regulated to ensure that the appropriate gene segments are rearranged during lymphocyte development. It has been proposed that regulation of substrate usage is determined by changes in accessibility of the DNA targets. We show that Rag-mediated recombination of an episomal substrate in cells is affected by its packaging into chromatin. Chromatinized substrates were inefficiently rearranged, and methylation further reduced recombination. Disruption of nu...

Cherry, Sara R.; Baltimore, David

1999-01-01

229

Reconstitution of recombination-associated DNA synthesis with human proteins  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The repair of DNA breaks by homologous recombination is a high-fidelity process, necessary for the maintenance of genome integrity. Thus, DNA synthesis associated with recombinational repair must be largely error-free. In this report, we show that human DNA polymerase delta (?) is capable of robust DNA synthesis at RAD51-mediated recombination intermediates dependent on the processivity clamp PCNA. Translesion synthesis polymerase eta (?) also extends these substrates, albeit far less proce...

Sneeden, Jessica L.; Grossi, Sara M.; Tappin, Inger; Hurwitz, Jerard; Heyer, Wolf-dietrich

2013-01-01

230

Non-native N-aroyl L-homoserine lactones are potent modulators of the quorum sensing receptor RpaR in Rhodopseudomonas palustris.  

Science.gov (United States)

Quorum sensing (QS) is a process by which bacteria use low-molecular-weight signaling molecules (or autoinducers) to assess their local population densities and alter gene expression levels at high cell numbers. Many Gram-negative bacteria use N-acyl L-homoserine lactones (AHLs) with aliphatic acyl groups as signaling molecules for QS. However, bacteria that utilize AHLs with aroyl acyl groups have been recently discovered; they include the metabolically versatile soil bacterium Rhodopseudomonas palustris, which uses p-coumaroyl HL (p-cAHL) as its QS signal. This autoinducer is especially unusual because its acyl group is believed to originate from a monolignol (i.e., p-coumarate) produced exogenously by plants in the R. palustris environment, rather than through the endogenous fatty acid biosynthesis pathway like other native AHLs. As such, p-cAHL could signal not only bacterial density, but also the availability of an exogenous plant-derived substrate and might even constitute an interkingdom signal. Like other Gram-negative bacteria, QS in R. palustris is controlled by the p-cAHL signal binding its cognate LuxR-type receptor, RpaR. We sought to determine if non-native aroyl HLs (ArHLs) could potentially activate or inhibit RpaR in R. palustris, and thereby modulate QS in this bacterium. Herein, we report the testing of a set of synthetic ArHLs for RpaR agonism and antagonism by using a R. palustris reporter strain. Several potent non-native RpaR agonists and antagonists were identified. Additionally, the screening data revealed that lower concentrations of ArHL are required to strongly agonize RpaR than to antagonize it. Structure-activity relationship analyses of the active ArHLs indicated that potent RpaR agonists tend to have sterically small substituents on their aryl groups, most notably in the ortho position. In turn, the most potent RpaR antagonists were based on either the phenylpropionyl HL (PPHL) or the phenoxyacetyl HL (POHL) scaffold, and many contained an electron-withdrawing group at either the meta or para positions of the aryl ring. To our knowledge, the compounds reported herein represent the first abiotic chemical modulators of RpaR, and more generally, the first abiotic ligands capable of intercepting QS in bacteria that utilize native ArHL signals. In view of the origins of the p-cAHL signal in R. palustris, the largely unknown role of QS in this bacterium, and R. palustris' unique environmental lifestyles, we anticipate that these compounds could be valuable as chemical probes to study QS in R. palustris in a range of fundamental and applied contexts. PMID:24281952

McInnis, Christine E; Blackwell, Helen E

2014-01-01

231

Conditional expression of RPA190, the gene encoding the largest subunit of yeast RNA polymerase I: effects of decreased rRNA synthesis on ribosomal protein synthesis.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The synthesis of ribosomal proteins (r proteins) under the conditions of greatly reduced RNA synthesis were studied by using a strain of the yeast Saccharomyces cerevisiae in which the production of the largest subunit (RPA190) of RNA polymerase I was controlled by the galactose promoter. Although growth on galactose medium was normal, the strain was unable to sustain growth when shifted to glucose medium. This growth defect was shown to be due to a preferential decrease in RNA synthesis caus...

1990-01-01

232

Comparative Genome Analysis of “Candidatus Phytoplasma australiense” (Subgroup tuf-Australia I; rp-A) and “Ca. Phytoplasma asteris” Strains OY-M and AY-WB? †  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The chromosome sequence of “Candidatus Phytoplasma australiense” (subgroup tuf-Australia I; rp-A), associated with dieback in papaya, Australian grapevine yellows in grapevine, and several other important plant diseases, was determined. The circular chromosome is represented by 879,324 nucleotides, a GC content of 27%, and 839 protein-coding genes. Five hundred two of these protein-coding genes were functionally assigned, while 337 genes were hypothetical proteins with unknown function. P...

Tran-nguyen, L. T. T.; Kube, M.; Schneider, B.; Reinhardt, R.; Gibb, K. S.

2008-01-01

233

Regulation of V(D)J recombination by nucleosome positioning at recombination signal sequences  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A key component in the regulation of V(D)J recombination is control of the accessibility of RAG proteins to recombination signal sequences (RSS). Nucleosomes are known to inhibit this accessibility. We show here that the signal sequence itself represses accessibility by causing nucleosome positioning over the RSS. This positioning is mediated, in vitro and in vivo, by the conserved nonamer of the RSS. Consistent with this strong positioning, nucleosomes at RSSs are resistant to remodelling by...

Baumann, Matthias; Mamais, Adamantios; Mcblane, Fraser; Xiao, Hua; Boyes, Joan

2003-01-01

234

PRODUCTION OF RECOMBINANT PROTEINS IN INSECT CELLS  

Directory of Open Access Journals (Sweden)

Full Text Available Among the wide range of methods and expression hosts available for the heterologous production of recombinant proteins, insect cells are ideal for the production of complex proteins requiring extensive post-translational modification. This review article provides an overview of the available insect-cell expression systems and their properties, focusing on the widely-used Baculovirus Expression Vector System (BEVS. We discuss the different strategies used to generate recombinant baculovirus vectors and show how advanced techniques for virus titer determination can accelerate the production of recombinant proteins. The stable transfection of insect cells is an alternative to BEVS which has recently been augmented with recombinase-mediated cassette exchange for site-specific gene integration. We consider the advantages and limitations of these techniques for the production of recombinant proteins in insect cells and compare them to other expression platforms.

Christian Kollewe

2013-01-01

235

DNA Detection Using Recombination Proteins  

Digital Repository Infrastructure Vision for European Research (DRIVER)

DNA amplification is essential to most nucleic acid testing strategies, but established techniques require sophisticated equipment or complex experimental procedures, and their uptake outside specialised laboratories has been limited. Our novel approach, recombinase polymerase amplification (RPA), couples isothermal recombinase-driven primer targeting of template material with strand-displacement DNA synthesis. It achieves exponential amplification with no need for pretreatment of sample DNA....

Piepenburg, Olaf; Williams, Colin H.; Stemple, Derek L.; Armes, Niall A.

2006-01-01

236

Diffusion controlled initial recombination  

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This work addresses nucleation rates in systems with strong initial recombination. Initial (or `geminate') recombination is a process where a dissociated structure (anion, vortex, kink etc.) recombines with its twin brother (cation, anti-vortex, anti-kink) generated in the same nucleation event. Initial recombination is important if there is an asymptotically vanishing interaction force instead of a generic saddle-type activation barrier. At low temperatures, initial recombi...

Christen, T.; Buttiker, M.

1997-01-01

237

DNA damage and homologous recombination signaling induced by thymidylate deprivation.  

Science.gov (United States)

DNA damage is accepted as a consequence of thymidylate deprivation induced by chemotherapeutic inhibitors of thymidylate synthase (TS), but the types of damage and signaling responses remain incompletely understood. Thymidylate deprivation increases dUTP and uracil in DNA, which is removed by base excision repair (BER). Because BER requires a synthesis step, strand break intermediates presumably accumulate. Thymidylate deprivation also induces cell cycle arrest during replication. Homologous recombination (HR) is a means of repairing persistent BER intermediates and collapsed replication forks. There are also intimate links between HR and S-phase checkpoint pathways. In this study, the goals were to determine the involvement of HR-associated proteins and DNA damage signaling responses to thymidylate deprivation. When RAD51, which is a central component of HR, was depleted by siRNA cells were sensitized to raltitrexed (RTX), which specifically inhibits TS. To our knowledge, this is the first demonstration in mammalian cells that depletion of RAD51 causes sensitivity to thymidylate deprivation. Activation of DNA damage signaling responses was examined following treatment with RTX. Phosphorylation of replication protein A (RPA2 subunit) and formation of damage-induced foci were strikingly evident following IC(50) doses of RTX. Induction was much more striking following RTX treatment than with hydroxyurea, which is commonly used to inhibit replication. RTX treatment also induced foci of RAD51, gamma-H2AX, phospho-Chk1, and phospho-NBS1, although the extent of co-localization with RPA2 foci varied. Collectively, the results suggest that HR and S-phase checkpoint signaling processes are invoked by thymidylate deprivation and influence cellular resistance to thymidylate deprivation. PMID:18773878

Yang, Zhengguan; Waldman, Alan S; Wyatt, Michael D

2008-10-15

238

Mismatch repair protein hMSH2-hMSH6 recognizes mismatches and forms sliding clamps within a D-loop recombination intermediate.  

Science.gov (United States)

High fidelity homologous DNA recombination depends on mismatch repair (MMR), which antagonizes recombination between divergent sequences by rejecting heteroduplex DNA containing excessive nucleotide mismatches. The hMSH2-hMSH6 heterodimer is the first responder in postreplicative MMR and also plays a prominent role in heteroduplex rejection. Whether a similar molecular mechanism underlies its function in these two processes remains enigmatic. We have determined that hMSH2-hMSH6 efficiently recognizes mismatches within a D-loop recombination initiation intermediate. Mismatch recognition by hMSH2-hMSH6 is not abrogated by human replication protein A (HsRPA) bound to the displaced single-stranded DNA (ssDNA) or by HsRAD51. In addition, ATP-bound hMSH2-hMSH6 sliding clamps that are essential for downstream MMR processes are formed and constrained within the heteroduplex region of the D-loop. Moreover, the hMSH2-hMSH6 sliding clamps are stabilized on the D-loop by HsRPA bound to the displaced ssDNA. Our findings reveal similarities and differences in hMSH2-hMSH6 mismatch recognition and sliding-clamp formation between a D-loop recombination intermediate and linear duplex DNA. PMID:24395779

Honda, Masayoshi; Okuno, Yusuke; Hengel, Sarah R; Martín-López, Juana V; Cook, Christopher P; Amunugama, Ravindra; Soukup, Randal J; Subramanyam, Shyamal; Fishel, Richard; Spies, Maria

2014-01-21

239

Smc5/6 maintains stalled replication forks in a recombination-competent conformation.  

Science.gov (United States)

The Smc5/6 structural maintenance of chromosomes complex is required for efficient homologous recombination (HR). Defects in Smc5/6 result in chromosome mis-segregation and fragmentation. By characterising two Schizosaccharomyces pombe smc6 mutants, we define two separate functions for Smc5/6 in HR. The first represents the previously described defect in processing recombination-dependent DNA intermediates when replication forks collapse, which leads to increased rDNA recombination. The second novel function defines Smc5/6 as a positive regulator of recombination in the rDNA and correlates mechanistically with a requirement to load RPA and Rad52 onto chromatin genome-wide when replication forks are stably stalled by nucleotide depletion. Rad52 is required for all HR repair, but Rad52 loading in response to replication fork stalling is unexpected and does not correlate with damage-induced foci. We propose that Smc5/6 is required to maintain stalled forks in a stable recombination-competent conformation primed for replication restart. PMID:19158664

Irmisch, Anja; Ampatzidou, Eleni; Mizuno, Ken'ichi; O'Connell, Matthew J; Murray, Johanne M

2009-01-21

240

A mitotic recombination system for mouse chromosome 17  

Science.gov (United States)

Mitotic recombination between homologous chromosomes is a genetic technique for mosaic analysis in model organisms. The general application of this technique in the mouse depends on establishment of effective recombination systems for individual chromosomes and reliable and sensitive methods for detection of recombination events. Here, we established a Cre/LoxP-mediated recombination system in mice for mosaic analysis of full-length chromosome 17. Cre-mediated germ-line recombination between the homologous chromosomes was observed with ?9% frequency in a progeny test. Mitotic recombination in somatic tissues was evaluated and scored in B and T lymphocytes with the aid of surface markers and fluorescent-activated cell sorting. We show that a lineage-specific Cre can induce mitotic recombination with a highly reproducible frequency of 0.5–1.0% in lymphoid progenitors. The recombination system established here allows for a simple and accurate detection and isolation of recombination events in live cells, making this system particularly attractive for mosaic analysis or mutagenesis studies in the immune system.

Sun, Lei; Wu, Xiaohui; Han, Min; Xu, Tian; Zhuang, Yuan

2008-01-01

 
 
 
 
241

A switch in the formation of alternative DNA loops modulates lambda site-specific recombination.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The virally encoded Xis protein is one of the components in the site-specific recombination reactions of bacteriophage lambda. It is required for excisive recombination and inhibits integrative recombination. The mechanism of Xis inhibition of the integration reaction was investigated by methylation protection assays (footprinting analyses) in conjunction with recombination assays. Xis is shown to mediate the formation of a specific attP looped structure involving cooperative and competitive ...

1991-01-01

242

A method for the prevention of thrombin-induced degradation of recombinant proteins  

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A new strategy to prevent degradation of recombinant proteins caused by non-specific cleavage by thrombin is described. We demonstrate that degradation due to non-specific cleavage of recombinant protein mediated by thrombin can be completely prevented by separation of thrombin from the recombinant protein on spin columns packed with heparin-sepharose. This method is generally applicable to all recombinant proteins that require the thrombin for the cleavage of affinity tags for purification. ...

Rajalingam, Dakshinamurthy; Kathir, Karuppanan Muthusamy; Ananthamurthy, Koteshwara; Adams, Paul D.; Kumar, Thallapuranam Krishnaswamy Suresh

2008-01-01

243

Screened Coulomb interaction calculations: cRPA implementation and applications to dynamical screening and self-consistency in uranium dioxide and cerium  

Science.gov (United States)

We report an implementation of the constrained random phase approximation (cRPA) method within the projector augmented-wave framework. It allows for the calculation of the screened interaction in the same Wannier orbitals as our recent DFT+U and DFT+DMFT implementations. We present calculations of the dynamical Coulomb screened interaction in uranium dioxide and ? and ? cerium on Wannier functions. We show that a self-consistent calculation of the static screened interaction in DFT+U together with a consistent Wannier basis is mandatory for ? cerium and uranium dioxide. We emphasize that a static approximation for the screened interaction in ? cerium is too drastic.

Amadon, Bernard; Applencourt, Thomas; Bruneval, Fabien

2014-03-01

244

Recombination in radar meteors  

Science.gov (United States)

Recombinations in the ionized columns generated by faint radar meteors are observed as: (1) A rapid loss of returned signal in the first few milliseconds after formation of the column; (2) an apparent absence of bright, low meteors; and (3) anomalies in apparent diffusion rates. Recombination at rates characteristic of dissociative recombination of ionized atmospheric molecules N2(+) and O2(+) is completely consistent with the observations. It appears either that the molecules are ionized in the initial formation of the ionized column or that there is rapid charge exchange. Recombination is a sufficient cause for the differences between diffusion measures and other atmospheric studies.

Southworth, R. B.

1973-01-01

245

Preparation and topology of the Mediator middle module  

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Mediator is the central coactivor complex required for regulated transcription by RNA polymerase (Pol) II. Mediator consists of 25 subunits arranged in the head, middle, tail and kinase modules. Structural and functional studies of Mediator are limited by the availability of protocols for the preparation of recombinant modules. Here, we describe protocols for obtaining pure endogenous and recombinant complete Mediator middle module from Saccharomyces cerevisiae that consists of seven subunits...

Koschubs, Tobias; Lorenzen, Kristina; Baumli, Sonja; Sandstro?m, Saana; Heck, Albert J. R.; Cramer, Patrick

2010-01-01

246

An N-terminal acidic region of Sgs1 interacts with Rpa70 and recruits Rad53 kinase to stalled forks  

Science.gov (United States)

DNA replication fork stalling poses a major threat to genome stability. This is counteracted in part by the intra-S phase checkpoint, which stabilizes arrested replication machinery, prevents cell-cycle progression and promotes DNA repair. The checkpoint kinase Mec1/ATR and RecQ helicase Sgs1/BLM contribute synergistically to fork maintenance on hydroxyurea (HU). Both enzymes interact with replication protein A (RPA). We identified and deleted the major interaction sites on Sgs1 for Rpa70, generating a mutant called sgs1-r1. In contrast to a helicase-dead mutant of Sgs1, sgs1-r1 did not significantly reduce recovery of DNA polymerase ? at HU-arrested replication forks. However, the Sgs1 R1 domain is a target of Mec1 kinase, deletion of which compromises Rad53 activation on HU. Full activation of Rad53 is achieved through phosphorylation of the Sgs1 R1 domain by Mec1, which promotes Sgs1 binding to the FHA1 domain of Rad53 with high affinity. We propose that the recruitment of Rad53 by phosphorylated Sgs1 promotes the replication checkpoint response on HU. Loss of the R1 domain increases lethality selectively in cells lacking Mus81, Slx4, Slx5 or Slx8.

Hegnauer, Anna Maria; Hustedt, Nicole; Shimada, Kenji; Pike, Brietta L; Vogel, Markus; Amsler, Philipp; Rubin, Seth M; van Leeuwen, Fred; Guenole, Aude; van Attikum, Haico; Thoma, Nicolas H; Gasser, Susan M

2012-01-01

247

The tumor suppressor PML specifically accumulates at RPA/Rad51-containing DNA damage repair foci but is nonessential for DNA damage-induced fibroblast senescence.  

Science.gov (United States)

The PML tumor suppressor has been functionally implicated in DNA damage response and cellular senescence. Direct evidence for such a role based on PML knockdown or knockout approaches is still lacking. We have therefore analyzed the irradiation-induced DNA damage response and cellular senescence in human and mouse fibroblasts lacking PML. Our data show that PML nuclear bodies (NBs) nonrandomly associate with persistent DNA damage foci in unperturbed human skin and in high-dose-irradiated cell culture systems. PML bodies do not associate with transient ?H2AX foci after low-dose gamma irradiation. Superresolution microscopy reveals that all PML bodies within a nucleus are engaged at Rad51- and RPA-containing repair foci during ongoing DNA repair. The lack of PML (i) does not majorly affect the DNA damage response, (ii) does not alter the efficiency of senescence induction after DNA damage, and (iii) does not affect the proliferative potential of primary mouse embryonic fibroblasts during serial passaging. Thus, while PML NBs specifically accumulate at Rad51/RPA-containing lesions and senescence-derived persistent DNA damage foci, they are not essential for DNA damage-induced and replicative senescence of human and murine fibroblasts. PMID:24615016

Münch, Sandra; Weidtkamp-Peters, Stefanie; Klement, Karolin; Grigaravicius, Paulius; Monajembashi, Shamci; Salomoni, Paolo; Pandolfi, Pier Paolo; Weißhart, Klaus; Hemmerich, Peter

2014-05-01

248

Resolution-of-identity approach to Hartree-Fock, hybrid density functionals, RPA, MP2, and \\textit{GW} with numeric atom-centered orbital basis functions  

CERN Multimedia

We present a computational framework that allows for all-electron Hartree-Fock (HF), hybrid density functionals, random-phase approximation (RPA), second-order M{\\o}ller-Plesset perturbation theory (MP2), and $GW$ calculations based on efficient and accurate numeric atomic-centered orbital (NAO) basis sets. The common feature in these approaches is that their key quantities are expressible in terms of products of single-particle basis functions, which can in turn be expanded in a set of auxiliary basis functions. This is a technique known as the "resolution of identity (RI)" which facilitates an efficient treatment of both the two-electron Coulomb repulsion integrals (required in all these approaches) as well as the linear response function (required for RPA and $GW$). We propose a simple prescription for constructing the auxiliary basis which can be applied regardless of whether the underlying radial functions have a specific analytical shape (e.g., Gaussian) or are numerically tabulated. We demonstrate the ...

Ren, Xinguo; Blum, Volker; Wieferink, Jürgen; Tkatchenko, Alexandre; Sanfilippo, Andrea; Reuter, Karsten; Scheffler, Matthias

2012-01-01

249

In vivo effects of anti-inflammatory agents on arachidonic acid (AA) metabolites in the pleural fluid of rats injured in a reverse passive Arthus (RPA) reaction  

International Nuclear Information System (INIS)

Leukotriene (LT)C_4-D_4, prostaglandin (PG)E_2, thromboxane B_2 (TXB), and 6-ketoprostaglandin F_1? (6-KP) were measured by radioimmunoassay in pleural fluid of rats immunologically injured in an RPA paradigm. Rats given intravenous BSA were injected intrapleurally 20 min. later with anti-BSA. Phenidone (30 mg/kg), indomethacin (0.3-100 mg/kg), dazoxiben (100 mg/kg), AA-861 (2,3,5 trimethyl-6(12 hydroxy-5,10-dodecadinyl)-1,4-benzoquinone; 100 mg/kg) or vehicle was given intragastrically 1 hr prior to injury. Pleural fluid samples were collected 1 hr after injury. Statistically significant (P < 0.05) reductions were found in fluid volume after phenidone, indomethacin, or AA-861 administration, in LTC_4-D_4 after phenidone and AA-861 treatment, in PGE_2 and 6-KP after phenidone and indomethacin treatment and in TXB after dazoxiben and indomethacin treatment. Dazoxiben significantly (p < 0.05) increased 6-KP. These data suggest that anti-inflammatory agents given in this in vivo RPA paradigm inhibited AA metabolism in a predictable manner. Also, drug administration was associated with changes in metabolite concentrations at additional pathway sites. Consequently, this paradigm may be a useful model in evaluating shifts in AA metabolism brought about by inflammatory responses and treatments

1986-03-05

250

An N-terminal acidic region of Sgs1 interacts with Rpa70 and recruits Rad53 kinase to stalled forks.  

Science.gov (United States)

DNA replication fork stalling poses a major threat to genome stability. This is counteracted in part by the intra-S phase checkpoint, which stabilizes arrested replication machinery, prevents cell-cycle progression and promotes DNA repair. The checkpoint kinase Mec1/ATR and RecQ helicase Sgs1/BLM contribute synergistically to fork maintenance on hydroxyurea (HU). Both enzymes interact with replication protein A (RPA). We identified and deleted the major interaction sites on Sgs1 for Rpa70, generating a mutant called sgs1-r1. In contrast to a helicase-dead mutant of Sgs1, sgs1-r1 did not significantly reduce recovery of DNA polymerase ? at HU-arrested replication forks. However, the Sgs1 R1 domain is a target of Mec1 kinase, deletion of which compromises Rad53 activation on HU. Full activation of Rad53 is achieved through phosphorylation of the Sgs1 R1 domain by Mec1, which promotes Sgs1 binding to the FHA1 domain of Rad53 with high affinity. We propose that the recruitment of Rad53 by phosphorylated Sgs1 promotes the replication checkpoint response on HU. Loss of the R1 domain increases lethality selectively in cells lacking Mus81, Slx4, Slx5 or Slx8. PMID:22820947

Hegnauer, Anna Maria; Hustedt, Nicole; Shimada, Kenji; Pike, Brietta L; Vogel, Markus; Amsler, Philipp; Rubin, Seth M; van Leeuwen, Fred; Guénolé, Aude; van Attikum, Haico; Thomä, Nicolas H; Gasser, Susan M

2012-09-12

251

Recombinant methods and materials  

Energy Technology Data Exchange (ETDEWEB)

This patent describes a method for stably effecting the insertion or deletion of a selected DNA sequence at a specific site in a viral genome. The method consists of: (1) isolating from the genome a linear DNA fragment comprising both (a) the specific site determined for insertion or deletion of selected DNA sequence and (b) flanking DNA sequences normally preceding and following the site; (2) preparing first and second altered genome fragments from the fragment isolated in step (1). (a) the first altered fragment comprising the fragment comprising a thymidine kinase gene in a position intermediate the ends of the fragment, and (b) the second altered fragment comprising the fragment having the selected DNA sequence inserted therein or deleted therefrom; (3) contacting the genome with the first altered fragment under conditions permitting recombination at sites of DNA sequence homology, selecting for a recombinant genome comprising the thymidine kinase gene, and isolating the recombinant genome; and (4) contacting the recombinant genome isolated in step (3) with the second altered fragment under conditions permitting recombination at sites of DNA sequence homology, selecting for a recombinant genome lacking the thymidine kinase gene, and isolating the recombinant genome product.

Roizman, B.; Post, L.E.

1988-09-06

252

Dielectronic recombination: an introduction  

International Nuclear Information System (INIS)

Atoms contained in hot plasmas such as those found in stellar coronae and controlled fusion devices are subject to intense bombardment by electrons whose energy distribution is characterized by the plasma temperature. Collisions can cause multiple ionization of the atom and the electrons may also recombine with the ion. The distribution of ion charge states in a plasma at a given temperature depends upon the competing rates of ionization and recombination. When an unbound electron recombines the gain in potential energy must be removed in some way. Two processes are known to be important in recombination: radiative recombination (RR) in which a photon is released whose energy is exactly equal to the potential energy gain, and dielectronic recombination (DR) in which a continuum electron excites a previously bound electron and in so doing loses just enough energy to be captured into a bound state (nl). The latter process results in a doubly excited ion in a lower charge state which may either auto-ionize or emit a photon resulting in a stabilized recombination. (Auth.)

1983-08-02

253

Recent dielectronic recombination experiments  

International Nuclear Information System (INIS)

New recombination experiments with merged cold beams of electrons and atomic ions have been carried out at the storage ring facilities TSR in Heidelberg, ESR in Darmstadt, and CRYRING in Stockholm. A brief overview is given on the recent activities in which the Giessen group was engaged. Topics of this research were dielectronic recombination (DR) of astrophysically relevant ions, recombination of highly charged ions with respect to cooling losses in storage rings, field effects on DR, search for interference effects in photorecombination of ions, correlation effects in DR of low-Z ions, spectroscopy of high-Z ions by DR, and lifetimes of metastable states deduced from DR experiments

1998-11-01

254

Recombination in Circulating Enteroviruses  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Recombination is a well-known phenomenon for enteroviruses. However, the actual extent of recombination in circulating nonpoliovirus enteroviruses is not known. We have analyzed the phylogenetic relationships in four genome regions, VP1, 2A, 3D, and the 5? nontranslated region (NTR), of 40 enterovirus B strains (coxsackie B viruses and echoviruses) representing 11 serotypes and isolated in 1981 to 2002 in the former Soviet Union states. In the VP1 region, strains of the same serotype expect...

2003-01-01

255

The use of recombinant DNA plasmids for the determination of DNA-repair and recombination in cultured mammalian cells.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Using the recombinant plasmid pSV2gpt and DNA transfer techniques, cell mediated DNA ligation and recombination of plasmid DNA have been demonstrated in four human cell lines. Data suggesting the involvement of a possible defect in the cellular equilibrium between ligation and exonuclease digestion of double strand DNA scissions in an ataxia-telangiectasia (A-T) cell line is discussed. The same A-T line was grossly proficient in DNA recombination but it will be necessary to distinguish betwee...

Cox, R.; Masson, W. K.; Debenham, P. G.; Webb, M. B.

1984-01-01

256

Towards higher accuracy and better frequency response with standard multi-hole probes in turbulence measurement with remotely piloted aircraft (RPA  

Directory of Open Access Journals (Sweden)

Full Text Available This study deals with the problem of turbulence measurement with small remotely piloted aircraft (RPA. It shows how multi-hole probes (MHPs can be used to measure fluctuating parts of the airflow in flight up to 20 Hz. Accurate measurement of the transient wind in the outdoor environment is needed for the estimation of the 3-D wind vector as well as turbulent fluxes of heat, momentum, water vapour, etc. In comparison to an established MHP system, experiments were done to show how developments of the system setup can improve data quality. The study includes a re-evaluation of the pneumatic tubing setup, the conversion from pressures to airspeed, the pressure transducers, and the data acquisition system. In each of these fields, the steps that were taken lead to significant improvements. A spectral analysis of airspeed data obtained in flight tests shows the capability of the system to measure atmospheric turbulence up to the desired frequency range.

N. Wildmann

2014-04-01

257

3D Radiation Therapy Boost Improves the Outcome of Whole Brain Radiation Therapy Treated RPA II Patients with One or Two Brain Metastases  

Science.gov (United States)

Purpose to evaluate the role of whole brain radiotherapy (WBRT) and radiation boost (RB) for 208 patients recursive partitioning analysis (RPA) II with 1 or 2 brain metastases (BM) at a single institution. Methods and Materials the dose of WBRT was 30 Gy (10 fractions of 3 Gy). One hundred thirty-two patients (63.5%) benefited from RB of 9 Gy in 3 fractions of 3 Gy at the metastatic site. Patients had 1 or 2 BM in 122 (58.7%) and 86 cases (41.3%), respectively. Results patients with one or two metastases had similar survival (4.6 and 5.1 months, respectively) (p = 0.4). Median overall survival (OS) for patients treated with WBRT and RB, and with WBRT alone was 5.9 and 3.7 months, respectively (p = 0.03). The 6-, 12- and 24-month OS rates after WBRT and RB were 48.5%, 25% and 10.6%, respectively, while WBRT alone resulted in OS rates of 34%, 22.4% and 3.2%, respectively (p = 0.03). After WBRT and RB, the 6-, 12- and 24-month local control rates were 92%, 82% and 67%, respectively, while they were 81.2%, 75% and 37.5%, respectively, after WBRT alone (p = 0.03). The 6-, 12- and 24-month brain control rates after WBRT and RB were 88.7%, 75.8% and 62%, respectively, and after WBRT alone they were 78.5%, 59% and 37.7%, respectively (p = 0.03). Conclusion additional boost delivered with 3D conformal radiotherapy improves local and brain control rates significantly as well as overall survival for RPA II patients with 1 or 2 unresectable BM.

Antoni, Delphine; Clavier, Jean-Baptiste; Pop, Marius; Schumacher, Catherine; Lefebvre, Francois; Noel, Georges

2014-01-01

258

3D Radiation Therapy Boost Improves the Outcome of Whole Brain Radiation Therapy Treated RPA II Patients with One or Two Brain Metastases  

Directory of Open Access Journals (Sweden)

Full Text Available Purpose: to evaluate the role of whole brain radiotherapy (WBRT and radiation boost (RB for 208 patients recursive partitioning analysis (RPA II with 1 or 2 brain metastases (BM at a single institution. Methods and Materials: the dose of WBRT was 30 Gy (10 fractions of 3 Gy. One hundred thirty-two patients (63.5% benefited from RB of 9 Gy in 3 fractions of 3 Gy at the metastatic site. Patients had 1 or 2 BM in 122 (58.7% and 86 cases (41.3%, respectively. Results: patients with one or two metastases had similar survival (4.6 and 5.1 months, respectively (p = 0.4. Median overall survival (OS for patients treated with WBRT and RB, and with WBRT alone was 5.9 and 3.7 months, respectively (p = 0.03. The 6-, 12- and 24-month OS rates after WBRT and RB were 48.5%, 25% and 10.6%, respectively, while WBRT alone resulted in OS rates of 34%, 22.4% and 3.2%, respectively (p = 0.03. After WBRT and RB, the 6-, 12- and 24-month local control rates were 92%, 82% and 67%, respectively, while they were 81.2%, 75% and 37.5%, respectively, after WBRT alone (p = 0.03. The 6-, 12- and 24-month brain control rates after WBRT and RB were 88.7%, 75.8% and 62%, respectively, and after WBRT alone they were 78.5%, 59% and 37.7%, respectively (p = 0.03. Conclusion: additional boost delivered with 3D conformal radiotherapy improves local and brain control rates significantly as well as overall survival for RPA II patients with 1 or 2 unresectable BM.

Delphine Antoni

2014-05-01

259

High-frequency, site-specific recombination between lactococcal and pAM beta 1 plasmid DNAs.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

In vivo recombination events involving the 75-kilobase lactose proteinase plasmid pCI301 of Lactococcus lactis subsp. lactis UC317 and the conjugative enterococcal plasmid pAM beta 1 were analyzed. A fragment, identified as containing the pCI301 recombination site, mediated greatly elevated levels of mobilization and recombination with pAM beta 1 when cloned in a nonmobilizable L. lactis-Escherichia coli shuttle vector. This latter recombination event was site and orientation specific on both...

Hayes, F.; Daly, C.; Fitzgerald, G. F.

1990-01-01

260

Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The phage lambda-derived Red recombination system is a powerful tool for making targeted genetic changes in Escherichia coli, providing a simple and versatile method for generating insertion, deletion, and point mutations on chromosomal, plasmid, or BAC targets. However, despite the common use of this system, the detailed mechanism by which lambda Red mediates double-stranded DNA recombination remains uncertain. Current mechanisms posit a recombination intermediate in which both 5? ends of ...

Mosberg, J. A.; Lajoie, M. J.; Church, G. M.

2010-01-01

 
 
 
 
261

Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by ?C31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. ?C31 integrase-mediated ...

Colloms, S. D.; Merrick, C. A.; Olorunniji, F. J.; Stark, W. M.; Smith, M. C. M.; Osbourn, A.; Keasling, J. D.; Rosser, S. J.

2013-01-01

262

Schizosaccharomyces pombe Mms1 channels repair of perturbed replication into Rhp51 independent homologous recombination.  

Science.gov (United States)

In both Schizosaccharomyces pombe and Saccharomyces cerevisiae, Mms22 and Mms1 form a complex with important functions in the response to DNA damage, loss of which leads to perturbations during replication. Furthermore, in S. cerevisiae, Mms1 has been suggested to function in concert with a Cullin-like protein, Rtt101/Cul8, a potential paralog of Cullin 4. We performed epistasis analysis between ?mms1 and mutants of pathways with known functions in genome integrity, and measured the recruitment of homologous recombination proteins to blocked replication forks and recombination frequencies. We show that, in S. pombe, the functions of Mms1 and the conserved components of the Cullin 4 ubiquitin ligase, Pcu4 and Ddb1, do not significantly overlap. Furthermore, unlike in S. cerevisiae, the function of the H3K56 acetylase Rtt109 is not essential for Mms1 function. We provide evidence that Mms1 function is particularly important when a single strand break is converted into a double strand break during replication. Genetic data connect Mms1 to a Mus81 and Rad22(Rad52) dependent, but Rhp51 independent, branch of homologous recombination. This is supported by results demonstrating that Mms1 is recruited to a site-specific replication fork barrier and that, in a ?mms1 strain, Rad22(Rad52) and RPA recruitment to blocked forks are reduced, whereas Rhp51 recruitment is unaffected. In addition, Mms1 appears to specifically promote chromosomal rearrangements in a recombination assay. These observations suggest that Mms1 acts to channel repair of perturbed replication into a particular sub-pathway of homologous recombination. PMID:21183410

Vejrup-Hansen, Rasmus; Mizuno, Ken'Ichi; Miyabe, Izumi; Fleck, Oliver; Holmberg, Christian; Murray, Johanne M; Carr, Antony M; Nielsen, Olaf

2011-03-01

263

Schizosaccharomyces pombe Mms1 channels repair of perturbed replication into Rhp51 independent homologous recombination  

DEFF Research Database (Denmark)

In both Schizosaccharomyces pombe and Saccharomyces cerevisiae, Mms22 and Mms1 form a complex with important functions in the response to DNA damage, loss of which leads to perturbations during replication. Furthermore, in S. cerevisiae, Mms1 has been suggested to function in concert with a Cullin-like protein, Rtt101/Cul8, a potential paralog of Cullin 4. We performed epistasis analysis between ¿mms1 and mutants of pathways with known functions in genome integrity, and measured the recruitment of homologous recombination proteins to blocked replication forks and recombination frequencies. We show that, in S. pombe, the functions of Mms1 and the conserved components of the Cullin 4 ubiquitin ligase, Pcu4 and Ddb1, do not significantly overlap. Furthermore, unlike in S. cerevisiae, the function of the H3K56 acetylase Rtt109 is not essential for Mms1 function. We provide evidence that Mms1 function is particularly important when a single strand break is converted into a double strand break during replication. Genetic data connect Mms1 to a Mus81 and Rad22(Rad52) dependent, but Rhp51 independent, branch of homologous recombination. This is supported by results demonstrating that Mms1 is recruited to a site-specific replication fork barrier and that, in a ¿mms1 strain, Rad22(Rad52) and RPA recruitment to blocked forks are reduced, whereas Rhp51 recruitment is unaffected. In addition, Mms1 appears to specifically promote chromosomal rearrangements in a recombination assay. These observations suggest that Mms1 acts to channel repair of perturbed replication into a particular sub-pathway of homologous recombination.

Vejrup-Hansen, Rasmus; Mizuno, Ken'Ichi

2011-01-01

264

Recombinant Protein Production by In Vivo Polymer Inclusion Display ?  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A novel approach to produce purified recombinant proteins was established. The target protein is produced as polyhydroxyalkanoate (PHA) synthase fusion protein, which mediates intracellular formation of PHA inclusions displaying the target protein. After isolation of the PHA inclusions, the pure target protein was released by simple enterokinase digestion.

2011-01-01

265

Three Body Recombination of Ultracold Dipoles to Weakly Bound Dimers  

Science.gov (United States)

We use universality in two-body dipolar physics to study three-body recombination. We present results for the universal structure of weakly bound two-dipole states that depend only on the s-wave scattering length (a). We study threshold three-body recombination rates into weakly bound dimer states as a function of the scattering length. A Fermi golden rule analysis is used to estimate rates for different events mediated by the dipole-dipole interaction and a phenomenological contact interaction. The three-body recombination rate in the limit where a?D contains terms which scale as a4, a2D2, and D4, where D is the dipolar length. When a?D, the three-body recombination rate scales as D4.

Ticknor, Christopher; Rittenhouse, Seth T.

2010-07-01

266

Three body recombination of ultracold dipoles to weakly bound dimers  

CERN Document Server

We use universality in two-body dipolar physics to study three-body recombination. We present results for the universal structure of weakly bound two-dipole states that depend only on the s-wave scattering length ($a$). We study threshold three-body recombination rates into weakly-bound dimer states as a function of the scattering length. A Fermi Golden rule analysis is used to estimate rates for different events mediated by the dipole-dipole interaction and a phenomenological contact interaction. The three-body recombination rate in the limit where $a\\gg D$ contains terms which scale as $a^{4}$, $a^{2}D^{2}$ and $D^{4}$, where $D$ is the dipolar length. When $a \\ll D$, the three-boby recombination rate scales as $D^4$.

Ticknor, Chris

2010-01-01

267

Recombinational DNA repair and human disease  

Energy Technology Data Exchange (ETDEWEB)

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.

Thompson, Larry H.; Schild, David

2002-11-30

268

Recombinational DNA repair and human disease  

International Nuclear Information System (INIS)

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

2002-11-30

269

Recombinant adeno-associated virus-mediated global anterograde delivery of glial cell line-derived neurotrophic factor to the spinal cord: comparison of rubrospinal and corticospinal tracts in the rat.  

Science.gov (United States)

Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of spinal lower motoneurons. Gene delivery is a promising strategy to deliver therapeutic molecules to these vulnerable cells. However, definition of an optimal route of delivery capable of accessing neurons over a considerable extent of the neuraxis represents a significant logistical problem. Intramuscular vector injections are not ideal as this approach would involve hundreds of injections to completely treat an ALS patient and also would be dependent on retrograde transport of the viral platform of choice. Alternatively, upper motoneurons could deliver trophic factors over considerable distances by anterograde transport after a relatively localized intracerebral injection. To test this approach, the present study was designed to compare the corticospinal (CST) and rubrospinal (RST) tracts for their ability to transport recombinant adeno-associated virus serotype 5 (rAAV5)-derived green fluorescent protein (GFP) or glial cell line-derived neurotrophic factor (GDNF) to the spinal cord. Unilateral injections of rAAV5-GFP into the red nucleus (RN) or motor cortex of normal rats produced GFP-positive fibers in the appropriate descending tracts extending to the lumbar spinal cord. For both tracts, GFP-positive axonal projections into the spinal gray matter were consistently observed. GDNF immunohistochemistry demonstrated that confirmed RN injections resulted in GDNF-positive fibers projecting into spinal gray matter as seen in the GFP group. In contrast, confirmed cortical rAAV5-GDNF injections resulted in less evident staining in spinal cord. Spinal cord GDNF levels were elevated at distances up to 72 mm from the injection sites, and confirmed that RST-related GDNF transport to spinal cord surpassed CST-associated delivery. PMID:18072858

Foust, Kevin D; Flotte, Terence R; Reier, Paul J; Mandel, Ronald J

2008-01-01

270

Recombinant vWF type 2A mutants R834Q and R834W show a defect in mediating platelet adhesion to collagen, independent of enhanced sensitivity to a plasma protease.  

Science.gov (United States)

Type 2A von Willebrand Disease (vWD) is characterized by the absence of high molecular weight von Willebrand factor (VWF) multimers in plasma which is caused by enhanced extracellular proteolysis or defective intracellular transport. We identified in vWD type 2A patients two mutations in the A2 domain at position 834 in which arginine (R) was substituted for glutamine (R834Q) or tryptophan (R834W). We reproduced these mutations in vWF cDNA and expressed the recombinant proteins in furin cDNA containing baby hamster kidney (fur-BHK) cells. The subunit composition and the multimeric structure of both mutants was similar to wild-type (WT) vWF. Characterization of mutant R834Q by ristocetin or botrocetin induced platelet binding, and by binding to heparin showed no abnormality. R834W had normal botrocetin induced platelet binding, but ristocetin induced platelet binding and binding to heparin were decreased. Under static conditions R834Q and R834W, at 10 microg/ml, bound equally well to collagen type III as WT-vWF. At high shear rate conditions both mutants supported platelet adhesion normally when coated to a glass surface or preincubated on collagen. When R834Q or R834W was added to the perfusate, adhesion to collagen type III was 50% of the WT-vWF value, which was not due to a decreased collagen binding under flow. A divalent cation dependent protease, purified from plasma, degraded the 2A mutants rapidly while WT-vWF was not affected. In conclusion, the mutations present in the A2 domain of vWF result in an enhanced proteolytic sensitivity to a divalent ion-dependent protease. When present in the perfusate, R834Q and R834W show a decrease in platelet adhesion to collagen type III under flow conditions, which is not caused by decreased binding of the mutant vWF to collagen or enhanced proteolysis. PMID:10404778

Lankhof, H; Damas, C; Schiphorst, M E; Ijsseldijk, M J; Bracke, M; Furlan, M; de Groot, P G; Sixma, J J; Vink, T

1999-06-01

271

SUMO Wrestles with Recombination  

Directory of Open Access Journals (Sweden)

Full Text Available DNA double-strand breaks (DSBs comprise one of the most toxic DNA lesions, as the failure to repair a single DSB has detrimental consequences on the cell. Homologous recombination (HR constitutes an error-free repair pathway for the repair of DSBs. On the other hand, when uncontrolled, HR can lead to genome rearrangements and needs to be tightly regulated. In recent years, several proteins involved in different steps of HR have been shown to undergo modification by small ubiquitin-like modifier (SUMO peptide and it has been suggested that deficient sumoylation impairs the progression of HR. This review addresses specific effects of sumoylation on the properties of various HR proteins and describes its importance for the homeostasis of DNA repetitive sequences. The article further illustrates the role of sumoylation in meiotic recombination and the interplay between SUMO and other post-translational modifications.

Lumír Krej?í

2012-07-01

272

Exploding foil recombination laser  

International Nuclear Information System (INIS)

In addition to experiments using the collisional-excitation approach to a soft x-ray laser, the authors tested a recombination-pumped laser scheme. Intense (I/sub L/ approx. 2 x 1014 W/cm2) 0.53-?m light of 100- to 200-ps duration from the Novette laser was used to fully ionize a magnesium exploding-foil target. After the peak of the laser pulse, the plasma cooled rapidly due to expansion, electron conduction, and radiation. In the regime of high electron density (approx. 1020 cm-3) and low electron temperature (approx. 100 eV), three-body recombination preferentially populates the upper levels of the hydrogen-like magnesium ion. The population of the lower levels is depleted by fast radiative decay. This process can result in a population inversion on the n = 4 to 3, 130-A transition

1985-06-01

273

The RPA nuclear continuum in "1"6O(e,e') reactions at low momentum transfer - decay on (e,e'p) and (e,e'n) reaction channels  

International Nuclear Information System (INIS)

A theoretical investigation has been performed of "1"6O(e,e') and "1"6O(e,e'x) reactions at low momentum transfer in the frame of a self-consistent HF and RPA theory with a SK3 interaction. Nuclear responses and their multipole components have been calculated in the whole energy-range for the two electron kinematics i) ?_i = 67 MeV and ? = 40"0, ii) ?_i = 130 MeV and ? = 50"0. The microscopic structure of HF and RPA resonating states in the energy continuum has been inferred from the calculation. Decay properties in the reaction channels (e,e'p) and (e,e'n) have been discussed in the two cases of a semidirect and a knockout reaction process. (orig.)

1987-01-01

274

Nonradiative recombination in semiconductors  

CERN Document Server

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

Abakumov, VN; Yassievich, IN

1991-01-01

275

Recombinant Human Enterovirus 71  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Two human enterovirus 71 (HEV71) isolates were identified from hand, foot and mouth disease patients with genome sequences that had high similarity to HEV71 (>93%) at 5´ UTR, P1, and P2 and coxsackievirus A16 (CV-A16, >85%) at P3 and 3´UTR. Intertypic recombination is likely to have occurred between HEV71 and CV-A16 or an as-yet to be described CV-A16-like virus.

2004-01-01

276

A Simplified Recombinant PSO  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Simplified forms of the particle swarm algorithm are very beneficial in contributing to understanding how a particle swarm optimization (PSO) swarm functions. One of these forms, PSO with discrete recombination, is extended and analyzed, demonstrating not just improvements in performance relative to a standard PSO algorithm, but also significantly different behavior, namely, a reduction in bursting patterns due to the removal of stochastic components from the update equations.

Dan Bratton; Tim Blackwell

2008-01-01

277

Radiative recombination rate coefficients  

International Nuclear Information System (INIS)

Radiative recombination rate coefficients obtained using the nonrelativistic dipole result of Stobbe for the RR cross section are compared to other nonrelativistic approaches. A good agreement is found with widely used formulas. The RR rate coefficients can be calculated for different beam temperatures and for arbitrary nl sublevels up to high Rydberg states. The computation technique used allows us a fast evaluation of the dipole matrix elements without any additional approximation. (orig.)

2000-01-01

278

Intercultural Mediation  

Directory of Open Access Journals (Sweden)

Full Text Available The Intercultural Mediator facilitates exchanges between people of different socio-cultural backgrounds and acts as a bridge between immigrants and national and local associations, health organizations, services and offices in order to foster integration of every single individual. As the use mediation increases, mediators are more likely to be involved in cross-cultural mediation, but only the best mediators have the opportunity to mediate cross border business disputes or international politics conflicts. This article attempts to provide a new perspective about the intercultural mediation.

Dragos Marian Radulescu

2012-11-01

279

Unraveling the mechanism of BRCA2 in homologous recombination  

Digital Repository Infrastructure Vision for European Research (DRIVER)

BRCA2 is the product of a breast cancer susceptibility gene in human and the founding member of an emerging family of proteins present throughout the eukaryotic domain that serve in homologous recombination. The function of BRCA2 in recombination is to control RAD51, a protein that catalyzes homologous pairing and DNA strand exchange. By physically interacting with both RAD51 and single-stranded DNA, BRCA2 mediates delivery of RAD51 preferentially to sites of ssDNA exposed as a result of DNA ...

Holloman, William K.

2011-01-01

280

Did the universe recombine?  

International Nuclear Information System (INIS)

The Zel'dovich-Sunyaev model-independent arguments for the existence of a neutral hydrogen phase is reviewed in light of new limits on the Compton y parameter from COBE. It is concluded that with baryon densities compatible with standard cosmological nucleosynthesis, the universe could have remained fully ionized throughout its history without producing a detectable spectral distortion. It is argued that it is unlikely that spectral observations of the cosmic microwave background will ever require the universe to have recombined for flat cosmologies. 22 refs

1991-01-01

 
 
 
 
281

Did the universe recombine  

Energy Technology Data Exchange (ETDEWEB)

The Zel'dovich-Sunyaev model-independent arguments for the existence of a neutral hydrogen phase is reviewed in light of new limits on the Compton y parameter from COBE. It is concluded that with baryon densities compatible with standard cosmological nucleosynthesis, the universe could have remained fully ionized throughout its history without producing a detectable spectral distortion. It is argued that it is unlikely that spectral observations of the cosmic microwave background will ever require the universe to have recombined for flat cosmologies. 22 refs.

Bartlett, J.G.; Stebbins, A. (California, University, Berkeley (USA) Toronto, University (Canada))

1991-04-01

282

Hypoxia-induced hypothermia mediated by GABA in the rostral parapyramidal area of the medulla oblongata.  

Science.gov (United States)

Hypoxia evokes a regulated decrease in the body core temperature (Tc) in a variety of animals. The neuronal mechanisms of this response include, at least in part, glutamatergic activation in the lateral preoptic area (LPO) of the hypothalamus. As the sympathetic premotor neurons in the medulla oblongata constitute a cardinal relay station in the descending neuronal pathway from the hypothalamus for thermoregulation, their inhibition can also be critically involved in the mechanisms of the hypoxia-induced hypothermia. Here, I examined the hypothesis that hypoxia-induced hypothermia is mediated by glutamate-responsive neurons in the LPO that activate GABAergic transmission in the rostral raphe pallidus (rRPa) and neighboring parapyramidal region (PPy) of the medulla oblongata in urethane-chloralose-anesthetized, neuromuscularly blocked, artificially ventilated rats. Unilateral microinjection of GABA (15nmol) into the rRPa and PPy regions elicited a prompt increase in tail skin temperature (Ts) and decreases in Tc, oxygen consumption rate (VO2), and heart rate. Next, when the GABAA receptor blocker bicuculline methiodide (bicuculline methiodide (BMI), 10pmol) alone was microinjected into the rRPa, it elicited unexpected contradictory responses: simultaneous increases in Ts, VO2 and heart rate and a decrease in Tc. Then, when BMI was microinjected bilaterally into the PPy, no direct effect on Ts was seen; and thermogenic and tachycardic responses were slight. However, pretreatment of the PPy with BMI, but not vehicle saline, greatly attenuated the hypothermic responses evoked by hypoxic (10%O2-90%N2, 5min) ventilation or bilateral microinjections of glutamate (5nmol, each side) into the LPO. The results suggest that hypoxia-induced hypothermia was mediated, at least in part, by the activation of GABAA receptors in the PPy. PMID:24607346

Osaka, T

2014-05-16

283

Cervical cancer isolate PT3, super-permissive for adeno-associated virus replication, over-expresses DNA polymerase ?, PCNA, RFC and RPA  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Adeno-associated virus (AAV type 2 is an important virus due to its use as a safe and effective human gene therapy vector and its negative association with certain malignancies. AAV, a dependo-parvovirus, autonomously replicates in stratified squamous epithelium. Such tissue occurs in the nasopharynx and anogenitals, from which AAV has been clinically isolated. Related autonomous parvoviruses also demonstrate cell tropism and preferentially replicate in oncogenically transformed cells. Combining these two attributes of parvovirus tropism, squamous and malignant, we assayed if AAV might replicate in squamous cervical carcinoma cell isolates. Results Three primary isolates (PT1-3 and two established cervical cancer cell lines were compared to normal keratinocytes (NK for their ability to replicate AAV. One isolate, PT3, allowed for high levels of AAV DNA replication and virion production compared to others. In research by others, four cellular components are known required for in vitro AAV DNA replication: replication protein A (RPA, replication factor C (RFC, proliferating cell nuclear antigen (PCNA, and DNA polymerase delta (POLD1. Thus, we examined PT3 cells for expression of these components by DNA microarray and real-time quantitative PCR. All four components were over-expressed in PT3 over two representative low-permissive cell isolates (NK and PT1. However, this super-permissiveness did not result in PT3 cell death by AAV infection. Conclusion These data, for the first time, provide evidence that these four cellular components are likely important for AAV in vivo DNA replication as well as in vitro. These data also suggest that PT3 will be a useful reagent for investigating the AAV-permissive transcriptome and AAV anti-cancer effect.

Melchert Russell B

2009-04-01

284

Calculation of Gamow-Teller ?-strength functions in the rubidium region in the RPA approximation with Nilsson-model wave functions  

International Nuclear Information System (INIS)

We calculate allowed Gamow-Teller and, in a few cases, Fermi ?-strength functions in a model that is applicable to studies of nuclei throughout the periodic system. For our first study we have selected a sequence of rubidium isotopes, namely 3789Rb-3799Rb. We develop a model that uses calculated Nilsson-model wave functions, spherical or deformed, as the case may be, as the starting point for determining the wave functions of the mother and daughter nuclei in the ?-decay. Pairings is treated in the BCS approximation. To account for the retardation of low-energy GT decay rates we add, as is customarily done, a simple residual interaction specific to GT decay, namely Vsub(GT)=:?1-?1+:; to the hamiltonian. This residual interaction is treated in the RPA approximation. The strength of the interaction is adjusted to get agreement between the calculated and experimental energy of the giant Gamow-Teller resonance for 208Pb and 144Sm. Since the present model is based on calculated wave functions and single-particle levels, studies of nuclei far from stability, where little experimental information is available, are more straightforward relative to calculations where 'experimental' levels are used. The model can treat deformed nuclei employing wave functions calculated to desired accuracy, within the framework of the model, for the deformed single-particle well. The calculations show that use of single-particle parameters appropriate to the region studied and taking deformation into account is important. We find good agreement between calculated and experimental spectra over the region studied, provided an appropriate choice of single-particle parameters and deformation is made. (orig.)

1984-04-16

285

Dielectronic recombination of heliumlike nickel  

International Nuclear Information System (INIS)

The dielectronic recombination excitation function for He-like Ni/sup 26+/ ions has been measured. The Ni/sup 26+/ ions were created and held in an electron beam ion trap, and K x rays were detected. Several features were observed, including resonant excitation of Ni/sup 26+/ x rays and the transition from dielectronic recombination to direct excitation at threshold. The cross section for the KLL dielectronic recombination resonances relative to radiative recombination has been measured, and agrees with theoretical cross sections calculated using the multiconfiguration Dirac-fock model

1989-05-01

286

Positive selection of FLP-mediated unequal sister chromatid exchange products in mammalian cells.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Site-specific recombination provides a powerful tool for studying gene function at predetermined chromosomal sites. Here we describe the use of a blasticidin resistance system to select for recombination in mammalian cells using the yeast enzyme FLP. The vector is designed so that site-specific recombination reconstructs the antibiotic resistance marker within the sequences flanked by the FLP target sites. This approach allows the detection of DNA excised by FLP-mediated recombination and fac...

Aladjem, M. I.; Brody, L. L.; O Gorman, S.; Wahl, G. M.

1997-01-01

287

Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks.  

Science.gov (United States)

The multifunctional Mre11-Rad50-Nbs1 (MRN) protein complex recruits ATM/Tel1 checkpoint kinase and CtIP/Ctp1 homologous recombination (HR) repair factor to double-strand breaks (DSBs). HR repair commences with the 5'-to-3' resection of DNA ends, generating 3' single-strand DNA (ssDNA) overhangs that bind Replication Protein A (RPA) complex, followed by Rad51 recombinase. In Saccharomyces cerevisiae, the Mre11-Rad50-Xrs2 (MRX) complex is critical for DSB resection, although the enigmatic ssDNA endonuclease activity of Mre11 and the DNA-end processing factor Sae2 (CtIP/Ctp1 ortholog) are largely unnecessary unless the resection activities of Exo1 and Sgs1-Dna2 are also eliminated. Mre11 nuclease activity and Ctp1/CtIP are essential for DSB repair in Schizosaccharomyces pombe and mammals. To investigate DNA end resection in Schizo. pombe, we adapted an assay that directly measures ssDNA formation at a defined DSB. We found that Mre11 and Ctp1 are essential for the efficient initiation of resection, consistent with their equally crucial roles in DSB repair. Exo1 is largely responsible for extended resection up to 3.1 kb from a DSB, with an activity dependent on Rqh1 (Sgs1) DNA helicase having a minor role. Despite its critical function in DSB repair, Mre11 nuclease activity is not required for resection in fission yeast. However, Mre11 nuclease and Ctp1 are required to disassociate the MRN complex and the Ku70-Ku80 nonhomologous end-joining (NHEJ) complex from DSBs, which is required for efficient RPA localization. Eliminating Ku makes Mre11 nuclease activity dispensable for MRN disassociation and RPA localization, while improving repair of a one-ended DSB formed by replication fork collapse. From these data we propose that release of the MRN complex and Ku from DNA ends by Mre11 nuclease activity and Ctp1 is a critical step required to expose ssDNA for RPA localization and ensuing HR repair. PMID:21931565

Langerak, Petra; Mejia-Ramirez, Eva; Limbo, Oliver; Russell, Paul

2011-09-01

288

Homologous Recombination via Synthesis-Dependent Strand Annealing in Yeast Requires the Irc20 and Srs2 DNA Helicases  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Synthesis-dependent strand-annealing (SDSA)-mediated homologous recombination replaces the sequence around a DNA double-strand break (DSB) with a copy of a homologous DNA template, while maintaining the original configuration of the flanking regions. In somatic cells at the 4n stage, Holliday-junction-mediated homologous recombination and nonhomologous end joining (NHEJ) cause crossovers (CO) between homologous chromosomes and deletions, respectively, resulting in loss of heterozygosity (LOH)...

2012-01-01

289

Spin dependent recombination  

International Nuclear Information System (INIS)

The spin dependent recombination (SDR) technique is used to observe the 29Si hyperfine spectra of radiation-induced Pb centers at the Si/SiO2 interface in a MOSFET. The Pb center is a paramagnetic, trivalent silicon defect that is the dominant radiation-induced interface state. The 29Si hyperfine spectra give detailed atomic scale information about the Pb center. The authors' SDR results show that the 29Si hyperfine spectra vary with surface potential. This result indicates that differences in the defect's local geometry lead to substantial differences in the defect's energy level. However, the 29Si hyperfine spectra are found to be relatively independent of the ionizing radiation dosage

1990-12-01

290

Recombinant glucose uptake system  

Energy Technology Data Exchange (ETDEWEB)

Recombinant organisms are disclosed that contain a pathway for glucose uptake other than the pathway normally utilized by the host cell. In particular, the host cell is one in which glucose transport into the cell normally is coupled to PEP production. This host cell is transformed so that it uses an alternative pathway for glucose transport that is not coupled to PEP production. In a preferred embodiment, the host cell is a bacterium other than Z. mobilis that has been transformed to contain the glf and glk genes of Z. mobilis. By uncoupling glucose transport into the cell from PEP utilization, more PEP is produced for synthesis of products of commercial importance from a given quantity of biomass supplied to the host cells.

Ingrahm, Lonnie O. (Gainesville, FL); Snoep, Jacob L. (Groede, NL); Arfman, Nico (Delft, NL)

1997-01-01

291

Dissociative recombination of dications  

International Nuclear Information System (INIS)

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 N2 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 cm3 s-1 at 300 K was extracted. Furthermore, we present new results on the CO2+ DR rate, and a summary and comparison of measured DR rate coefficients for both the singly and doubly-charged ions of CO, CO2, and N2 is presented

2003-07-08

292

Dielectronic recombination lines of C+  

International Nuclear Information System (INIS)

The present paper presents atomic data generated to investigate the recombination lines of C II in the spectra of planetary nebulae. These data include energies of bound and autoionizing states, oscillator strengths and radiative transition probabilities, autoionization probabilities, and recombination coefficients. The R-matrix method of electron scattering theory was used to describe the C2+ plus electron system

2013-11-01

293

The Karyopherin Kap142p/Msn5p Mediates Nuclear Import and Nuclear Export of Different Cargo Proteins  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We have identified a novel pathway for protein import into the nucleus. Although the product of Saccharomyces cerevisiae gene MSN5 was previously shown to function as a karyopherin (Kap) for nuclear export of various proteins, we discovered a nuclear import pathway mediated by Msn5p (also referred to as Kap142p). We have purified from yeast cytosol a complex containing Kap142p and the trimeric replication protein A (RPA), which is required for multiple aspects of DNA metabolism, including DNA...

2001-01-01

294

Recombinant lentivector as a genetic immunization vehicle for antitumor immunity  

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Encouraged by remarkable successes in preventing infectious diseases and by the well established potential of immune system for controlling tumor growth, active therapeutic immunization approaches hold great promise for treating malignant tumors. In recent years, engineered recombinant viral vectors have been carefully examined as genetic immunization vehicles and have been demonstrated to induce potent T cell mediated immune responses that can control tumor growth. Very recent efforts sugges...

2007-01-01

295

Somatic and Germinal Recombination of a Direct Repeat in Arabidopsis  

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Homologous recombination between a pair of directly repeated transgenes was studied in Arabidopsis. The test construct included two different internal, non-overlapping deletion alleles of npt (neomycin phosphotransferase) flanking an active HPT (hygromycin phosphotransferase) gene. This construct was introduced into Arabidopsis by agrobacterium-mediated transformation with selection for resistance to hygromycin, and two independent single-insert lines were analyzed. Selection for active NPT b...

Assaad, F. F.; Signer, E. R.

1992-01-01

296

Similarity of Recombinant Human Perlecan Domain 1 by Alternative Expression Systems Bioactive Heterogenous Recombinant Human Perlecan D1  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Heparan sulfate glycosaminoglycans are diverse components of certain proteoglycans and are known to interact with growth factors as a co-receptor necessary to induce signalling and growth factor activity. In this report we characterize heterogeneously glycosylated recombinant human perlecan domain 1 (HSPG2 abbreviated as rhPln.D1 synthesized in either HEK 293 cells or HUVECs by transient gene delivery using either adenoviral or expression plasmid technology. Results By SDS-PAGE analysis following anion exchange chromatography, the recombinant proteoglycans appeared to possess glycosaminoglycan chains ranging, in total, from 6 kDa to >90 kDa per recombinant. Immunoblot analysis of enzyme-digested high Mr rhPln.D1 demonstrated that the rhPln.D1 was synthesized as either a chondroitin sulfate or heparan sulfate proteoglycan, in an approximately 2:1 ratio, with negligible hybrids. Secondary structure analysis suggested helices and sheets in both recombinant species. rhPln.D1 demonstrated binding to rhFGF-2 with an apparent kD of 2 ± 0.2 nM with almost complete susceptibility to digestion by heparinase III in ligand blot analysis but not to chondroitinase digestion. Additionally, we demonstrate HS-mediated binding of both rhPln.D1 species to several other GFs. Finally, we corroborate the augmentation of FGF-mediated cell activation by rhPln.D1 and demonstrate mitogenic signalling through the FGFR1c receptor. Conclusions With importance especially to the emerging field of DNA-based therapeutics, we have shown here that proteoglycan synthesis, in different cell lines where GAG profiles typically differ, can be directed by recombinant technology to produce populations of bioactive recombinants with highly similar GAG profiles.

Ellis April L

2010-09-01

297

Failure patterns by prognostic group as determined by recursive partitioning analysis (RPA) of 1547 on four radiation therapy oncology group studies in operable non-small cell lung cancer (NSCLC)  

International Nuclear Information System (INIS)

Purpose: To identify groups of patients who might benefit from more aggressive systemic or local treatment based on failure patterns when unresectable NSCLC was treated by radiation therapy alone. Methods: 1547 patients from 4 RTOG trials treated by RT alone were analyzed for the patterns of first failure by PRA class which was defined by prognostic factors, e.g., stage, KPS, weight loss, pleural effusion, age. All patients were AJCC stage II, IIIA or IIIB with KPS of at least 50 and n previous radiotherapy or chemotherapy for their NSCLC. Progressions in the primary (within irradiated fields), thorax (outside irradiated area), brain and distant metastasis other than brain were compared (two-sided) for each failure category by RPA. Results: The RPA classes are four distinct subgroups that had significantly different median survivals of 12.6, 8.3, 6.2 and 3.3 months for classes I, II, III and IV respectively (all groups p=0.0002). Pair comparison showed that RPA I vs IV p<0.0001, I vs III p=0.006, II vs IV p<0.0001, and III vs IV p=0.06. Conclusions: These results suggest the burden of disease and physiologic compromise in class IV patients are sufficient to cause death before specific sites of failure can be discerned. Site specific treatment strategies (intensive local therapy, combination chemotherapy, prophylactic cranial irradiation) may lead to improved outcome in class I and II, but are unlikely to alter outcome in class III and IV

1997-01-01

298

Yeast oligo-mediated genome engineering (YOGE).  

Science.gov (United States)

High-frequency oligonucleotide-directed recombination engineering (recombineering) has enabled rapid modification of several prokaryotic genomes to date. Here, we present a method for oligonucleotide-mediated recombineering in the model eukaryote and industrial production host Saccharomyces cerevisiae , which we call yeast oligo-mediated genome engineering (YOGE). Through a combination of overexpression and knockouts of relevant genes and optimization of transformation and oligonucleotide designs, we achieve high gene-modification frequencies at levels that only require screening of dozens of cells. We demonstrate the robustness of our approach in three divergent yeast strains, including those involved in industrial production of biobased chemicals. Furthermore, YOGE can be iteratively executed via cycling to generate genomic libraries up to 10 (5) individuals at each round for diversity generation. YOGE cycling alone or in combination with phenotypic selections or endonuclease-based negative genotypic selections can be used to generate modified alleles easily in yeast populations with high frequencies. PMID:24160921

DiCarlo, James E; Conley, Andrew J; Penttilä, Merja; Jäntti, Jussi; Wang, Harris H; Church, George M

2013-12-20

299

Delayed recombination and standard rulers  

International Nuclear Information System (INIS)

Measurements of baryonic acoustic oscillations (BAOs) in galaxy surveys have been recognized as a powerful tool for constraining dark energy. However, this method relies on the knowledge of the size of the acoustic horizon at recombination derived from cosmic microwave background (CMB) anisotropy measurements. This estimate is typically derived assuming a standard recombination scheme; additional radiation sources can delay recombination altering the cosmic ionization history and the cosmological inferences drawn from CMB and BAO data. In this paper we quantify the effect of delayed recombination on the determination of dark energy parameters from future BAO surveys such as the Baryon Oscillation Spectroscopic Survey and the Wide-Field Multi-Object Spectrograph. We find the impact to be small but still not negligible. In particular, if recombination is nonstandard (to a level still allowed by CMB data), but this is ignored, future surveys may incorrectly suggest the presence of a redshift-dependent dark energy component. On the other hand, in the case of delayed recombination, adding to the analysis one extra parameter describing deviations from standard recombination does not significantly degrade the error bars on dark energy parameters and yields unbiased estimates. This is due to the CMB-BAO complementarity.

2009-02-15

300

Restrictions Limiting the Generation of DNA Double Strand Breaks during Chromosomal V(D)J Recombination  

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Antigen receptor loci are composed of numerous variable (V), diversity (D), and joining (J) gene segments, each flanked by recombination signal sequences (RSSs). The V(D)J recombination reaction proceeds through RSS recognition and DNA cleavage steps making it possible for multiple DNA double strand breaks (DSBs) to be introduced at a single locus. Here we use ligation-mediated PCR to analyze DNA cleavage intermediates in thymocytes from mice with targeted RSS mutations at the endogenous TCR?...

Tillman, Robert E.; Wooley, Andrea L.; Hughes, Maureen M.; Wehrly, Tara D.; Swat, Wojciech; Sleckman, Barry P.

2002-01-01

 
 
 
 
301

Strand breaks without DNA rearrangement in V (D)J recombination.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Somatic gene rearrangement of immunoglobulin and T-cell receptor genes [V(D)J recombination] is mediated by pairs of specific DNA sequence motifs termed signal sequences. In experiments described here, retroviral vectors containing V(D)J rearrangement cassettes in which the signal sequences had been altered were introduced into wild-type and scid (severe combined immune deficiency) pre-B cells and used to define intermediates in the V(D)J recombination pathway. The scid mutation has previousl...

Hendrickson, E. A.; Liu, V. F.; Weaver, D. T.

1991-01-01

302

Role of Blm and collaborating factors in recombination and survival following replication stress in Ustilago maydis  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Inactivation of the structural gene for the RecQ family member, BLM in human, Sgs1 in budding yeast, or Rqh1 in fission yeast leads to inappropriate recombination, chromosome abnormalities, and disturbed replication fork progression. Studies with yeasts have demonstrated that auxiliary gene functions can contribute in overlapping ways with Sgs1 or Rqh1 to circumvent or overcome lesions in DNA caused by certain genotoxic agents. In the combined absence of these functions, recombination-mediate...

Mao, Ninghui; Kojic, Milorad; Holloman, William K.

2009-01-01

303

Mutations affecting RNA polymerase I-stimulated exchange and rDNA recombination in yeast  

International Nuclear Information System (INIS)

HOT1 is a cis-acting recombination-stimulatory sequence isolated from the rDNA repeat unit of yeast. The ability of HOT1 to stimulate mitotic exchange appears to depend on its ability to promote high levels of RNA polymerase I transcription. A qualitative colony color sectoring assay was developed to screen for trans-acting mutations that alter the activity of HOT1. Both hypo-recombination and hyper-recombination mutants were isolated. Genetic analysis of seven HOT1 recombination mutants (hrm) that decrease HOT1 activity shows that they behave as recessive nuclear mutations and belong to five linkage groups. Three of these mutations, hrm1, hrm2, and hrm3, also decrease rDNA exchange but do not alter recombination in the absence of HOT1. Another mutation, hrm4, decreases HOT1-stimulated recombination but does not affect rDNA recombination or exchange in the absence of HOT1. Two new alleles of RAD52 were also isolated using this screen. With regard to HOT1 activity, rad52 is epistatic to all four hrm mutations indicating that the products of the HRM genes and of RAD52 mediate steps in the same recombination pathway. Finding mutations that decrease both the activity of HOT1 and exchange in the rDNA supports the hypothesis that HOT1 plays a role in rDNA recombination

1991-01-01

304

Double strand interaction is the predominant pathway for intermolecular recombination of adeno-associated viral genomes  

International Nuclear Information System (INIS)

Intermolecular recombination is the foundation for dual vector mediated larger gene transfer by recombinant adeno-associated virus (rAAV). To identify precursors for intermolecular recombination, we sequentially infected skeletal muscle with AAV LacZ trans-splicing viruses. At 1 month postinfection, nearly all inputting single-strand (ss) AAV genomes were cleared out in muscle. If ss-ss interaction is absolutely required for intermolecular recombination, LacZ expression from sequential infection will be negligible to that from coinfection. Interestingly, expression from sequential infection reached ?50% of that from coinfection at the 1-month time-point in BL6 mice. In immune deficient SCID mice, expression from sequential infection was comparable to that from coinfection at the 4- and 13-month time points. Our results suggest that ds interaction represents the predominant pathway for AAV intermolecular recombination

2003-08-15

305

Low Efficiency of Homology-Facilitated Illegitimate Recombination during Conjugation in Escherichia coli  

Science.gov (United States)

Homology-facilitated illegitimate recombination has been described in three naturally competent bacterial species. It permits integration of small linear DNA molecules into the chromosome by homologous recombination at one end of the linear DNA substrate, and illegitimate recombination at the other end. We report that homology-facilitated illegitimate recombination also occurs in Escherichia coli during conjugation with small non-replicative plasmids, but at a low frequency of 3×10?10 per recipient cell. The fate of linear DNA in E. coli is either RecBCD-dependent degradation, or circularisation by ligation, and integration into the chromosome by single crossing-over. We also report that the observed single crossing-overs are recA-dependent, but essentially recBCD, and recFOR independent. This suggests that other, still unknown, proteins may act as mediator for the loading of RecA on DNA during single crossing-over recombination in E. coli.

Amarir-Bouhram, Jihane; Goin, Melodie; Petit, Marie-Agnes

2011-01-01

306

Recombineering: genetic engineering in bacteria using homologous recombination.  

Science.gov (United States)

The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. It then presents support protocols that describe several two-step selection/counter-selection methods of making genetic alterations without leaving any unwanted changes in the targeted DNA, and a method for retrieving onto a plasmid a genetic marker (cloning by retrieval) from the Escherichia coli chromosome or a co-electroporated DNA fragment. Additional protocols describe methods to screen for unselected mutations, removal of the defective prophage from recombineering strains, and other useful techniques. Curr. Protoc. Mol. Biol. 106:1.16.1-1.16.39. © 2014 by John Wiley & Sons, Inc. PMID:24733238

Thomason, Lynn C; Sawitzke, James A; Li, Xintian; Costantino, Nina; Court, Donald L

2014-01-01

307

Purification of recombinant laccase from Trametes versicolor in Pichia methanolica and its use for the decolorization of anthraquinone dye.  

Science.gov (United States)

A recombinant laccase from Trametes versicolor in Pichia methanolica was produced constitutively in a defined medium. The recombinant laccase was purified using ultrafiltration, anion-exchange chromatography, and gel filtration. The molecular weight of the purified laccase was estimated as 64 kDa by SDS-PAGE. The purified recombinant laccase decolorized more than 90% of Remazol Brilliant Blue R (RBBR) initially at 80 mg l(-1) after 16 h at 45 degrees C and pH 5 when 25 U laccase ml(-1) was used. The purified recombinant laccase could efficiently decolorize RBBR without additional redox mediators. PMID:18688574

Guo, Mei; Lu, Fuping; Liu, Minyao; Li, Tuoping; Pu, Jun; Wang, Na; Liang, Peng; Zhang, Chenyun

2008-12-01

308

Recombinant Lactobacillus plantarum inhibits house dust mite-specific T-cell responses.  

Science.gov (United States)

Recent evidence suggests that chronic exposure to lactobacilli, which are part of the normal intestinal flora, inhibits the development of allergic disorders. Allergy is mediated by Th2 cells, which produce high levels of IL4 and IL5, and suppressive effects of lactic acid bacteria on the development of allergy have been attributed to their Th1-inducing properties. On the other hand, lactic acid bacteria have also been shown to suppress autoimmune disorders which are mediated by Th1 cells producing high levels of IFNgamma. To study this apparent discrepancy, the immunomodulatory potential of lactobacilli was evaluated using recombinants that express an immunodominant T-cell epitope of Der p 1 of house dust mites. Mucosal immunization of C57BL/6 J mice with such recombinants resulted in the induction of T cells which produced low amounts of IFNgamma. Immunization with the house dust mite peptide followed by treatment with recombinant Lactobacillus plantarum resulted in the inhibition of both IFNgamma and IL5 production. The effect on IFNgamma production was shown to be a non-specific effect of L. plantarum. The effect on IL5 production, however, was only observed when the recombinant expressing the Der p 1 peptide, but not the control recombinant, was used for treatment. Neither of the recombinants had an effect on the antibody response. Taken together, these data suggest that recombinant L. plantarum may be a suitable candidate for the treatment of allergic disorders. PMID:11678893

Kruisselbrink, A; Heijne Den Bak-Glashouwer, M J; Havenith, C E; Thole, J E; Janssen, R

2001-10-01

309

Hydrogen recombiner development at AECL  

International Nuclear Information System (INIS)

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 pressure of hydrogen. The recombiner also reacts carbon monoxide, in the presence of hydrogen, at approximately the same rate as the hydrogen. The catalyst materials and wet-proofing are unaffected by radiation or high temperatures. Large scale tests confirm self-start behavior and demonstrate strong mixing, irrespective of recombiner placement. (author)

1997-03-01

310

Recombinant snake venom prothrombin activators  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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 ...

2013-01-01

311

Eukaryotes arose after genetic recombination  

Directory of Open Access Journals (Sweden)

Full Text Available Division of ancestral prokaryotic pragenome into two circular double-stranded DNA molecules by genetic recombination, is a base for future separate evolution of nuclear and mitochondrial gene compartment. This suggests monophyletic origin of both, mitochondrion and nucleus. Presumed organism which genome undergoes genetic recombination has to be searched among an aerobic, oxygen nonproducing, archaeon with no rigid cell wall, but a plasma membrane. Plastid evolves from an aerobic, oxygen producing protoeukaryote, after mitoplastid genome duplication and subsequent functional segregation.

Stupar Milanko R.

2006-01-01

312

Homologous recombination between transfected DNAs.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

An extensive analysis of the fate and structure of polyomavirus-plasmid recombinant molecules transfected into Rat-1 cells has revealed that the DNA often becomes integrated within transformed cell DNA in a head-to-tail tandem arrangement. This occurs independently of the replicative capacity of the transforming DNA and is facilitated by the use of large quantities of DNA during transfection. These observations have led us to suggest that head-to-tail tandems are formed by homologous recombin...

1983-01-01

313

Two-center dielectronic recombination  

CERN Document Server

In the presence of a neighboring atom, electron-ion recombination can proceed resonantly via excitation of an electron in the atom, with subsequent relaxation through radiative decay. It is shown that this two-center dielectronic process can largely dominate over single-center radiative recombination at internuclear distances as large as several nanometers. The relevance of the predicted process is demonstrated by using examples of water-dissolved alkali cations and warm dense matter.

Müller, C; López-Urrutia, J R Crespo; Harman, Z

2010-01-01

314

Delayed recombination and cosmic parameters  

International Nuclear Information System (INIS)

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, ns, 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 1? to R=1.734±0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: ??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.

2008-09-15

315

Monitoring and Evaluation of Smolt Migration in the Columbia Basin : Volume VII : Evaluation of the Compliance Testing Framework for RPA Improvement as Stated in the 2000 Federal Columbia River Power System (FCRPS) Biological Opinion.  

Energy Technology Data Exchange (ETDEWEB)

Using the pre-2000 reach survival probabilities reported in the 2000 FCRPS Biological Opinion (BO) for three selected stocks: yearling and sub-yearling chinook and steelhead, power curves were constructed for each of the two statistical hypothesis tests suggested in the BO. These power calculation results were interpreted in terms of the ability of the statistical tests to correctly identify the true states of recovery (i.e., fail or succeed in fulfilling RPA expectations). The proposed one-sided tests have a moderate to low probability of correctly assessing the true status of the recovery by the years 2005 and 2008. The relatively poor odds of making the correct decision with the BO proposed Tests 1 and 2 suggest alternative decision rules need to be investigated and developed for assessing RPA compliance. Therefore, we propose to immediately examine alternative decision rules that might maximize the likelihood of correct decisions while minimizing the prospect of incorrect decisions. The Bayesian analysis will incorporate scientific/biological knowledge/expertise.

Skalski, John R.; Ngouenet, Roger F.

2001-05-01

316

Structure of the Mediator subunit Cyclin C and subunit interaction studies within the Mediator head module  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The Mediator of transcriptional regulation is the central coactivator that enables a response of RNA polymerase II to activators and repressors. It is conserved from yeast to human and consists of 25 subunits in yeast that are organized in four modules called head, middle, tail, and CDK8/Cyclin C module. Despite its central role in transcription the functional mechanism remains enigmatic. To overcome the lack of detailed structural data on the Mediator a recombinant expression system was esta...

Ho?ppner, Sabine

2005-01-01

317

Pairing and recombination features during meiosis in Cebus paraguayanus (Primates: Platyrrhini  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Among neotropical Primates, the Cai monkey Cebus paraguayanus (CPA presents long, conserved chromosome syntenies with the human karyotype (HSA as well as numerous C+ blocks in different chromosome pairs. In this study, immunofluorescence (IF against two proteins of the Synaptonemal Complex (SC, namely REC8 and SYCP1, two recombination protein markers (RPA and MLH1, and one protein involved in the pachytene checkpoint machinery (BRCA1 was performed in CPA spermatocytes in order to analyze chromosome meiotic behavior in detail. Results Although in the vast majority of pachytene cells all autosomes were paired and synapsed, in a small number of nuclei the heterochromatic C-positive terminal region of bivalent 11 remained unpaired. The analysis of 75 CPA cells at pachytene revealed a mean of 43.22 MLH1 foci per nucleus and 1.07 MLH1 foci in each CPA bivalent 11, always positioned in the region homologous to HSA chromosome 21. Conclusion Our results suggest that C blocks undergo delayed pairing and synapsis, although they do not interfere with the general progress of pairing and synapsis.

Garcia-Cruz Raquel

2009-06-01

318

Assessing the function of homologous recombination DNA repair in malignant pleural effusion (MPE) samples.  

Science.gov (United States)

Background:Patients with malignant pleural effusions (MPEs) generally have advanced disease with poor survival and few therapeutic options. Cells within MPEs may be used to stratify patients for targeted therapy. Targeted therapy with poly(ADP ribose) polymerase inhibitors (PARPi) depends on identifying homologous recombination DNA repair (HRR)-defective cancer cells. We aimed to determine the feasibility of assaying HRR status in MPE cells.Methods:A total of 15 MPE samples were collected from consenting patients with non-small-cell lung cancer (NSCLC), mesothelioma and ovarian and breast cancer. Primary cultures were confirmed as epithelial by pancytokeratin, and HRR status was determined by the detection of ?H2AX and RAD51 foci following a 24-h exposure to rucaparib, by immunofluorescence microscopy. Massively parallel next-generation sequencing of DNA repair genes was performed on cultured MPE cells.Results:From 15 MPE samples, 13 cultures were successfully established, with HRR function successfully determined in 12 cultures. Four samples - three NSCLC and one mesothelioma - were HRR defective and eight samples - one NSCLC, one mesothelioma, one sarcomatoid, one breast and four ovarian cancers - were HRR functional. No mutations in DNA repair genes were associated with HRR status, but there was probable loss of heterozygosity of FANCG, RPA1 and PARP1.Conclusions:HRR function can be successfully detected in MPE cells demonstrating the potential to stratify patients for targeted therapy with PARPi. PMID:24867690

Patterson, M J; Sutton, R E; Forrest, I; Sharrock, R; Lane, M; Kaufmann, A; O'Donnell, R; Edmondson, R J; Wilson, B T; Curtin, N J

2014-07-01

319

Axion mediation  

Science.gov (United States)

We explore the possibility that supersymmetry breaking is mediated to the Standard Model sector through the interactions of a generalized axion multiplet that gains a F-term expectation value. Using an effective field theory framework we enumerate the most general possible set of axion couplings and compute the Standard Model sector soft-supersymmetry-breaking terms. Unusual, non-minimal spectra, such as those of both natural and split supersymmetry are easily implemented. We discuss example models and low-energy spectra, as well as implications of the particularly minimal case of mediation via the QCD axion multiplet. We argue that if the Peccei-Quinn solution to the strong-CP problem is realized in string theory then such axion-mediation is generic, while in a field theory model it is a natural possibility in both DFSZ- and KSVZ-like regimes. Axion mediation can parametrically dominate gravity-mediation and is also cosmologically beneficial as the constraints arising from axino and gravitino overproduction are reduced. Finally, in the string context, axion mediation provides a motivated mechanism where the UV completion naturally ameliorates the supersymmetric flavor problem.

Baryakhtar, Masha; Hardy, Edward; March-Russell, John

2013-07-01

320

Endonuclease G: A role for the enzyme in recombination and cellular proliferation  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Our earlier studies had suggested that endonuclease G (EndoG), a member of the evolutionarily conserved DNA/RNA nonspecific ???-Me-finger nuclease family, functioned in the a sequence-mediated segment inversion observed during herpes simplex virus 1 replication. To test this hypothesis, we used RNA interference to reduce the level of EndoG in mammalian cells in culture. Reduction of EndoG produced a small but statistically significant decrease in a sequence-mediated recombination, suggesti...

Huang, Ke-jung; Ku, Chia-chi; Lehman, I. Robert

2006-01-01

 
 
 
 
321

Recombinant snake venom prothrombin activators.  

Science.gov (United States)

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 for additional cofactors, but does not discriminate non-carboxylated prothrombin from biologically active ?-carboxylated prothrombin. Here we report that recombinant trocarin and oscutarin could not efficiently generate thrombin without additional protein co-factors. We confirm that both trocarin and oscutarin are similar to human coagulation Factor X (FX), explaining the need for additional cofactors. Sequencing of a genomic fragment containing 7 out of the 8 exons coding for oscutarin further confirmed the similarity to human FX. PMID:23111318

Lövgren, Ann

2013-01-01

322

Inhomogeneous recombinations during cosmic reionization  

CERN Document Server

By depleting the ionizing photon budget available to expand cosmic HII regions, recombining systems (or Lyman limit systems) can have a large impact during (and following) cosmic reionization. Unfortunately, directly resolving such structures in large-scale reionization simulations is computationally impractical. Instead, here we implement a sub-grid prescription for tracking inhomogeneous recombinations in the intergalactic medium. Building on previous work parameterizing photo-heating feedback on star-formation, we present large-scale, semi-numeric reionization simulations which self-consistently track the local (sub-grid) evolution of both sources and sinks of ionizing photons. Our simple, single-parameter model naturally results in both an extended reionization and a modest, slowly-evolving emissivity, consistent with observations. Recombinations are instrumental in slowing the growth of large HII regions, and damping the rapid rise of the ionizing background in the late stages of (and following) reioniza...

Sobacchi, Emanuele

2014-01-01

323

Recombination effect in nuclear multifragmentation  

International Nuclear Information System (INIS)

Hot nuclear systems formed in intermediate-energy heavy-ion collisions may disassemble into a number of fragments either by sequential binary decay or one-step prompt multifragmentation. Any pair of these fragments during dynamical evolution may come very close to one another and recombine. The fused complex may undergo further fragmentation if sufficiently excited. Using the transition-state model for binary decay and the microcanonical model for prompt multifragmentation, the effect of this recombination is studied for a number of observables. ((orig.))

1995-04-17

324

Repair and recombination of X-irradiated plasmids in Xenopus laevis oocytes  

International Nuclear Information System (INIS)

Plasmid DNA substrates were X-irradiated and injected into the nuclei of Xenopus laevis oocytes. After incubation for 20 h, DNA was recovered from the oocytes and analyzed simultaneously for repair and for intermolecular homologous recombination by electrophoresis and bacterial transformation. Oocyte-mediated repair of DNA strand breaks was observed with both methods. Using a repair-deficient mutant Escherichia coli strain and its repair-proficient parent as hosts for the transformation assay, we also demonstrated that oocytes repaired oxidative-type DNA base damage induced by X-rays. X-irradiation of a circular DNA stimulated its potential to recombine with a homologous linear partner. Recombination products were detected directly by Southern blot hybridization and as bacterial transformant clones expressing two antibiotic resistance markers originally carried separately on the two substrates. The increase in recombination was dependent on X-ray dose. There is some suggestion that lesions other than double-strand breaks contribute to the stimulation of oocyte-mediated homologous recombination. In summary, oocytes have considerable capacity to repair X-ray-induced damage, and some X-ray lesions stimulate homologous recombination in these cells

1990-01-01

325

Genetically Engineered Poxviruses for Recombinant Gene Expression, Vaccination, and Safety  

Science.gov (United States)

Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing genes within the cytoplasm of mammalian cells. As a research tool, recombinant vaccinia viruses are used to synthesize and analyze the structure--function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of recombinant vaccinia virus has been realized in the form of an effective oral wild-life rabies vaccine, although no product for humans has been licensed. A genetically altered vaccinia virus that is unable to replicate in mammalian cells and produces diminished cytopathic effects retains the capacity for high-level gene expression and immunogenicity while promising exceptional safety for laboratory workers and potential vaccine recipients.

Moss, Bernard

1996-10-01

326

Global divergence of microbial genome sequences mediated by propagating fronts  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We model the competition between homologous recombination and point mutation in microbial genomes, and present evidence for two distinct phases, one uniform, the other genetically diverse. Depending on the specifics of homologous recombination, we find that global sequence divergence can be mediated by fronts propagating along the genome, whose characteristic signature on genome structure is elucidated, and apparently observed in closely related Bacillus strains. Front propagation provides an...

Vetsigian, Kalin; Goldenfeld, Nigel

2005-01-01

327

Axion Mediation  

CERN Document Server

We explore the possibility that supersymmetry breaking is mediated to the Standard Model sector through the interactions of a generalized axion multiplet that gains a F-term expectation value. Using an effective field theory framework we enumerate the most general possible set of axion couplings and compute the Standard Model sector soft-supersymmetry-breaking terms. Unusual, non-minimal spectra, such as those of both natural and split supersymmetry are easily implemented. We discuss example models and low-energy spectra, as well as implications of the particularly minimal case of mediation via the QCD axion multiplet. We argue that if the Peccei-Quinn solution to the strong-CP problem is realized in string theory then such axion-mediation is generic, while in a field theory model it is a natural possibility in both DFSZ- and KSVZ-like regimes. Axion mediation can parametrically dominate gravity-mediation and is also cosmologically beneficial as the constraints arising from axino and gravitino overproduction ...

Baryakhtar, Masha; March-Russell, John

2013-01-01

328

Inhomogeneous recombinations during cosmic reionization  

Science.gov (United States)

By depleting the ionizing photon budget available to expand cosmic H II regions, recombining systems (or Lyman limit systems) can have a large impact during (and following) cosmic reionization. Unfortunately, directly resolving such structures in large-scale reionization simulations is computationally impractical. Instead, here we implement a subgrid prescription for tracking inhomogeneous recombinations in the intergalactic medium. Building on previous work parametrizing photoheating feedback on star formation, we present large-scale, seminumeric reionization simulations which self-consistently track the local (subgrid) evolution of both sources and sinks of ionizing photons. Our simple, single-parameter model naturally results in both an extended reionization and a modest, slowly evolving emissivity, consistent with observations. Recombinations are instrumental in slowing the growth of large H II regions, and damping the rapid rise of the ionizing background in the late stages of (and following) reionization. As a result, typical H II regions are smaller by factors of ˜2 to 3 throughout reionization. The large-scale (k ? 0.2 Mpc-1) ionization power spectrum is suppressed by factors of ?2-3 in the second half of reionization. Therefore properly modelling recombinations is important in interpreting virtually all reionization observables, including upcoming interferometry with the redshifted 21cm line. Consistent with previous works, we find the clumping factor of ionized gas to be C H II ˜ 4 at the end of reionization.

Sobacchi, Emanuele; Mesinger, Andrei

2014-05-01

329

Recombination in immunoglobulin gene loci  

Directory of Open Access Journals (Sweden)

Full Text Available Gene network of the lymphoid cell differentiation coordinates precisely the recombination process in immunoglobulin gene loci. In our opinion, cellular microRNAs can contribute to the allelic exclusion through microRNA-directed DNA methylation and participate in retargeting recombinases activity from the gene loci of heavy immunoglobulin chains to the gene loci of light chains

Komisarenko S. V.

2009-02-01

330

AID-driven deletion causes immunoglobulin heavy chain locus suicide recombination in B cells.  

Science.gov (United States)

Remodeling of immunoglobulin genes by activation-induced deaminase (AID) is required for affinity maturation and class-switch recombination in mature B lymphocytes. In the immunoglobulin heavy chain locus, these processes are predominantly controlled by the 3' cis-regulatory region. We now show that this region is transcribed and undergoes AID-mediated mutation and recombination around phylogenetically conserved switchlike DNA repeats. Such recombination, which we term locus suicide recombination, deletes the whole constant region gene cluster and thus stops expression of the immunoglobulin of the B cell surface, which is critical for B cell survival. The frequency of this event is approaching that of class switching and makes it a potential regulator of B cell homeostasis. PMID:22539552

Péron, Sophie; Laffleur, Brice; Denis-Lagache, Nicolas; Cook-Moreau, Jeanne; Tinguely, Aurélien; Delpy, Laurent; Denizot, Yves; Pinaud, Eric; Cogné, Michel

2012-05-18

331

Decolorization of Alizarin Red and other synthetic dyes by a recombinant laccase from Pichia pastoris.  

Science.gov (United States)

A cDNA encoding for a laccase was isolated from the white-rot fungus Lenzites gibbosa by RT-PCR and expressed in the Pichia pastoris. The laccase native signal peptide efficiently directed the secretion of the recombinant laccase in an active form. Factors influencing laccase expression, such as pH, cultivation temperature, copper concentration and methanol concentration, were optimized. The recombinant enzyme was purified to electrophoretic homogeneity, and was estimated to have a MW of ~61.5 kDa. The purified enzyme behaved similarly to the native laccase produced by L. gibbosa and efficiently decolorized Alizarin Red, Neutral Red, Congo Red and Crystal Violet, without the addition of redox mediators. The decolorization capacity of this recombinant enzyme suggests that it could be a useful biocatalyst for the treatment of dye-containing effluents. This study is the first report on the synthetic dye decolorization by a recombinant L. gibbosa laccase. PMID:24078122

Zheng, Miaomiao; Chi, Yujie; Yi, Hongwei; Shao, Shuli

2014-01-01

332

Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for V(D)J recombination  

Digital Repository Infrastructure Vision for European Research (DRIVER)

DNA-dependent protein kinase (DNA-PK) is utilized in both DNA double-strand break repair (DSBR) and V(D)J recombination, but the mechanism by which this multiprotein complex participates in these proces­ses is unknown. To evaluate the importance of DNA-PK-mediated protein phosphorylation in DSBR and V(D)J recombination, we assessed the effects of the phosphatidyl inositol 3-kinase inhibitor wortmannin on the repair of ionizing radiation-induced DNA double-strand breaks and V(D)J recombinatio...

Kienker, Laura J.; Shin, Euy Kyun; Meek, Katheryn

2000-01-01

333

Glycoengineering approach to half-life extension of recombinant biotherapeutics.  

Science.gov (United States)

The potential for protein-engineered biotherapeutics is enormous, but pharmacokinetic modulation is a major challenge. Manipulating pharmacokinetics, biodistribution, and bioavailability of small peptide/protein units such as antibody fragments is a major pharmaceutical ambition, illustrated by the many chemical conjugation and recombinant fusion approaches being developed. We describe a recombinant approach that leads to successful incorporation of polysialic acid, PSA for the first time, onto a therapeutically valuable protein. This was achieved by protein engineering of the PSA carrier domain of NCAM onto single-chain Fv antibody fragments (one directed against noninternalizing carcinoembryonic antigen-CEA and one against internalizing human epidermal growth factor receptor-2-HER2). This created novel polysialylated antibody fragments with desired pharmacokinetics. Production was achieved in human embryonic kidney cells engineered to express human polysialyltransferase, and the recombinant, glycosylated product was successfully fractionated by ion-exchange chromatography. Polysialylation was verified by glycosidase digestion and mass spectrometry, which showed the correct glycan structures and PSA chain length similar to that of native NCAM. Binding was demonstrated by ELISA and surface plasmon resonance and on live cells by flow cytometry and confocal immunofluorescence. Unexpectedly, polysialylation inhibited receptor-mediated endocytosis of the anti-HER2 scFv. Recombinant polysialylation led to an estimated 3-fold increase in hydrodynamic radius, comparable to PEGylation, leading to an almost 30-fold increase in blood half-life and a similar increase in blood exposure. This increase in bioavailability led to a 12-fold increase in tumor uptake by 24 h. In summary, recombinant polysialylation of antibody fragments in our system is a novel and feasible approach applicable for pharmacokinetic modulation, and may have wider applications. PMID:22681552

Chen, Chen; Constantinou, Antony; Chester, Kerry A; Vyas, Bijal; Canis, Kevin; Haslam, Stuart M; Dell, Anne; Epenetos, Agamemnon A; Deonarain, Mahendra P

2012-08-15

334

Antibacterial activity of recombinant murine beta interferon.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Recombinant murine beta interferon was protective and therapeutic for mice against Listeria monocytogenes infection in vivo. The recombinant murine beta interferon caused enhanced H2O2 release by macrophages in vivo, but not in vitro.

Fujiki, T.; Tanaka, A.

1988-01-01

335

The ?0 polarization and the recombination mechanism  

International Nuclear Information System (INIS)

We use the recombination and the Thomas Precession Model to obtain a prediction for the ?0 polarization in the p+p??0+X reaction. We study the effect of the recombination function on the ?0 polarization

1997-03-15

336

A Genomewide Screen for Suppressors of Alu-Mediated Rearrangements Reveals a Role for PIF1  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Alu-mediated rearrangement of tumor suppressor genes occurs frequently during carcinogenesis. In breast cancer, this mechanism contributes to loss of the wild-type BRCA1 allele in inherited disease and to loss of heterozygosity in sporadic cancer. To identify genes required for suppression of Alu-mediated recombination we performed a genomewide screen of a collection of 4672 yeast gene deletion mutants using a direct repeat recombination assay. The primary screen and subsequent analysis ident...

2012-01-01

337

Strand exchange activity of human recombination protein Rad52  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Repair of double-strand breaks is essential for the maintenance of genome integrity and cell survival. In eukaryotes, double-strand-break repair by homologous recombination requires the Rad52 group of proteins. Human Rad52 protein (HsRad52)-mediated annealing of complementary strands has been studied in detail, but little has been reported on the recombinase activities of HsRad52. For this study, we purified HsRad52 from Escherichia coli. DNase I protection experiments indicated that HsRad52 ...

Kumar, Jaspal K.; Gupta, Ravindra C.

2004-01-01

338

Ribonuclease activity and RNA binding of recombinant human Dicer  

Digital Repository Infrastructure Vision for European Research (DRIVER)

RNA silencing phenomena, known as post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double-stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. Here, we report cloning and expression of the 218 kDa human Dicer, and characterization of its ribonuclease activity and dsRNA-binding properties. The recombinant enzyme generated ?21–23 nucleotide products from dsRNA. Processing of the microRNA let-...

Provost, Patrick; Dishart, David; Doucet, Johanne; Frendewey, David; Samuelsson, Bengt; Ra?dmark, Olof

2002-01-01

339

Recombination sites in plasmid drug resistance gene amplification.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The resistance plasmid NR1 derivative pRR330 consists of a neomycin-kanamycin resistance gene (neo-kan) flanked by directly repeated sequences of both insertion element IS1 DNA (768 base pairs) and 840 base pairs of DNA which are a part of the chloramphenicol acetyltransferase (cam) gene. Most Escherichia coli cell populations that were cultured in high neomycin concentrations carried plasmids whose neo-kan gene amplification was mediated either by IS1 DNA or by cam DNA as homologous recombin...

Peterson, B. C.; Rownd, R. H.

1985-01-01

340

Comparative analysis of right element mutant lox sites on recombination efficiency in embryonic stem cells  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Cre-mediated site-specific integrative recombination in mouse embryonic stem (ES cells is a useful tool for genome engineering, allowing precise and repeated site-specific integration. To promote the integrative reaction, a left element/right element (LE/RE mutant strategy using a pair of lox sites with mutations in the LE or RE of the lox sequence has previously been developed. Recombination between LE and RE mutant lox produces a wild-type loxP site as well as an LE+RE double mutant lox site, which has mutations in both sides and less affinity to Cre, resulting in stable integration. We previously demonstrated successful integrative recombination using lox71 (an LE mutant and lox66 (an RE mutant in ES cells. Recently, other LE/RE mutant lox sites showing higher recombination efficiency in Escherichia coli have been reported. However, their recombination efficiency in mammalian cells remains to be analyzed. Results Using ES cells, we compared six RE mutant lox sites, focusing on their recombination efficiency with lox71. All of the RE mutant lox sites showed similar recombination efficiency. We then analyzed the stability of the recombined product, i.e., the LE+RE double mutant lox site, under continuous and strong Cre activity in ES cells. Two RE mutants, loxJTZ17 and loxKR3, produced more stable LE+RE double mutant lox than did the lox66/71 double mutant. Conclusion The two mutant RE lox sites, loxJTZ17 and loxKR3, are more suitable than lox66 for Cre-mediated integration or inversion in ES cells.

Araki Masatake

2010-03-01

 
 
 
 
341

Pharmacology of recombinant or genetically engineered drugs  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Recombinant technology or genetic engineering is a modern method used for the synthesis of therapeutic agents. The central theme of recombinant technology is the process of "gene cloning" which consists of the production of a defined fragment of DNA and its propagation and amplification in a suitable host cell. Drugs developed by recombinant technology or genetic engineering are known as biologics, biopharmaceuticals, recombinant DNA expressed products, bioengineered, or genetically engineere...

Kishore Kamal; Krishan Pawan

2009-01-01

342

Oligonucleotide Recombination in Gram-Negative Bacteria  

Digital Repository Infrastructure Vision for European Research (DRIVER)

This report describes several key aspects of a novel form of RecA-independent homologous recombination. We found that synthetic single stranded DNA oligonucleotides (oligos) introduced into bacteria by transformation can site-specifically recombine with bacterial chromosomes in the absence of any additional phage encoded functions. Oligo recombination was tested in four genera of Gram-negative bacteria and in all cases evidence for recombination was apparent. The experiments presented here we...

Swingle, Bryan; Markel, Eric; Costantino, Nina; Bubunenko, Mikhail G.; Cartinhour, Samuel; Court, Donald L.

2010-01-01

343

Homology requirements for recombination in Escherichia coli.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The DNA sequence homology required for recombination in Escherichia coli has been determined by measuring the recombination frequency between insulin DNA in a miniplasmid pi VX and a homologous sequence in a bacteriophage lambda vector. A minimum of approximately equal to 20 base pairs in a completely homologous segment is required for significant recombination. There is an exponential increase in the frequency of recombination when the length of homologous DNA is increased from 20 base pairs...

Watt, V. M.; Ingles, C. J.; Urdea, M. S.; Rutter, W. J.

1985-01-01

344

Population inversion in recombining hydrogen plasma  

International Nuclear Information System (INIS)

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)

1978-01-01

345

The effect of a single recombination event  

DEFF Research Database (Denmark)

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 of the effect of a recombination event is the genealogical type of the event and whether SNP variation is present that can reveal the genealogical consequences of the recombination event. Recombination events that only change some branch lengths in the genealogy have a very small, but detectable, effect. The more lineages left when the recombination event occurs, the larger effect it has, implying that it is mainly young recombination events that we detect when estimating the rate. If the population is growing, though, more lineages are present back in time and relatively more ancient recombination events may leave a stronger effect on data. We also investigate the amount of recombination events expected to be shared by two populations as a function of their separation time and explicitly model the European and African population in at attempt to survey how large an effect recombination events shared by these two populations are expected to contribute compared to the effect of private recombination events

Schierup, Mikkel Heide; Jensen, Thomas Mailund

346

Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL*S?  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The RAD51 protein is a central player in homologous recombinational repair. The RAD51B protein is one of five RAD51 paralogs that function in the homologous recombinational repair pathway in higher eukaryotes. In the present study, we found that the human EVL (Ena/Vasp-like) protein, which is suggested to be involved in actin-remodeling processes, unexpectedly binds to the RAD51 and RAD51B proteins and stimulates the RAD51-mediated homologous pairing and strand exchang...

Takaku, Motoki; Machida, Shinichi; Hosoya, Noriko; Nakayama, Shugo; Takizawa, Yoshimasa; Sakane, Isao; Shibata, Takehiko; Miyagawa, Kiyoshi; Kurumizaka, Hitoshi

2009-01-01

347

The Saccharomyces cerevisiae DNA recombination and repair functions of the RAD52 epistasis group inhibit Ty1 transposition.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

RNA transcribed from the Saccharomyces cerevisiae retrotransposon Ty1 accumulates to a high level in mitotically growing haploid cells, yet transposition occurs at very low frequencies. The product of reverse transcription is a linear double-stranded DNA molecule that reenters the genome by either Ty1-integrase-mediated insertion or homologous recombination with one of the preexisting genomic Ty1 (or delta) elements. Here we examine the role of the cellular homologous recombination functions ...

Rattray, A. J.; Shafer, B. K.; Garfinkel, D. J.

2000-01-01

348

Recombination in Enteroviruses Is a Biphasic Replicative Process Involving the Generation of Greater-than Genome Length 'Imprecise' Intermediates  

Science.gov (United States)

Recombination in enteroviruses provides an evolutionary mechanism for acquiring extensive regions of novel sequence, is suggested to have a role in genotype diversity and is known to have been key to the emergence of novel neuropathogenic variants of poliovirus. Despite the importance of this evolutionary mechanism, the recombination process remains relatively poorly understood. We investigated heterologous recombination using a novel reverse genetic approach that resulted in the isolation of intermediate chimeric intertypic polioviruses bearing genomes with extensive duplicated sequences at the recombination junction. Serial passage of viruses exhibiting such imprecise junctions yielded progeny with increased fitness which had lost the duplicated sequences. Mutations or inhibitors that changed polymerase fidelity or the coalescence of replication complexes markedly altered the yield of recombinants (but did not influence non-replicative recombination) indicating both that the process is replicative and that it may be possible to enhance or reduce recombination-mediated viral evolution if required. We propose that extant recombinants result from a biphasic process in which an initial recombination event is followed by a process of resolution, deleting extraneous sequences and optimizing viral fitness. This process has implications for our wider understanding of ‘evolution by duplication’ in the positive-strand RNA viruses.

Lowry, Kym; Woodman, Andrew; Cook, Jonathan; Evans, David J.

2014-01-01

349

Phase II Radiation Therapy Oncology Group trial of conventional radiation therapy followed by treatment with recombinant interferon-? for supratentorial glioblastoma: Results of RTOG 9710  

International Nuclear Information System (INIS)

Purpose: The aim of this study was to determine whether recombinant human interferon ?-1a (rhIFN-?), when given after radiation therapy, improves survival in glioblastoma. Methods and Materials: After surgery, 109 patients with newly diagnosed supratentorial glioblastoma were enrolled and treated with radiation therapy (60 Gy). A total of 55 patients remained stable after radiation and were treated with rhIFN-? (6 MU/day i.m., 3 times/week). Outcomes were compared with Radiation Therapy Oncology Group glioma historical database. Results: RhIFN-? was well tolerated, with 1 Grade 4 toxicity and 8 other patients experiencing Grade 3 toxicity. Median survival time (MST) of the 55 rhIFN-?-treated patients was 13.4 months. MST for the 34 rhIFN-?-treated in RPA Classes III and IV was 16.9 vs. 12.4 months for historical controls (hazard ratio [HR] = 1.27, 95% confidence interval [CI] = 0.89-1.81). There was also a trend toward improved survival across all RPA Classes comparing the 55 rhIFN-? treated patients and 1,658 historical controls (HR = 1.24, 95% CI = 0.94-1.63). The high rate of early failures (54/109) after radiation and before initiation of rhIFN-? was likely caused by stricter interpretation of early radiographic changes in the current study. Matched-pair and intent-to-treat analyses performed to try to address this bias showed no difference in survival between study patients and controls. Conclusion: RhIFN-? given after conventional radiation therapy was well tolerated, with a trend toward survival benefit in patients who remained stable after radiation therapy. These data suggest that rhIFN-? warrants further evaluation in additional studies, possibly in combination with current temozolomide-based regimens

2006-11-01

350

Recombination lasing in heliumlike silicon  

International Nuclear Information System (INIS)

A major goal of current X-ray laser research is the achievement of gain in the 23.3 A-43.7 A wavelength region, known as the ''water window.'' Silicon is the lowest atomic number element for which all the heliumlike 3-2 transitions lie in this region. The authors examine the fundamental kinetics of recombination lasing in this species and conclude that the Si XIII 1s3d/sup 1/D/sub 2/-1s2rho/sup 1/P/sub 1/ line at 39.1 A is an attractive candidate for recombination-pumped lasing. Attainment of gain in this line is somewhat more energetically favorable than for the hydrogenic Al XIII 3-2 transitions, but radiative trapping may be somewhat more troublesome than for H-like Al

1988-01-01

351

The CMB bispectrum from recombination  

CERN Document Server

We compute the cosmic microwave background temperature bispectrum generated by nonlinearities at recombination on all scales. We use CosmoLib$2^{\\rm nd}$, a numerical Boltzmann code at second-order to compute CMB 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 per cent 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 $l_{\\rm max} =2000$, its signal-to-noise is $S/N=0.47$ and will bias a local signal by $f_{\\rm NL}^{\\rm loc}\\simeq 0.82$.

Huang, Zhiqi

2012-01-01

352

Recombination Fluorescence in Ultracold Neutral Plasmas  

International Nuclear Information System (INIS)

We present the first measurements and simulations of recombination fluorescence from ultracold neutral calcium plasmas. This method probes three-body recombination at times less than 1 ?s, shorter than previously published time scales. For the lowest initial electron temperatures, the recombination rate scales with the density as n02.2, significantly slower than the predicted n03. Recombination fluorescence opens a new diagnostic window in ultracold plasmas. In most cases it probes deeply bound level populations that depend critically on electron energetics. However, a perturbation in the calcium 4snd Rydberg series allows our fluorescence measurements to probe the population in weakly bound levels that result just after recombination

2008-08-15

353

Interface recombination influence on carrier transport  

International Nuclear Information System (INIS)

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)

2013-02-01

354

To the theory of dissociative recombination  

International Nuclear Information System (INIS)

An analytical expression for cross section of dissociative recombination of electron and molecular ion has been obtained. The Morse potential has been choosen as an internuclear potential of both ground electron state of the molecular ion and resonance molecular state. To obtain the recombination coefficient, averaging of dissociative recombination cross section in the Maxwell distribution has been performed in consequence of which the recombination coefficient temperature dependence, ? approximately Tsup(-1/2), has been determined. The temperature dependence of the dissociative recombination which well agrees with experiment has been calculated for the e"+NO"+ process as an example

1982-01-01

355

Inhibited Recombination of Charged Magnetoexcitons  

CERN Multimedia

Time-resolved photoluminescence measurements show that the decay time for charged excitons in a GaAs two-dimensional electron gas increases by an order of magnitude at high magnetic fields. Unlike neutral excitons, the charged exciton center-of-mass is spatially confined in a ``magnetically-adjustable quantum dot'' by the cyclotron orbit and the quantum well. The inhibited recombination is explained by a reduced phase coherence volume of the magnetically-confined charged excitons.

Okamura, H; Sundaram, M; Gossard, A C

1998-01-01

356

Characterization of new recombinant noroviruses  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Noroviruses are important etiologic agents of acute gastroenteritis and show great genetic diversity. To characterize more fully previously detected strains that could not be assigned unequivocally to one particular genotype based on the RNA polymerase, we have sequenced a region in the capsid gene and, in some cases, in the junction between open reading frame 1 (ORF1) and ORF2. The results allowed us to identify several recombinant noroviruses: GGHb viruses were detected for the first time i...

Ambert Balay, K.; Bon, F.; Le Guyader, Soizick; Pothier, P.; Kohli, E.

2005-01-01

357

Characterization of New Recombinant Noroviruses  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Noroviruses are important etiologic agents of acute gastroenteritis and show great genetic diversity. To characterize more fully previously detected strains that could not be assigned unequivocally to one particular genotype based on the RNA polymerase, we have sequenced a region in the capsid gene and, in some cases, in the junction between open reading frame 1 (ORF1) and ORF2. The results allowed us to identify several recombinant noroviruses: GGIIb viruses were detected for the first time ...

Ambert-balay, K.; Bon, F.; Le Guyader, F.; Pothier, P.; Kohli, E.

2005-01-01

358

Microbial factories for recombinant pharmaceuticals  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Abstract Most of the hosts used to produce the 151 recombinant pharmaceuticals so far approved for human use by the Food and Drug Administration (FDA) and/or by the European Medicines Agency (EMEA) are microbial cells, either bacteria or yeast. This fact indicates that despite the diverse bottlenecks and obstacles that microbial systems pose to the efficient production of functional mammalian proteins, namely lack or unconventional post-translational modifications, proteolytic insta...

Ferrer-Miralles Neus; Domingo-Espín Joan; Corchero José; Vázquez Esther; Villaverde Antonio

2009-01-01

359

Inhibited Recombination of Charged Magnetoexcitons  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Time-resolved photoluminescence measurements show that the decay time for charged excitons in a GaAs two-dimensional electron gas increases by an order of magnitude at high magnetic fields. Unlike neutral excitons, the charged exciton center-of-mass is spatially confined in a ``magnetically-adjustable quantum dot'' by the cyclotron orbit and the quantum well. The inhibited recombination is explained by a reduced phase coherence volume of the magnetically-confined charged exc...

Okamura, H.; Heiman, D.; Sundaram, M.; Gossard, A. C.

1998-01-01

360

Recombinations of Busy Beaver Machines  

CERN Multimedia

Many programmers belive that Turing-based machines cannot think. We also believe in this, however it is interesting to note that the most sophisticated machines are not programmed by human beings. We have only discovered them. In this paper, using well-known Busy Beaver and Placid Platypus machines, we generate further very similar, but not exactly the same machines. We have found a recombinated BB_5 machine which can make 70.740.809 steps before halting.

Bátfai, Norbert

2009-01-01

 
 
 
 
361

Comparison of Adjuvant Efficacy of Herpes Simplex Virus Type 1 Recombinant Viruses Expressing TH1 and TH2 Cytokine Genes  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The adjuvant effects of cytokines in humoral and cell-mediated immunity to herpes simplex virus type 1 (HSV-1) have been examined in mice using HSV-1 recombinant viruses expressing murine interleukin-2 (IL-2), IL-4, or gamma interferon (IFN-?) gene. Groups of naive BALB/c mice were immunized intraperitoneally with one or three doses of the HSV-1 recombinant viruses expressing IL-2, IL-4, or IFN-? or with parental control virus. Despite similar replication kinetics, these three recombinant v...

Osorio, Yanira; Ghiasi, Homayon

2003-01-01

362

Nondisjunction of chromosome 15: Origin and recombination  

Energy Technology Data Exchange (ETDEWEB)

Thirty-two cases of uniparental disomy (UPD), ascertained from Prader-Willi syndrome patients (N=27) and Angelman syndrome patients (N-5), are used to investigate the pattern of recombination associated with nondisjunction of chromosome 15. In addition, the meiotic stage of nondisjunction is inferred by using markers mapping near the centromere. Two basic approaches to the analysis of recombination in specific pairwise intervals along the chromosome. This method shows a significant reduction in recombination for two of five intervals examined. Second, the observed frequency of each recombinant class (i.e., zero, one, two, three, or more observable crossovers) is compared with expected values. This is useful for testing whether the reduction in recombination can be attributed solely to a proportion of cases with no recombination at all (because of asynapsis), with the remaining groups showing normal recombination (or even excess recombination), or whether recombination is uniformly reduced. Analysis of maternal UPD(15) data shows a slight reduction in the multiple-recombinant classes, with a corresponding increase in both the zero- and one-recombinant classes over expected values. The majority, more than 82%, of the extra chromosomes in maternal UPD(15) cases are due to meiotic I nondisjunction events. In contrast, more paternal UPD(15) cases so far examined appear to have a postzygotic origin of the extra paternal chromosome. 33 refs., 1 fig., 7 tabs.

Robinson, W.P.; Bernasconi, F.; Schinzel, A.A.; Mutirangura, A.; Ledbetter, D.H. (Baylor College of Medicine, Houston, TX (United States)); Langlois, S. (Univ. of Britisch Columbia, Vancouver (Canada)); Morris, M.A.; Malcolm, S.

1993-09-01

363

Homologous Recombination in Negative Sense RNA Viruses  

Directory of Open Access Journals (Sweden)

Full Text Available Recombination is an important process that influences biological evolution at many different levels. More and more homologous recombination events have been reported among negative sense RNA viruses recently. While sporadic authentic examples indicate that homologous recombination does occur, recombination seems to be generally rare or even absent in most negative sense RNA viruses, and most of the homologous recombination events reported in the literature were likely generated artificially due to lab contamination or inappropriate bioinformatics methods. Homologous recombination in negative sense RNA viruses should be reported with caution in the future, and only after stringent quality control efforts. Moreover, co-infection experiments should be performed to confirm whether recombination can occur.

Michael Worobey

2011-08-01

364

OsHUS1 Facilitates Accurate Meiotic Recombination in Rice.  

Science.gov (United States)

Meiotic recombination normally takes place between allelic sequences on homologs. This process can also occur between non-allelic homologous sequences. Such ectopic interaction events can lead to chromosome rearrangements and are normally avoided. However, much remains unknown about how these ectopic interaction events are sensed and eliminated. In this study, using a screen in rice, we characterized a homolog of HUS1 and explored its function in meiotic recombination. In Oshus1 mutants, in conjunction with nearly normal homologous pairing and synapsis, vigorous, aberrant ectopic interactions occurred between nonhomologous chromosomes, leading to multivalent formation and subsequent chromosome fragmentation. These ectopic interactions relied on programed meiotic double strand breaks and were formed in a manner independent of the OsMER3-mediated interference-sensitive crossover pathway. Although early homologous recombination events occurred normally, the number of interference-sensitive crossovers was reduced in the absence of OsHUS1. Together, our results indicate that OsHUS1 might be involved in regulating ectopic interactions during meiosis, probably by forming the canonical RAD9-RAD1-HUS1 (9-1-1) complex. PMID:24901798

Che, Lixiao; Wang, Kejian; Tang, Ding; Liu, Qiaoquan; Chen, Xiaojun; Li, Yafei; Hu, Qing; Shen, Yi; Yu, Hengxiu; Gu, Minghong; Cheng, Zhukuan

2014-06-01

365

Telomerase-null survivor screening identifies novel telomere recombination regulators.  

Science.gov (United States)

Telomeres are protein-DNA structures found at the ends of linear chromosomes and are crucial for genome integrity. Telomeric DNA length is primarily maintained by the enzyme telomerase. Cells lacking telomerase will undergo senescence when telomeres become critically short. In Saccharomyces cerevisiae, a very small percentage of cells lacking telomerase can remain viable by lengthening telomeres via two distinct homologous recombination pathways. These "survivor" cells are classified as either Type I or Type II, with each class of survivor possessing distinct telomeric DNA structures and genetic requirements. To elucidate the regulatory pathways contributing to survivor generation, we knocked out the telomerase RNA gene TLC1 in 280 telomere-length-maintenance (TLM) gene mutants and examined telomere structures in post-senescent survivors. We uncovered new functional roles for 10 genes that affect the emerging ratio of Type I versus Type II survivors and 22 genes that are required for Type II survivor generation. We further verified that Pif1 helicase was required for Type I recombination and that the INO80 chromatin remodeling complex greatly affected the emerging frequency of Type I survivors. Finally, we found the Rad6-mediated ubiquitination pathway and the KEOPS complex were required for Type II recombination. Our data provide an independent line of evidence supporting the idea that these genes play important roles in telomere dynamics. PMID:23390378

Hu, Yan; Tang, Hong-Bo; Liu, Ning-Ning; Tong, Xia-Jing; Dang, Wei; Duan, Yi-Min; Fu, Xiao-Hong; Zhang, Yang; Peng, Jing; Meng, Fei-Long; Zhou, Jin-Qiu

2013-01-01

366

Vaccination of Tamarin with Recombinant Vaccinia Virus Expressing Epstein Barr Virus (EBV Latent Proteins  

Directory of Open Access Journals (Sweden)

Full Text Available The aim of this work was to see whether tamarin immunisation with recombinant vaccinia virus expressing Epstein Barr Virus latent proteins could prime T cells which were, on activation, able to inhibit the outgrowth of Epstein Barr virus transformed cells in vitro. The vaccination appeared to be successful as all vaccinated tamarins developed vaccinia lesions. However, the vaccination protocol did not elicit a cell-mediated response capable of inhibiting the outgrowth of autologous Lymphoblastoid Cell Lines (LCLs as seen in the tamarin infected with whole EBV, even though the recombinant vaccinia viruses used expressed the antigens commonly recognised by sero positive humans.

Suzan Finerty

2004-09-01

367

Bacteriophage recombination systems and biotechnical applications.  

Science.gov (United States)

Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, ?, and N15, the integrase from the Streptomyces phage, ?C31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed. PMID:24442504

Nafissi, Nafiseh; Slavcev, Roderick

2014-04-01

368

Shu1 promotes homolog bias of meiotic recombination in Saccharomyces cerevisiae.  

Science.gov (United States)

Homologous recombination occurs closely between homologous chromatids with highly ordered recombinosomes through RecA homologs and mediators. The present study demonstrates this relationship during the period of "partner choice" in yeast meiotic recombination. We have examined the formation of recombination intermediates in the absence or presence of Shu1, a member of the PCSS complex, which also includes Psy3, Csm2, and Shu2. DNA physical analysis indicates that Shu1 is essential for promoting the establishment of homolog bias during meiotic homologous recombination, and the partner choice is switched by Mek1 kinase activity. Furthermore, Shu1 promotes both crossover (CO) and non-crossover (NCO) pathways of meiotic recombination. The inactivation of Mek1 kinase allows for meiotic recombination to progress efficiently, but is lost in homolog bias where most doublestrand breaks (DSBs) are repaired via stable intersister joint molecules. Moreover, the Srs2 helicase deletion cells in the budding yeast show slightly reduced COs and NCOs, and Shu1 promotes homolog bias independent of Srs2. Our findings reveal that Shu1 and Mek1 kinase activity have biochemically distinct roles in partner choice, which in turn enhances the understanding of the mechanism associated with the precondition for homolog bias. PMID:24213600

Hong, Soogil; Kim, Keun Pil

2013-11-01

369

Heterogeneity in recombinant protein production  

DEFF Research Database (Denmark)

A crucial step in biotechnology is the scale-up process. Normally, lab scale verification and optimization of production processes and strains are performed in small reactors with perfect mixing and hence the cells experience a homogenous environment. The gradients that occur in industrial scale bioreactors are often not taken into consideration in these experiments. Gradients occur due to insufficient mixing in the reactor, and affect the process in a variety of ways. When cells travel through the reactor and encounter different substrate concentrations, oxygen availability, pH, temperature, etc. the cell physiology is affected. Cells are stressed, and this may severely affect growth, by-product accumulation, biomass yield and recombinant product yield. The stress caused by exposure to divergent microenvironments, genetic differences of individual cells, differing cell cycle stage and cell age, all contribute to make a population in a fermenter heterogeneous, resulting in cell-to-cell variation in physiological parameters of the microbial culture. Our study aims at investigating how population heterogeneity and recombinant protein production is affected by environmental gradients in bioreactors. For this purpose, a Saccharomyces cerevisiae strain, that functions as a protein production reporter, has been developed. A heterologous protein has been tagged with a fluorescent protein providing a way to measure the amount of heterologous protein produced by the cells on single cell level. Gradients are simulated in small bioreactors and the population heterogeneity can be visualised by analysing single cells with flow cytometry. This can give new insights to cell physiology and recombinant protein production at the industrial scale.

Schalén, Martin; Johanson, Ted

2012-01-01

370

Selenomethionine labeling of recombinant proteins.  

Science.gov (United States)

Selenomethionine incorporation is a standard method for determining the phases in protein crystallography by single- or multiwavelength anomalous dispersion. Recombinant expression of selenomethionine-containing protein in non-auxotrophic Pichia pastoris strains yield an incorporation of about 50%. The expression of a mutated variant of Penicillium minioluteum dextranase in P. pastoris is used to illustrate the method utilized to obtain selenomethionyl-substituted protein and to show the phasing power of the acquired anomalous signal. The dextranase structure was solved using the anomalous signal achieved from 50% selenomethionine incorporation. PMID:17951642

Larsson, Anna M; Jones, T Alwyn

2007-01-01

371

Recombinant DNA technology in apple.  

Science.gov (United States)

This review summarizes the achievements of almost 20 years of recombinant DNA technology applied to apple, grouping the research results into the sections: developing the technology, insect resistance, fungal disease resistance, self-incompatibility, herbicide resistance, fire blight resistance, fruit ripening, allergens, rooting ability, and acceptance and risk assessment. The diseases fire blight, caused by Erwinia amylovora, and scab, caused by Venturia inaequalis, were and still are the prime targets. Shelf life improvement and rooting ability of rootstocks are also relevant research areas. The tools to create genetically modified apples of added value to producers, consumers, and the environment are now available. PMID:17522823

Gessler, Cesare; Patocchi, Andrea

2007-01-01

372

Recombinant Holotoxoid Vaccine against Botulism?  

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The botulinum neurotoxins (BoNT) are the most toxic proteins for humans and designated “Category A Select Agents.” The current vaccine against botulism is in limited supply, and there is a need to develop new vaccine strategies. A recombinant BoNT/A toxoid was produced in Clostridium botulinum that contained a double amino acid substitution, R363A Y365F (termed BoNT/ARYM). BoNT/ARYM was noncatalytic for SNAP25 and nontoxic for mice. Immunization with BoNT/ARYM protected mice from challeng...

Pier, Christina L.; Tepp, William H.; Bradshaw, Marite; Johnson, Eric A.; Barbieri, Joseph T.; Baldwin, Michael R.

2008-01-01

373

Inhibition of DNA topoisomerase II may trigger illegitimate recombination in living cells: experiments with a model system.  

Science.gov (United States)

We have developed a plasmid test system to study recombination in vitro and in mammalian cells in vivo, and to analyze the possible role of DNA topoisomerase II. The system is based on a plasmid construct containing an inducible marker gene ccdB ("killer" (KIL) gene) whose product is lethal for bacterial cells, flanked by two different potentially recombinogenic elements. The plasmids were subjected to recombinogenic conditions in vitro or in vivo after transient transfection into COS-1 cells, and subsequently transformed into E. coli which was then grown in the presence of the ccdB gene inducer. Hence, all viable colonies contained recombinant plasmids since only recombination between the flanking regions could remove the KIL gene. Thus, it was possible to detect recombination events and to estimate their frequency. We found that the frequency of topoisomerase II-mediated recombination in vivo is significantly higher than in a minimal in vitro system. The presence of VM-26, an inhibitor of the religation step of the topoisomerase II reaction, increased the recombination frequency by 60%. We propose that cleavable complexes of topoisomerase II are either not religated, triggering error-prone repair of the DNA breaks, or are incorrectly religated resulting in strand exchange. We also studied the influence of sequences known to contain preferential breakpoints for recombination in vivo after chemotherapy with topoisomerase II-targeting drugs, but no preferential stimulation of recombination by these sequences was detected in this non-chromosomal context. PMID:16676353

Umanskaya, Olga N; Lebedeva, Svetlana S; Gavrilov, Alexey A; Bystritskiy, Andrey A; Razin, Sergey V

2006-10-01

374

Electronic recombination in a nonequilibrium plasma  

International Nuclear Information System (INIS)

We study the recombination in a neutral, fully ionized, anisotropic nonequilibrium plasma with applications to electronic cooling of heavy ion beams in a storage ring. These plasmas are characterized by low particle densities and extreme temperature differences between the free electrons and ions. Starting from a microscopic master equation for the coefficient of recombination, collective effects and three body recombination processes are considered. Because of the small density only photon-like collective modes are important for the induced recombination. As the ion temperature is high initially, it is necessary to include the movement of the ions in the calculation of the recombination coefficient. In a cooler plasma recombination into the high-lying states is cut off by the Stark effect due to the transient electric fields which appear after a Lorentz transformation of the external transversal magnetic fields to the rest frame of the beam. (orig.)

1988-01-01

375

Recombination fluorescence in ultracold neutral plasmas  

CERN Document Server

We present the first measurements and simulations of recombination fluorescence in ultracold neutral plasmas. In contrast with previous work, experiment and simulation are in significant disagreement. Comparison with a recombination model suggests that the disagreement could be due to the high energy portion of the electron energy distribution or to large energy changes in electron/Rydberg scattering. Recombination fluorescence opens a new diagnostic window in ultracold plasmas because it probes the deeply-bound Rydberg levels, which depend critically on electron energetics.

Bergeson, S D

2007-01-01

376

Density dependence of dielectronic recombination in selenium  

Energy Technology Data Exchange (ETDEWEB)

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/sup 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/sup 3//sec at 1.0 keV, which is to be compared with the zero-density value of 2.8 x 10/sup -11/ cm/sup 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/sup 3//sec, which is substantially reduced from the zero-density result of 3.3 x 10/sup -11/ cm/sup 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)).

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

1986-09-01

377

Recombinant DNA production of spider silk proteins.  

Science.gov (United States)

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. PMID:24119078

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

2013-11-01

378

Density dependence of dielectronic recombination in selenium  

International Nuclear Information System (INIS)

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)

1986-01-01

379

Engineered interphase chromosome loops guide intrachromosomal recombination  

Digital Repository Infrastructure Vision for European Research (DRIVER)

How large-scale topologies regulate interphase chromosome function remains an important question in eukaryotic cell biology. Looped structures are thought to modulate transcription by pairing promoters with distant control elements and to orchestrate intrachromosomal recombination events by pairing appropriate recombination partners. To explore the effects of chromosomal topology on intrachromosomal recombination, distinct loop geometries were engineered into chromosome III of the budding ye...

2001-01-01

380

Two RNA polymerase I subunits control the binding and release of Rrn3 during transcription.  

Science.gov (United States)

Rpa34 and Rpa49 are nonessential subunits of RNA polymerase I, conserved in species from Saccharomyces cerevisiae and Schizosaccharomyces pombe to humans. Rpa34 bound an N-terminal region of Rpa49 in a two-hybrid assay and was lost from RNA polymerase in an rpa49 mutant lacking this Rpa34-binding domain, whereas rpa34Delta weakened the binding of Rpa49 to RNA polymerase. rpa34Delta mutants were caffeine sensitive, and the rpa34Delta mutation was lethal in a top1Delta mutant and in rpa14Delta, rpa135(L656P), and rpa135(D395N) RNA polymerase mutants. These defects were shared by rpa49Delta mutants, were suppressed by the overexpression of Rpa49, and thus, were presumably mediated by Rpa49 itself. rpa49 mutants lacking the Rpa34-binding domain behaved essentially like rpa34Delta mutants, but strains carrying rpa49Delta and rpa49-338::HIS3 (encoding a form of Rpa49 lacking the conserved C terminus) had reduced polymerase occupancy at 30 degrees C, failed to grow at 25 degrees C, and were sensitive to 6-azauracil and mycophenolate. Mycophenolate almost fully dissociated the mutant polymerase from its ribosomal DNA (rDNA) template. The rpa49Delta and rpa49-338::HIS3 mutations had a dual effect on the transcription initiation factor Rrn3 (TIF-IA). They partially impaired its recruitment to the rDNA promoter, an effect that was bypassed by an N-terminal deletion of the Rpa43 subunit encoded by rpa43-35,326, and they strongly reduced the release of the Rrn3 initiation factor during elongation. These data suggest a dual role of the Rpa49-Rpa34 dimer during the recruitment of Rrn3 and its subsequent dissociation from the elongating polymerase. PMID:18086878

Beckouet, Frédéric; Labarre-Mariotte, Sylvie; Albert, Benjamin; Imazawa, Yukiko; Werner, Michel; Gadal, Olivier; Nogi, Yasuhisa; Thuriaux, Pierre

2008-03-01

 
 
 
 
381

Microbial factories for recombinant pharmaceuticals  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Most of the hosts used to produce the 151 recombinant pharmaceuticals so far approved for human use by the Food and Drug Administration (FDA and/or by the European Medicines Agency (EMEA are microbial cells, either bacteria or yeast. This fact indicates that despite the diverse bottlenecks and obstacles that microbial systems pose to the efficient production of functional mammalian proteins, namely lack or unconventional post-translational modifications, proteolytic instability, poor solubility and activation of cell stress responses, among others, they represent convenient and powerful tools for recombinant protein production. The entering into the market of a progressively increasing number of protein drugs produced in non-microbial systems has not impaired the development of products obtained in microbial cells, proving the robustness of the microbial set of cellular systems (so far Escherichia coli and Saccharomyces cerevisae developed for protein drug production. We summarize here the nature, properties and applications of all those pharmaceuticals and the relevant features of the current and potential producing hosts, in a comparative way.

Domingo-Espín Joan

2009-03-01

382

Nebular Abundances from Recombination Lines  

Science.gov (United States)

Ionic abundances, deduced from intrinsically more reliable optical recombination lines (ORLs) are presented for ten planetary nebulae (PN) and the Hii region M 42. For all ionic species studied, the ORL ionic abundances relative to H, Xi+ / H+, are consistently higher than those deduced from collisionally excited lines (CELs). The ratios of ORL and CEL abundances vary from source to source and cover a wide range of 1-15. Observational uncertainties, grossly underestimated recombination rates, blending by unknown lines or radiative fluorescence as the cause of the discrepancy can be ruled out. While the ORL abundances appear secure and the evidence points to the traditional CEL method being at fault, the standard scenario of temperature and/or density fluctuations fails to explain quantitatively all available data, particularly the apparent low CEL abundances derived from IR fine-structure lines. The presence of high-density, H-deficient, low-temperature condensations embedded in a diffuse nebula of ``normal'' abundances, i.e. a nebula of the type of a ``born-again'' PN such as A 30, may provide a possible solution for those extreme nebulae where the ORL abundances are a factor of ten or more higher than derived from CELs.

Liu, X.-W.; Barlow, M. J.; Danziger, I. J.; Storey, P. J.

383

Experimental recombination rates for highly charged ions  

Energy Technology Data Exchange (ETDEWEB)

Recent studies of recombination between free electrons and highly charged ions using electron coolers of heavy-ion storage rings have produced accurate rate coefficients of interest for plasma modeling and diagnostics. Some surprises were discovered which can lead to revisions of recombination models. With bare ions one finds at low energy a strong and puzzling deviation from radiative recombination theory. Dielectronic recombination with C3+, N4+ show that jj coupling gives essential contributions to the cross section also for light ions. (author)

Reinhold Schuch [Dept. of Atomic Physics, Stockholm Univ., Frescativ., Stockholm (Sweden)

2000-01-01

384

Experimental recombination rates for highly charged ions  

International Nuclear Information System (INIS)

Recent studies of recombination between free electrons and highly charged ions using electron coolers of heavy-ion storage rings have produced accurate rate coefficients of interest for plasma modeling and diagnostics. Some surprises were discovered which can lead to revisions of recombination models. With bare ions one finds at low energy a strong and puzzling deviation from radiative recombination theory. Dielectronic recombination with C3+, N4+) show that jj coupling gives essential contributions to the cross section also for light ions. (author)

2000-01-01

385

Consequences of recombination on traditional phylogenetic analysis.  

DEFF Research Database (Denmark)

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 (mtDNA 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 not be immediately detectable in a data set. The phylogenies when recombination is present superficially resemble phylogenies for sequences from an exponentially growing population. However, exponential growth has a different effect on statistics such as Tajima's D. Furthermore, ignoring recombination leads to a large overestimation of the substitution rate heterogeneity and the loss of the molecular clock. These results are discussed in relation to viral and mtDNA data sets. Udgivelsesdato: 2000-Oct

Schierup, M H; Hein, J

2000-01-01

386

Induction of intrachromosomal homologous recombination in human cells by raltitrexed, an inhibitor of thymidylate synthase.  

Science.gov (United States)

Thymidylate deprivation brings about "thymineless death" in prokaryotes and eukaryotes. Although the precise mechanism for thymineless death has remained elusive, inhibition of the enzyme thymidylate synthase (TS), which catalyzes the de novo synthesis of TMP, has served for many years as a basis for chemotherapeutic strategies. Numerous studies have identified a variety of cellular responses to thymidylate deprivation, including disruption of DNA replication and induction of DNA breaks. Since stalled or collapsed replication forks and strand breaks are generally viewed as being recombinogenic, it is not surprising that a link has been demonstrated between recombination induction and thymidylate deprivation in bacteria and lower eukaryotes. A similar connection between recombination and TS inhibition has been suggested by studies done in mammalian cells, but the relationship between recombination and TS inhibition in mammalian cells had not been demonstrated rigorously. To gain insight into the mechanism of thymineless death in mammalian cells, in this work we undertook a direct investigation of recombination in human cells treated with raltitrexed (RTX), a folate analog that is a specific inhibitor of TS. Using a model system to study intrachromosomal homologous recombination in cultured fibroblasts, we provide definitive evidence that treatment with RTX can stimulate accurate recombination events in human cells. Gene conversions not associated with crossovers were specifically enhanced several-fold by RTX. Additional experiments demonstrated that recombination events provoked by a double-strand break (DSB) were not impacted by treatment with RTX, nor was error-prone DSB repair via nonhomologous end-joining. Our work provides evidence that thymineless death in human cells is not mediated by corruption of DSB repair processes and suggests that an increase in chromosomal recombination may be an important element of cellular responses leading to thymineless death. PMID:18603020

Waldman, Barbara Criscuolo; Wang, Yibin; Kilaru, Kasturi; Yang, Zhengguan; Bhasin, Alaukik; Wyatt, Michael D; Waldman, Alan S

2008-10-01

387

BRCA1 controls homologous recombination at Tus/Ter-stalled mammalian replication forks.  

Science.gov (United States)

Replication fork stalling can promote genomic instability, predisposing to cancer and other diseases. Stalled replication forks may be processed by sister chromatid recombination (SCR), generating error-free or error-prone homologous recombination (HR) outcomes. In mammalian cells, a long-standing hypothesis proposes that the major hereditary breast/ovarian cancer predisposition gene products, BRCA1 and BRCA2, control HR/SCR at stalled replication forks. Although BRCA1 and BRCA2 affect replication fork processing, direct evidence that BRCA gene products regulate homologous recombination at stalled chromosomal replication forks is lacking, due to a dearth of tools for studying this process. Here we report that the Escherichia coli Tus/Ter complex can be engineered to induce site-specific replication fork stalling and chromosomal HR/SCR in mouse cells. Tus/Ter-induced homologous recombination entails processing of bidirectionally arrested forks. We find that the Brca1 carboxy (C)-terminal tandem BRCT repeat and regions of Brca1 encoded by exon 11-two Brca1 elements implicated in tumour suppression-control Tus/Ter-induced homologous recombination. Inactivation of either Brca1 or Brca2 increases the absolute frequency of 'long-tract' gene conversions at Tus/Ter-stalled forks, an outcome not observed in response to a site-specific endonuclease-mediated chromosomal double-strand break. Therefore, homologous recombination at stalled forks is regulated differently from homologous recombination at double-strand breaks arising independently of a replication fork. We propose that aberrant long-tract homologous recombination at stalled replication forks contributes to genomic instability and breast/ovarian cancer predisposition in BRCA mutant cells. PMID:24776801

Willis, Nicholas A; Chandramouly, Gurushankar; Huang, Bin; Kwok, Amy; Follonier, Cindy; Deng, Chuxia; Scully, Ralph

2014-06-26

388

Observation of interference between dielectronic recombination and radiative recombination in highly charged uranium ions  

International Nuclear Information System (INIS)

We have performed high-resolution measurements of photon excitation functions for radiative recombination and dielectronic recombination in highly charged uranium ions. The data show evidence for quantum interference between the two processes in the vicinity of the KLL resonances

1995-01-02

389

Recombination hotspots and host susceptibility modulate the adaptive value of recombination during maize streak virus evolution  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Abstract Background Maize streak virus -strain A (MSV-A; Genus Mastrevirus, Family Geminiviridae), the maize-adapted strain of MSV that causes maize streak disease throughout sub-Saharan Africa, probably arose between 100 and 200 years ago via homologous recombination between two MSV strains adapted to wild grasses. MSV recombination experiments and analyses of natural MSV recombination patterns have revealed that this recombination event entailed t...

Monjane Adérito L; van der Walt Eric; Varsani Arvind; Rybicki Edward P; Martin Darren P

2011-01-01

390

The fission yeast FANCM ortholog directs non-crossover recombination during meiosis.  

Science.gov (United States)

The formation of healthy gametes depends on programmed DNA double-strand breaks (DSBs), which are each repaired as a crossover (CO) or non-crossover (NCO) from a homologous template. Although most of these DSBs are repaired without giving COs, little is known about the genetic requirements of NCO-specific recombination. We show that Fml1, the Fanconi anemia complementation group M (FANCM)-ortholog of Schizosaccharomyces pombe, directs the formation of NCOs during meiosis in competition with the Mus81-dependent pro-CO pathway. We also define the Rad51/Dmc1-mediator Swi5-Sfr1 as a major determinant in biasing the recombination process in favor of Mus81, to ensure the appropriate amount of COs to guide meiotic chromosome segregation. The conservation of these proteins from yeast to humans suggests that this interplay may be a general feature of meiotic recombination. PMID:22723423

Lorenz, Alexander; Osman, Fekret; Sun, Weili; Nandi, Saikat; Steinacher, Roland; Whitby, Matthew C

2012-06-22

391

Metabolic responses to recombinant bioprocesses in Escherichia coli  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Escherichia coli has been widely used for the production of recombinant proteins. However, the unbalances between host metabolism and recombinant biosynthesis continue to hamper the efficiency of these recombinant bioprocesses. The additional drainage of biosynthetic precursors toward recombinant processes burdens severely the metabolism of cells that, ultimately, elicits a series of stress responses, reducing biomass growth and recombinant protein production. Several strategies to overco...

Carneiro, S.; Ferreira, E. C.; Rocha, I.

2013-01-01

392

Mediation of mouse natural cytotoxic activity by tumour necrosis factor  

Science.gov (United States)

Natural cell-mediated cytotoxic activity in the mouse has been associated with two types of effector cells, the natural killer (NK) cell and the natural cytotoxic (NC) cell, which seem to differ with regard to their patterns of target selectivity, cell surface characteristics and susceptibility to regulatory factors1. During studies on the mechanism of action of cytotoxic molecules, it became evident that WEHI-164, the prototype NC target cell, was highly susceptible to direct lysis by both human and mouse recombinant tumour necrosis factor (TNF). Here we show that NC, but not NK activity mediated by normal splenocytes, is abrogated by rabbit antibodies to recombinant and natural TNF, respectively. Thus, the cell-mediated activity defined as NC is due to release of TNF by normal spleen cells and does not represent a unique natural effector mechanism.

Ortaldo, John R.; Mason, Llewellyn H.; Mathieson, Bonnie J.; Liang, Shu-Mei; Flick, David A.; Herberman, Ronald B.

1986-06-01

393

Dielectronic recombination lines of C{sup +}  

Energy Technology Data Exchange (ETDEWEB)

The present paper presents atomic data generated to investigate the recombination lines of C II in the spectra of planetary nebulae. These data include energies of bound and autoionizing states, oscillator strengths and radiative transition probabilities, autoionization probabilities, and recombination coefficients. The R-matrix method of electron scattering theory was used to describe the C{sup 2+} plus electron system.

Sochi, Taha, E-mail: taha.sochi@kcl.ac.uk; Storey, Peter J.

2013-11-15

394

Recombinant organisms for production of industrial products  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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...

Adrio, Jose-luis; Demain, Arnold L.

2010-01-01

395

RNAi and heterochromatin repress centromeric meiotic recombination  

DEFF Research Database (Denmark)

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 essential in most species for proper homologue segregation. Nevertheless, recombination is repressed specifically in and around the centromeres of chromosomes, apparently because rare centromeric (or pericentromeric) recombination events, when they do occur, can disrupt proper segregation and lead to genetic disabilities, including birth defects. The basis by which centromeric meiotic recombination is repressed has been largely unknown. We report here that, in fission yeast, RNAi functions and Clr4-Rik1 (histone H3 lysine 9 methyltransferase) are required for repression of centromeric recombination. 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.

Ellermeier, Chad; Higuchi, Emily C

2010-01-01

396

Electron-ion recombination at low energy  

International Nuclear Information System (INIS)

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.)

1993-01-01

397

Telomeric recombination induced by dysfunctional telomeres  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Telomeric recombination has been observed in telomerase-negative alternative lengthening of telomeres in human cancer cells and following telomerase inhibition or gene deletion. This study shows that telomeric recombination mechanisms can also be activated by dysfunctional telomeres without telomerase inhibition in telomerase-positive cells.

Brault, Marie Eve; Autexier, Chantal

2011-01-01

398

Fundamental Studies of Recombinant Hydrogenases  

Energy Technology Data Exchange (ETDEWEB)

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.

Adams, Michael W

2014-01-25

399

Tackling Heterogeneity: A Leaf Disc-Based Assay for the High-Throughput Screening of Transient Gene Expression in Tobacco  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Transient Agrobacterium-mediated gene expression assays for Nicotiana tabacum (N. tabacum) are frequently used because they facilitate the comparison of multiple expression constructs regarding their capacity for maximum recombinant protein production. However, for three model proteins, we found that recombinant protein accumulation (rpa) was significantly influenced by leaf age and leaf position effects. The ratio between the highest and lowest amount of protein accumulation (max/min ratio) ...

Piotrzkowski, Natalia; Schillberg, Stefan; Rasche, Stefan

2012-01-01

400

Roles for NBS1 in alternative nonhomologous end joining of V(D)J recombination intermediates  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Recent work has highlighted the importance of alternative, error-prone mechanisms for joining DNA double-strand breaks (DSB) in mammalian cells. These noncanonical, non-homologous end joining (NHEJ) pathways threaten genomic stability but remain poorly characterized. The RAG post-cleavage complex normally prevents V(D)J recombination-associated DSBs from accessing alternative NHEJ. Because the MRE11/RAD50/NBS1 complex localizes to RAG-mediated DSBs and possesses DNA end tethering, processing ...

Deriano, Ludovic; Stracker, Travis H.; Baker, Annalee; Petrini, John H. J.; Roth, David B.

2009-01-01

 
 
 
 
401

Recombinant Escherichia coli Strain Produces a ZZ Domain Displaying Biopolyester Granules Suitable for Immunoglobulin G Purification?  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The immunoglobulin G (IgG) binding ZZ domain of protein A from Staphylococcus aureus was fused to the N terminus of the polyhydroxyalkanoate (PHA) synthase from Cupriavidus necator. The fusion protein was confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and mediated formation of ZZ domain-displaying PHA granules in recombinant Escherichia coli. The IgG binding capacity of isolated granules was assessed using enzyme-linked immunosorbent assay and could ...

2006-01-01

402

Recombinant norovirus-specific scFv inhibit virus-like particle binding to cellular ligands  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Abstract Background Noroviruses cause epidemic outbreaks of gastrointestinal illness in all age-groups. The rapid onset and ease of person-to-person transmission suggest that inhibitors of the initial steps of virus binding to susceptible cells have value in limiting spread and outbreak persistence. We previously generated a monoclonal antibody (mAb) 54.6 that blocks binding of recombinant norovirus-like particles (VLP) to Caco-2 intestinal cells and inhibits VLP-mediated hem...

Ettayebi Khalil; Hardy Michele E

2008-01-01

403

The Purified and Recombinant Legionella pneumophila Chaperonin Alters Mitochondrial Trafficking and Microfilament Organization?  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A portion of the total cellular pool of the Legionella pneumophila chaperonin, HtpB, is found on the bacterial cell surface, where it can mediate invasion of nonphagocytic cells. HtpB continues to be abundantly produced and released by internalized L. pneumophila and may thus have postinvasion functions. We used here two functional models (protein-coated beads and expression of recombinant proteins in CHO cells) to investigate the competence of HtpB in mimicking early intracellular traffickin...

Chong, Audrey; Lima, Celia A.; Allan, David S.; Nasrallah, Gheyath K.; Gardun?o, Rafael A.

2009-01-01

404

Suppression by human recombinant gamma interferon of in vitro macrophage nonopsonic and opsonic phagocytosis and killing.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Although gamma interferon (IFN-gamma) exerts profound effects on the state of activation of macrophages, its influence on receptor-mediated phagocytosis and killing of extracellular bacteria is poorly understood. Human monocytes cultured in the presence of human recombinant IFN-gamma exhibited an enhanced capacity to produce superoxide anion. Although these cells bound greater numbers of particles via Fc receptors, their capacity to phagocytose by these receptors or to bind or ingest particle...

Speert, D. P.; Thorson, L.

1991-01-01

405

Stable expression and characterization of N-terminal tagged recombinant human bone morphogenetic protein 15  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Oocyte-derived growth factors are critically involved in multiple ovarian processes via paracrine actions. Although recombinant proteins have been applied to dissect the physiological functions of these factors, variation of activities among different protein preparations remains an issue. To further elucidate the roles of one of these growth factors, bone morphogenetic protein 15 (BMP15), in mediating oocyte-regulated molecular and cellular events and to explore its potential clinical applic...

Li, Qinglei; Rajanahally, Saneal; Edson, Mark A.; Matzuk, Martin M.

2009-01-01

406

Adherence to respiratory epithelia by recombinant Escherichia coli expressing Klebsiella pneumoniae type 3 fimbrial gene products.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We examined the role of Klebsiella fimbrial types 1 and 3 in mediating adherence to human buccal and tracheal cells and to lung tissue sections. We found that clinical isolates of Klebsiella pneumoniae producing type 3 fimbriae and Escherichia coli HB101 containing a recombinant plasmid encoding expression of Klebsiella type 3 fimbriae (pFK10) demonstrated increased adherence to tracheal cells, trypsinized buccal cells, and lung tissue sections, in contrast to nonfimbriate and to type 1 fimbr...

Hornick, D. B.; Allen, B. L.; Horn, M. A.; Clegg, S.

1992-01-01

407

Recombinational Repair Is Critical for Survival of Escherichia coli Exposed to Nitric Oxide  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Nitric oxide (NO?) is critical to numerous biological processes, including signal transduction and macrophage-mediated immunity. In this study, we have explored the biological effects of NO?-induced DNA damage on Escherichia coli. The relative importance of base excision repair, nucleotide excision repair (NER), and recombinational repair in preventing NO?-induced toxicity was determined. E. coli strains lacking either NER or DNA glycosylases (including those that repair alkylation dama...

Spek, Erik J.; Wright, Teresa L.; Stitt, Molly S.; Taghizadeh, Nazbeh R.; Tannenbaum, Steven R.; Marinus, Martin G.; Engelward, Bevin P.

2001-01-01

408

Erlotinib attenuates homologous recombinational repair of chromosomal breaks in human breast cancer cells  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The epidermal growth factor receptor family (EGFR) has been implicated in a number of cancers, including breast, and its members have become the target of novel cancer therapies. In this report, we show a novel link between erlotinib, a potent EGFR inhibitor, DNA damage, and homology-directed recombinational repair (HDR) in human breast cancer cells. Erlotinib suppresses HDR. This is not secondary to erlotinib-mediated changes in cell cycle and is associated with increased ?-H2AX foci, which...

Li, Liping; Wang, Hong; Yang, Eddy S.; Arteaga, Carlos L.; Xia, Fen

2008-01-01

409

Intrachromosomal Recombination within the vsp Locus of Mycoplasma bovis Generates a Chimeric Variable Surface Lipoprotein Antigen  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A family of 13 related but divergent vsp genes was recently found in the chromosome of the bovine pathogen Mycoplasma bovis. The vsp genomic locus was shown to undergo high-frequency rearrangements and to mediate phenotypic switching of variable lipoprotein antigens (Vsps) on the mycoplasma cell surface. Here we report that the vsp gene repertoire is subject to changes. Genetic analysis of M. bovis clonal isolates displaying distinct Vsp phenotypes showed that an intergenic recombination even...

Lysnyansky, Inessa; Ron, Yael; Sachse, Konrad; Yogev, David

2001-01-01

410

Recombination analysis reveals a double recombination event in hepatitis E virus  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Recombination of Hepatitis E Virus (HEV has rarely been reported. In the present study, phylogenetic and recombination analyses were performed on 134 complete HEV genomes. Three potentially significant recombination events, including both intra-genotype and one inter-genotype, were identified by recombination detection analysis. Recombination events I and II occurred intra-genotype and inter-genotype, respectively, among three isolates, including the lineage represented by CHN-XJ-SW13 (GU119961, swine isolate, E067-SIJ05C (AB369690, human isolate, and JJT-Kan (AB091394, human isolate, and lead to the recombinant swine isolate swCH31 (DQ450072. Recombination event III occurred between the lineage represented by the NA1 (M73218 and K52-87 (L25595, which resulted in the recombinant Xingjiang-1 (D11092. Our analyses proved that that recombination could occur between human and swine HEV strains, double recombination events existed in HEV, and recombination event could happen within ORF2 region of HEV. These results will provide valuable hints for future research on HEV diversity.

Shao Shihe

2010-06-01

411

Containment air circulation for optimal hydrogen recombination  

International Nuclear Information System (INIS)

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)

1997-03-01

412