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1

RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells.  

UK PubMed Central (United Kingdom)

The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role of RPA in homologous recombination in assembly of the RAD51 and RAD52 proteins. Furthermore, our data suggest that replacement of RPA with the RAD51 and RAD52 proteins is affected by checkpoint signalling.

Sleeth KM; Sřrensen CS; Issaeva N; Dziegielewski J; Bartek J; Helleday T

2007-10-01

2

RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells.  

Science.gov (United States)

The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role of RPA in homologous recombination in assembly of the RAD51 and RAD52 proteins. Furthermore, our data suggest that replacement of RPA with the RAD51 and RAD52 proteins is affected by checkpoint signalling. PMID:17765923

Sleeth, Kate M; Sřrensen, Claus Storgaard; Issaeva, Natalia; Dziegielewski, Jaroslaw; Bartek, Jiri; Helleday, Thomas

2007-08-15

3

A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination.  

UK PubMed Central (United Kingdom)

Double-stranded DNA breaks (DSBs) induce a phosphorylation-mediated signaling cascade, but the role of phosphatases in this pathway remains unclear. Here we show that human protein phosphatase 4 (PP4) dephosphorylates replication protein A (RPA) subunit RPA2, regulating its role in the DSB response. PP4R2, a regulatory subunit of PP4, mediates the DNA damage-dependent association between RPA2 and the PP4C catalytic subunit. PP4 efficiently dephosphorylates phospho-RPA2 in vitro, and silencing PP4R2 in cells alters the kinetics and pattern of RPA2 phosphorylation. Depletion of PP4R2 impedes homologous recombination (HR) via inefficient loading of the essential HR factor RAD51, causing an extended G2-M checkpoint and hypersensitivity to DNA damage. Cells expressing phosphomimetic RPA2 mutants have a comparable phenotype, suggesting that PP4-mediated dephosphorylation of RPA2 is necessary for an efficient DNA-damage response. These observations provide new insight into the role and regulation of RPA phosphorylation in HR-mediated repair.

Lee DH; Pan Y; Kanner S; Sung P; Borowiec JA; Chowdhury D

2010-03-01

4

A PP4 phosphatase complex dephosphorylates RPA2 to facilitate DNA repair via homologous recombination.  

Science.gov (United States)

Double-stranded DNA breaks (DSBs) induce a phosphorylation-mediated signaling cascade, but the role of phosphatases in this pathway remains unclear. Here we show that human protein phosphatase 4 (PP4) dephosphorylates replication protein A (RPA) subunit RPA2, regulating its role in the DSB response. PP4R2, a regulatory subunit of PP4, mediates the DNA damage-dependent association between RPA2 and the PP4C catalytic subunit. PP4 efficiently dephosphorylates phospho-RPA2 in vitro, and silencing PP4R2 in cells alters the kinetics and pattern of RPA2 phosphorylation. Depletion of PP4R2 impedes homologous recombination (HR) via inefficient loading of the essential HR factor RAD51, causing an extended G2-M checkpoint and hypersensitivity to DNA damage. Cells expressing phosphomimetic RPA2 mutants have a comparable phenotype, suggesting that PP4-mediated dephosphorylation of RPA2 is necessary for an efficient DNA-damage response. These observations provide new insight into the role and regulation of RPA phosphorylation in HR-mediated repair. PMID:20154705

Lee, Dong-Hyun; Pan, Yunfeng; Kanner, Shlomo; Sung, Patrick; Borowiec, James A; Chowdhury, Dipanjan

2010-02-14

5

Replication protein A (RPA): the eukaryotic SSB.  

UK PubMed Central (United Kingdom)

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that is highly conserved in eukaryotes. RPA plays essential roles in many aspects of nucleic acid metabolism, including DNA replication, nucleotide excision repair, and homologous recombination. In this review, we provide a comprehensive overview of RPA structure and function and highlight the more recent developments in these areas. The last few years have seen major advances in our understanding of the mechanism of RPA binding to DNA, including the structural characterization of the primary DNA-binding domains (DBD) and the identification of two secondary DBDs. Moreover, evidence indicates that RPA utilizes a multistep pathway to bind single-stranded DNA involving a particular molecular polarity of RPA, a mechanism that is apparently used to facilitate origin denaturation. In addition to its mechanistic roles, RPA interacts with many key factors in nucleic acid metabolism, and we discuss the critical nature of many of these interactions to DNA metabolism. RPA is a phosphorylation target for DNA-dependent protein kinase (DNA-PK) and likely the ataxia telangiectasia-mutated gene (ATM) protein kinase, and recent observations are described that suggest that RPA phosphorylation plays a significant modulatory role in the cellular response to DNA damage.

Iftode C; Daniely Y; Borowiec JA

1999-01-01

6

Opposing roles for two molecular forms of replication protein A in Rad51-Rad54-mediated DNA recombination in Plasmodium falciparum.  

UK PubMed Central (United Kingdom)

UNLABELLED: The bacterial RecA protein and its eukaryotic homologue Rad51 play a central role in the homologous DNA strand exchange reaction during recombination and DNA repair. Previously, our lab has shown that PfRad51, the Plasmodium falciparum homologue of Rad51, exhibited ATPase activity and promoted DNA strand exchange in vitro. In this study, we evaluated the catalytic functions of PfRad51 in the presence of putative interacting partners, especially P. falciparum homologues of Rad54 and replication protein A. PfRad54 accelerated PfRad51-mediated pairing between single-stranded DNA (ssDNA) and its homologous linear double-stranded DNA (dsDNA) in the presence of 0.5 mM CaCl2. We also present evidence that recombinant PfRPA1L protein serves the function of the bacterial homologue single-stranded binding protein (SSB) in initiating homologous pairing and strand exchange activity. More importantly, the function of PfRPA1L was negatively regulated in a dose-dependent manner by PfRPA1S, another RPA homologue in P. falciparum. Finally, we present in vivo evidence through comet assays for methyl methane sulfonate-induced DNA damage in malaria parasites and accompanying upregulation of PfRad51, PfRad54, PfRPA1L, and PfRPA1S at the level of transcript and protein needed to repair DNA damage. This study provides new insights into the role of putative Rad51-interacting proteins involved in homologous recombination and emphasizes the physiological role of DNA damage repair during the growth of parasites. IMPORTANCE: Homologous recombination plays a major role in chromosomal rearrangement, and Rad51 protein, aided by several other proteins, plays a central role in DNA strand exchange reaction during recombination and DNA repair. This study reports on the characterization of the role of P. falciparum Rad51 in homologous strand exchange and DNA repair and evaluates the functional contribution of PfRad54 and PfRPA1 proteins. Data presented here provide mechanistic insights into DNA recombination and DNA damage repair mechanisms in this parasite. The importance of these research findings in future work will be to investigate if Rad51-dependent mechanisms are involved in chromosomal rearrangements during antigenic variation in P. falciparum. A prominent determinant of antigenic variation, the extraordinary ability of the parasite to rapidly change its surface molecules, is associated with var genes, and antigenic variation presents a major challenge to vaccine development.

Gopalakrishnan AM; Kumar N

2013-01-01

7

The self consistent RPA  

International Nuclear Information System (INIS)

Following the equation of motion method and the variation principle we derive the self consistent RPA equations including a generalized mean field equation coupled to the RPA fluctuations. We show some of its properties in three exactly solvable models of particle-hole particle-particle and superfluid correlations. (Author)

1998-01-01

8

Activity of the Rhodopseudomonas palustris p-coumaroyl-homoserine lactone-responsive transcription factor RpaR.  

UK PubMed Central (United Kingdom)

The Rhodopseudomonas palustris transcriptional regulator RpaR responds to the RpaI-synthesized quorum-sensing signal p-coumaroyl-homoserine lactone (pC-HSL). Other characterized RpaR homologs respond to fatty acyl-HSLs. We show here that RpaR functions as a transcriptional activator, which binds directly to the rpaI promoter. We developed an RNAseq method that does not require a ribosome depletion step to define a set of transcripts regulated by pC-HSL and RpaR. The transcripts include several noncoding RNAs. A footprint analysis showed that purified His-tagged RpaR (His(6)-RpaR) binds to an inverted repeat element centered 48.5 bp upstream of the rpaI transcript start site, which we mapped by S1 nuclease protection and primer extension analyses. Although pC-HSL-RpaR bound to rpaI promoter DNA, it did not bind to the promoter regions of a number of RpaR-regulated genes not in the rpaI operon. This indicates that RpaR control of these other genes is indirect. Because the RNAseq analysis allowed us to track transcript strand specificity, we discovered that there is pC-HSL-RpaR-activated antisense transcription of rpaR. These data raise the possibility that this antisense RNA or other RpaR-activated noncoding RNAs mediate the indirect activation of genes in the RpaR-controlled regulon.

Hirakawa H; Oda Y; Phattarasukol S; Armour CD; Castle JC; Raymond CK; Lappala CR; Schaefer AL; Harwood CS; Greenberg EP

2011-05-01

9

Role of replication protein A in double holliday junction dissolution mediated by the BLM-Topo III?-RMI1-RMI2 protein complex.  

UK PubMed Central (United Kingdom)

The conserved BTR complex, composed of the Bloom's syndrome helicase (BLM), topoisomerase III?, RMI1, and RMI2, regulates homologous recombination in favor of non-crossover formation via the dissolution of the double Holliday Junction (dHJ). Here we show enhancement of the BTR-mediated dHJ dissolution reaction by the heterotrimeric single-stranded DNA binding protein replication protein A (RPA). Our results suggest that RPA acts by sequestering a single-stranded DNA intermediate during dHJ dissolution. We provide evidence that RPA physically interacts with RMI1. The RPA interaction domain in RMI1 has been mapped, and RMI1 mutants impaired for RPA interaction have been generated. Examination of these mutants ascertains the significance of the RMI1-RPA interaction in dHJ dissolution. Our results thus implicate RPA as a cofactor of the BTR complex in dHJ dissolution.

Xue X; Raynard S; Busygina V; Singh AK; Sung P

2013-05-01

10

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

11

Activity of the Rhodopseudomonas palustris p-coumaroyl-homoserine lactone-responsive transcription factor RpaR.  

Science.gov (United States)

The Rhodopseudomonas palustris transcriptional regulator RpaR responds to the RpaI-synthesized quorum-sensing signal p-coumaroyl-homoserine lactone (pC-HSL). Other characterized RpaR homologs respond to fatty acyl-HSLs. We show here that RpaR functions as a transcriptional activator, which binds directly to the rpaI promoter. We developed an RNAseq method that does not require a ribosome depletion step to define a set of transcripts regulated by pC-HSL and RpaR. The transcripts include several noncoding RNAs. A footprint analysis showed that purified His-tagged RpaR (His(6)-RpaR) binds to an inverted repeat element centered 48.5 bp upstream of the rpaI transcript start site, which we mapped by S1 nuclease protection and primer extension analyses. Although pC-HSL-RpaR bound to rpaI promoter DNA, it did not bind to the promoter regions of a number of RpaR-regulated genes not in the rpaI operon. This indicates that RpaR control of these other genes is indirect. Because the RNAseq analysis allowed us to track transcript strand specificity, we discovered that there is pC-HSL-RpaR-activated antisense transcription of rpaR. These data raise the possibility that this antisense RNA or other RpaR-activated noncoding RNAs mediate the indirect activation of genes in the RpaR-controlled regulon. PMID:21378182

Hirakawa, Hidetada; Oda, Yasuhiro; Phattarasukol, Somsak; Armour, Christopher D; Castle, John C; Raymond, Christopher K; Lappala, Colin R; Schaefer, Amy L; Harwood, Caroline S; Greenberg, E Peter

2011-03-04

12

Self consistent RPA for superfluid Fermi systems  

Science.gov (United States)

Self consistent RPA, a formalism generalizing usual RPA such as to incorporate fully RPA groundstate correlations, is extended to superfluid or superconducting Fermi systems. An application to the seniority model demonstrates strong improvement over the standard mean-field RPA scheme.

Dukelsky, J.; Schuck, P.

1996-02-01

13

Tamoxifen-inducible Cre-mediated recombination in adipocytes.  

UK PubMed Central (United Kingdom)

To generate a mouse line which allows inducible, Cre/loxP-dependent recombination in adipocytes, we used RedE/RedT-mediated recombineering to insert the CreER(T)˛-transgene, which encodes a fusion protein of Cre and a mutated tamoxifen-responsive estrogen receptor, into the start codon of the adipocyte-specific Adipoq gene. Adipoq encodes adiponectin, an adipokine specifically expressed in differentiated adipocytes. Tamoxifen treatment induced almost complete recombination in white adipose tissue of the AdipoqCreER(T)˛ mouse line (97%-99%), while no recombination was seen in vehicle-treated animals. Recombination in brown adipose tissue was about 15%, whereas other organs and tissues did not undergo recombination. In addition, mice expressing CreER(T)˛ in adipocytes did not show any alterations of metabolic functions like glucose tolerance, lipolysis, or energy expenditure compared to control mice. Therefore the AdipoqCreER(T)˛ mouse line will be a valuable tool for studying the consequences of a temporally controlled deletion of floxed genes in white adipose tissue.

Sassmann A; Offermanns S; Wettschureck N

2010-10-01

14

Replication protein A (RPA) phosphorylation prevents RPA association with replication centers.  

Science.gov (United States)

Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2(D)) or alanine (RPA2(A)). Although RPA2(D) was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2(D) mutant was selectively unable to associate with replication centers in vivo. In cells containing greatly reduced levels of endogenous RPA2, RPA2(D) again did not localize to replication sites, indicating that the defect in supporting chromosomal DNA replication is not due to competition with the wild-type protein. Use of phosphospecific antibodies demonstrated that endogenous hyperphosphorylated RPA behaves similarly to RPA2(D). In contrast, under DNA damage or replication stress conditions, RPA2(D), like RPA2(A) and wild-type RPA2, was competent to associate with DNA damage foci as determined by colocalization with gamma-H2AX. We conclude that RPA2 phosphorylation prevents RPA association with replication centers in vivo and potentially serves as a marker for sites of DNA damage. PMID:14966274

Vassin, Vitaly M; Wold, Marc S; Borowiec, James A

2004-03-01

15

Replication protein A (RPA) phosphorylation prevents RPA association with replication centers.  

UK PubMed Central (United Kingdom)

Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2(D)) or alanine (RPA2(A)). Although RPA2(D) was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2(D) mutant was selectively unable to associate with replication centers in vivo. In cells containing greatly reduced levels of endogenous RPA2, RPA2(D) again did not localize to replication sites, indicating that the defect in supporting chromosomal DNA replication is not due to competition with the wild-type protein. Use of phosphospecific antibodies demonstrated that endogenous hyperphosphorylated RPA behaves similarly to RPA2(D). In contrast, under DNA damage or replication stress conditions, RPA2(D), like RPA2(A) and wild-type RPA2, was competent to associate with DNA damage foci as determined by colocalization with gamma-H2AX. We conclude that RPA2 phosphorylation prevents RPA association with replication centers in vivo and potentially serves as a marker for sites of DNA damage.

Vassin VM; Wold MS; Borowiec JA

2004-03-01

16

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

17

On BLM helicase in recombination-mediated telomere maintenance.  

Science.gov (United States)

Bloom syndrome (BS) is an extremely rare, autosomal recessive genetic syndrome of humans. Patients with BS are predisposed to almost all forms of cancer and also display premature aging phenotypes. These patients are diagnosed in the clinics by hyper-recombination phenotype that is manifested by high rates of sister chromatid exchange. The gene mutated in BS, designated BLM, lies on chromosome 15q26.1 and encodes a RecQ-like ATP-dependent 3'-5' helicase, which functions in DNA double-strand break repair processes such as non-homologous end joining, homologous recombination-mediated repair, resolution of stalled replication forks and synthesis-dependent strand annealing, although its precise functions at the telomeres are speculative. Recently it has been suggested that the BLM helicase may play important roles in Telomerase-independent forms of telomere elongation or alternative lengthening of telomeres (ALT). A mechanism that although provides cells with a window of opportunity to save ends of their chromosomes, puts these Telomerase (-/-) cells under continuous stress. BLM localization within ALT-associated PML nuclear bodies in telomerase-negative immortalized cell lines and its interaction with the telomere-specific proteins strengthens that suggestion. Here, I begin by outlining features common to all RecQ helicases. I, then, survey evidences that implicate possible roles of BLM helicase in this recombination-mediated mechanism of telomere elongation. PMID:23268311

Rezazadeh, Sarallah

2012-12-26

18

On BLM helicase in recombination-mediated telomere maintenance.  

UK PubMed Central (United Kingdom)

Bloom syndrome (BS) is an extremely rare, autosomal recessive genetic syndrome of humans. Patients with BS are predisposed to almost all forms of cancer and also display premature aging phenotypes. These patients are diagnosed in the clinics by hyper-recombination phenotype that is manifested by high rates of sister chromatid exchange. The gene mutated in BS, designated BLM, lies on chromosome 15q26.1 and encodes a RecQ-like ATP-dependent 3'-5' helicase, which functions in DNA double-strand break repair processes such as non-homologous end joining, homologous recombination-mediated repair, resolution of stalled replication forks and synthesis-dependent strand annealing, although its precise functions at the telomeres are speculative. Recently it has been suggested that the BLM helicase may play important roles in Telomerase-independent forms of telomere elongation or alternative lengthening of telomeres (ALT). A mechanism that although provides cells with a window of opportunity to save ends of their chromosomes, puts these Telomerase (-/-) cells under continuous stress. BLM localization within ALT-associated PML nuclear bodies in telomerase-negative immortalized cell lines and its interaction with the telomere-specific proteins strengthens that suggestion. Here, I begin by outlining features common to all RecQ helicases. I, then, survey evidences that implicate possible roles of BLM helicase in this recombination-mediated mechanism of telomere elongation.

Rezazadeh S

2013-04-01

19

Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (R...

Vassin, Vitaly M.; Wold, Marc S.; Borowiec, James A.

20

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...

 
 
 
 
21

Bistability-mediated carrier recombination at light-induced boron-oxygen complexes in silicon.  

UK PubMed Central (United Kingdom)

A first-principles study of the BO2 complex in B-doped Czochralski Si reveals a defect-bistability-mediated carrier recombination mechanism, which contrasts with the standard fixed-level Shockley-Read-Hall model of recombination. An O2 dimer distant from B causes only weak carrier recombination, which nevertheless drives O2 diffusion under light to form the BO2 complex. Although BO2 and O2 produce nearly identical defect levels in the band gap, the recombination at BO2 is substantially faster than at O2 because the charge state of the latter inhibits the hole capture step of recombination.

Du MH; Branz HM; Crandall RS; Zhang SB

2006-12-01

22

Bistability-Mediated Carrier Recombination at Light-Induced Boron-Oxygen Complexes in Silicon  

Science.gov (United States)

A first-principles study of the BO2 complex in B-doped Czochralski Si reveals a defect-bistability-mediated carrier recombination mechanism, which contrasts with the standard fixed-level Shockley-Read-Hall model of recombination. An O2 dimer distant from B causes only weak carrier recombination, which nevertheless drives O2 diffusion under light to form the BO2 complex. Although BO2 and O2 produce nearly identical defect levels in the band gap, the recombination at BO2 is substantially faster than at O2 because the charge state of the latter inhibits the hole capture step of recombination.

Du, Mao-Hua; Branz, Howard M.; Crandall, Richard S.; Zhang, S. B.

2006-12-01

23

Transduction of cellular neo mRNA by retrovirus-mediated recombination.  

UK PubMed Central (United Kingdom)

Transduction of cellular oncogenes by retroviruses is thought to be a multistep process, involving transcriptional activation of a cellular gene by upstream proviral integration and joining of cellular DNA to retroviral transcriptional signals, followed by copackaging and recombination with a helper virus genome during reverse transcription. To examine the molecular mechanism of the reverse transcriptase-mediated recombination, we introduced into mouse fibroblast cells a variety of constructs in which the neo selectable marker was joined to flanking retroviruslike or cell-like sequences. After superinfection and copackaging with a replication-competent Mo-MuLVsupF virus, the formation of recombinant neo transducing viruses was assessed in a second round of virus infection by the ability to confer G418 resistance to infected cells. Our results showed that recombinant neo proviruses were generated from neo RNA containing either a 5' or 3' retroviral end, implying that one recombination event with helper virus RNA was sufficient to incorporate the neo gene into proviral DNA. Recombination occurred with an apparent frequency of 10(-4) to 10(-5) per replication cycle in the absence of homology between the two recombining partners. This frequency, however, increased at least 100-fold if homology was provided at the site of recombination. Our results support the hypothesis that neo-transducing viruses arise via reverse transcriptase-mediated recombination of RNA rather than by recombination proceeding through DNA intermediates. Unexpectedly, removal of the retroviral packaging site psi reduced the number of neo recombinants only slightly. Our data indicated that although RNAs lacking the psi site are poorly packaged into virions, those RNAs that are included in the virions undergo frequent recombination, even if there is no selection for recombination. Many of the neo recombinants formed with the psi- constructs had undergone additional recombinations and often incorporated the psi site from the helper RNA.

Stuhlmann H; Dieckmann M; Berg P

1990-12-01

24

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 dif...

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

25

Reconstitution of recombination-associated DNA synthesis with human proteins.  

UK PubMed Central (United Kingdom)

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 processively. The single-stranded DNA binding protein RPA facilitates recombination-mediated DNA synthesis by increasing the efficiency of primer utilization, preventing polymerase stalling at specific sequence contexts, and overcoming polymerase stalling caused by topological constraint allowing the transition to a migrating D-loop. Our results support a model whereby the high-fidelity replicative DNA polymerase ? performs recombination-associated DNA synthesis, with translesion synthesis polymerases providing a supportive role as in normal replication.

Sneeden JL; Grossi SM; Tappin I; Hurwitz J; Heyer WD

2013-05-01

26

Involvement of Escherichia coli DNA Replication Proteins in Phage Lambda Red-Mediated Homologous Recombination.  

UK PubMed Central (United Kingdom)

The Red recombination system of bacteriophage lambda is widely used for genetic engineering because of its ability to promote recombination between bacterial chromosomes or plasmids and linear DNA species introduced by electroporation. The process is known to be intimately tied to replication, but the cellular functions which participate with Red in this process are largely unknown. Here two such functions are identified: the GrpE-DnaK-DnaJ chaperone system, and DNA polymerase I. Mutations in either function are found to decrease the efficiency of Red recombination. grpE and dnaJ mutations which greatly decrease Red recombination with electroporated DNA species have only small effects on Red-mediated transduction. This recombination event specificity suggests that the involvement of GrpE-DnaJ-DnaK is not simply an effect on Red structure or stability.

Poteete AR

2013-01-01

27

Recombinant avian adeno-associated virus-mediated oviduct-specific expression of recombinant human tissue kallikrein.  

UK PubMed Central (United Kingdom)

Human tissue kallikrein (hK1) plays an important role in regulation of blood pressure, electrolyte and glucose transport, and renal function. To evaluate the feasibility of viral vector-mediated expression of recombinant human tissue kallikrein (rhK1) in the egg white of laying hens, human tissue kallikrein gene (hKLK1) cDNA-expression cassette was subcloned into avian adeno-associated virus (AAAV) transfer vector pAITR and transfected into AAV-293 cells with AAAV helper vector pcDNA-ARC and adenovirus helper vector pHelper. The recombinant viral particles with a typical AAAV morphology and relatively high titer were generated and identified by PCR and electron microscopy. After 1 intravenous injection of each laying hen with 2 x 10(10) viral particles, oviduct-specific expression of hKLK1 cDNA was demonstrated by reverse transcription-PCR. Secretion of rhK1 into the egg white was detected by enzymatic assay from d 2, reaching the highest level of 107 U/mL in wk 3, and lasted for more than 6 wk after injection. Western blotting showed that the oviduct-expressed rhK1 had the same molecular mass with the natural enzyme. These data suggest that rAAAV can mediate high level and long-lasting transgene expression in oviduct cells, and the established expression system is useful for production of other recombinant proteins.

Wang AP; Sun HC; Wang JY; Wang YJ; Yuan WF

2008-04-01

28

Recombinant avian adeno-associated virus-mediated oviduct-specific expression of recombinant human tissue kallikrein.  

Science.gov (United States)

Human tissue kallikrein (hK1) plays an important role in regulation of blood pressure, electrolyte and glucose transport, and renal function. To evaluate the feasibility of viral vector-mediated expression of recombinant human tissue kallikrein (rhK1) in the egg white of laying hens, human tissue kallikrein gene (hKLK1) cDNA-expression cassette was subcloned into avian adeno-associated virus (AAAV) transfer vector pAITR and transfected into AAV-293 cells with AAAV helper vector pcDNA-ARC and adenovirus helper vector pHelper. The recombinant viral particles with a typical AAAV morphology and relatively high titer were generated and identified by PCR and electron microscopy. After 1 intravenous injection of each laying hen with 2 x 10(10) viral particles, oviduct-specific expression of hKLK1 cDNA was demonstrated by reverse transcription-PCR. Secretion of rhK1 into the egg white was detected by enzymatic assay from d 2, reaching the highest level of 107 U/mL in wk 3, and lasted for more than 6 wk after injection. Western blotting showed that the oviduct-expressed rhK1 had the same molecular mass with the natural enzyme. These data suggest that rAAAV can mediate high level and long-lasting transgene expression in oviduct cells, and the established expression system is useful for production of other recombinant proteins. PMID:18340000

Wang, A P; Sun, H C; Wang, J Y; Wang, Y J; Yuan, W F

2008-04-01

29

Homologous recombination-mediated cloning and manipulation of genomic DNA regions using Gateway and recombineering systems  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Employing genomic DNA clones to characterise gene attributes has several advantages over the use of cDNA clones, including the presence of native transcription and translation regulatory sequences as well as a representation of the complete repertoire of potential splice variants encoded by the gene. However, working with genomic DNA clones has traditionally been tedious due to their large size relative to cDNA clones and the presence, absence or position of particular restriction enzyme sites that may complicate conventional in vitro cloning procedures. Results To enable efficient cloning and manipulation of genomic DNA fragments for the purposes of gene expression and reporter-gene studies we have combined aspects of the Gateway system and a bacteriophage-based homologous recombination (i.e. recombineering) system. To apply the method for characterising plant genes we developed novel Gateway and plant transformation vectors that are of small size and incorporate selectable markers which enable efficient identification of recombinant clones. We demonstrate that the genomic coding region of a gene can be directly cloned into a Gateway Entry vector by recombineering enabling its subsequent transfer to Gateway Expression vectors. We also demonstrate how the coding and regulatory regions of a gene can be directly cloned into a plant transformation vector by recombineering. This construct was then rapidly converted into a novel Gateway Expression vector incorporating cognate 5' and 3' regulatory regions by using recombineering to replace the intervening coding region with the Gateway Destination cassette. Such expression vectors can be applied to characterise gene regulatory regions through development of reporter-gene fusions, using the Gateway Entry clones of GUS and GFP described here, or for ectopic expression of a coding region cloned into a Gateway Entry vector. We exemplify the utility of this approach with the Arabidopsis PAP85 gene and demonstrate that the expression profile of a PAP85::GUS transgene highly corresponds with native PAP85 expression. Conclusion We describe a novel combination of the favourable attributes of the Gateway and recombineering systems to enable efficient cloning and manipulation of genomic DNA clones for more effective characterisation of gene function. Although the system and plasmid vectors described here were developed for applications in plants, the general approach is broadly applicable to gene characterisation studies in many biological systems.

Rozwadowski Kevin; Yang Wen; Kagale Sateesh

2008-01-01

30

Application of RPA in afforestation in Guyuan  

UK PubMed Central (United Kingdom)

This paper describes the application of RPA in Guyuan of forestation. The result shows that can raise the abilities of drought resistance and grwoth, and efficiently prevent the harm of mouses. 1~(st) and 2~(nd). The high-growth of Larix sp. in planting years are 8.36cm and 22.08cm, 2.0cm and 13.33cm taller than control. The death rate by Zokor in 2~(nd) planting year is 1.0%, The death rate by drought is 4.0% after dipping in RPA slurry. The death rate by Zokor is 33.0%, by drought is 20.0% in control. The defending effect of RPA to drought and mouses is up to 89.5%

Yang Xuejun; Han Chongxuan; Li Jiguang; Wang Mingchun; Zhang Hongli; Yang Qinge; He Yan

2004-01-01

31

Entropy and Effective temperature in second RPA dynamics  

International Nuclear Information System (INIS)

[en] 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

1989-01-01

32

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.

1999-01-01

33

The Skyrme energy functional and RPA calculations  

International Nuclear Information System (INIS)

A prescription to derive the particle-hole interaction from Skyrme's forces is given. Self-consistent RPA calculations in 16O and 40Ca show that no spin-flip instability appears, large discrepancies between experimental and calculated spectra are attributed to the single particle energies. The importance of self-consistency is established. (Auth.).

1976-01-01

34

Hed1 regulates Rad51-mediated recombination via a novel mechanism  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Two RecA orthologs, Rad51 and Dmc1, mediate homologous recombination in meiotic cells. During budding yeast meiosis, Hed1 coordinates the actions of Rad51 and Dmc1 by down-regulating Rad51 activity. It is thought that Hed1-dependent attenuation of Rad51 facilitates formation of crossovers that are n...

Busygina, Valeria; Sehorn, Michael G.; Shi, Idina Y.; Tsubouchi, Hideo; Roeder, G. Shirleen; Sung, Patrick

35

Recombination-mediated genetic engineering of Plasmodium berghei DNA.  

UK PubMed Central (United Kingdom)

DNA of Plasmodium berghei is difficult to manipulate in Escherichia coli by conventional restriction and ligation methods due to its high content of adenine and thymine (AT) nucleotides. This limits our ability to clone large genes and to generate complex vectors for modifying the parasite genome. We here describe a protocol for using lambda Red recombinase to modify inserts of a P. berghei genomic DNA library constructed in a linear, low-copy, phage-derived vector. The method uses primer extensions of 50 bp, which provide sufficient homology for an antibiotic resistance marker to recombine efficiently with a P. berghei genomic DNA insert in E. coli. In a subsequent in vitro Gateway reaction the bacterial marker is replaced with a cassette for selection in P. berghei. The insert is then released and used for transfection. The basic techniques we describe here can be adapted to generate highly efficient vectors for gene deletion, tagging, targeted mutagenesis, or genetic complementation with larger genomic regions.

Pfander C; Anar B; Brochet M; Rayner JC; Billker O

2013-01-01

36

Cre recombinase recombination gene and Cre/loxP-mediated transgenosis safe plant expression vector  

UK PubMed Central (United Kingdom)

The invention discloses a Cre recombinase recombination gene and a Cre/loxP-mediated transgenic safe plant expression vector, relates to a Cre recombinase recombination gene and a plant expression vector and solves the problems of the traditional method for removing marker genes in transgenic plants by the traditional Cre/loxP locus specific recombination system has the problem that the deleting efficiency is reduced because the Cre/loxP locus specific recombination system has the reversibility. The crein gene sequence is as shown in SEQ ID NO:1. The vector contains a maker gene bar, a crein gene, a heat shock promoter hsp and a forming type promoter CaMV35S. The invention can improve the safety of the transgenic plants.

DIAN CHEN; YONG WANG; YAYING XU

37

Replication Protein A (RPA) Hampers the Processive Action of APOBEC3G Cytosine Deaminase on Single-Stranded DNA  

Science.gov (United States)

Background Editing deaminases have a pivotal role in cellular physiology. A notable member of this superfamily, APOBEC3G (A3G), restricts retroviruses, and Activation Induced Deaminase (AID) generates antibody diversity by localized deamination of cytosines in DNA. Unconstrained deaminase activity can cause genome-wide mutagenesis and cancer. The mechanisms that protect the genomic DNA from the undesired action of deaminases are unknown. Using the in vitro deamination assays and expression of A3G in yeast, we show that replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, severely inhibits the deamination activity and processivity of A3G. Principal Findings/Methodology We found that mutations induced by A3G in the yeast genomic reporter are changes of a single nucleotide. This is unexpected because of the known property of A3G to catalyze multiple deaminations upon one substrate encounter event in vitro. The addition of recombinant RPA to the oligonucleotide deamination assay severely inhibited A3G activity. Additionally, we reveal the inverse correlation between RPA concentration and the number of deaminations induced by A3G in vitro on long ssDNA regions. This resembles the “hit and run” single base substitution events observed in yeast. Significance Our data suggest that RPA is a plausible antimutator factor limiting the activity and processivity of editing deaminases in the model yeast system. Because of the similar antagonism of yeast RPA and human RPA with A3G in vitro, we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance.

Lada, Artem G.; Prakash, Aishwarya; Borgstahl, Gloria E. O.; Rogozin, Igor B.; Pavlov, Youri I.

2011-01-01

38

Replication protein A (RPA) hampers the processive action of APOBEC3G cytosine deaminase on single-stranded DNA.  

UK PubMed Central (United Kingdom)

BACKGROUND: Editing deaminases have a pivotal role in cellular physiology. A notable member of this superfamily, APOBEC3G (A3G), restricts retroviruses, and Activation Induced Deaminase (AID) generates antibody diversity by localized deamination of cytosines in DNA. Unconstrained deaminase activity can cause genome-wide mutagenesis and cancer. The mechanisms that protect the genomic DNA from the undesired action of deaminases are unknown. Using the in vitro deamination assays and expression of A3G in yeast, we show that replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, severely inhibits the deamination activity and processivity of A3G. PRINCIPAL FINDINGS/METHODOLOGY: We found that mutations induced by A3G in the yeast genomic reporter are changes of a single nucleotide. This is unexpected because of the known property of A3G to catalyze multiple deaminations upon one substrate encounter event in vitro. The addition of recombinant RPA to the oligonucleotide deamination assay severely inhibited A3G activity. Additionally, we reveal the inverse correlation between RPA concentration and the number of deaminations induced by A3G in vitro on long ssDNA regions. This resembles the "hit and run" single base substitution events observed in yeast. SIGNIFICANCE: Our data suggest that RPA is a plausible antimutator factor limiting the activity and processivity of editing deaminases in the model yeast system. Because of the similar antagonism of yeast RPA and human RPA with A3G in vitro, we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance.

Lada AG; Waisertreiger IS; Grabow CE; Prakash A; Borgstahl GE; Rogozin IB; Pavlov YI

2011-01-01

39

Recombinant adenovirus-mediated gene transfer suppresses experimental arthritis  

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Full Text Available Collagen Induced Arthritis (CIA) is a widely studied animal model to develop and test novel therapeutic approaches for treating Rheumatoid Arthritis (RA) in humans. Soluble Cytotoxic T-Lymphocyte Antigen 4 (CTLA4-Ig), which binds B7 molecule on antigen presenting cells and blocks CD28 mediated T-lymphocyte activation, has been shown to ameliorate experimental autoimmune diseases such as lupus, diabetes and CIA. Objective of our research was to investigate in vivo the effectiveness of blocking the B7/CD28 T-lymphocyte co-stimulatory pathway, utilizing a gene transfer technology, as a therapeutic strategy against CIA. Replication-deficient adenoviruses encoding a chimeric CTLA4-Ig fusion protein, or ?-galactosidase as control, have been injected intravenously once at arthritis onset. Disease activity has been monitored by the assessment of clinical score, paw thickness and type II collagen (CII) specific cellular and humoral immune responses for 21 days. The adenovirally delivered CTLA4-Ig fusion protein at a dose of 2×108 pfu suppressed established CIA, whereas the control ?-galactosidase did not significantly affect the disease course. CII-specific lymphocyte proliferation, IFNg production and anti-CII antibodies were significantly reduced by CTLA4-Ig treatment. Our results demonstrate that blockade of the B7/CD28 co-stimulatory pathway by adenovirus-mediated CTLA4-Ig gene transfer is effective in treating established CIA suggesting its potential in treating RA.

E. Quattrocchi; M. Feldmann

2011-01-01

40

A recurrent translocation is mediated by homologous recombination between HERV-H elements  

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Full Text Available Abstract Background Chromosome rearrangements are caused by many mutational mechanisms; of these, recurrent rearrangements can be particularly informative for teasing apart DNA sequence-specific factors. Some recurrent translocations are mediated by homologous recombination between large blocks of segmental duplications on different chromosomes. Here we describe a recurrent unbalanced translocation casued by recombination between shorter homologous regions on chromosomes 4 and 18 in two unrelated children with intellectual disability. Results Array CGH resolved the breakpoints of the 6.97-Megabase (Mb) loss of 18q and the 7.30-Mb gain of 4q. Sequencing across the translocation breakpoints revealed that both translocations occurred between 92%-identical human endogenous retrovirus (HERV) elements in the same orientation on chromosomes 4 and 18. In addition, we find sequence variation in the chromosome 4 HERV that makes one allele more like the chromosome 18 HERV. Conclusions Homologous recombination between HERVs on the same chromosome is known to cause chromosome deletions, but this is the first report of interchromosomal HERV-HERV recombination leading to a translocation. It is possible that normal sequence variation in substrates of non-allelic homologous recombination (NAHR) affects the alignment of recombining segments and influences the propensity to chromosome rearrangement.

Hermetz Karen E; Surti Urvashi; Cody Jannine D; Rudd M Katharine

2012-01-01

 
 
 
 
41

Toward a consistent RHA-RPA  

International Nuclear Information System (INIS)

The authors examine the RPA based on a relativistic Hartree approximation description for nuclear ground states. This model includes contributions from the negative energy sea at the 1-loop level. They emphasize consistency between the treatment of the ground state and the RPA. This consistency is important in the description of low-lying collective levels but less important for the longitudinal (e, e') quasi-elastic response. They also study the effect of imposing a 3-momentum cutoff on negative energy sea contributions. A cutoff of twice the nucleon mass improves agreement with observed spin orbit splittings in nuclei compared to the standard infinite cutoff results, an effect traceable to the fact that imposing the cutoff reduces m*/m. The cutoff is much less important than consistency in the description of low-lying collective levels. The cutoff model provides excellent agreement with quasi-elastic (e, e') data.

1991-01-01

42

Ground-state correlations beyond RPA  

Energy Technology Data Exchange (ETDEWEB)

An approach is proposed which includes ground-state correlations beyond RPA. This is achieved by using a renormalized quasiboson approximation with reference to the correlated ground state of the system, rather than to the uncorrelated Hartree-Fock one, and by expressing the particle-particle and hole-hole pair operators in terms of the particle-hole ones. The set of the so-obtained nonlinear equations for the one-phonon energy and amplitudes are first solved within the Lipkin-Meshkov-Glick model and the results are compared with those of previous approaches as well as with the exact ones. Applications to realistic nuclei, starting from fully self-consistent HF + RPA calculations with a Skyrme force are also presented. ((orig.))

Catara, F. (Dipartimento di Fisica, Universita di Catania, Catania (Italy) Istituto Nazionale di Fisica Nucleare, Sezione di Catania, Corso Italia 57, I-95129 Catania (Italy)); Dinh Dang, N. (Istituto Nazionale di Fisica Nucleare, Sezione di Catania, Corso Italia 57, I-95129 Catania (Italy)); Sambataro, M. (Istituto Nazionale di Fisica Nucleare, Sezione di Catania, Corso Italia 57, I-95129 Catania (Italy))

1994-10-17

43

Ground-state correlations beyond RPA  

International Nuclear Information System (INIS)

[en] An approach is proposed which includes ground-state correlations beyond RPA. This is achieved by using a renormalized quasiboson approximation with reference to the correlated ground state of the system, rather than to the uncorrelated Hartree-Fock one, and by expressing the particle-particle and hole-hole pair operators in terms of the particle-hole ones. The set of the so-obtained nonlinear equations for the one-phonon energy and amplitudes are first solved within the Lipkin-Meshkov-Glick model and the results are compared with those of previous approaches as well as with the exact ones. Applications to realistic nuclei, starting from fully self-consistent HF + RPA calculations with a Skyrme force are also presented. ((orig.))

1994-10-17

44

Continuum RPA calculation of escape widths  

Energy Technology Data Exchange (ETDEWEB)

Particle-hole partial decay widths are calculated within the continuum RPA exactly, i.e. without any further approximation, in a square well plus Coulomb potential and using a separable residual interaction. The results are compared with the ones obtained by making pole expansions of the single-particle Green functions (Berggren and Mittag-Leffler). It is found that the Berggren and Mittag-Leffler expansions give results in good agreement with the 'exact' ones. (orig.).

Vertse, T. (Inst. of Nuclear Research, Hungarian Academy of Sciences, Debrecen (Hungary)); Curutchet, P.; Liotta, R.J. (Manne Siegbahn Inst. of Physics, Stockholm (Sweden)); Bang, J. (Niels Bohr Inst., Copenhagen (Denmark)); Giai, N. van (Inst. de Physique Nucleaire, 91 - Orsay (France))

1991-07-25

45

Continuum RPA calculation of escape widths  

International Nuclear Information System (INIS)

[en] Particle-hole partial decay widths are calculated within the continuum RPA exactly, i.e. without any further approximation, in a square well plus Coulomb potential and using a separable residual interaction. The results are compared with the ones obtained by making pole expansions of the single-particle Green functions (Berggren and Mittag-Leffler). It is found that the Berggren and Mittag-Leffler expansions give results in good agreement with the 'exact' ones. (orig.)

1991-07-25

46

RPA calculations with Gaussian expansion method  

International Nuclear Information System (INIS)

The Gaussian expansion method (GEM) is applied to calculations of the nuclear excitations in the random-phase approximation (RPA). We adopt the mass-independent basis-set that is successful in the mean-field calculations. The RPA results obtained by the GEM are compared with those obtained by several other available methods in Ca isotopes, by using a density-dependent contact interaction along with the Woods-Saxon single-particle states. It is confirmed that energies, transition strengths and widths of their distribution are described by the GEM with good precision, for the 1-, 2+ and 3- collective states. The GEM is then applied to the self-consistent RPA calculations with the finite-range Gogny D1S interaction. The spurious center-of-mass motion is well separated from the physical states in the E1 response, and the energy-weighted sum rules for the isoscalar transitions are fulfilled reasonably well. Properties of low-energy transitions in 60Ca are investigated in some detail.

2009-09-15

47

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

UK PubMed Central (United Kingdom)

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 structural biology experiments revealed that caffeine interfered with a pivotal step in homologous recombination, homologous joint molecule formation, through increasing interactions of the RAD51 nucleoprotein filament with non-homologous DNA. Our results suggest that recombination pathways dependent on extensive homology search are caffeine-sensitive and stress the importance of considering direct checkpoint-independent mechanisms in the interpretation of the effects of caffeine on DNA repair.

Zelensky AN; Sanchez H; Ristic D; Vidic I; van Rossum-Fikkert SE; Essers J; Wyman C; Kanaar R

2013-07-01

48

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

Science.gov (United States)

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 structural biology experiments revealed that caffeine interfered with a pivotal step in homologous recombination, homologous joint molecule formation, through increasing interactions of the RAD51 nucleoprotein filament with non-homologous DNA. Our results suggest that recombination pathways dependent on extensive homology search are caffeine-sensitive and stress the importance of considering direct checkpoint-independent mechanisms in the interpretation of the effects of caffeine on DNA repair. PMID:23666627

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

2013-05-10

49

Expression of human clotting factor IX mediated by various recombinant lentiviral vectors in hemophilia B mice  

UK PubMed Central (United Kingdom)

Hemophilia B is an X-linked disorder due to a partial or complete deficiency of clotting factor IX activity in the plasma. Conventional therapy depends on the infusion of human plasma or F IX concentration. The therapeutic effect is temporary and periodic infusions are required. In addition, such therapy is associated with significant risks of AIDs and hepatitis. Thus, it approves beneficial to develop an alternative therapy for hemophilia B. In this study, two various recombinant lentiviral vectors with hF IX gene were constructed. The recombinant FUXW vector was regulated by ubiquitin promoter and the recombinant FAXW vector was regulated by ABP promoter (liver specific) respectively. High titer of recombinant lentivirus was prepared and purified from 293T cells mediated by transient cotransfection of three plasmids with calcium phosphate. Recombinant FUXW virus and FAXW virus was cotransfected to hemophilia B mice by injection and hydrodynamics-based administration via tail vein. Serum hF IX antigen was detected in all treated mice, and the levels of F IX antigen showed dose-responses, the high peak level of hF IX was 45 ng/mL, and its expression lasted for more than 60 d. The expression level of FAXW group was higher than that of FUXW group. There was no significant difference of expression level between injection and hydrodynamics-based administration via tail vein. These data offered a promising result for further study.

Gong Juli; Chen Xiaoguang; Zhu Huanzhang; Xue Jinglun

2004-01-01

50

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

Science.gov (United States)

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 repetitive manner, unwinding a short length of duplex DNA followed by rapid reannealing and reinitiation of unwinding in several successions. Our results show that a monomeric BLM can 'measure' how many base pairs it has unwound, and once it has unwound a critical length, it reverses the unwinding reaction through strand switching and translocating on the opposing strand. Repetitive unwinding persisted even in the presence of hRPA, and interaction between wild-type BLM and hRPA was necessary for unwinding reinitiation on hRPA-coated DNA. The reported activities may facilitate BLM processing of stalled replication forks and illegitimately formed recombination intermediates. PMID:19165145

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

2009-01-22

51

Recombiner  

International Nuclear Information System (INIS)

Burnable gases in a reactor container are delivered by a blower, and oxygen and hydrogen in the burnable gases are heated by a heater. Oxygen and hydrogen are combined by a recombiner into steams, and a gas/liquid two phase flow during the steam formation is cooled by a cooler. Then, the gas/liquid two phase flow is separated by a gas/liquid separator, and liquefied water is introduced into a suppression chamber to control the concentration of the burnable gases released into the container. In this case, dynamic equipments such as a blower, a cooler and a gas/liquid separator in the recombiner are disposed fixedly, and static equipments, i.e., only the portions of the heater and the recombiner are combined integrally so as to be portable. With such a constitution, complexity due to common use of the portion is mitigated without degradation of reliability, and operation for the maintenance and handling can be conducted flexibly. (T.M.).

1990-12-25

52

Recombinant CTFR detection in CF tracheal epithelial cells following in vitro liposomeme-mediated gene transfer.  

UK PubMed Central (United Kingdom)

The efficacy of CFTR gene transfer mediated by cationic liposomes Dc-Chol/DOPE into cystic fibrosis (CF) tracheal epithelial cells carrying defective processing mutations (S549N/N1303K), was assessed by studying mRNA and protein expression of the recombinant product. Appreciable levels of mRNA transcripts were detected 48 h after transfection, while complete translocation of the recombinant CFTR to the apical membrane of epithelial cells was observed after 72 h following transfection. Our results suggest that in vitro restoration of a normal CFTR processing and migration to the cell plasmalemma requires 72 h at least as demonstrated by immunocyto-fluorescence using the monoclonal antibody MATG 1016. These findings are relevant onto gene transfer phase I clinical studies.

Colosimo A; Scarpino S; Sangiuolo F; Sario SD; Mossa G; Novelli G; Dallapiccola B

1997-07-01

53

Recombinant CTFR detection in CF tracheal epithelial cells following in vitro liposomeme-mediated gene transfer.  

Science.gov (United States)

The efficacy of CFTR gene transfer mediated by cationic liposomes Dc-Chol/DOPE into cystic fibrosis (CF) tracheal epithelial cells carrying defective processing mutations (S549N/N1303K), was assessed by studying mRNA and protein expression of the recombinant product. Appreciable levels of mRNA transcripts were detected 48 h after transfection, while complete translocation of the recombinant CFTR to the apical membrane of epithelial cells was observed after 72 h following transfection. Our results suggest that in vitro restoration of a normal CFTR processing and migration to the cell plasmalemma requires 72 h at least as demonstrated by immunocyto-fluorescence using the monoclonal antibody MATG 1016. These findings are relevant onto gene transfer phase I clinical studies. PMID:19856289

Colosimo, A; Scarpino, S; Sangiuolo, F; Sario, S D; Mossa, G; Novelli, G; Dallapiccola, B

1997-07-01

54

Mcm2, but Not Rpa, Is a Component of the Mammalian Early G1-Phase Prereplication Complex  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Previous experiments in Xenopus egg extracts identified what appeared to be two independently assembled prereplication complexes (pre-RCs) for DNA replication: the stepwise assembly of ORC, Cdc6, and Mcm onto chromatin, and the FFA-1–mediated recruitment of RPA into foci on chromatin. We have invest...

Dimitrova, Daniela S.; Todorov, Ivan T.; Melendy, Thomas; Gilbert, David M.

55

A consistent approximation scheme beyond RPA for bosons  

Energy Technology Data Exchange (ETDEWEB)

In this paper, we develop a consistent extension of RPA for bosonic systems. In order to illustrate the method, we consider the case of the anharmonic oscillator. We compare our results with those obtained in mean-field and standard RPA approaches, with the exact ones and show that they are very close to the exact ones. (authors)

Davesne, D.; Oertel, M.; Hansen, H. [IPN Lyon, 43 Bd du 11 Novembre 1918, F-69622 Villeurbanne Cedex (France)

2002-07-01

56

Some applications of renormalized RPA in bosonic field theories  

Energy Technology Data Exchange (ETDEWEB)

We present some applications of the renormalized RPA in bosonic field theories. We first present some developments for the explicit calculation of the total energy in {phi}{sup 4} theory and discuss its phase structure in 1 + 1 dimensions. We also demonstrate that the Goldstone theorem is satisfied in the O(N) model within the renormalized RPA. (authors)

Hansen, H. [IPN Lyon, 43 Bd. du 11 Novembre 1918, F-69622 Villeurbanne Cedex (France)]|[Centro de Fysica Teorica, Departamento de Fysica da Universidade de Coimbra, 3004-516 Coimbra (Portugal); Chanfray, G. [IPN Lyon, 43 Bd. du 11 Novembre 1918, F-69622 Villeurbanne Cedex (France)

2003-07-01

57

Some applications of renormalized RPA in bosonic field theories  

Energy Technology Data Exchange (ETDEWEB)

We present some applications of the renormalized RPA in bosonic field theories. We first present some developments for the explicit calculation of the total energy in {phi}{sup 4} theory and discuss its phase structure in 1+1 dimensions. We also demonstrate that the Goldstone theorem is satisfied in the O(N) model within the renormalized RPA. (orig.)

Hansen, H. [IPN Lyon, 43 Bd. du 11 Novembre 1918, F-69622, Villeurbanne Cedex (France); Departamento de Fisica da Universidade de Coimbra, Centro de Fisica Teorica, P-3004-516, Coimbra (Portugal); Chanfray, G. [IPN Lyon, 43 Bd. du 11 Novembre 1918, F-69622, Villeurbanne Cedex (France)

2004-06-01

58

Solving RPA eigenvalue equation in real-space  

Energy Technology Data Exchange (ETDEWEB)

We present a computational method to solve RPA eigenvalue equation employing a uniform grid representation in the three-dimensional Cartesian coordinate. The conjugate gradient method, an iterative method for a generalized eigenvalue problem, is used for this purpose. We apply the present method to Hartree-Fock +RPA calculation with BKN-like interaction and Skyrme interaction. (author)

Muta, Atsushi [Tokyo Institute of Polytechnics, Faculty of Women College, Atsugi, Kanagawa (Japan); Iwata, Jun-ichi; Hashimoto, Yukio; Yabana, Kazuhiro [Tsukuba Univ., Institute of Physics, Tsukuba, Ibaraki (Japan)

2002-09-01

59

One-step construction of lentiviral reporter using Red-mediated recombination.  

UK PubMed Central (United Kingdom)

Current approaches to generate lentiviral vectors, which have been used extensively for gene therapy, are time consuming and require a large expenditure. Here, we directly clone the full length myosin light chain kinase cDNA into enhanced green fluorescence protein (EGFP)-fused pLenti6/V5 expression vector in just one step with the use of Red-mediated recombination system, allowing for rapid and effective cloning of lentiviral expression vectors. In addition, the simultaneous expression of EGFP reporter provides a convenient monitoring mean for host cell infection and for localization of the target proteins.

Zha J; Chen X; Li C; Zhu M; Ding G; He W

2011-11-01

60

Species-specific variation of RPA-interacting protein (RIP) splice isoforms.  

UK PubMed Central (United Kingdom)

Replication Protein A (RPA) is a single stranded DNA-binding protein involved in DNA metabolic activities such as replication, repair, and recombination. RPA-Interacting Protein alpha (RIPalpha) was originally identified as a nuclear transporter of RPA in Xenopus. The human RIPalpha gene encodes several splice isoforms, of which hRIPalpha and hRIPbeta are the major translation products in vivo. However, limited information is available about the alternative splicing of RIPalpha in eukaryotes, apart from that in humans. In this study, we examined the alternative splicing of RIPalpha in the Drosophila, Xenopus, and mouse system. We showed that the number of splice isoforms of RIPalpha was species-specific, and displayed a tendency to increase in higher eukaryotes. Moreover, a mouse ortholog of hRIPbeta, mRIPbeta2, was not SUMOylated, in contrast to hRIPbeta. Based on these results, we suggest that the RIPalpha gene gains more splice isoforms and additional modifications after molecular evolution. [BMB reports 2009; 42(1): 22-27].

Kim K; Lee EJ; Lee SH; Seo T; Jang IS; Park J; Lee JH

2009-01-01

 
 
 
 
61

Extended RPA study of nuclear collective phenomena  

International Nuclear Information System (INIS)

A fully microscopic study of nuclear collective phenomena is presented within the framework of an extended RPA which includes 1p-1h and 2p-2h excitations in a consistent way. This theory allows us to obtain a very realistic description of various excitation spectra. As a result, a strong evidence of correlation effects beyond mean-field theory emerges. The effective interaction used is a G-matrix derived from the meson-exchange potential. The extended theory introduces also additional correlations which screen the long-large part of the effective interaction. This effect significantly enhances the stability of the ground state against density fluctuations. In this connection a possible importance of relativistic effects is also discussed. 99 refs., 19 figs., 5 tabs. (author)

1987-01-01

62

RPA correction to the optical potential  

Directory of Open Access Journals (Sweden)

Full Text Available In studies of nucleon elastic scattering, a correction to the microscopic optical potential built from Melbourne g-matrix was found to be necessary at low nucleon incident energy [1,2]. Indeed, at energies lower than 60 MeV, the absorption generated from Melbourne g-matrix is too weak within 25%. Coupling to collective excited states of the target nucleus are not included in the g-matrix and could explain the missing absorption. We propose to calculate this correction to the optical potential using the Gogny D1S effective nucleon-nucleon interaction in the coupling to excited states of the target. We use the Random Phase Approximation (RPA) description of the excited states of the target with the same interaction.

Blanchon G.; Dupuis M.; Mau N. Vinh; Bauge E.

2010-01-01

63

Subtraction method and stability condition in the extended RPA theories  

CERN Document Server

The extended RPA theories are analyzed from the point of view of the problem of stability of their solutions. Three kinds of such theories are considered: the second RPA and two versions of the quasiparticle-phonon coupling model within the time-blocking approximation: the model including 1p1h*phonon configurations and the two-phonon model. It is shown that stability is ensured by making use of the subtraction method proposed previously to solve double counting problem in these theories. This enables one to generalize the famous Thouless theorem proved in the case of the RPA. These results are illustrated by an example of schematic model.

Tselyaev, V I

2013-01-01

64

Nuclear spin-isospin excitation by extended RPA models  

Energy Technology Data Exchange (ETDEWEB)

Nuclear responses of spin-isospin modes are studied with microscopic structure models. The RPA model based on the quasi-boson approximation has been extended by restoring the commutation relations of fermions in the equation-of-motion formalism. It is shown that the extended model avoids a collapse of RPA, and gives more stable results than the usual RPA on nuclear spin-isospin responses. In order to perform more reliable nuclear structure calculations, a very efficient shell-model program has been developed for fp-shell nuclei, by taking a TJ-coupled basis and the Davidson algorithm for matrix diagonalization. (author)

Muto, Kazuo [Tokyo Inst. of Tech. (Japan). Dept. of Physics

1996-06-01

65

Protein tag-mediated conjugation of oligonucleotides to recombinant affinity binders for proximity ligation.  

UK PubMed Central (United Kingdom)

While antibodies currently play a dominant role as affinity reagents in biological research and for diagnostics, a broad range of recombinant proteins are emerging as promising alternative affinity reagents in detection assays and quantification. DNA-mediated affinity-based assays, such as immuno-PCR and proximity ligation assays (PLA), use oligonucleotides attached to affinity reagents as reporter molecules. Conjugation of oligonucleotides to affinity reagents generally employs chemistries that target primary amines or cysteines. Because of the random nature of these processes neither the number of oligonucleotides conjugated per molecule nor their sites of attachment can be accurately controlled for affinity reagents with several available amines and cysteines. Here, we present a straightforward and convenient approach to functionalize recombinant affinity reagents for PLA by expressing the reagents as fusion partners with SNAP protein tags. This allowed us to conjugate oligonucleotides in a site-specific fashion, yielding precisely one oligonucleotide per affinity reagent. We demonstrate this method using designed ankyrin repeat proteins (DARPins) recognizing the tumor antigen HER2 and we apply the conjugates in different assay formats. We also show that SNAP or CLIP tags, expressed as fusion partners of transfected genes, allow oligonucleotide conjugations to be performed in fixed cells, with no need for specific affinity reagents. The approach is used to demonstrate induced interactions between the fusion proteins FKBP and FRB by allowing the in situ conjugated oligonucleotides to direct the production of templates for localized rolling circle amplification reactions.

Gu GJ; Friedman M; Jost C; Johnsson K; Kamali-Moghaddam M; Plückthun A; Landegren U; Söderberg O

2013-01-01

66

Attachment Site Selection and Identity in Bxb1 Serine Integrase-Mediated Site-Specific Recombination  

Science.gov (United States)

Phage-encoded serine integrases mediate directionally regulated site-specific recombination between short attP and attB DNA sites without host factor requirements. These features make them attractive for genome engineering and synthetic genetics, although the basis for DNA site selection is poorly understood. Here we show that attP selection is determined through multiple proofreading steps that reject non-attP substrates, and that discrimination of attP and attB involves two critical site features: the outermost 5–6 base pairs of attP that are required for Int binding and recombination but antagonize attB function, and the “discriminators” at positions ?15/+15 that determine attB identity but also antagonize attP function. Thus, although the attachment sites differ in length and sequence, only two base changes are needed to convert attP to attL, and just two more from attL to attB. The opposing effect of site identifiers ensures that site schizophrenia with dual identities does not occur.

Singh, Shweta; Ghosh, Pallavi; Hatfull, Graham F.

2013-01-01

67

Ground state correlations beyond RPA in finite Fermi systems  

International Nuclear Information System (INIS)

Difficulties in the standard Random Phase Approximation (RPA) are discussed. Previous attempt to eliminate the inconsistency due to uncorrelated HF ground state are shortly reviewed and some results are presented. A new approach is proposed to go further towards a self-consistent RPA. Finally some results on metallic clusters are presented, using a simplified version of the approach which requires less computational effort. It represents a significant improvement over the methods used so far.

1996-01-01

68

RPA correlations and nuclear densities in relativistic mean field approach  

International Nuclear Information System (INIS)

The relativistic mean field approach (RMF) is well known for describing accurately binding energies and nucleon distributions in atomic nuclei throughout the nuclear chart. The random phase approximation (RPA) built on top of the RMF is also a good framework for the study of nuclear excitations. Here, we examine the consequences of long range correlations brought about by the RPA on the neutron and proton densities as given by the RMF approach. (authors)

2007-01-01

69

Regression of established pulmonary metastases and subcutaneous tumor mediated by the systemic administration of high-dose recombinant interleukin 2  

Energy Technology Data Exchange (ETDEWEB)

Incubation of resting lymphoid cells with recombinant interleukin 2 (IL-2) in vitro leads to the generation of lymphokine activated killer (LAK) cells capable of lysing fresh tumor cell suspensions in short-term chromium-release assays. Previous studies have demonstrated that the injection of LAK cells plus low doses of recombinant IL-2 were capable of inhibiting the growth of pulmonary metastases. The authors have now explored the ability of high doses of recombinant IL-2, administered systemically, to generate LAK cells in vivo, and to mediate antitumor effects directly. Doses of 100,000 U recombinant IL-2 administered intraperitoneally approximately every 8 h for 5 d were capable of dramatically inhibiting established 3-d pulmonary metastases from the MCA-105 and MCA-106 syngeneic sarcomas and the syngeneic B16 melanoma in C57BL/6 mice. Grossly visible metastases present at 10 d after tumor injection also underwent regression following IL-2 therapy. All antitumor effects of the systemic administration of recombinant IL-2 were eliminated if mice received prior treatment with 500 rad total body irradiation. The administration of high doses of recombinant IL-2 was also capable of inhibiting the growth of 3-d established subcutaneous tumors from the MCA-105 sarcoma, and of mediating the inhibition of growth and regression of established palpable subcutaneous MCA-105 sarcomas. The ready availability of high doses of recombinant human IL-2, and the demonstration of antitumor effects seen in animal models have led us to the initiation of the clinical trials of recombinant IL-2 in humans.

Rosenberg, S.A.; Mule, J.J.; Spiess, P.J.; Reichert, C.M.; Schwarz, S.L.

1985-05-01

70

Mediators of systemic inflammatory response syndrome and the role of recombinant activated protein C in sepsis syndrome.  

UK PubMed Central (United Kingdom)

The systemic inflammatory response syndrome, the host's response to infection involves a series of cascading events that mobilize a series of mediators involving the immune system, complement, and the coagulation cascade. Although the initial focus of mediators is to limit infection, this cascade may run amok and cause the development of hypotension, vascular instability, and disseminated intravascular coagulation, leading to morbidity and mortality in the host. Several therapeutic trials have focused on the modulation of these mediators, but use of recombinant human activated protein C in patients with severe sepsis is the only one that has shown a benefit in clinical trials.

Kak V

2011-12-01

71

Mediators of systemic inflammatory response syndrome and the role of recombinant activated protein C in sepsis syndrome.  

Science.gov (United States)

The systemic inflammatory response syndrome, the host's response to infection involves a series of cascading events that mobilize a series of mediators involving the immune system, complement, and the coagulation cascade. Although the initial focus of mediators is to limit infection, this cascade may run amok and cause the development of hypotension, vascular instability, and disseminated intravascular coagulation, leading to morbidity and mortality in the host. Several therapeutic trials have focused on the modulation of these mediators, but use of recombinant human activated protein C in patients with severe sepsis is the only one that has shown a benefit in clinical trials. PMID:22054759

Kak, Vivek

2011-09-09

72

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

73

SITE-SPECIFIC RECOMBINATION FOR PLANT GENETIC ENGINEERING: STRATEGY FOR AGRO-MEDIATED GENE STACKING  

Science.gov (United States)

The precise rearrangement of DNA in planta can be achieved through site-specific recombination. For the past decade and a half, laboratory experiments have shown that site-specific recombination can delete genomic DNA, regulate gene expression, recombine chromosomes, and target new DNA into designat...

74

FLP-mediated recombination of FRT sites in the maize genome.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Molecular evidence is provided for genomic recombinations in maize cells induced by the yeast FLP/FRT site-specific recombination system. The FLP protein recombined FRT sites previously integrated into the maize genome leading to excision of a selectable marker, the neo gene. NPTII activity was not ...

Lyznik, L A; Rao, K V; Hodges, T K

75

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

International Nuclear Information System (INIS)

[en] 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

2009-01-01

76

Cre-mediated recombination efficiency and transgene expression patterns of three retinal bipolar cell-expressing Cre transgenic mouse lines.  

UK PubMed Central (United Kingdom)

PURPOSE: Retinal bipolar cells, comprising multiple types, play an essential role in segregating visual information into multiple parallel pathways in the retina. The ability to manipulate gene expression in specific bipolar cell type(s) in the retina is important for understanding the molecular basis of their normal physiological functions and diseases/disorders. The Cre/LoxP recombination system has become an important tool for allowing gene manipulation in vivo, especially with the increasing availability of cell- and tissue-specific Cre transgenic mouse lines. Detailed in vivo examination of the Cre/LoxP recombination efficiency and the transgene expression patterns for cell- and tissue-specific Cre transgenic mouse lines is essential for evaluating their utility. In this study, we investigated the Cre-mediated recombination efficiency and transgene expression patterns of retinal bipolar cell-expressing Cre transgenic lines by crossing with a Cre reporter mouse line and through Cre-dependent recombinant adeno-associated virus (rAAV) vector-mediated transgene delivery. METHODS: Three retinal bipolar cell-expressing Cre-transgenic mouse lines, 5-HTR2a-cre, Pcp2-cre, and Chx10-cre, were crossed with a strong Cre reporter mouse line that expresses a red fluorescent protein variant, tdTomato. rAAV2 vectors carrying a double-floxed inverted open-reading frame sequence encoding channelrhodopsin-2-mCherry (ChR2-mCherry) driven by a ubiquitous neuronal EF1? or a ubiquitous CMV promoter with a rAAV2 capsid mutation (Y444F) were injected into the intravitreal space of the eyes. Immunohistochemistry using retinal bipolar cell type-specific markers was performed to examine Cre-mediated recombination efficiency and the transgene expression patterns in bipolar cells in retinal whole mounts and vertical sections. RESULTS: For the 5-HTR2a-cre and Pcp2-cre mouse lines, the expression pattern of the Cre-mediated recombination by crossing the reporter line largely resembled the expression pattern of Cre. The bipolar cells showing Cre-mediated recombination in the 5-HTR2a-cre line and the Pcp2-cre line were predominantly type 4 cone bipolar cells and rod bipolar cells, respectively. For the Chx10-cre mouse line, the expression pattern of the Cre-mediated recombination by crossing the reporter line was different from that of Cre. The Cre-mediated transgene expression in retinal bipolar cells in the Chx10-cre line was not observed by crossing with the reporter mouse line but through Cre-dependent rAAV vector delivery. A rAAV2 vector with the combination of a CMV promoter and the Y444F capsid mutation achieved Cre-dependent transgene expression in retinal bipolar cells. CONCLUSIONS: The retinal bipolar cell-expressing Cre-transgenic lines and the Cre-dependent rAAV vector reported in this study could be valuable tools for gene targeting and manipulation in retinal bipolar cells in mice.

Lu Q; Ivanova E; Ganjawala TH; Pan ZH

2013-01-01

77

RPA phosphorylation facilitates mitotic exit in response to mitotic DNA damage  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Human replication protein A (RPA) becomes phosphorylated on the RPA2 subunit by cyclin B-Cdc2 during mitosis, although the functional role of this modification is unclear. We find that this modification stimulates RPA2 to become hyperphosphorylated in response to mitotic DNA damage caused by bleomyc...

Anantha, Rachel William; Sokolova, Elena; Borowiec, James A.

78

Mitotic crisis: The unmasking of a novel role for RPA  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Mitotic DNA damage is a constant threat to genomic integrity, yet understanding of the cellular responses to this stress remain incomplete. Recent work by Anantha et al. (PNAS 2008; 105:12903–8) has found surprising evidence that RPA, the primary eukaryotic single-stranded DNA-binding protein, can s...

Anantha, Rachel William; Borowiec, James A.

79

Recombinant adenovirus-mediated intestinal trefoil factor gene therapy for burn-induced intestinal mucosal injury.  

UK PubMed Central (United Kingdom)

Intestinal trefoil factor (ITF) is a small polypeptide with potential medical values whose main pharmacological effects are to alleviate gastrointestinal mucosal injury caused by various injury factors and promote the repair of damaged mucosa. However, its low yield limits its application. The purpose of our study was to construct a recombinant adenoviral vector containing the hITF gene and observe the therapeutic effect of burn-induced intestinal mucosal injury using in vitro and in vivo analysis. First, a recombinant shuttle plasmid was constructed by ligating a pAdTrack-CMV vector with a full-length hITF gene containing a signal peptide and the mature peptide, followed by the recombinant Ad-hITF adenovirus vector after linearization and homologous recombination with the backbone plasmid in the competent BJ5183 strain. Second, the hITF expression level was detected using reverse transcription polymerase chain reaction and western blotting after Ad-hITF infection of colon cancer HT-29 cells. The recombinant adenovirus significantly promoted cell migration in an in vitro wounding model. Finally, we confirmed that the recombinant adenovirus could significantly expedite the healing of intestinal mucosal injury after establishing a mouse model in which severe burns were stimulated and the recombinant adenovirus was delivered by intragastric injection. In summary, we constructed a recombinant adenoviral vector containing the hITF gene and confirmed its role in promoting repair of the intestinal mucosa. Our study provides a novel way to treat burn-induced intestinal mucosal injury.

Sun Y; Zhu Y; Wang L; Mao X; Peng X; Peng Y

2013-01-01

80

Self-consistent RPA based on a many-body vacuum  

International Nuclear Information System (INIS)

Self-Consistent RPA is extended in a way so that it is compatible with a variational ansatz for the ground-state wave function as a fermionic many-body vacuum. Employing the usual equation-of-motion technique, we arrive at extended RPA equations of the Self-Consistent RPA structure. In principle the Pauli principle is, therefore, fully respected. However, the correlation functions entering the RPA matrix can only be obtained from a systematic expansion in powers of some combinations of RPA amplitudes. We demonstrate for a model case that this expansion may converge rapidly.

2011-01-01

 
 
 
 
81

Agrobacterium tumefaciens-mediated transformation for investigation of somatic recombination in the fungal pathogen Armillaria mellea.  

UK PubMed Central (United Kingdom)

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. Investigations of the mechanisms and adaptive consequences of somatic recombination are, however, hampered by the lack of a method to reliably synthesize somatic recombinants. Here we report the first genetic transformation system for the genus Armillaria. We transformed A. mellea with selective markers for use in diploid-haploid matings to reliably synthesize somatic recombinants. This was accomplished with Agrobacterium tumefaciens carrying pBGgHg, which carries the hygromycin phosphotransferase gene (hph). hph was integrated into transformants, as evidenced by serial transfer to selective media, PCR, reverse transcription-PCR (RT-PCR), and Southern hybridization. Nuclear and mitochondrial markers were developed to genotype synthesized mycelia. In matings between a wild-type diploid and hygromycin-resistant haploids (transgenic), we identified recombinant, hygromycin-resistant diploids and, additionally, hygromycin-resistant triploids, all with the mitochondrial haplotype of the haploid partner. Our approach created no mycelium in which the haploid nucleus was replaced by the diploid nucleus, the typical outcome of diploid-haploid matings in Armillaria. This genetic transformation system, in combination with new markers to track chromosomal and cytoplasmic inheritance in A. mellea, will advance research aimed at characterizing the significance of somatic recombination in the ecology of this important fungus.

Baumgartner K; Fujiyoshi P; Foster GD; Bailey AM

2010-12-01

82

Agrobacterium tumefaciens-mediated transformation for investigation of somatic recombination in the fungal pathogen Armillaria mellea.  

Science.gov (United States)

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. Investigations of the mechanisms and adaptive consequences of somatic recombination are, however, hampered by the lack of a method to reliably synthesize somatic recombinants. Here we report the first genetic transformation system for the genus Armillaria. We transformed A. mellea with selective markers for use in diploid-haploid matings to reliably synthesize somatic recombinants. This was accomplished with Agrobacterium tumefaciens carrying pBGgHg, which carries the hygromycin phosphotransferase gene (hph). hph was integrated into transformants, as evidenced by serial transfer to selective media, PCR, reverse transcription-PCR (RT-PCR), and Southern hybridization. Nuclear and mitochondrial markers were developed to genotype synthesized mycelia. In matings between a wild-type diploid and hygromycin-resistant haploids (transgenic), we identified recombinant, hygromycin-resistant diploids and, additionally, hygromycin-resistant triploids, all with the mitochondrial haplotype of the haploid partner. Our approach created no mycelium in which the haploid nucleus was replaced by the diploid nucleus, the typical outcome of diploid-haploid matings in Armillaria. This genetic transformation system, in combination with new markers to track chromosomal and cytoplasmic inheritance in A. mellea, will advance research aimed at characterizing the significance of somatic recombination in the ecology of this important fungus. PMID:20952653

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

2010-10-15

83

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

Science.gov (United States)

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. Investigations of the mechanisms and adaptive consequences of somatic recombination are, however, hampered by the lack of a method to reliably synthesize somatic recombinants. Here we report the first genetic transformation system for the genus Armillaria. We transformed A. mellea with selective markers for use in diploid-haploid matings to reliably synthesize somatic recombinants. This was accomplished with Agrobacterium tumefaciens carrying pBGgHg, which carries the hygromycin phosphotransferase gene (hph). hph was integrated into transformants, as evidenced by serial transfer to selective media, PCR, reverse transcription-PCR (RT-PCR), and Southern hybridization. Nuclear and mitochondrial markers were developed to genotype synthesized mycelia. In matings between a wild-type diploid and hygromycin-resistant haploids (transgenic), we identified recombinant, hygromycin-resistant diploids and, additionally, hygromycin-resistant triploids, all with the mitochondrial haplotype of the haploid partner. Our approach created no mycelium in which the haploid nucleus was replaced by the diploid nucleus, the typical outcome of diploid-haploid matings in Armillaria. This genetic transformation system, in combination with new markers to track chromosomal and cytoplasmic inheritance in A. mellea, will advance research aimed at characterizing the significance of somatic recombination in the ecology of this important fungus.

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

2010-01-01

84

phiC31 integrase-mediated site-specific recombination in barley.  

UK PubMed Central (United Kingdom)

The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F(1), even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity.

Kapusi E; Kempe K; Rubtsova M; Kumlehn J; Gils M

2012-01-01

85

Negative control of cell size in the cyanobacterium Synechococcus elongatus PCC 7942 by the essential response regulator RpaB.  

UK PubMed Central (United Kingdom)

The essential NblS-RpaB pathway for photosynthesis regulation and acclimatization to a variety of environmental conditions is the most conserved two-component system in cyanobacteria. To get insights into the RpaB implication in cell homeostasis we investigated the phenotypic impact of altering expression of the essential rpaB gene of Synechococcus elongatus PCC 7942 and determined the in vivo levels of the RpaB and RpaB~P polypeptides. Our results implicate non-phosphorylated RpaB in controlling cell length and shape and suggest that intrinsic regulation may be important to prevent drastic variations in RpaB levels and activity.

Moronta-Barrios F; Espinosa J; Contreras A

2013-03-01

86

RPA instabilities in finite nuclei at low density  

Energy Technology Data Exchange (ETDEWEB)

Early development of the instabilities in a dilute nuclear source is investigated using a finite temperature quantal Random Phase Approximation (RPA) approach for different systems. The growth rates of the unstable collective modes are determined by solving a dispersion relation. Calculations indicate that, for an expanding source, unstable modes show a transition from surface to volume character. (author). 15 refs. Submitted to Nuclear Physics (US).

Jacquot, B.; Chomaz, Ph. [Grand Accelerateur National d`Ions Lourds (GANIL), 14 - Caen (France); Colonna, M. [Grand Accelerateur National d`Ions Lourds (GANIL), 14 - Caen (France)]|[Catania Univ. (Italy). Lab. Nazionale del Sud; Ayik, S. [Tennessee Technological Univ., Cookeville, TN (United States)]|[Middle East Technical Univ., Ankara (Turkey)

1996-07-01

87

Identification of transglutaminase-mediated deamidation sites in a recombinant alpha-gliadin by advanced mass-spectrometric methodologies.  

UK PubMed Central (United Kingdom)

Celiac disease is a permanent immune-mediated food intolerance triggered by ingestion of wheat gliadins in genetically susceptible individuals. It has been reported that tissue transglutaminase plays an important role in the onset of celiac disease by converting specific glutamine residues within gliadin fragments into glutamic acid residues. This process increases binding affinity of gliadin peptides to HLA-DQ2/DQ8 molecules, thus enhancing the immune response. The aim of the present study was to achieve a detailed structural characterization of modifications induced by transglutaminase on gliadin peptides. Therefore, structural analyses were carried out on a recombinant alpha-gliadin and on a panel of 26 synthetic peptides, overlapping the complete protein sequence. Modified glutamine residues were identified by means of advanced mass-spectrometric methodologies on the basis of MALDI-TOF-MS and tandem mass spectrometry. Results led to the identification of 19 of 94 glutamine residues present in the recombinant alpha-gliadin, which were converted into glutamic acid residues by a transglutaminase-mediated reaction. This allowed us to achieve a global view of the modifications induced by the enzyme on this protein. Furthermore, results gathered could likely be utilized as relevant information for a better understanding of processes leading to T-cell recognition of gliadin peptides involved in celiac disease.

Mazzeo MF; De Giulio B; Senger S; Rossi M; Malorni A; Siciliano RA

2003-11-01

88

Protection against intestinal amebiasis by a recombinant vaccine is transferable by T cells and mediated by gamma interferon.  

UK PubMed Central (United Kingdom)

We have previously shown that vaccination with purified Entamoeba histolytica Gal/GalNAc lectin or recombinant subunits can protect mice from intestinal amebiasis upon intracecal challenge. In this study, we demonstrated with adoptive-transfer experiments that this lectin vaccine protection is mediated by T cells but not serum. The cell-mediated immune (CMI) response was characterized by significant gamma interferon (IFN-gamma), interleukin 12 (IL-12), IL-2, IL-10, and IL-17 production. To move toward a human vaccine, we switched to a recombinant protein and tested a range of adjuvants and routes appropriate for humans. We found that subcutaneous delivery of LecA with IDRI's adjuvant system EM014 elicited a potent Th1-type CMI profile and provided significant protection, as measured by culture negativity (79% efficacy); intranasal immunization with cholera toxin provided 56% efficacy; and alum induced a Th2-type response that protected 62 to 68% of mice. Several antibody and CMI cytokine responses were examined for correlates of protection, and prechallenge IFN-gamma(+) or IFN-gamma-, IL-2-, and tumor necrosis factor alpha-triple-positive CD4 cells in blood were statistically associated with protection. To test the role of IFN-gamma in LecA-mediated protection, we neutralized IFN-gamma in LecA-immunized mice and found that it abrogated the protection conferred by vaccination. These data demonstrate that CMI is sufficient for vaccine protection from intestinal amebiasis and reveal an important role for IFN-gamma, even in the setting of alum.

Guo X; Barroso L; Becker SM; Lyerly DM; Vedvick TS; Reed SG; Petri WA Jr; Houpt ER

2009-09-01

89

Recombinant Wolbachia heat shock protein 60 (HSP60) mediated immune responses in patients with lymphatic filariasis.  

Science.gov (United States)

Wolbachia, an endosymbiont present in filarial nematodes, have been implicated in a variety of roles, including the worm development and survival. Elucidation of the role of Wolbachia in filarial nematode biology and pathogenesis has become the focus of many studies and its contribution to parasite survival or immune response is still unclear. Recombinant Wolbachia HSP60 decreases T cell activation and lymphoproliferation in filarial infected people compared to endemic controls as observed by the assessment of T cell activation markers and cytokine responses in the peripheral blood mononuclear cells. Reduced T cell activation may be linked to T regulatory cell activity since it is associated with increased expression of CTLA4 and CD25 on CD4(+) T cells in filarial infected group upon stimulation with recombinant Wolbachia HSP60. In addition, elevated interleukin-10 and TGF-? cytokines corroborate the reduced CD4(+) T cell activation and interferon-? observed upon recombinant Wolbachia HSP60 stimulation in filarial patients. Hence, these findings indicate that Wolbachia HSP60 may also contribute to the immune modulation seen in filarial patients. PMID:21827871

Shiny, Chandanapurath; Krushna, Nagampalli S A; Babu, Subash; Elango, S; Manokaran, Guruswamy; Narayanan, Rangarajan Badri

2011-07-23

90

Mitotic crisis: the unmasking of a novel role for RPA.  

UK PubMed Central (United Kingdom)

Mitotic DNA damage is a constant threat to genomic integrity, yet understanding of the cellular responses to this stress remain incomplete. Recent work by Anantha et al. (2008; PNAS 105:12903-8) has found surprising evidence that RPA, the primary eukaryotic single-stranded DNA-binding protein, can stimulate the ability of cells to exit mitosis into a 2N G(1) phase. Along with providing additional discussion of this study, we review evidence suggesting that DNA replication and repair factors can modulate mitotic transit by acting through Polo-like kinase-1 (Plk1) and the centrosome. 'A crisis unmasks everyone.'-Mason Cooley, U.S. aphorist.

Anantha RW; Borowiec JA

2009-02-01

91

Mitotic crisis: the unmasking of a novel role for RPA.  

Science.gov (United States)

Mitotic DNA damage is a constant threat to genomic integrity, yet understanding of the cellular responses to this stress remain incomplete. Recent work by Anantha et al. (2008; PNAS 105:12903-8) has found surprising evidence that RPA, the primary eukaryotic single-stranded DNA-binding protein, can stimulate the ability of cells to exit mitosis into a 2N G(1) phase. Along with providing additional discussion of this study, we review evidence suggesting that DNA replication and repair factors can modulate mitotic transit by acting through Polo-like kinase-1 (Plk1) and the centrosome. 'A crisis unmasks everyone.'-Mason Cooley, U.S. aphorist. PMID:19176996

Anantha, Rachel William; Borowiec, James A

2009-02-21

92

Finite temperature RPA in symmetric nuclear matter with Skyrme interactions  

Energy Technology Data Exchange (ETDEWEB)

We investigate the RPA response for thermally excited nuclear matter interacting through Skyrme interactions. Closed analytical expressions are obtained for the dynamic susceptibility in each spin-isopspin channel. We compute the strength as a function of energy, transferred momentum and temperature, and examine the evolution of collective states, when present. The energy weighted sum rules M{sub k}, for k = -1, 1 and 3 are also shown to possess explicit expressions as functions of both momentum and temperature. It is seen that thermal effects on the susceptibility are as important as dynamical ones associated to momentum transfer, at least for temperatures as high as 20% of the Fermi energy. (orig.).

Hernandez, E.S. [Buenos Aires Univ. (Argentina). Dept. de Fisica; Navarro, J. [IFIC (Centre Mixt CSIC Universitat de Valencia), Facultat de Fisica, 46.100 Burjassot (Spain); Polls, A. [Departament d`Estructura i Constituents de la Materia, Facultat de Fisica, Universitat de Barcelona, 08028 Barcelona (Spain); Ventura, J. [Departament d`Estructura i Constituents de la Materia, Facultat de Fisica, Universitat de Barcelona, 08028 Barcelona (Spain)

1996-01-22

93

Recombination of a 3-chlorobenzoate catabolic plasmid from Alcaligenes eutrophus NH9 mediated by direct repeat elements.  

UK PubMed Central (United Kingdom)

Alcaligenes eutrophus NH9 was isolated from soil. This strain can utilize 3-chlorobenzoate (3-CB) as a sole source of carbon and energy. Most of the 3-CB-negative segregants had lost one of the plasmids present in the parent strain. The genes for catabolism of 3-CB were located within a 9.2-kb SacI fragment of this plasmid (pENH91). The genes were found to hybridize with genes for components of the modified ortho cleavage pathway from Pseudomonas putida. In one of the 3-CB-negative segregants, the plasmid had undergone the deletion of a segment with a size of about 12.5 kb that covered the catabolic genes. The deletion event seemed to be the result of reciprocal recombination between two highly homologous sequences with sizes of 2.5 kb that were present as a direct repeat at the two ends of the region that included the catabolic genes. Nucleotide sequence analysis of homologous fragments revealed a structure that resembled an insertion sequence and relatedness to IS21. During repeated subculturing of NH9 on liquid media with 3-CB, the culture was taken over by a derivative strain (designated NH9A) in which the degradative plasmid carried a duplicate copy of the 12.5-kb region that contained the catabolic genes. The duplication of these genes seemed again to have been mediated by recombination between the direct repeat sequences.

Ogawa N; Miyashita K

1995-11-01

94

Recombinant derivatives of the human high-mobility group protein HMGB2 mediate efficient nonviral gene delivery.  

UK PubMed Central (United Kingdom)

Certain natural peptides and proteins of mammalian origin are able to bind and condense plasmid DNA, a prerequisite for the formation of transfection-competent complexes that facilitate nonviral gene delivery. Here we have generated recombinant derivatives of the human high-mobility group (HMG) protein HMGB2 and investigated their potential as novel protein-based transfection reagents. A truncated form of HMGB2 encompassing amino acids 1-186 of the molecule was expressed in Escherichia coli at high yield. This HMGB2186 protein purified from bacterial lysates was able to condense plasmid DNA in a concentration-dependent manner, and mediated gene delivery into different established tumor cell lines more efficiently than poly(l-lysine). By attaching, via gene fusion, additional functional domains such as the HIV-1 TAT protein transduction domain (TAT(PTD)-HMGB2186), the nuclear localization sequence of the simian virus 40 (SV40) large T-antigen (SV40(NLS)-HMGB2186), or the importin-beta-binding domain (IBB) of human importin-alpha (IBB-HMGB2186), chimeric fusion proteins were produced which displayed markedly improved transfection efficiency. Addition of chloroquine strongly enhanced gene transfer by all four HMGB2186 derivatives studied, indicating cellular uptake of protein-DNA complexes via endocytosis. The IBB-HMGB2186 molecule in the presence of the endosomolytic reagent was the most effective. Our results show that recombinant derivatives of human HMGB2 facilitate efficient nonviral gene delivery and may become useful reagents for applications in gene therapy.

Sloots A; Wels WS

2005-08-01

95

An African horse sickness virus serotype 4 recombinant canarypox virus vaccine elicits specific cell-mediated immune responses in horses.  

Science.gov (United States)

A recombinant canarypox virus vectored vaccine co-expressing synthetic genes encoding outer capsid proteins, VP2 and VP5, of African horse sickness virus (AHSV) serotype 4 (ALVAC(®)-AHSV4) has been demonstrated to fully protect horses against homologous challenge with virulent field virus. Guthrie et al. (2009) detected weak and variable titres of neutralizing antibody (ranging from <10 to 40) 8 weeks after vaccination leading us to hypothesize that there could be a participation of cell mediated immunity (CMI) in protection against AHSV4. The present study aimed at characterizing the CMI induced by the experimental ALVAC(®)-AHSV4 vaccine. Six horses received two vaccinations twenty-eight days apart and three horses remained unvaccinated. The detection of VP2/VP5 specific IFN-? responses was assessed by enzyme linked immune spot (ELISpot) assay and clearly demonstrated that all ALVAC(®)-AHSV4 vaccinated horses developed significant IFN-? production compared to unvaccinated horses. More detailed immune responses obtained by flow cytometry demonstrated that ALVAC(®)-AHSV4 vaccinations induced immune cells, mainly CD8(+) T cells, able to recognize multiple T-epitopes through all VP2 and only the N-terminus sequence of VP5. Neither VP2 nor VP5 specific IFN-? responses were detected in unvaccinated horses. Overall, our data demonstrated that an experimental recombinant canarypox based vaccine induced significant CMI specific for both VP2 and VP5 proteins of AHSV4. PMID:22763149

El Garch, H; Crafford, J E; Amouyal, P; Durand, P Y; Edlund Toulemonde, C; Lemaitre, L; Cozette, V; Guthrie, A; Minke, J M

2012-06-15

96

An African horse sickness virus serotype 4 recombinant canarypox virus vaccine elicits specific cell-mediated immune responses in horses.  

UK PubMed Central (United Kingdom)

A recombinant canarypox virus vectored vaccine co-expressing synthetic genes encoding outer capsid proteins, VP2 and VP5, of African horse sickness virus (AHSV) serotype 4 (ALVAC(®)-AHSV4) has been demonstrated to fully protect horses against homologous challenge with virulent field virus. Guthrie et al. (2009) detected weak and variable titres of neutralizing antibody (ranging from <10 to 40) 8 weeks after vaccination leading us to hypothesize that there could be a participation of cell mediated immunity (CMI) in protection against AHSV4. The present study aimed at characterizing the CMI induced by the experimental ALVAC(®)-AHSV4 vaccine. Six horses received two vaccinations twenty-eight days apart and three horses remained unvaccinated. The detection of VP2/VP5 specific IFN-? responses was assessed by enzyme linked immune spot (ELISpot) assay and clearly demonstrated that all ALVAC(®)-AHSV4 vaccinated horses developed significant IFN-? production compared to unvaccinated horses. More detailed immune responses obtained by flow cytometry demonstrated that ALVAC(®)-AHSV4 vaccinations induced immune cells, mainly CD8(+) T cells, able to recognize multiple T-epitopes through all VP2 and only the N-terminus sequence of VP5. Neither VP2 nor VP5 specific IFN-? responses were detected in unvaccinated horses. Overall, our data demonstrated that an experimental recombinant canarypox based vaccine induced significant CMI specific for both VP2 and VP5 proteins of AHSV4.

El Garch H; Crafford JE; Amouyal P; Durand PY; Edlund Toulemonde C; Lemaitre L; Cozette V; Guthrie A; Minke JM

2012-09-01

97

Endogenous signal peptides efficiently mediate the secretion of recombinant proteins in Pichia pastoris.  

UK PubMed Central (United Kingdom)

By predicting the potential signal peptides from proteins that are naturally secreted by Pichia pastoris, we identified three possible endogenous signal peptides: Scw, Dse and Exg. We compared their capability to mediate the secretion of enhanced green fluorescent protein (EGFP) and Candida antarctica lipase B (CALB) with that of the Saccharomyces cerevisiae ?-factor prepro-signal. EGFP entered the secretory pathway of P. pastoris and was efficiently secreted into the culture medium by all three endogenous peptides. Further, these three putative endogenous signal peptides were also effective in secreting CALB. These endogenous signal peptides thus have the potential to mediate the efficient secretion of heterologous proteins in P. pastoris.

Liang S; Li C; Ye Y; Lin Y

2013-01-01

98

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 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 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 (more) 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.

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

1999-08-01

99

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

Directory of Open Access Journals (Sweden)

Full Text Available 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 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.

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

1999-01-01

100

Hapten mediated display and pairing of recombinant antibodies accelerates assay assembly for biothreat countermeasures.  

UK PubMed Central (United Kingdom)

A bottle-neck in recombinant antibody sandwich immunoassay development is pairing, demanding protein purification and modification to distinguish captor from tracer. We developed a simple pairing scheme using microliter amounts of E. coli osmotic shockates bearing site-specific biotinylated antibodies and demonstrated proof of principle with a single domain antibody (sdAb) that is both captor and tracer for polyvalent Marburgvirus nucleoprotein. The system could also host pairs of different sdAb specific for the 7 botulinum neurotoxin (BoNT) serotypes, enabling recognition of the cognate serotype. Inducible supE co-expression enabled sdAb populations to be propagated as either phage for more panning from repertoires or expressed as soluble sdAb for screening within a single host strain. When combined with streptavidin-g3p fusions, a novel transdisplay system was formulated to retrofit a semi-synthetic sdAb library which was mined for an anti-Ebolavirus sdAb which was immediately immunoassay ready, thereby speeding up the recombinant antibody discovery and utilization processes.

Sherwood LJ; Hayhurst A

2012-01-01

 
 
 
 
101

Sequential and synergistic modification of human RPA stimulates chromosomal DNA repair.  

UK PubMed Central (United Kingdom)

The activity of human replication protein A (RPA) in DNA replication and repair is regulated by phosphorylation of the middle RPA2 subunit. It has previously been shown that up to nine different N-terminal residues are modified in vivo and in response to genotoxic stress. Using a novel antibody against phospho-Ser(29), a moiety formed by cyclin-Cdk, we observed that RPA2 was phosphorylated during mitosis in nonstressed cells. Robust phosphorylation of Ser(29) was also seen in interphase cells following treatment with the DNA-damaging agent camptothecin, a rare example of stress stimulating the modification of a repair factor by cyclin-Cdk. RPA2 phosphorylation is regulated both in cis and trans. Cis-phosphorylation follows a preferred pathway. (That is, the initial modification of Ser(33) by ATR stimulates subsequent phosphorylation of Cdk sites Ser(23) and Ser(29)). These events then facilitate modification of Thr(21) and extreme N-terminal sites Ser(4) and Ser(8), probably by DNA-PK. Our data also indicate that the phosphorylation of one RPA molecule can influence the phosphorylation of other RPA molecules in trans. Cells in which endogenous RPA2 was "replaced" with a double S23A/S29A-RPA2 mutant were seen to have an abnormal cell cycle distribution both in normal and in stressed cells. Such cells also showed aberrant DNA damage-dependent RPA foci and had persistent staining of gammaH2AX following DNA damage. Our data indicate that RPA phosphorylation facilitates chromosomal DNA repair. We postulate that the RPA phosphorylation pattern provides a means to regulate the DNA repair pathway utilized.

Anantha RW; Vassin VM; Borowiec JA

2007-12-01

102

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

103

Efficient inducible Cre-mediated recombination in Tcf21 cell lineages in the heart and kidney.  

UK PubMed Central (United Kingdom)

Tcf21 is a Class II bHLH family member with essential roles in the formation of the lungs, kidneys, gonads, spleen, and heart. Here, we report the utility of a mouse line with targeted insertion of a tamoxifen-inducible Cre recombinase, MerCreMer at the Tcf21 locus. This mouse line will permit the inducible expression of Cre recombinase in Tcf21-expressing cells. Using ROSA26 reporter mice, we show that Cre recombinase is specifically and robustly activated in multiple Tcf21-expressing tissues during embryonic and postnatal development. The expression profile in the kidney is particularly dynamic with the ability to cause recombination in mesangial cells at one time of induction and podocytes at another time. These features make the Tcf21-driven inducible Cre line (Tcf21(iCre) ) a valuable genetic tool for spatiotemporal gene function analysis and lineage tracing of cells in the heart, kidney, cranial muscle, and gonads.

Acharya A; Baek ST; Banfi S; Eskiocak B; Tallquist MD

2011-11-01

104

Efficient inducible Cre-mediated recombination in Tcf21 cell lineages in the heart and kidney.  

Science.gov (United States)

Tcf21 is a Class II bHLH family member with essential roles in the formation of the lungs, kidneys, gonads, spleen, and heart. Here, we report the utility of a mouse line with targeted insertion of a tamoxifen-inducible Cre recombinase, MerCreMer at the Tcf21 locus. This mouse line will permit the inducible expression of Cre recombinase in Tcf21-expressing cells. Using ROSA26 reporter mice, we show that Cre recombinase is specifically and robustly activated in multiple Tcf21-expressing tissues during embryonic and postnatal development. The expression profile in the kidney is particularly dynamic with the ability to cause recombination in mesangial cells at one time of induction and podocytes at another time. These features make the Tcf21-driven inducible Cre line (Tcf21(iCre) ) a valuable genetic tool for spatiotemporal gene function analysis and lineage tracing of cells in the heart, kidney, cranial muscle, and gonads. PMID:21432986

Acharya, Asha; Baek, Seung Tae; Banfi, Serena; Eskiocak, Banu; Tallquist, Michelle D

2011-08-18

105

Giant resonances in nuclei and clusters of different shape: the separable RPA  

International Nuclear Information System (INIS)

The self-consistent RPA (SRPA) with separable residual interaction is applied to the description of E? giant resonances (multipole collective oscillations of valence electrons) in nuclei (metal clusters) of different shape. the method drastically simplifies RPA calculations, providing nevertheless reasonable agreement with available experimental data. (author)

1998-01-01

106

RPA calculation of Gamow-Teller and M1 states includind ?-hole excitation  

International Nuclear Information System (INIS)

The self-consistent RPA model with Skyrme type interactions is extended to include ?-hole excitations. Energies and transition strengths of Gamow-Teller and MI states are studied systematically. We find that the combined effects of RPA correlations and ?-hole excitation decrease the transition strengths by about 35%. (orig.).

1982-01-01

107

RPA phosphorylation facilitates mitotic exit in response to mitotic DNA damage.  

UK PubMed Central (United Kingdom)

Human replication protein A (RPA) becomes phosphorylated on the RPA2 subunit by cyclin B-Cdc2 during mitosis, although the functional role of this modification is unclear. We find that this modification stimulates RPA2 to become hyperphosphorylated in response to mitotic DNA damage caused by bleomycin treatment. Cells in which endogenous RPA2 was replaced by a mutant subunit lacking both Cdc2 sites had a significant defect in mitotic release into a 2N G(1) phase after exposure to bleomycin. An increased percentage of these mutant cells also was positive initially for cyclin B expression and BubR1 chromatin staining, indicative of an extended spindle assembly checkpoint. The mutant cells that experienced mitotic DNA damage also underwent apoptosis at higher levels than cells expressing the WT subunit. Even so, we did not find the mutation had any dramatic effects on the level of DNA repair in mitosis. Cells lacking ATM (a checkpoint factor and RPA2 kinase) also were severely defective in mitotic exit and were unable to support RPA hyperphosphorylation after mitotic DNA damage. Although checkpoint 1 effector kinase (Chk1) had a more complex role, inhibition of Chk1 activity with UCN-01 also reduced mitotic exit. Chk1 activation and mitotic RPA hyperphosphorylation were found to be independent events. Our results demonstrate that mitotic RPA hyperphosphorylation facilitates release of cells from a damaged mitosis into a 2N G(1) phase, thereby increasing cell viability.

Anantha RW; Sokolova E; Borowiec JA

2008-09-01

108

Some fundamental aspects of RPA approach and its relation with the nucleus structure  

International Nuclear Information System (INIS)

The RPA equations are obtained by the linearization of motion equation and by the time dependent Hartree-Fock method; one gives the explict relation between RPA vacuum and H-F vacuum and a complete discussion about spurious states. Finally, the authors present the cranking method and some examples. (author)

1976-01-01

109

A West Nile virus (WNV) recombinant canarypox virus vaccine elicits WNV-specific neutralizing antibodies and cell-mediated immune responses in the horse.  

Science.gov (United States)

Successful vaccination against West Nile virus (WNV) requires induction of both neutralizing antibodies and cell-mediated immune responses. In this study, we have assessed the ability of a recombinant ALVAC-WNV vaccine (RECOMBITEK WNV) to elicit neutralizing antibodies and virus-specific cell-mediated immune responses in horses. In addition, we examined whether prior exposure to ALVAC-WNV vaccine would inhibit B and cell-mediated immune responses against the transgene product upon subsequent booster immunizations with the same vaccine. The results demonstrated that the recombinant ALVAC-WNV vaccine induced neutralizing antibodies and prM/E insert-specific IFN-gamma(+) producing cells against WNV in vaccinated horses. Prior exposure to ALVAC-WNV vaccine did not impair the ability of horses to respond to two subsequent booster injections with the same vaccine, although anti-vector-specific antibody and cell-mediated immune responses were induced in vaccinated horses. This report describes, for the first time, the induction of antigen-specific cell-mediated responses following vaccination with an ALVAC virus recombinant vaccine encoding WNV antigens. Moreover, we showed that both WNV-specific IFN-gamma producing cells and anti-WNV neutralizing antibody responses, are not inhibited by subsequent vaccinations with the same vector vaccine. PMID:18372050

El Garch, H; Minke, J M; Rehder, J; Richard, S; Edlund Toulemonde, C; Dinic, S; Andreoni, C; Audonnet, J C; Nordgren, R; Juillard, V

2008-02-16

110

A West Nile virus (WNV) recombinant canarypox virus vaccine elicits WNV-specific neutralizing antibodies and cell-mediated immune responses in the horse.  

UK PubMed Central (United Kingdom)

Successful vaccination against West Nile virus (WNV) requires induction of both neutralizing antibodies and cell-mediated immune responses. In this study, we have assessed the ability of a recombinant ALVAC-WNV vaccine (RECOMBITEK WNV) to elicit neutralizing antibodies and virus-specific cell-mediated immune responses in horses. In addition, we examined whether prior exposure to ALVAC-WNV vaccine would inhibit B and cell-mediated immune responses against the transgene product upon subsequent booster immunizations with the same vaccine. The results demonstrated that the recombinant ALVAC-WNV vaccine induced neutralizing antibodies and prM/E insert-specific IFN-gamma(+) producing cells against WNV in vaccinated horses. Prior exposure to ALVAC-WNV vaccine did not impair the ability of horses to respond to two subsequent booster injections with the same vaccine, although anti-vector-specific antibody and cell-mediated immune responses were induced in vaccinated horses. This report describes, for the first time, the induction of antigen-specific cell-mediated responses following vaccination with an ALVAC virus recombinant vaccine encoding WNV antigens. Moreover, we showed that both WNV-specific IFN-gamma producing cells and anti-WNV neutralizing antibody responses, are not inhibited by subsequent vaccinations with the same vector vaccine.

El Garch H; Minke JM; Rehder J; Richard S; Edlund Toulemonde C; Dinic S; Andreoni C; Audonnet JC; Nordgren R; Juillard V

2008-06-01

111

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

112

Inhibition of corneal neovascularization by recombinant adenovirus-mediated sFlk-1 expression  

International Nuclear Information System (INIS)

The interaction of vascular endothelial growth factor (VEGF) and its receptors (Flt-1, Flk-1/KDR) is correlated with neovascularization in the eyes. Therefore, blocking the binding of VEGF and the corresponding receptor has become critical for inhibiting corneal neovascularization. In this study, we have expressed the cDNA for sFlk-1 under the control of cytomegalovirus immediate-early promoter (CMV) from an E1/partial E3 deleted replication defective recombinant adenovirus, and Ad.sflk-1 expression was determined by Western blotting. We have shown that conditioned media from Ad.sflk-1-infected ARPE-19 cells significantly reduced VEGF-induced human umbilical vein endothelial cells (HUVEC) and murine endothelial cells (SVEC) proliferation in vitro compared with the control vector. In vivo, adenoviral vectors expressing green fluorescent protein alone (Ad.GFP) were utilized to monitor gene transfer to the cornea. Moreover, in the models of corneal neovascularization, the injection of Ad.sflk-1 (108 PFU) into the anterior chamber could significantly inhibit angiogenic changes compared with Ad.null-injected and vehicle-injected models. Immunohistochemical analysis showed that corneal endothelial cells and corneal stroma of cauterized rat eyes were efficiently transduced and expressed sFlk-1. These results not only support that adenoviral vectors are capable of high-level transgene expression but also demonstrate that Ad.sflk-1 gene therapy might be a feasible approach for inhibiting the development of corneal neovascularization.

2000-01-00

113

A high-throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Cre-loxP recombination refers to the process of site-specific recombination mediated by two loxP sequences and the Cre recombinase protein. Transgenic experiments exploit integrative recombination, where a donor plasmid carrying a loxP site and DNA of interest integrate into a recipient loxP site in a target genome. Unfortunately, integrative recombination is highly inefficient because the insert is flanked by two loxP sites, which themselves become targets for Cre and lead to subsequent excision of the insert. A small number of mutations have been discovered in parts of the loxP sequence, specifically the spacer and inverted repeat segments, that increase the efficiency of integrative recombination. In this study we introduce a high-throughput in vitro assay to rapidly detect novel loxP spacer mutants and describe the sequence characteristics of successful recombinants. Results We created synthetic loxP oligonucleotides that contained a combination of inverted repeat mutations (the lox66 and lox71 mutations) and mutant spacer sequences, degenerate at 6 of the 8 positions. After in vitro Cre recombination, 3,124 recombinant clones were identified by sequencing. Included in this set were 31 unique, novel, self-recombining sequences. Using network visualization tools, we recognized 12 spacer sets with restricted promiscuity. We observed that increased guanine content at all spacer positions save for position 8 resulted in increased recombination. Interestingly, recombination between identical spacers was not preferred over non-identical spacers. We also identified a set of 16 pairs of loxP spacers that reacted at least twice with another spacer, but not themselves. Further, neither the wild-type P1 phage loxP sequence nor any of the known loxP spacer mutants appeared to be kinetically favoured by Cre recombinase. Conclusion This study approached loxP spacer mutant screening in an unbiased manner, assuming nothing about candidate loxP sites save for the conserved 4 and 5 spacer positions. Candidate sites were free to recombine with any other sequence in the pool of all possible sites. The subset of loxP sites identified here are candidates for in vivo serial recombination as they have already demonstrated limited promiscuity with other loxP spacer and stability in the presence of Cre.

Missirlis Perseus I; Smailus Duane E; Holt Robert A

2006-01-01

114

Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress.  

Science.gov (United States)

ATR is an essential kinase activated in response to DNA-replication stress, with a known target being the RPA2 subunit of human replication protein A (RPA). We find that S33-RPA2 phosphorylation by ATR occurs primarily in the late-S and G2 phases, probably at sites of residual stalled DNA-replication forks, with S33-P-RPA2 contained within nuclear repair centers. Although cells in which endogenous RPA2 was ;replaced' with an RPA2 protein with mutations T21A and S33A (T21A/S33A-RPA) had normal levels of DNA replication under non-stress conditions, the mutant cells were severely deficient in the amount of DNA synthesis occurring during replication stress. These cells also had abnormally high levels of chromatin-bound RPA, indicative of increased amounts of single-stranded DNA (ssDNA) and showed defective recovery from stress. Cells replaced with the mutant RPA2 also generated G1 cells with a broader DNA distribution and high levels of apoptosis following stress, compared with cells expressing wild-type RPA2. Surprisingly, cells expressing the wild-type RPA2 subunit had increased levels of stress-dependent DNA breaks. Our data demonstrate that RPA phosphorylation at the T21 and S33 sites facilitates adaptation of a DNA-replication fork to replication stress. PMID:19843584

Vassin, Vitaly M; Anantha, Rachel William; Sokolova, Elena; Kanner, Shlomo; Borowiec, James A

2009-10-20

115

Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress.  

UK PubMed Central (United Kingdom)

ATR is an essential kinase activated in response to DNA-replication stress, with a known target being the RPA2 subunit of human replication protein A (RPA). We find that S33-RPA2 phosphorylation by ATR occurs primarily in the late-S and G2 phases, probably at sites of residual stalled DNA-replication forks, with S33-P-RPA2 contained within nuclear repair centers. Although cells in which endogenous RPA2 was ;replaced' with an RPA2 protein with mutations T21A and S33A (T21A/S33A-RPA) had normal levels of DNA replication under non-stress conditions, the mutant cells were severely deficient in the amount of DNA synthesis occurring during replication stress. These cells also had abnormally high levels of chromatin-bound RPA, indicative of increased amounts of single-stranded DNA (ssDNA) and showed defective recovery from stress. Cells replaced with the mutant RPA2 also generated G1 cells with a broader DNA distribution and high levels of apoptosis following stress, compared with cells expressing wild-type RPA2. Surprisingly, cells expressing the wild-type RPA2 subunit had increased levels of stress-dependent DNA breaks. Our data demonstrate that RPA phosphorylation at the T21 and S33 sites facilitates adaptation of a DNA-replication fork to replication stress.

Vassin VM; Anantha RW; Sokolova E; Kanner S; Borowiec JA

2009-11-01

116

Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress  

Digital Repository Infrastructure Vision for European Research (DRIVER)

ATR is an essential kinase activated in response to DNA-replication stress, with a known target being the RPA2 subunit of human replication protein A (RPA). We find that S33-RPA2 phosphorylation by ATR occurs primarily in the late-S and G2 phases, probably at sites of residual stalled DNA-replica...

Vassin, Vitaly M.; Anantha, Rachel William; Sokolova, Elena; Kanner, Shlomo; Borowiec, James A.

117

Recombinant derivatives of the human high-mobility group protein HMGB2 mediate efficient nonviral gene delivery.  

Science.gov (United States)

Certain natural peptides and proteins of mammalian origin are able to bind and condense plasmid DNA, a prerequisite for the formation of transfection-competent complexes that facilitate nonviral gene delivery. Here we have generated recombinant derivatives of the human high-mobility group (HMG) protein HMGB2 and investigated their potential as novel protein-based transfection reagents. A truncated form of HMGB2 encompassing amino acids 1-186 of the molecule was expressed in Escherichia coli at high yield. This HMGB2186 protein purified from bacterial lysates was able to condense plasmid DNA in a concentration-dependent manner, and mediated gene delivery into different established tumor cell lines more efficiently than poly(l-lysine). By attaching, via gene fusion, additional functional domains such as the HIV-1 TAT protein transduction domain (TAT(PTD)-HMGB2186), the nuclear localization sequence of the simian virus 40 (SV40) large T-antigen (SV40(NLS)-HMGB2186), or the importin-beta-binding domain (IBB) of human importin-alpha (IBB-HMGB2186), chimeric fusion proteins were produced which displayed markedly improved transfection efficiency. Addition of chloroquine strongly enhanced gene transfer by all four HMGB2186 derivatives studied, indicating cellular uptake of protein-DNA complexes via endocytosis. The IBB-HMGB2186 molecule in the presence of the endosomolytic reagent was the most effective. Our results show that recombinant derivatives of human HMGB2 facilitate efficient nonviral gene delivery and may become useful reagents for applications in gene therapy. PMID:16098203

Sloots, Arjen; Wels, Winfried S

2005-08-01

118

Plasmid DNA delivery into MDA-MB-453 cells mediated by recombinant Her-NLS fusion protein  

Directory of Open Access Journals (Sweden)

Full Text Available Sivakumar Jeyarajan*, Jennifer Xavier*, N Madhusudhana Rao, Vijaya GopalCentre for Cellular and Molecular Biology, Council for Scientific and Industrial Research, Hyderabad, Andhra Pradesh, India; *These authors contributed equally to this workAbstract: A major rate-limiting step in nonviral gene delivery is the entry of nucleic acids across various membrane barriers and eventually into the nucleus where it must be transcribed. Cell-penetrating peptides and proteins are employed to generate formulations that overcome these challenges to facilitate DNA delivery into cells efficiently. However, these are limited by their inability to deliver nucleic acids selectively due to lack of specificity because they deliver to both cancer and normal cells. In this study, through modular design, we generated a recombinant fusion protein designated as Her-nuclear localization sequence (Her-NLS), where heregulin-a (Her), a targeting moiety, was cloned in frame with cationic NLS peptide to obtain a cell-specific targeting biomolecule for nucleic acid delivery. The heregulin-a1 isoform possesses the epidermal growth factor-like domain and binds to HER2/3 heterodimers which are overexpressed in certain breast cancers. Purified recombinant Her-NLS fusion protein binds plasmid DNA and specifically transfects MDA-MB-453 cells overexpressing the epidermal growth factor receptors HER2/3 in vitro. The approach described would also permit replacement of heregulin ligand with other targeting moieties that would be suited to cell-specific nucleic acid delivery mediated via receptor-ligand interactions.Keywords: cell targeting, nucleic acid delivery, nuclear localization sequences, heregulin, transfection, HER2 receptors

Sivakumar Jeyarajan; Jennifer Xavier; N Madhusudhana Rao; et al

2010-01-01

119

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.

Leonhard Wouter N; Roelfsema Jeroen H; Lantinga-van Leeuwen Irma S; Breuning Martijn H; Peters Dorien JM

2008-01-01

120

Continuum fluid dynamics and RPA for giant resonances  

International Nuclear Information System (INIS)

Under the restriction of irrotational flow the macroscopic fluid-dynamical (FD) theory of giant resonances based on the generalized scaling approach can be used to calculate excitations in the continuum. For this purpose a self-consistent description of the ground state with correct asymptotic properties for large r is required. Starting with a spherical Hartree-Fock ground state obtained from a simple density-dependent force the results (strength functions, velocity fields and transition densities) for FD and self-consistent RPA are systematically compared. The best agreement is obtained for high-lying collective states (in particular the isovector monopole) which are generated by strong residual particle-hole interactions. The significance of transverse components of the scaling field is studied by comparing polarizability sum rules msub(-1) in the different models. (orig.).

1984-01-01

 
 
 
 
121

Stability of thermal HFB and dissipative thermal RPA  

CERN Document Server

It is shown that, as for a Nilsson + pairing model, the extended stability condition of the thermal Hartree-Fock-Bogoliubov (THFB) solution coincides with the one of the thermal RPA (TRPA) solution unless the pairing constant G is too large. As possible extensions of the TRPA equation in alternative ways describing thermal fluctuation effect, the extended TRPA (ETRPA) and the dissipative TRPA (DTRPA) are discussed. Furthermore, the general microscopic framework of the TRPA predicts the saturation and decrease of giant resonance width in high temperature limit, i.e. the fragmentation width GAMMA sub f propor to(kT) sup ( sup - sup 3 sup ( sup 2 sup ) sup ) and the spreading width GAMMA suparrow down propor to(kT) sup ( sup - sup 1 sup ( sup 2 sup ) sup ).

Tanabe, K

1999-01-01

122

Solitary BioY Proteins Mediate Biotin Transport into Recombinant Escherichia coli.  

UK PubMed Central (United Kingdom)

Energy-coupling factor (ECF) transporters form a large group of vitamin uptake systems in prokaryotes. They are composed of highly diverse, substrate-specific, transmembrane proteins (S units), a ubiquitous transmembrane protein (T unit), and homo- or hetero-oligomeric ABC ATPases. Biotin transporters represent a special case of ECF-type systems. The majority of the biotin-specific S units (BioY) is known or predicted to interact with T units and ABC ATPases. About one-third of BioY proteins, however, are encoded in organisms lacking any recognizable T unit. This finding raises the question of whether these BioYs function as transporters in a solitary state, a feature ascribed to certain BioYs in the past. To address this question in living cells, an Escherichia coli K-12 derivative deficient in biotin synthesis and devoid of its endogenous high-affinity biotin transporter was constructed as a reference strain. This organism is particularly suited for this purpose because components of ECF transporters do not naturally occur in E. coli K-12. The double mutant was viable in media containing either high levels of biotin or a precursor of the downstream biosynthetic path. Importantly, it was nonviable on trace levels of biotin. Eight solitary bioY genes of proteobacterial origin were individually expressed in the reference strain. Each of the BioYs conferred biotin uptake activity on the recombinants, which was inferred from uptake assays with [(3)H]biotin and growth of the cells on trace levels of biotin. The results underscore that solitary BioY transports biotin across the cytoplasmic membrane.

Finkenwirth F; Kirsch F; Eitinger T

2013-09-01

123

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

Energy Technology Data Exchange (ETDEWEB)

We evaluated the feasibility of non-invasive imaging of recombinant adenovirus-mediated human sodium-iodide symporter (hNIS) gene expression by {sup 99m}TcO{sub 4}{sup -} scintigraphy in skeletal muscle of rats. Replication-defective recombinant adenovirus encoding hNIS gene [Rad-CMV-hNIS 5 x 10{sup 7}, 2 x 10{sup 8} or 1 x 10{sup 9} plaque forming units (pfu)] or {beta}-galactosidase gene (Rad-CMV-LacZ 1 x 10{sup 9} 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 {sup 99m}TcO{sub 4}{sup -} (1.85 MBq). An additional two rats injected with 1 x 10{sup 9} pfu of Rad-CMV-hNIS underwent {sup 99m}TcO{sub 4}{sup -} scintigraphy with sodium perchlorate. After the imaging studies, rats were sacrificed for assessment of the biodistribution of {sup 99m}TcO{sub 4}{sup -} and measurement of hNIS mRNA expression. In all the rats injected with 1 x 10{sup 9} pfu of Rad-CMV-hNIS, hNIS expression was successfully imaged by {sup 99m}TcO{sub 4}{sup -} 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 {sup 99m}TcO{sub 4}{sup -} was retained in the liver (p<0.001) and the right muscle (p<0.05), with the highest uptake in rats injected with 1 x 10{sup 9} 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 10{sup 9} pfu of Rad-CMV-hNIS (p<0.05), with a positive correlation with the imaging counts (r=0.810, p<0.05) and the biodistribution (r=0.847, p<0.001). Hot spots in rats injected with 1 x 10{sup 9} pfu of Rad-CMV-hNIS were specifically inhibited by sodium perchlorate. This study illustrated that {sup 99m}TcO{sub 4}{sup -} scintigraphy can monitor Rad-CMV-hNIS-mediated gene expression in skeletal muscle of rats, non-invasively and quantitatively. (orig.)

Yang, Hyun Suk; Park, Seong-Wook [Department of Internal Medicine (Cardiology), Asan Medical Center, University of Ulsan College of Medicine, 388-1 Pungnap-dong, Songpa-gu, 138-736, Seoul (Korea); Lee, Heuiran; Kim, Sung Jin [Department of Microbiology, University of Ulsan College of Medicine, Seoul (Korea); Lee, Won Woo [Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seongnam (Korea); Department of Nuclear Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea); Yang, You-Jung; Moon, Dae Hyuk [Department of Nuclear Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul (Korea)

2004-09-01

124

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.)

2004-01-01

125

The potential of recombinant vesicular stomatitis virus-mediated virotherapy against metastatic colon cancer.  

UK PubMed Central (United Kingdom)

Colorectal cancer (CRC) is the fourth most frequently diagnosed cancer and the second leading cause of cancer-related mortality in the United States. The liver and lung are the most common sites of distant metastasis of CRC. The approval of newer chemotherapeutic agents such as oxaliplatin, irinotecan, bevacizumab, cetuximab and panitumumab has significantly improved survival, yet the majority of patients still succumb to the disease in less than 2 years. Novel therapeutic agents that can provide significant clinical benefit for metastatic CRC patients are needed. Oncolytic vesicular stomatitis virus (VSV) is a promising tool as a cancer therapeutic agent. In this study, we examined the feasibility of repeated intravenous infusions of rVSV in multiple CRC lung metastases, compared with repeated hepatic arterial administration in multifocal CRC liver metastasis in immune competent rats. We established a multifocal liver metastases model or the multiple lung metastases model using a CRC cell line, RCN-H4, implanted into syngeneic F344/DuCrj rats. 4.0x10(6) plaque-forming units (pfu) of recombinant VSV vectors expressing mutant (L289A) Newcastle disease virus fusion protein [rVSV-NDV/F(L289A)] were administered 3 times for 3 consecutive days locally via the hepatic artery for liver metastases or systemically via the penial vein for lung metastases. In the liver metastasis model, significantly enhanced survival was observed with rVSV-NDV/F(L289A)-treated rats (P=0.0196). Median survival was 110 and 25 days, respectively. In addition, 4 out of 7 of the rVSV-NDV/F(L289A)-treated rats demonstrated long-term survival exceeding 100 days. The long-term surviving rats were sacrificed to evaluate for residual malignancy. Liver tumors were not detected. In the lung metastasis model, median survival was 10 [VSV-NDV/F(L289A)-treated rats] and 7 days (control). Although survival was significantly prolonged (P<0.001), none of the rats achieved long-term survival. VSV virotherapy has potential for CRC liver and lung metastases, although systemic venous delivery is much less effective than locoregional delivery such as hepatic arterial infusion.

Yamaki M; Shinozaki K; Sakaguchi T; Meseck M; Ebert O; Ohdan H; Woo SL

2013-02-01

126

In vitro and in vivo activities of recombinant anthrax protective antigen co-expressed with thioredoxin in Escherichia coli.  

UK PubMed Central (United Kingdom)

Because of the central role it plays in the formation of lethal toxin and edema toxin, protective antigen (PA) is the principal target for the development of vaccines against anthrax. In the present study, we explored and compared the in vitro and in vivo activities of recombinant anthrax protective antigen (rPA) and receptor binding domain of protective antigen (PA4). As a result, the fully soluble rPA and PA4 proteins were successfully expressed in Escherichia coli by co-expression with thioredoxin (Trx), and the rPA was active in forming cytotoxic lethal toxins, indicating that the rPA protein retains a functionally biological activity. Furthermore, immunization with rPA protein induced stronger PA-specific immune responses in mice than PA4 protein. The protection elicited by immunization with PA4 suggests the presence of common neutralizing epitopes between rPA and PA4, but the immunization with rPA protein induced stronger neutralizing antibodies and protective levels against challenge with the B. anthracis strain A16R than the PA4 protein. The sera neutralizing antibodies titers correlated well with anti-PA group ELISA antibodies titers and the in vivo protective potency. Based on the results of cell cytotoxicity assays and the observed immune responses and protective potency, we concluded that the soluble rPA protein retains the in vitro and in vivo functionally biological activity and can be developed into a highly effective human subunit vaccine candidate against anthrax.

Ma Y; Yu YZ; Zhu YF; Xu Q; Sun ZW

2013-07-01

127

RPA coordinates DNA end resection and prevents formation of DNA hairpins.  

UK PubMed Central (United Kingdom)

Replication protein A (RPA) is an essential eukaryotic single-stranded DNA binding protein with a central role in DNA metabolism. RPA directly participates in DNA double-strand break repair by stimulating 5'-3' end resection by the Sgs1/BLM helicase and Dna2 endonuclease in vitro. Here we investigated the role of RPA in end resection in vivo, using a heat-inducible degron system that allows rapid conditional depletion of RPA in Saccharomyces cerevisiae. We found that RPA depletion eliminated both the Sgs1-Dna2- and Exo1-dependent extensive resection pathways and synergized with mre11? to prevent end resection. The short single-stranded DNA tails formed in the absence of RPA were unstable due to 3' strand loss and the formation of fold-back hairpin structures that required resection initiation and Pol32-dependent DNA synthesis. Thus, RPA is required to generate ssDNA, and also to protect ssDNA from degradation and inappropriate annealing that could lead to genome rearrangements.

Chen H; Lisby M; Symington LS

2013-05-01

128

Recombinant sendai virus-mediated gene transfer to mouse pancreatic stem cells.  

UK PubMed Central (United Kingdom)

Efficient gene transfer into stem cells is essential for the basic research and for therapeutic applications in gene-modified regenerative medicine. Adenovirus (AdV) vectors, one of the most commonly used types of vectors, can mediate high, albeit transient, levels of expression of the transgene in pancreatic stem/progenitor cells. However, high multiplicity of infection (MOI) with AdV vectors can result in cellular toxicity. Therefore, AdV vectors have been of limited usefulness in clinical applications. In this study, we investigated the in vitro gene transfer efficiency of Sendai virus (SeV) vectors, a paramyxovirus vector that can efficiently introduce foreign genes without toxicity into several cell types, including pancreatic stem cells. The dose-dependent GFP expression of pancreatic stem cells transfected with SeV vectors after 48 h of culture at 37 degrees C was observed. The transfection of pancreatic stem cells with SeV vectors and AdV vectors results in equal expression of the transgene (GFP expression) in the cells after 48 h of culture at 37 degrees C. Although the transfection of pancreatic stem cells with AdV vectors at high MOIs was cytotoxic, transfection with SeV vectors at high MOIs was rarely cytotoxic. In addition, pancreatic stem cells transfected with SeV maintained their differentiation ability. These data suggest that SeV could provide advantages with respect to safety issues in gene-modified regenerative medicine.

Oishi K; Noguchi H; Yukawa H; Inoue M; Takagi S; Iwata H; Hasegawa M; Hayashi S

2009-01-01

129

Recombinant leech antiplatelet protein prevents collagen-mediated platelet aggregation but not collagen graft thrombosis in baboons.  

UK PubMed Central (United Kingdom)

Leech antiplatelet protein (LAPP) is a specific inhibitor of collagen-induced human platelet aggregation and adhesion to collagen under static conditions. Recombinant LAPP (rLAPP) and L-366,763 (acetylated-Cys-Asn-Pro-Arg-Gly-Asp-Cys-NH2), a peptidyl fibrinogen receptor antagonist, were evaluated in an anesthetized baboon thrombosis model using a collagen-coated graft segment of an arteriovenous shunt to elicit thrombus formation. Animals were randomized to receive systemic intravenous administration of rLAPP (100 micrograms.kg-1 x min-1; n = 5), L-366,763 (8.5 micrograms.kg-1 x min-1; n = 3), or saline (n = 3). Despite complete and selective inhibition of type I collagen-induced ex vivo aggregation of platelets, rLAPP had no significant effect on the rate or the extent of 111-In-labeled platelet deposition onto the collagen graft and no effect on template bleeding time. In contrast, L-366,763 completely prevented platelet deposition, maintained blood flow, and significantly prolonged bleeding time at the dosage that inhibited ex vivo aggregation in response to all agonists studied. In this study, the absence of an antithrombotic benefit of rLAPP contrasted sharply with the efficacy of the fibrinogen receptor antagonist. These results demonstrate that specific inhibition of collagen-mediated platelet aggregation alone is not sufficient to prevent platelet-dependent thrombosis in this baboon model.

Schaffer LW; Davidson JT; Siegl PK; Gould RJ; Nutt RF; Brady SF; Connolly TM

1993-11-01

130

Recombinant leech antiplatelet protein prevents collagen-mediated platelet aggregation but not collagen graft thrombosis in baboons.  

Science.gov (United States)

Leech antiplatelet protein (LAPP) is a specific inhibitor of collagen-induced human platelet aggregation and adhesion to collagen under static conditions. Recombinant LAPP (rLAPP) and L-366,763 (acetylated-Cys-Asn-Pro-Arg-Gly-Asp-Cys-NH2), a peptidyl fibrinogen receptor antagonist, were evaluated in an anesthetized baboon thrombosis model using a collagen-coated graft segment of an arteriovenous shunt to elicit thrombus formation. Animals were randomized to receive systemic intravenous administration of rLAPP (100 micrograms.kg-1 x min-1; n = 5), L-366,763 (8.5 micrograms.kg-1 x min-1; n = 3), or saline (n = 3). Despite complete and selective inhibition of type I collagen-induced ex vivo aggregation of platelets, rLAPP had no significant effect on the rate or the extent of 111-In-labeled platelet deposition onto the collagen graft and no effect on template bleeding time. In contrast, L-366,763 completely prevented platelet deposition, maintained blood flow, and significantly prolonged bleeding time at the dosage that inhibited ex vivo aggregation in response to all agonists studied. In this study, the absence of an antithrombotic benefit of rLAPP contrasted sharply with the efficacy of the fibrinogen receptor antagonist. These results demonstrate that specific inhibition of collagen-mediated platelet aggregation alone is not sufficient to prevent platelet-dependent thrombosis in this baboon model. PMID:8218100

Schaffer, L W; Davidson, J T; Siegl, P K; Gould, R J; Nutt, R F; Brady, S F; Connolly, T M

1993-11-01

131

Generation of Nkx2.2:lacZ mice using recombination-mediated cassette exchange technology.  

Science.gov (United States)

Nkx2.2 encodes a homeodomain transcription factor required for the correct specification and/or differentiation of cells in the pancreas, intestine, and central nervous system (CNS). To follow the fate of cells deleted for Nkx2.2 within these tissues, we generated Nkx2.2:lacZ knockin mice using a recombination-mediated cassette exchange (RMCE) approach. Expression analysis of lacZ and/or ?-galactosidase in Nkx2.2(lacZ/+) heterozygote embryos and adults demonstrates that lacZ faithfully recapitulates endogenous Nkx2.2 expression. Furthermore, the Nkx2.2(lacZ/lacZ) homozygous embryos display phenotypes indistinguishable from the previously characterized Nkx2.2(-/-) strain. LacZ expression analyses in the Nkx2.2(lacZ/lacZ) homozygous embryos indicate that Nkx2.2-expressing progenitor cells within the pancreas are generated in their normal numbers and are not mislocalized within the pancreatic ductal epithelium or developing islets. In the CNS of Nkx2.2(lacZ/lacZ) embryos, LacZ-expressing cells within the ventral P3 progenitor domain display different migration properties depending on the developmental stage and their respective differentiation potential. PMID:22539496

Arnes, Luis; Leclerc, Kevin; Friel, Jessica M; Hipkens, Susan B; Magnuson, Mark A; Sussel, Lori

2012-05-19

132

Recombinant sendai virus-mediated gene transfer to adipose tissue-derived stem cells (ASCs).  

Science.gov (United States)

Adipose tissue-derived stem cells (ASCs) are expected to have clinical applications as well as other stem cells, because ASCs can be obtained safely from adult donors and used in autologous therapies without concern about rejection and the need for immunosuppression. However, the use of gene transfer with Sendai virus (SeV) vectors, which can efficiently introduce foreign genes without toxicity into several cells, with ASCs has not yet been investigated. This study documents on the use of SeV vectors for gene transfer to ASCs. The dose-dependent GFP expression of ASCs transfected with SeV vectors after 48 h of culture at 37 degrees C was first evaluated. Next, the cellular toxicity of ASCs transfected with SeV vectors was verified. In addition, SeV vectors were compared with adenovirus (AdV) vectors. Finally, the time-dependent GFP expression of ASCs transfected with SeV vectors was evaluated. The results showed that transfection of ASCs with SeV vectors results in more efficient expression of transgene (GFP expression) in the ASCs than with AdV vectors after 48 h of culture at 37 degrees C. Moreover, while the transfection of ASCs with AdV vectors at high MOIs was cytotoxic (a lot of transfected cells died) that of ASCs with SeV vectors at high MOIs was not necessarily cytotoxic. In addition, the preservation of multilineage ASCs transfected with SeV was observed. In conclusion, this is the first report describing the successful use of SeV-mediated gene transfer in ASCs, and the results indicate that SeV may thus provide advantages with respect to safety issues in gene therapy. PMID:18468234

Yukawa, Hiroshi; Noguchi, Hirofumi; Oishi, Koichi; Miyazaki, Takamichi; Kitagawa, Yasuo; Inoue, Makoto; Hasegawa, Mamoru; Hayashi, Shuji

2008-01-01

133

Recombinant sendai virus-mediated gene transfer to adipose tissue-derived stem cells (ASCs).  

UK PubMed Central (United Kingdom)

Adipose tissue-derived stem cells (ASCs) are expected to have clinical applications as well as other stem cells, because ASCs can be obtained safely from adult donors and used in autologous therapies without concern about rejection and the need for immunosuppression. However, the use of gene transfer with Sendai virus (SeV) vectors, which can efficiently introduce foreign genes without toxicity into several cells, with ASCs has not yet been investigated. This study documents on the use of SeV vectors for gene transfer to ASCs. The dose-dependent GFP expression of ASCs transfected with SeV vectors after 48 h of culture at 37 degrees C was first evaluated. Next, the cellular toxicity of ASCs transfected with SeV vectors was verified. In addition, SeV vectors were compared with adenovirus (AdV) vectors. Finally, the time-dependent GFP expression of ASCs transfected with SeV vectors was evaluated. The results showed that transfection of ASCs with SeV vectors results in more efficient expression of transgene (GFP expression) in the ASCs than with AdV vectors after 48 h of culture at 37 degrees C. Moreover, while the transfection of ASCs with AdV vectors at high MOIs was cytotoxic (a lot of transfected cells died) that of ASCs with SeV vectors at high MOIs was not necessarily cytotoxic. In addition, the preservation of multilineage ASCs transfected with SeV was observed. In conclusion, this is the first report describing the successful use of SeV-mediated gene transfer in ASCs, and the results indicate that SeV may thus provide advantages with respect to safety issues in gene therapy.

Yukawa H; Noguchi H; Oishi K; Miyazaki T; Kitagawa Y; Inoue M; Hasegawa M; Hayashi S

2008-01-01

134

Recombinant GroEL enhances protective antigen-mediated protection against Bacillus anthracis spore challenge.  

Science.gov (United States)

The fatal inhalation infection caused by Bacillus anthracis results from a complex pathogenic cycle involving release of toxins by bacteria that germinate from spores. Currently available vaccines against anthrax consist of protective antigen (PA), one of the anthrax toxin components. However, these PA-based vaccines are only partially protective against spore challenge in mice. This shows that exclusive elicitation of high anti-PA titer does not directly correlate with protection. Here, we demonstrate that inclusion of GroEL of B. anthracis with PA elicits enhanced protection against anthrax spore challenge in mice. GroEL was included as it has been reported to be present both on the exosporium and in the secretome in addition to the cell surface of B. anthracis. It has also been found protective against other pathogens. In the present study, immunization with GroEL alone was also potent enough to induce high humoral and cell-mediated response and significantly prolonged the mean time to death in spore-challenged mice. As a surface antigen, opsonization of spores with anti-GroEL IgG showed increased uptake of treated spores and therefore accelerated rate of spore destruction by phagocytic cells leading to the protection of mice. We found that GroEL was able to enhance nitric oxide release from lymphocytes and also reduce bacterial load from the organs, probably through the activation of macrophages and over-expression of certain innate immunity receptors. Therefore, the present study emphasizes that GroEL is an effective immunomodulator against B. anthracis infection. PMID:23263010

Sinha, Kanchan; Bhatnagar, Rakesh

2012-12-21

135

Recombinant GroEL enhances protective antigen-mediated protection against Bacillus anthracis spore challenge.  

UK PubMed Central (United Kingdom)

The fatal inhalation infection caused by Bacillus anthracis results from a complex pathogenic cycle involving release of toxins by bacteria that germinate from spores. Currently available vaccines against anthrax consist of protective antigen (PA), one of the anthrax toxin components. However, these PA-based vaccines are only partially protective against spore challenge in mice. This shows that exclusive elicitation of high anti-PA titer does not directly correlate with protection. Here, we demonstrate that inclusion of GroEL of B. anthracis with PA elicits enhanced protection against anthrax spore challenge in mice. GroEL was included as it has been reported to be present both on the exosporium and in the secretome in addition to the cell surface of B. anthracis. It has also been found protective against other pathogens. In the present study, immunization with GroEL alone was also potent enough to induce high humoral and cell-mediated response and significantly prolonged the mean time to death in spore-challenged mice. As a surface antigen, opsonization of spores with anti-GroEL IgG showed increased uptake of treated spores and therefore accelerated rate of spore destruction by phagocytic cells leading to the protection of mice. We found that GroEL was able to enhance nitric oxide release from lymphocytes and also reduce bacterial load from the organs, probably through the activation of macrophages and over-expression of certain innate immunity receptors. Therefore, the present study emphasizes that GroEL is an effective immunomodulator against B. anthracis infection.

Sinha K; Bhatnagar R

2013-04-01

136

In vivo features of signal transduction by the essential response regulator RpaB from Synechococcus elongatus PCC 7942.  

UK PubMed Central (United Kingdom)

The NblS-RpaB signalling pathway, the most conserved two-component system in cyanobacteria, regulates photosynthesis and acclimatization to a variety of environmental conditions and is involved in negative regulation of high-light-induced genes. However, relevant regulatory details of the NblS-RpaB signalling pathway remain to be elucidated. We recently showed that the response regulator RpaB is regulated by specific (de)phosphorylation from the histidine kinase NblS and that RpaB and its phosphorylatable residue Asp56 are both required for viability of Synechococcus elongatus PCC 7942. We show here that the phosphorylated form of RpaB is present in cells growing under standard laboratory conditions and that high light stress affected the ratio of phosphorylated to non-phosphorylated RpaB. It also decreased the amount of rpaB transcripts without appreciably changing the total levels of RpaB. Quantitative Western blotting and confocal microscopy analyses were consistent with RpaB being a very abundant regulator, with nucleoid localization. A genetically engineered RpaB-GFP (green fluorescent protein) fusion protein rescued lethality of the rpaB null mutant, indicating that it was functional. This is, to our knowledge, the first study demonstrating in a cyanobacterium, and for a two-component response regulator, that the in vivo ratio of phosphorylated to non-phosphorylated protein changes in response to environmental conditions.

Moronta-Barrios F; Espinosa J; Contreras A

2012-05-01

137

Characterization of Fabry mice treated with recombinant adeno-associated virus 2/8-mediated gene transfer  

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Full Text Available Abstract Background Enzyme replacement therapy (ERT) with ?-galactosidase A (?-Gal A) is currently the most effective therapeutic strategy for patients with Fabry disease, a lysosomal storage disease. However, ERT has limitations of a short half-life, requirement for frequent administration, and limited efficacy for patients with renal failure. Therefore, we investigated the efficacy of recombinant adeno-associated virus (rAAV) vector-mediated gene therapy for a Fabry disease mouse model and compared it with that of ERT. Methods A pseudotyped rAAV2/8 vector encoding ?-Gal A cDNA (rAAV2/8-hAGA) was prepared and injected into 18-week-old male Fabry mice through the tail vein. The ?-Gal A expression level and globotriaosylceramide (Gb3) levels in the Fabry mice were examined and compared with Fabry mice with ERT. Immunohistochemical and ultrastructural studies were conducted. Results Treatment of Fabry mice with rAAV2/8-hAGA resulted in the clearance of accumulated Gb3 in tissues such as liver, spleen, kidney, heart, and brain with concomitant elevation of ?-Gal A enzyme activity. Enzyme activity was elevated for up to 60 weeks. In addition, expression of the ?-Gal A protein was identified in the presence of rAAV2/8-hAGA at 6, 12, and 24 weeks after treatment. ?-Gal A activity was significantly higher in the mice treated with rAAV2/8-hAGA than in Fabry mice that received ERT. Along with higher ?-Gal A activity in the kidney of the Fabry mice treated with gene therapy, immunohistochemical studies showed more ?-Gal A expression in the proximal tubules and glomerulus, and less Gb3 deposition in Fabry mice treated with this gene therapy than in mice given ERT. The ?-gal A gene transfer significantly reduced the accumulation of Gb3 in the tubules and podocytes of the kidney. Electron microscopic analysis of the kidneys of Fabry mice also showed that gene therapy was more effective than ERT. Conclusions The rAAV2/8-hAGA mediated ?-Gal A gene therapy provided improved efficiency over ERT in the Fabry disease mouse model. Furthermore, rAAV2/8-hAGA-mediated expression showed a greater effect in the kidney than ERT.

Choi Jin-Ok; Lee Mi; Park Hae-Young; Jung Sung-Chul

2010-01-01

138

The anti-HBV effect mediated by a novel recombinant eukaryotic expression vector for IFN-?  

Science.gov (United States)

Background Chronic hepatitis B is a primary cause of liver-related death. Interferon alpha (IFN-?) is able to inhibit the replication of hepadnavirus, and the sustained and stable expression of IFN-? at appropriate level may be beneficial to HBV clearance. With the development of molecular cloning technology, gene therapy plays a more and more important role in clinical practice. In light of the findings, an attempt to investigate the anti-HBV effects mediated by a eukaryotic expression plasmid (pSecTagB-IFN-?) in vitro was carried out. Methods HBV positive cell line HepG2.2.15 and its parental cell HepG2 were transfected with pSecTagB-IFN-? or empty plasmid by using Lipofectamine™ 2000 reagent. The expression levels of IFN-? were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA methods. The effects of pSecTagB-IFN-? on HBV mRNA, DNA and antigens were analyzed by real-time fluorescence quantitative PCR (qRT-PCR) and ELISA assays. RT-PCR, qRT-PCR and western blot were employed to investigate the influence of pSecTagB-IFN-? on IFN-?-induced signal pathway. Furthermore, through qRT-PCR and ELISA assays, the suppressive effects of endogenously expressed IFN-? and the combination with lamivudine on HBV were also examined. Results pSecTagB-IFN-? could express efficiently in hepatoma cells, and then inhibited HBV replication, characterized by the decrease of HBV S gene (HBs) and HBV C gene (HBc) mRNA, the reduction of HBV DNA load, and the low contents of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). Mechanism research showed that the activation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signal pathway, the up-regulation of IFN-?-induced antiviral effectors and double-stranded (ds) RNA sensing receptors by delivering pSecTagB-IFN-?, could be responsible for these phenomena. Furthermore, pSecTagB-IFN-? vector revealed effectively anti-HBV effect than exogenously added IFN-?. Moreover, lamivudine combined with endogenously expressed IFN-? exhibited stronger anti-HBV effect than with exogenous IFN-?. Conclusion Our results showed that endogenously expressed IFN-? can effectively and persistently inhibit HBV replication in HBV infected cells. These observations opened a promising way to design new antiviral genetic engineering drugs based on IFN-?.

2013-01-01

139

Upregulation of class I major histocompatibility complex antigens by interferon gamma is necessary for T-cell-mediated elimination of recombinant adenovirus-infected hepatocytes in vivo.  

Science.gov (United States)

Recombinant adenoviruses are attractive vehicles for liver-directed gene therapy because of the high efficiency with which they transfer genes to hepatocytes in vivo. First generation recombinant adenoviruses deleted of E1 sequences also express recombinant and early and late viral genes, which lead to development of destructive cellular immune responses. Previous studies indicated that class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTLs) play a major role in eliminating virus-infected cells. The present studies utilize mouse models to evaluate the role of T-helper cells in the primary response to adenovirus-mediated gene transfer to the liver. In vivo ablation of CD4+ cells or interferon gamma (IFN-gamma) was sufficient to prevent the elimination of adenovirus-transduced hepatocytes, despite the induction of a measurable CTL response. Mobilization of an effective TH1 response as measured by in vitro proliferation assays was associated with substantial upregulation of MHC class I expression, an effect that was prevented in IFN-gamma-deficient animals. These results suggest that elimination of virus-infected hepatocytes in a primary exposure to recombinant adenovirus requires both induction of antigen-specific CTLs as well as sensitization of the target cell by TH1-mediated activation of MHC class I expression. PMID:7638177

Yang, Y; Xiang, Z; Ertl, H C; Wilson, J M

1995-08-01

140

Upregulation of class I major histocompatibility complex antigens by interferon gamma is necessary for T-cell-mediated elimination of recombinant adenovirus-infected hepatocytes in vivo.  

UK PubMed Central (United Kingdom)

Recombinant adenoviruses are attractive vehicles for liver-directed gene therapy because of the high efficiency with which they transfer genes to hepatocytes in vivo. First generation recombinant adenoviruses deleted of E1 sequences also express recombinant and early and late viral genes, which lead to development of destructive cellular immune responses. Previous studies indicated that class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTLs) play a major role in eliminating virus-infected cells. The present studies utilize mouse models to evaluate the role of T-helper cells in the primary response to adenovirus-mediated gene transfer to the liver. In vivo ablation of CD4+ cells or interferon gamma (IFN-gamma) was sufficient to prevent the elimination of adenovirus-transduced hepatocytes, despite the induction of a measurable CTL response. Mobilization of an effective TH1 response as measured by in vitro proliferation assays was associated with substantial upregulation of MHC class I expression, an effect that was prevented in IFN-gamma-deficient animals. These results suggest that elimination of virus-infected hepatocytes in a primary exposure to recombinant adenovirus requires both induction of antigen-specific CTLs as well as sensitization of the target cell by TH1-mediated activation of MHC class I expression.

Yang Y; Xiang Z; Ertl HC; Wilson JM

1995-08-01

 
 
 
 
141

Reduction of the RPA eigenvalue problem and a generalized Cholesky decomposition for real-symmetric matrices  

Energy Technology Data Exchange (ETDEWEB)

The particular symmetry of the random-phase-approximation (RPA) matrix has been utilized in the past to reduce the RPA eigenvalue problem into a symmetric-matrix problem of half the dimension. The condition of positive-definiteness of at least one of the matrices has been imposed (where A and B are the sub-matrices of the RPA matrix) so that, e.g., its square root can be found by Cholesky decomposition. In this work, alternative methods are pointed out to reduce the RPA problem to a real (not symmetric, in general) problem of half the dimension, with the condition of positive-definiteness relaxed. One of the methods relies on a generalized Cholesky decomposition, valid for non-singular real symmetric matrices. The algorithm is described and a corresponding routine in C is given. (authors)

Papakonstantinou, P. [Technische Univ. Darmstadt, Institut fur Kernphysik (Germany)

2007-04-15

142

Reduction of the RPA eigenvalue problem and a generalized Cholesky decomposition for real-symmetric matrices  

International Nuclear Information System (INIS)

The particular symmetry of the random-phase-approximation (RPA) matrix has been utilized in the past to reduce the RPA eigenvalue problem into a symmetric-matrix problem of half the dimension. The condition of positive-definiteness of at least one of the matrices has been imposed (where A and B are the sub-matrices of the RPA matrix) so that, e.g., its square root can be found by Cholesky decomposition. In this work, alternative methods are pointed out to reduce the RPA problem to a real (not symmetric, in general) problem of half the dimension, with the condition of positive-definiteness relaxed. One of the methods relies on a generalized Cholesky decomposition, valid for non-singular real symmetric matrices. The algorithm is described and a corresponding routine in C is given. (authors)

2007-01-01

143

Reduction of the RPA eigenvalue problem and a generalized Cholesky decomposition for real-symmetric matrices  

CERN Document Server

The particular symmetry of the random-phase-approximation (RPA) matrix has been utilized in the past to reduce the RPA eigenvalue problem into a symmetric-matrix problem of half the dimension. The condition of positive definiteness of at least one of the matrices A+-B has been imposed (where A and B are the submatrices of the RPA matrix) so that, e.g., its square root can be found by Cholesky decomposition. In this work, alternative methods are pointed out to reduce the RPA problem to a real (not symmetric, in general) problem of half the dimension, with the condition of positive definiteness relaxed. One of the methods relies on a generalized Cholesky decomposition, valid for non-singular real symmetric matrices. The algorithm is described and a corresponding routine in C is given.

Papakonstantinou, P

2007-01-01

144

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

International Nuclear Information System (INIS)

[en] 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)[pt] A Aproximacao Randonica de Fase (RPA), usada no tratamento de vibracoes nucleares de pequena amplitude incluindo os continua particula-buraco, e manipulada em termos de tecnicas previamente desenvolvidas para o trato de ressonancias de particula-simples em estruturas de reacao teoricas. Uma hierarquia de aproximacoes interpretaveis e obtida e propoe-se um processo simples de aproximacao que envolve um esforco numerico semelhante ao envolvido em calculos standard de RPA discretas. (Autor)

1987-01-01

145

Escherichia coli MW005: lambda Red-mediated recombineering and copy-number induction of oriV-equipped constructs in a single host  

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Full Text Available Abstract Background Escherichia coli strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the DNA is difficult to isolate in high yield and purity. To overcome this limitation vectors, e.g. pCC1FOS, have been constructed that contain the additional replication origin, oriV, which permits copy-number to be induced transiently when propagated in a suitable host strain, e.g. EPI300, that supplies the cognate trans-replication protein TrfA. Previously, we used EL350 and EPI300 sequentially to recombineer oriV-equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone. To eliminate these intervening DNA isolation and transformation steps we retrofitted EL350 with a PBAD-driven trfA gene generating strain MW005 that supports, independently, both recombineering and copy-number induction. Results The PBAD-driven copy of cre in EL350 was replaced seamlessly with a copy of trfA, PCR-amplified from EPI300 chromosomal DNA, to generate MW005. This new strain has been used to both generate, via recombineering, a number of reporter gene fusions directly from pCC1FOS-based Caenorhabditis elegans genomic clones and to transiently induce copy-number of fosmid and BAC clones prior to DNA preparation. Conclusions By retrofitting EL350, an established 'recombineering' E. coli strain, with a tightly regulated copy of trfA we have produced a new strain, MW005, which combines recombineering capacity with the useful ability to transiently induce copy-number of oriV-equipped clones. By coupling these two steps in a single strain, use of MW005 will enable the more rapid recombineering-mediated production of recombinant clones in the yield and quality necessary for many downstream purposes.

Westenberg Marcel; Bamps Sophie; Soedling Helen; Hope Ian A; Dolphin Colin T

2010-01-01

146

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

147

XerCD-mediated site-specific recombination leads to loss of the 57-kilobase gonococcal genetic island.  

UK PubMed Central (United Kingdom)

Most strains of Neisseria gonorrhoeae carry the 57-kb gonococcal genetic island (GGI), as do a few strains of Neisseria meningitidis. The GGI is inserted into the chromosome at the dif site (difA) and is flanked by a partial repeat of the dif site (difB). Since dif is a sequence recognized by the site-specific recombinases XerC and XerD and the GGI shows evidence of horizontal acquisition, we hypothesized that the GGI may be acquired or lost by XerCD-mediated site-specific recombination. We show that while the GGI flanked by wild-type dif sites, difA and difB, is not readily lost from the gonococcal chromosome, the substitution of difB with another copy of difA allows the frequent excision and loss of the GGI. In mutants carrying two difA sites (difA(+) difA(+)), the GGI can be detected as an extrachromosomal circle that exists transiently. A mutation of xerD diminished GGI excision from the chromosome of a difA(+) difA(+) strain, while mutations in recA or type IV secretion genes had no effect on the loss of the GGI. These data indicate that the GGI is maintained by the replication of the chromosome and that GGI excision and loss are dependent upon the dif sequence and xerD. The detection of a circular form of the GGI in a wild-type strain suggests that GGI excision may occur naturally and could function to facilitate GGI transfer. These data suggest a model of GGI excision and loss explaining the absence of the GGI from some gonococcal strains and the maintenance of variant GGIs in some gonococcal and meningococcal isolates.

Domínguez NM; Hackett KT; Dillard JP

2011-01-01

148

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

UK PubMed Central (United Kingdom)

Acute lymphoblastic leukemia (ALL) accounts for approximately 25% of pediatric malignancies. Of interest, the incidence of ALL is observed approximately 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 approximately 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 to girls.

Meissner B; Bartram T; Eckert C; Trka J; Panzer-Grümayer R; Hermanova I; Ellinghaus E; Franke A; Möricke A; Schrauder A; Teigler-Schlegel A; Dörge P; von Stackelberg A; Basso G; Bartram CR; Kirschner-Schwabe R; Bornhäuser B; Bourquin JP; Cazzaniga G; Hauer J; Attarbaschi A; Izraeli S; Zaliova M; Cario G; Zimmermann M; Avigad S; Sokalska-Duhme M; Metzler M; Schrappe M; Koehler R; Kronnie GT; Stanulla M

2013-09-01

149

Nuclear collective excitations using correlated realistic interactions: the role of explicit RPA correlations  

CERN Multimedia

We examine to which extent correlated realistic nucleon-nucleon interactions, derived within the Unitary Correlation Operator Method (UCOM), can describe nuclear collective motion in the framework of first-order random-phase approximation (RPA). To this end we employ the correlated Argonne V18 interaction in calculations within the so-called "Extended" RPA (ERPA) and investigate the response of closed-shell nuclei. The ERPA is a renormalized RPA version which considers explicitly the depletion of the Fermi sea due to long-range correlations and thus allows us to examine how these affect the excitation spectra. It is found that the effect on the properties of giant resonances is rather small. Compared to the standard RPA, where excitations are built on top of the uncorrelated Hartree-Fock (HF) ground state, their centroid energies decrease by up to 1 MeV, approximately, in the isovector channel. The isoscalar response is less affected in general. Thus, the disagreement between our previous UCOM-based RPA calcu...

Papakonstantinou, P; Roth, R

2006-01-01

150

Beyond RPA correlation energies: Evaluation of model exchange-correlation kernels  

Science.gov (United States)

The adiabatic-connection fluctuation-dissipation theorem (ACFDT) has drawn considerable attention in describing van der Waals (vdW) dispersion interactions. Under the random phase approximation (RPA), the EXX/RPA method yields the correct asymptotic behavior at large distances. However, for many advanced materials, e.g., organic/inorganic interfaces and molecular crystals, it is important to capture the short-range dispersion interaction within several angstrom. Because RPA pair distribution function is incorrect at short distances, the contribution of the exchange-correlation kernel has to be included properly. In this work, we implemented several model exchange-correlation kernels in the framework of ACFDT. Special attention was paid to develop non-local kernels suitable for inhomogeneous electronic systems. The performance of the exchange-correlation kernels were evaluated for both bulk and molecular systems.

Lu, Deyu

2013-03-01

151

Electrostatic switches that mediate the pH-dependent conformational change of "short" recombinant human pseudocathepsin D.  

UK PubMed Central (United Kingdom)

Human cathepsin D (hCatD) is an aspartic peptidase with a low pH optimum. X-ray crystal structures have been solved for an active, low pH (pH 5.1) form (CatD(lo)) [Baldwin, E. T., Bhat, T. N., Gulnik, S., Hosur, M. V., Sowder, R. C., Cachau, R. E., Collins, J., Silva, A. M., and Erickson, J. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6796-6800] and an inactive, high pH (pH 7.5) form (CatD(hi)) [Lee, A. Y., Gulnik, S. V., and Erickson, J. W. (1998) Nat. Struct. Biol. 5, 866-871]. It has been suggested that ionizable switches involving the carboxylate side chains of E5, E180, and D187 may mediate the reversible interconversion between CatD(hi) and CatD(lo) and that Y10 stabilizes CatD(hi) [Lee, A. Y., Gulnik, S. V., and Erickson, J. W. (1998) Nat. Struct. Biol. 5, 866-871]. To test these hypotheses, we generated single point mutants in "short" recombinant human pseudocathepsin D (srCatD), a model kinetically similar to hCatD [Beyer, B. M., and Dunn, B. M. (1996) J. Biol. Chem. 271, 15590-15596]. E180Q, Y10F, and D187N exhibit significantly higher kcat/Km values (2-, 3-, and 6-fold, respectively) at pH 3.7 and 4.75 compared to srCatD, indicating that these residues are important in stabilizing the CatD(hi). E5Q exhibits a 2-fold lower kcat/Km compared to srCatD at both pH values, indicating the importance of E5 in stabilizing the CatD(lo). Accordingly, full time-course "pH-jump" (pH 5.5-4.75) studies of substrate hydrolysis indicate that E180Q, D187N, and Y10F have shorter kinetic lag phases that represent the change from CatD(hi) to CatD(lo) compared to srCatD and E5Q. Intrinsic tryptophan fluorescence reveals that the variants have a native-like structure over the pH range of our assays. The results indicate that E180 and D187 participate as an electrostatic switch that initiates the conformational change of CatD(lo) to CatD(hi) and Y10 stabilizes CatD(hi) by hydrogen bonding to the catalytic Asp 33. E5 appears to play a less significant role as an ionic switch that stabilizes CatD(lo).

Goldfarb NE; Lam MT; Bose AK; Patel AM; Duckworth AJ; Dunn BM

2005-12-01

152

Simulation of large-scale production of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification system.  

Science.gov (United States)

Inteins are self-cleavable proteins that under reducing conditions can be cleaved from a recombinant target protein. Industrially, an intein-based system could potentially reduce production costs of recombinant proteins by facilitating a highly selective affinity purification using an inexpensive substrate such as chitin. In this study, SuperPro Designer was used to simulate the large-scale recovery of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification process based on the commercially available IMPACT system. The intein process was also compared with a conventional process simulated by SuperPro. The intein purification process initially simulated was significantly more expensive than the conventional process, primarily owing to the properties of the chitin resin and high reducing-agent (dithiothreitol [DTT]) raw material cost. The intein process was sensitive to the chitin resin binding capacity, cleavage efficiency of the intein fusion protein, the size of the target protein relative to the intein tag, and DTT costs. An optimized intein purification process considerably reduced costs by simulating an improved chitin resin and alternative reducing agents. Thus, to realize the full potential of intein purification processes, research is needed to improve the properties of chitin resin and to find alternative, inexpensive raw materials. PMID:16118465

Sharma, Shamik S; Chong, Shaorong; Harcum, Sarah W

2005-08-01

153

Simulation of large-scale production of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification system.  

UK PubMed Central (United Kingdom)

Inteins are self-cleavable proteins that under reducing conditions can be cleaved from a recombinant target protein. Industrially, an intein-based system could potentially reduce production costs of recombinant proteins by facilitating a highly selective affinity purification using an inexpensive substrate such as chitin. In this study, SuperPro Designer was used to simulate the large-scale recovery of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification process based on the commercially available IMPACT system. The intein process was also compared with a conventional process simulated by SuperPro. The intein purification process initially simulated was significantly more expensive than the conventional process, primarily owing to the properties of the chitin resin and high reducing-agent (dithiothreitol [DTT]) raw material cost. The intein process was sensitive to the chitin resin binding capacity, cleavage efficiency of the intein fusion protein, the size of the target protein relative to the intein tag, and DTT costs. An optimized intein purification process considerably reduced costs by simulating an improved chitin resin and alternative reducing agents. Thus, to realize the full potential of intein purification processes, research is needed to improve the properties of chitin resin and to find alternative, inexpensive raw materials.

Sharma SS; Chong S; Harcum SW

2005-08-01

154

I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.  

UK PubMed Central (United Kingdom)

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 M; Simon-Chazottes D; Vandormael-Pournin S; Soueid J; Langa F; Cohen-Tannoudji M; Bernard BA; Panthier JJ

2012-01-01

155

R-loop mediated transcription-associated recombination in trf4? mutants reveals new links between RNA surveillance and genome integrity.  

UK PubMed Central (United Kingdom)

To get further insight into the factors involved in the maintenance of genome integrity we performed a screening of Saccharomyces cerevisiae deletion strains inducing hyperrecombination. We have identified trf4, a gene encoding a non-canonical polyA-polymerase involved in RNA surveillance, as a factor that prevents recombination between DNA repeats. We show that trf4? confers a transcription-associated recombination phenotype that is mediated by the nascent mRNA. In addition, trf4? also leads to an increase in the mutation frequency. Both genetic instability phenotypes can be suppressed by overexpression of RNase H and are exacerbated by overexpression of the human cytidine deaminase AID. These results suggest that in the absence of Trf4 R-loops accumulate co-transcriptionally increasing the recombination and mutation frequencies. Altogether our data indicate that Trf4 is necessary for both mRNA surveillance and maintenance of genome integrity, serving as a link between RNA and DNA metabolism in S. cerevisiae.

Gavaldá S; Gallardo M; Luna R; Aguilera A

2013-01-01

156

Thermal entanglement in fully connected spin systems and its RPA description  

CERN Multimedia

We examine the thermal pairwise entanglement in a symmetric system of $n$ spins fully connected through anisotropic $XYZ$-type couplings embedded in a transverse magnetic field. We consider both the exact evaluation together with that obtained with the static path + random phase approximation (RPA) and the ensuing mean field + RPA. The latter is shown to provide an accurate analytic description of both the parallel and antiparallel thermal concurrence in large systems. We also analyze the limit temperature for pairwise entanglement, which is shown to increase for large fields and to decrease logarithmically with increasing $n$. Special finite size effects are as well discussed.

Matera, Juan Mauricio; Canosa, Norma

2011-01-01

157

Fowlpox virus recombinants expressing HPV-16 E6 and E7 oncogenes for the therapy of cervical carcinoma elicit humoral and cell-mediated responses in rabbits  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Around half million new cases of cervical cancer arise each year, making the development of an effective therapeutic vaccine against HPV a high priority. As the E6 and E7 oncoproteins are expressed in all HPV-16 tumour cells, vaccines expressing these proteins might clear an already established tumour and support the treatment of HPV-related precancerous lesions. Methods Three different immunisation regimens were tested in a pre-clinical trial in rabbits to evaluate the humoral and cell-mediated responses of a putative HPV-16 vaccine. Fowlpoxvirus (FP) recombinants separately expressing the HPV-16 E6 (FPE6) and E7 (FPE7) transgenes were used for priming, followed by E7 protein boosting. Results All of the protocols were effective in eliciting a high antibody response. This was also confirmed by interleukin-4 production, which increased after simultaneous priming with both FPE6 and FPE7 and after E7 protein boost. A cell-mediated immune response was also detected in most of the animals. Conclusion These results establish a preliminary profile for the therapy with the combined use of avipox recombinants, which may represent safer immunogens than vaccinia-based vectors in immuno-compromised individuals, as they express the transgenes in most mammalian cells in the absence of a productive replication.

Radaelli Antonia; Pozzi Eleana; Pacchioni Sole; Zanotto Carlo; Morghen Carlo

2010-01-01

158

The use of FLP-mediated recombination for the functional analysis of an effector gene family in the biotrophic smut fungus Ustilago maydis.  

UK PubMed Central (United Kingdom)

*In the Ustilago maydis genome, several novel secreted effector proteins are encoded by gene families. Because of the limited number of selectable markers, the ability to carry out sequential gene deletions has limited the analysis of effector gene families that may have redundant functions. *Here, we established an inducible FLP-mediated recombination system in U. maydis that allows repeated rounds of gene deletion using a single selectable marker (Hyg(R)). To avoid genome rearrangements via FRT sites remaining in the genome after excision, different mutated FRT sites were introduced. *The FLP-mediated selectable marker-removal technique was successfully applied to delete a family of 11 effector genes (eff1) using five sequential rounds of recombination. We showed that expression of all 11 genes is up-regulated during the biotrophic phase. Strains carrying deletions of 9 or all 11 genes showed a significant reduction in virulence, and this phenotype could be partially complemented by the introduction of different members from the gene family, demonstrating redundancy. *The establishment of the FLP/FRT system in a plant pathogenic fungus paves the way for analyzing multigene families with redundant functions.

Khrunyk Y; Münch K; Schipper K; Lupas AN; Kahmann R

2010-09-01

159

Rec-Mediated Recombinational Hot Spot Activity in Bacteriophage Lambda II. a Mutation Which Causes Hot Spot Activity  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Crosses have been performed which identify phage mutants (chi) which cause recombinational hot spot activity in ?. The hot spot activity is found in crosses of red- gam- chi- strains in rec+ hosts; in the crosses reported here, both the chi- mutations and the hot spot are located near the right end ...

Lam, Stephen T.; Stahl, Mary M.; McMilin, Kenneth D.; Stahl, Franklin W.

160

GENE INSERTION AND REPLACEMENT IN SCHIZOSACCHAROMYCES POMBE MEDIATED BY THE STREPTOMYCES BACTERIOPHAGE C31 SITE-SPECIFIC RECOMBINATION SYSTEM  

Science.gov (United States)

The Streptomyces bacteriophage íC31 site-specific recombination system was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to...

 
 
 
 
161

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

162

Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We report that during activation of the simian virus 40 (SV40) pre-replication complex, SV40 T antigen (Tag) helicase actively loads replication protein A (RPA) on emerging single-stranded DNA (ssDNA). This novel loading process requires physical interaction of Tag origin DNA-binding domain (OBD) wi...

Jiang, Xiaohua; Klimovich, Vitaly; Arunkumar, Alphonse I; Hysinger, Erik B; Wang, Yingda; Ott, Robert D; Guler, Gulfem D

163

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

International Nuclear Information System (INIS)

[en] 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

164

On the dynamics of polymer mixtures in solution using the RPA  

International Nuclear Information System (INIS)

The dynamics of polymer mixtures and copolymers in solution is investigated using the Random Phase Approximation (RPA). It is shown that the known results for the intermediate scattering functions are recovered in the Rouse limit only. If hydrodynamic interaction is not negligible, a discrepancy appears. This discrepancy can be observed by combining static and dynamic scattering experiments. (author). 10 refs.

1989-01-01

165

[Differentiation of adipose-derived stem cells induced by recombinant adenovirus's containing fibers derived from B-group serotype 35-mediated bone morphogenetic protein].  

UK PubMed Central (United Kingdom)

OBJECTIVE: To investigate the differentiation of the adipose-derived adult stem cell (ADASC) induced by the recombinant adenovirus's containing fibers derived from B-group serotype 35 (rAd5/F35)-mediated human bone morphogenetic protein 7 (hBMP-7) gene and to explore a new cell source for the bone tissue engineering. METHODS: The hBMP-7 gene was amplified with the pcDNA1.1/AMP-hBMP-7 plasmid as a formwork. After the purification, the gene fragment was cloned into the pDC316 carrier for the recombination of the plasmid of pDC316-hBMP-7. The 293 cells were co-transfected by the skeleton plasmid of pBHG-fiber5/35 and the shuttle plasmid of pDC316-hBMP-7, and the recombinant plasmid of Ad5/F35-hBMP-7 was obtained; the recombinant plasmid of Ad5/ F35-enhancd green fluorescent protein(EGFP) was obtained by the similar method. The rat ADASCs were cultured and transfected by the Ad5/F35-hBMP-7 plasmid and the Ad5/F35-EGFP plasmid, respectively; the remaining untransfected ADASC were used as the controls. The morphology and the growth pattern of the transfected cells were evaluated. The transcription and the expression of the transfected genes and the steogenic phenotypes such as calcium nodules and osteocalcin were evaluated by ELISA. RESULTS: The identification of PCR and enzyme cutting showed that the construction of the recombinant Ad5/F35-hBMP-7 plasmid could be confirmed. The transfection rate of the ADASC by the Ad5/F35-EGFP plasmid was determined to be greater than 90%. The hBMP-7 gene in the transfected ADASC could express the corresponding protein, and the formation of the calcium nodules could be found in the induced group. The electron microscopy showed that there was a calcium element in the cytoplasm, the alkaline phosphatase result was positive, and the expression of osteocalcin was increased. CONCLUSION: The rAd5/F35-hBMP-7 gene can promote the differentiation of the adipose-derived adult stem cells to the osteoblasts in the bone tissue engineering.

Kang Y; Liao W; Yuan Z

2007-02-01

166

[Production of l-ephedrine and d-pseudoephedrine in recombined yeasts obtained by argon ion implantation mediated Ephedra genome DNA transformation].  

Science.gov (United States)

The genome DNA from Ephedra glauca was randomly transferred into Hansenula anomala, respectively, by argon ion bombardment. Then, after screening by the motheds of bromothymol blue indicator selection, slant cultivation, l-ephedrine and d-pseudoephedrine copper chromic salt qualitative test and RP-HPLC determination, 3 strains, producing recombined yeasts were obtained, which can use glucose as a carbon source, NaNO3 as nitrogen source and be genetically stable. After cultivated in liquid medium for 72 hours and analyzed by the RP-HPLC, the recombined strains can produce l-ephedrine 11.87 mg/L and d-pseudoephedrine 4.11 mg/L excellular, d-pseudoephedrine 294.86 mg/g dry cell incellular, but l-ephedrine not detected incellular. The transformation efficiency of Ephedra genome DNA transferred into yeasts via argon ion bombardment was 0.65%. The effects of Ephedra genome DNA macromolecule integrity on yeast transformation system were discussed. The results shown that DNA macromolecule with integrated structure used as exogenous donor can obtain higher transformation efficiency than DNA macromolecule random fragments by ion implantation mediated DNA transformation. It was inferred that biosynthesis of l-ephedrine and the d-pseudoephedrin were controlled by linked together genes or gene clusters. PMID:18062271

Mao, Pei-Hong; Ma, Xiang-Dong; Jin, Xiang; Yang, Hong-Mei; Lou, Kai

2007-10-01

167

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

UK PubMed Central (United Kingdom)

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 LN; Li Y; Shi LZ; Hwang PY; He J; Wang H; Razavian N; Berns MW; Wu X

2013-05-01

168

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; Lee Hsin-Fang; Pettit Steven C; Zaro Jennica L; Huang Ning; Shen Wei-Chiang

2012-01-01

169

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

UK PubMed Central (United Kingdom)

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 D; Lee HF; Pettit SC; Zaro JL; Huang N; Shen WC

2012-01-01

170

TCreERT2, a transgenic mouse line for temporal control of Cre-mediated recombination in lineages emerging from the primitive streak or tail bud.  

UK PubMed Central (United Kingdom)

The study of axis extension and somitogenesis has been greatly advanced through the use of genetic tools such as the TCre mouse line. In this line, Cre is controlled by a fragment of the T (Brachyury) promoter that is active in progenitor cells that reside within the primitive streak and tail bud and which give rise to lineages emerging from these tissues as the embryonic axis extends. However, because TCre-mediated recombination occurs early in development, gene inactivation can result in an axis truncation that precludes the study of gene function in later or more posterior tissues. To address this limitation, we have generated an inducible TCre transgenic mouse line, called TCreERT2, that provides temporal control, through tamoxifen administration, in all cells emerging from the primitive streak or tail bud throughout development. TCreERT2 activity is mostly silent in the absence of tamoxifen and, in its presence, results in near complete recombination of emerging mesoderm from E7.5 through E13.5. We demonstrate the utility of the TCreERT2 line for determining rate of posterior axis extension and somite formation, thus providing the first in vivo tool for such measurements. To test the usefulness of TCreERT2 for genetic manipulation, we demonstrate that an early deletion of ß-Catenin via TCreERT2 induction phenocopies the TCre-mediated deletion of ß-Catenin defect, whereas a later induction bypasses this early phenotype and produces a similar defect in more caudal tissues. TCreERT2 provides a useful and novel tool for the control of gene expression of emerging embryonic lineages throughout development.

Anderson MJ; Naiche LA; Wilson CP; Elder C; Swing DA; Lewandoski M

2013-01-01

171

Cultured mast cells from asthmatic patients and controls respond with similar sensitivity to recombinant Der P2 induced, IgE-mediated activation  

DEFF Research Database (Denmark)

The function of cultured mast cells may depend on genetic or environmental influence on the stem cell donor. This study investigates whether asthma or atopy in the donor influenced the growth and sensitivity of mast cells cultured from asthma patients and healthy controls under identical conditions. Mast cells were cultured from peripheral blood from twelve patients with an objectively confirmed asthma diagnosis and eight healthy subjects. During the last 2 weeks of culture, mast cells were incubated with IL-4 and 80 kU/L recombinant human IgE containing two clones (7%+7%) specific for mite allergen Der p2. The sensitivity of IgE-mediated activation of mast cells was investigated as Fc?RI-mediated up-regulation of CD63. Ten subjects were atopic, defined as a positive skin prick test (>3 mm) to at least one of ten common allergens. After activation with recombinant Der p2, the maximum CD63 Median Fluorescence Intensity was 20 456 ± 1640 (SE) for asthma patients and 22 275 ± 1971 (SE) for controls (ns). The fraction of CD63 positive cells was 54.4% in asthma patients and 48.4% in controls (ns). The allergen concentration inducing 50% of the maximal CD63 response was similar in asthma patients (-0.4795 log ng/mL ± 0.092 (SE)) and controls (-0.6351 log ng/mL ± 0.083, ns) and in atopic and non-atopic subjects. When cultured, sensitised and activated under identical conditions, mast cells from allergic asthmatics and healthy controls respond similar. Activation of cultured mast cells appears to depend on culture conditions (IL-4, IgE) rather than on donor status as atopy and asthma. This article is protected by copyright. All rights reserved.

Krohn, Inge Kortekaas; Sverrild, Asger

2013-01-01

172

Effects of recombinant adenovirus mediated retinoblastoma gene 94 combined with ?-ray on growth of esophageal carcinoma cells  

International Nuclear Information System (INIS)

Objective: To study the combined effect of exogenous recombinant adenovirus-medicated retinoblastoma gene 94 (Ad-Rb94) combined with ?-ray on the growth of esophageal carcinoma cells. Methods: Cell culture were randomly divided into 5 groups: control group, recombinant adenovirus vector containing ?-galactosidase gene (Ad-LacZ) group, Ad-Rb94 group, ?-ray radiation group and Ad-Rb94 combined with ?-ray radiation group. EC109 cells were transfected by Ad-Rb94 and exposed to 4 Gy 137Cs ?-ray irradiation 6 hours after transfection. The inhibition rate of EC109 cells were detected by MTT assay. Results: EC109 cells transfected with Ad-Rb94 group, ?-ray radiation group and Ad-Rb94 combined with ?-ray radiation group were all inhibited. The inhibition rate of Ad-Rb94 combined with ?ray radiation group reached (40.30%±4.2%), significantly higher than Ad-Rb94 group (18.3±0.4%) and ?-ray radiation group (27.40%±2.9%) (?2=7.91, ?2=5.82, P2=5.12, P

2010-01-01

173

COM-1 promotes homologous recombination during Caenorhabditis elegans meiosis by antagonizing Ku-mediated non-homologous end joining.  

UK PubMed Central (United Kingdom)

Successful completion of meiosis requires the induction and faithful repair of DNA double-strand breaks (DSBs). DSBs can be repaired via homologous recombination (HR) or non-homologous end joining (NHEJ), yet only repair via HR can generate the interhomolog crossovers (COs) needed for meiotic chromosome segregation. Here we identify COM-1, the homolog of CtIP/Sae2/Ctp1, as a crucial regulator of DSB repair pathway choice during Caenorhabditis elegans gametogenesis. COM-1-deficient germ cells repair meiotic DSBs via the error-prone pathway NHEJ, resulting in a lack of COs, extensive chromosomal aggregation, and near-complete embryonic lethality. In contrast to its yeast counterparts, COM-1 is not required for Spo11 removal and initiation of meiotic DSB repair, but instead promotes meiotic recombination by counteracting the NHEJ complex Ku. In fact, animals defective for both COM-1 and Ku are viable and proficient in CO formation. Further genetic dissection revealed that COM-1 acts parallel to the nuclease EXO-1 to promote interhomolog HR at early pachytene stage of meiotic prophase and thereby safeguards timely CO formation. Both of these nucleases, however, are dispensable for RAD-51 recruitment at late pachytene stage, when homolog-independent repair pathways predominate, suggesting further redundancy and/or temporal regulation of DNA end resection during meiotic prophase. Collectively, our results uncover the potentially lethal properties of NHEJ during meiosis and identify a critical role for COM-1 in NHEJ inhibition and CO assurance in germ cells.

Lemmens BB; Johnson NM; Tijsterman M

2013-01-01

174

Immunogenicity in mice of anthrax recombinant protective antigen in the presence of aluminum adjuvants.  

Science.gov (United States)

The only US-licensed anthrax vaccine for human use, as well as several experimental vaccines containing solely purified recombinant protective antigen (rPA), are formulated using aluminum hydroxide (Al(OH)3) as an adjuvant. It has been suggested that effective adjuvanticity of aluminum salts for protein antigens depends, at least partially, on the degree of adsorption of the antigen to the adjuvant. On the other hand, the ease of antigen desorption from the adjuvant in a quantitative fashion may facilitate the assessment of vaccine characteristics in the laboratory. In this regard, aluminum phosphate (AlPO4), although deemed a "weaker" adjuvant than Al(OH)3, appears superior to the latter. To investigate the possibility of formulating rPA vaccines with AlPO4, as well as the significance of the adsorption of this antigen to the aluminum salt for adjuvanticity, we studied the effect of AlPO4 and Al(OH)3 on the induction of anti-rPA antibodies in mice. In a first immunization experiment the adjuvanticity of AlPO4 combined with rPA was examined. Antibodies against rPA were measured using an ELISA. Results indicated that AlPO4 is able to significantly increase the antibody response to rPA, irrespective of its degree of adsorption to the adjuvant. Based on these results, in a second experiment mice were immunized twice, with different formulations of rPA containing either AlPO4 or Al(OH)3, and rPA-antibodies were measured using ELISA and an in vitro toxin neutralization assay. Comparable immune responses to rPA were obtained with both aluminum salts. Additionally, results with AlPO4 as adjuvant confirmed that, in this mouse model, binding of the protein to the adjuvant is not essential for adjuvanticity, whereas the amount of adjuvant has an influence on the antibody response induced. PMID:15734073

Berthold, Inge; Pombo, Maria-Luz; Wagner, Leslie; Arciniega, Juan L

2005-03-14

175

Immunogenicity in mice of anthrax recombinant protective antigen in the presence of aluminum adjuvants.  

UK PubMed Central (United Kingdom)

The only US-licensed anthrax vaccine for human use, as well as several experimental vaccines containing solely purified recombinant protective antigen (rPA), are formulated using aluminum hydroxide (Al(OH)3) as an adjuvant. It has been suggested that effective adjuvanticity of aluminum salts for protein antigens depends, at least partially, on the degree of adsorption of the antigen to the adjuvant. On the other hand, the ease of antigen desorption from the adjuvant in a quantitative fashion may facilitate the assessment of vaccine characteristics in the laboratory. In this regard, aluminum phosphate (AlPO4), although deemed a "weaker" adjuvant than Al(OH)3, appears superior to the latter. To investigate the possibility of formulating rPA vaccines with AlPO4, as well as the significance of the adsorption of this antigen to the aluminum salt for adjuvanticity, we studied the effect of AlPO4 and Al(OH)3 on the induction of anti-rPA antibodies in mice. In a first immunization experiment the adjuvanticity of AlPO4 combined with rPA was examined. Antibodies against rPA were measured using an ELISA. Results indicated that AlPO4 is able to significantly increase the antibody response to rPA, irrespective of its degree of adsorption to the adjuvant. Based on these results, in a second experiment mice were immunized twice, with different formulations of rPA containing either AlPO4 or Al(OH)3, and rPA-antibodies were measured using ELISA and an in vitro toxin neutralization assay. Comparable immune responses to rPA were obtained with both aluminum salts. Additionally, results with AlPO4 as adjuvant confirmed that, in this mouse model, binding of the protein to the adjuvant is not essential for adjuvanticity, whereas the amount of adjuvant has an influence on the antibody response induced.

Berthold I; Pombo ML; Wagner L; Arciniega JL

2005-03-01

176

Augmentation of NK activity and/or macrophage-mediated cytotoxicity in the liver by biological response modifiers including human recombinant interleukin 2.  

UK PubMed Central (United Kingdom)

Administration of several biological response modifiers (BRMs) to mice strongly augmented natural killer (NK) activity of leukocytes isolated from the liver. This augmentation of NK activity was induced by two synthetic molecules (MVE-2 and poly ICLC), by two BRMs of bacterial origin (formalin-fixed Propionibacterium acnes: P. acnes and a streptococcal cell wall preparation designated OK-432), as well as a single injection of human recombinant interleukin-2 (hrIL 2). All of these BRMs augmented NK activity in the liver to a greater degree than in the spleen. In addition, adherent leukocytes (greater than 90% macrophages) isolated from the liver following P. acnes administration also exhibited augmented macrophage-mediated cytotoxicity. This cytotoxicity was characterized as macrophage mediated and distinguished from NK activity, on the basis of adherence purification, kinetics of cytotoxicity, and target cell selectivity. The results demonstrate that a variety of BRMs induce augmented natural immunity in the liver and suggest that such organ-associated immune responses may play an important role in the antimetastatic effects of BRMs.

Zhang SR; Salup RR; Urias PE; Twilley TA; Talmadge JE; Herberman RB; Wiltrout RH

1986-01-01

177

Evaluation in mice of the capillary leak syndrome (CLS) mediated by the systemic administration of recombinant interleukin-2 (IL-2)  

Energy Technology Data Exchange (ETDEWEB)

Since a CLS with interstitial pulmonary edema has been the major toxicity of IL-2 administration in humans, the authors studied this CLS in mice by quantitating the IL-2 mediated, tissue extravasation (Ex) of intravenously injected /sup 125/I-bovine serum albumin (BSA). Mice received saline (HBSS) or 200,000 U of IL-2 intraperitoneally thrice daily from day 0-6 before tissues were counted. A permeability index (PI) was calculated by dividing the mean counts per minute (CPM) from tissues of treated mice by those from controls. In a representative experiment, increased BSA Ex was noted in the lungs of mice treated with IL-2 when compared with HBSS (6187 +/- 141 and 638 +/- 64 CPM +/- SEM, respectively; p2 < .001; PI = 9.7). Other tissues with increased BSA Ex included the liver, spleen, kidneys and mesenteric lymph nodes (PI = 6.7, 10.0, 6.3, 6.0, respectively). BSA Ex, which did not occur with the excipient control, was dependent upon the dose and the duration of IL-2. Serial lung weights showed dramatic increases in water weight induced by IL-2. Treatment of mice with radiation (500R), cyclophosphamide, or cortisone acetate significantly reduced IL-2 mediated BSA Ex. Thus, IL-2 induced a generalized CLS which is mediated directly or indirectly by cellular mechanisms.

Ettinghausen, S.E.; Rosenstein, M.; Rosenberg, S.A.

1986-03-01

178

Effects of self-consistency violation in Hartree-Fock RPA calculations for nuclear giant resonances revisited  

International Nuclear Information System (INIS)

We provide accurate assessments of the consequences of violations of self-consistency in Hartree-Fock-(HF) based random-phase approximation (RPA) calculations of the centroid energy Ecen of isoscalar and isovector giant resonances of multipolarities L=0-3 in a wide range of nuclei. This is done by carrying out highly accurate HF-RPA calculations neglecting the particle-hole (p-h) spin-orbit or Coulomb interaction in the RPA and comparing with the fully self-consistent HF-RPA results. We find that the shifts in the value of Ecen because of self-consistency violation associated with the spin-orbit and Coulomb interactions are comparable or larger than the current experimental errors in Ecen.

2006-01-01

179

Effects of self-consistency violation in Hartree-Fock RPA calculations for nuclear giant resonances revisited  

CERN Multimedia

We provide accurate assessments of the consequences of violations of self-consistency in Hartree-Fock (HF) based random phase approximation (RPA) calculations of the centroid energy $E_{cen}$ of isoscalar and isovector giant resonances of multi-polarities $L=0-3$ in a wide range of nuclei. This is done by carrying out highly accurate HF-RPA calculations neglecting the particle-hole (ph) spin-orbit or Coulomb interaction in the RPA and comparing with the fully self-consistent HF-RPA results. We find that the shifts in the value of $E_{cen}$ due to self-consistency violation associated with the spin-orbit and Coulomb interactions are comparable or larger than the current experimental errors in $E_{cen}$.

Sil, T; Reinhard, P G; Shlomo, S; Sil, Tapas

2006-01-01

180

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

 
 
 
 
181

Accuracy of basis-set extrapolation schemes for DFT-RPA correlation energies in molecular calculations  

CERN Multimedia

We construct a reference benchmark set for atomic and molecular random-phase-approximation (RPA) correlation energies in a density functional theory (DFT) framework at the complete basis set limit. This set is used to evaluate the accuracy of some popular extrapolation schemes for RPA all-electron molecular calculations. The results indicate that for absolute energies accurate results, clearly outperforming raw data, are achievable with two-point extrapolation schemes based on quintuple- and sextuple-zeta basis sets. Moreover, we show that results in good agreement with the benchmark can also be also obtained by using a semiempirical extrapolation procedure based on quadruple- and quintuple-zeta basis sets. Finally, we analyze the performance of different extrapolation schemes for atomization energies.

Fabiano, E; 10.1007/s00214-012-1278-8

2013-01-01

182

Resonance and quasi-free electron scattering on 12C in self-consistent RPA theorie  

International Nuclear Information System (INIS)

On the basis of a self-consistent RPA theory with a SK3 nucleon force, the 12C(e, e') reaciton is evaluated from particle threshold up to 100 MeV including both the giant resonance and the quasi-free scattering region. For momentum transfer q approx.= 1 fm-1, our results yield considerable electric and magnetic multipole strengths in the continuum above the giant resonances. (orig.).

1983-01-01

183

Self-Consistent RPA from a Coupled Cluster Wave Function Perspective  

CERN Document Server

Self-Consistent RPA is rederived in a consistent way with the help of the Coupled Cluster ground state wave function truncated at the two body level. An exact killing operator for this wave function is introduced allowing for a detailed discussion of the approximation scheme. Several exactly solvable models are reanalyzed under this new perspective giving raise to a quantitative evaluation of the performances of this many body method.

Jemai, M; Schuck, P

2013-01-01

184

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

185

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

186

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

Science.gov (United States)

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.; Lorenzana, J.

2005-04-01

187

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

International Nuclear Information System (INIS)

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. (authors)

2002-01-01

188

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

189

Giant resonances studied in continuum-RPA and in GCM-approach  

International Nuclear Information System (INIS)

[en] Giant resonances in 16O, 40Ca and 208Pb have been calculated by using a RPA-wavefunction which takes into account the evaporation of a particle into the continuum. The widths of the resonances can therfore be obtained directly from the cross section plots. A density dependent delta-interaction with parameters fitted in the lead region to electromagnetic properties was used. (Auth.)

1975-02-24

190

Longitudinal and transverse form factors of /sup 12/C(e,e') in continuum RPA-SK3 theory  

International Nuclear Information System (INIS)

The separated structure functions for the reaction /sup 12/C(e,e') in the momentum transfer region 200 MeV/c?q?550 MeV/c are analysed in the frame of a continuum self-consistent RPA theory with a SK3 nucleon-nucleon force. The role of the RPA correlations is pointed out in the comparison with the HF predictions.

1984-01-01

191

5' --> 3' molecular polarity of human replication protein A (hRPA) binding to pseudo-origin DNA substrates.  

UK PubMed Central (United Kingdom)

Human replication protein A (hRPA) was previously seen to efficiently bind a 48 bp simian virus 40 (SV40) "pseudo-origin" (PO) substrate that mimics a DNA structure found within the SV40 T antigen-origin (ori) complex. To understand the role of hRPA during the initiation of replication, we examined the PO sequence and structure requirements for hRPA interaction. Binding and unwinding were found to be most efficient when both strands of the central 8 nt single-stranded DNA (ssDNA) bubble region contained a polypyrimidine structure, with these activities proportionately reduced when the bubble region was replaced with a purine tract on one or both strands. Examination of the importance of the two duplex flanks indicates that the early gene side contains a DNA structural feature located one duplex turn from the bubble whose mutation significantly affects the affinity of hRPA for the substrate. When present in the context of ori, mutation of this sequence was seen to have significant effects on SV40 DNA replication in vitro and on the denaturation of ori, indicating that origin activity can be modulated by cis-acting elements which alter the hRPA binding affinity. Use of fork and overhang substrates containing 8 nt pyrimidine or purine arms demonstrates that hRPA binding to DNA involves a particular molecular polarity in which initial hRPA binding occurs on the 5' side of a ssDNA substrate, and then extends in the 3' direction to create a stably bound hRPA. These data have implications on the mechanism of the initiation of eukaryotic DNA replication as well as on the sites of nascent strand synthesis within the origin.

Iftode C; Borowiec JA

2000-10-01

192

The trypanosomatid-specific N terminus of RPA2 is required for RNA polymerase I assembly, localization, and function.  

UK PubMed Central (United Kingdom)

African trypanosomes are the only organisms known to use RNA polymerase I (pol I) to transcribe protein-coding genes. These genes include VSG, which is essential for immune evasion and is transcribed from an extranucleolar expression site body (ESB). Several trypanosome pol I subunits vary compared to their homologues elsewhere, and the question arises as to how these variations relate to pol I function. A clear example is the N-terminal extension found on the second-largest subunit of pol I, RPA2. Here, we identify an essential role for this region. RPA2 truncation leads to nuclear exclusion and a growth defect which phenocopies single-allele knockout. The N terminus is not a general nuclear localization signal (NLS), however, and it fails to accumulate unrelated proteins in the nucleus. An ectopic NLS is sufficient to reinstate nuclear localization of truncated RPA2, but it does not restore function. Moreover, NLS-tagged, truncated RPA2 has a different subnuclear distribution to full-length protein and is unable to build stable pol I complexes. We conclude that the RPA2 N-terminal extension does not have a role exclusive to the expression of protein-coding genes, but it is essential for all pol I functions in trypanosomes because it directs trypanosomatid-specific interactions with RPA1.

Daniels JP; Gull K; Wickstead B

2012-05-01

193

PCR-mediated recombination: a general method applied to construct chimeric infectious molecular clones of plasma-derived HIV-1 RNA.  

UK PubMed Central (United Kingdom)

A PCR-based approach was developed that provides a powerful tool for engineering recombinant molecules without reliance on restriction sites. DNA sequences were first amplified by high-fidelity PCR using Pfu polymerase; they were then used both as 'megaprimers' and templates in subsequent asymmetric long PCR amplifications to form chimeric clones. To demonstrate the technique, we constructed chimeric full-length HIV-1 clones derived from reverse-transcribed plasma viral RNA and proviral LTRs. Biologic characterization of these clones showed that most were infectious in tissue culture and sequence analysis demonstrated an error rate of only one base change in 20 kb of DNA sequence. For PCR-mediated recombination, it is necessary to know the sequence of the 3' and 5' overlapping regions of the desired PCR products. This method may be extended to include construction of chimeras between any DNA fragments lacking sequence homology. Such chimeras may be constructed by introducing overlapping sequences to one of the fragments. To ensure that unwanted mutations have not been introduced into the clones constructed by this method, each clone should be sequenced. Our results demonstrate that by using a high-fidelity polymerase and highly controlled PCR conditions, the PCR-introduced error rate can be greatly minimized. This new procedure may be used to construct infectious chimeras of HIV or SIV for studies of vaccines and pathogenesis. Moreover, the method is designed to exchange viral genes at precise boundaries to study individual gene products from different HIV genomes. It can also be used to construct expression vectors for production of specific proteins or delivery vectors for gene transfer and gene therapy. Finally, the technique described here provides a versatile tool to transfer genes or gene fragments from different sources for genetic investigation and engineering.

Fang G; Weiser B; Visosky A; Moran T; Burger H

1999-02-01

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.  

UK PubMed Central (United Kingdom)

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 (P<0.05). Notably, goats inoculated with rAd-F-H developed significantly higher VNA titers (P<0.05) and stronger cell-mediated immune responses than did goats inoculated with rAd-F or rAd-H alone. The results suggest that the three rAds might be attractive candidate differentiating infected from vaccinated animals (DIVA) vaccines for preventing PPRV infection. Notably, the rAd-F-H expressing F-H fusion protein is likely the most potent candidate of the rAds.

Wang Y; Liu G; Chen Z; Li C; Shi L; Li W; Huang H; Tao C; Cheng C; Xu B; Li G

2013-07-01

195

Recombinant adeno-associated virus-mediated transfer of shRNA against Notch3 ameliorates hepatic fibrosis in rats.  

UK PubMed Central (United Kingdom)

Liver fibrosis, a wound healing process following all kinds of liver injuries, is characterized by excessive deposition of extracellular matrix (ECM). Our previous study revealed that Notch3 might participate in liver fibrogenesis by regulating the activation of hepatic stellate cells (HSCs). The aim of this study was to assess the effects of Notch3 shRNA on hepatic fibrosis in a rat model induced by carbon tetrachloride (CCl4) and to clarify the mechanisms underlying those effects. Recombinant adeno-associated virus type 1 (rAAV1) vector carrying Notch3 shRNA (rAAV1-Notch3-shRNA) was generated and transferred to rat livers via the tail vein. The expression of Notch3, Jagged1, Hes1 and ?-SMA were detected by real-time RT-PCR and immunofluorescence. The effects of rAAV1-Notch3-shRNA on fibrosis was investigated by pathological and immunohistochemical examination. Our findings showed that Notch3, Jagged1, Hes1 and ?-SMA were downregulated. This downregulation was accompanied by improved hepatic fibrosis after the inhibition of Notch3 in vivo. rAAV1-Notch3-shRNA treatment reversed the epithelial-mesenchymal transition (EMT) in fibrotic livers by decreasing the expression of transforming growth factor ?1 (TGF-?1) and vimentin in a line with the increased expression of E-cadherin. The inhibition of Notch3 was not found to play a role in hepatocyte proliferation. Rather, it inhibited hepatocyte apoptosis in vivo to some extent. The results of the present study suggest that the inhibition of Notch3 can protect hepatocytes from undergoing apoptosis and attenuate liver fibrogenesis. This may be a viable therapeutic option for hepatic fibrosis.

Zheng SP; Chen YX; Guo JL; Qi D; Zheng SJ; Zhang SL; Weng ZH

2013-06-01

196

Evaluation in mice of the capillary leak syndrome (CLS) mediated by the systemic administration of recombinant interleukin-2 (IL-2)  

International Nuclear Information System (INIS)

Since a CLS with interstitial pulmonary edema has been the major toxicity of IL-2 administration in humans, the authors studied this CLS in mice by quantitating the IL-2 mediated, tissue extravasation (Ex) of intravenously injected 125I-bovine serum albumin (BSA). Mice received saline (HBSS) or 200,000 U of IL-2 intraperitoneally thrice daily from day 0-6 before tissues were counted. A permeability index (PI) was calculated by dividing the mean counts per minute (CPM) from tissues of treated mice by those from controls. In a representative experiment, increased BSA Ex was noted in the lungs of mice treated with IL-2 when compared with HBSS (6187 +/- 141 and 638 +/- 64 CPM +/- SEM, respectively; p2

1986-03-01

197

Control of gene expression with small molecules: biotin-mediated acylation of targeted lysine residues in recombinant yeast.  

UK PubMed Central (United Kingdom)

Chemical inducers of dimerization (CIDs) are powerful tools for controlling diverse cellular processes. These small molecules typically form strong noncovalent interactions with proteins. We report a related approach involving covalent acylation of a specific lysine residue of a target protein by the small molecule biotin. To control protein-protein interactions with biotin, the biotin protein ligase BirA from E. coli was coexpressed in yeast with a streptavidin-LexA fusion protein and Avitag or BCCP biotin acceptor peptides fused to the B42 activation domain. The addition of biotin (10 nM) resulted in BirA-mediated biotinylation of the biotin acceptor protein, recruitment to LexA DNA sites, and maximal activation of reporter gene expression in this yeast tribrid system. The high potency, low toxicity, and low molecular weight of biotin as a covalent CID are attractive properties for controlling cellular processes.

Athavankar S; Peterson BR

2003-12-01

198

Control of gene expression with small molecules: biotin-mediated acylation of targeted lysine residues in recombinant yeast.  

Science.gov (United States)

Chemical inducers of dimerization (CIDs) are powerful tools for controlling diverse cellular processes. These small molecules typically form strong noncovalent interactions with proteins. We report a related approach involving covalent acylation of a specific lysine residue of a target protein by the small molecule biotin. To control protein-protein interactions with biotin, the biotin protein ligase BirA from E. coli was coexpressed in yeast with a streptavidin-LexA fusion protein and Avitag or BCCP biotin acceptor peptides fused to the B42 activation domain. The addition of biotin (10 nM) resulted in BirA-mediated biotinylation of the biotin acceptor protein, recruitment to LexA DNA sites, and maximal activation of reporter gene expression in this yeast tribrid system. The high potency, low toxicity, and low molecular weight of biotin as a covalent CID are attractive properties for controlling cellular processes. PMID:14700632

Athavankar, Sonalee; Peterson, Blake R

2003-12-01

199

Retroviral-mediated expression of recombinant Fancc enhances the repopulating ability of Fancc-/- hematopoietic stem cells and decreases the risk of clonal evolution.  

UK PubMed Central (United Kingdom)

Fanconi anemia (FA) is a chromosomal instability disorder characterized by a progressive bone marrow (BM) failure and an increased incidence of myeloid leukemias. Children with FA are currently being enrolled in clinical trials to evaluate the safety of retroviral-mediated gene transfer. Previously, we used Fancc(-/-) mice to show that Fancc(-/-) hematopoietic stem cells (HSCs) have a profound defect in repopulating ability. Here, we examined whether retroviral-mediated gene transfer of recombinant Fancc (rFancc) would restore the repopulating ability of Fancc(-/-) HSC to wild-type levels. Fancc(-/-) HSCs transduced with a retrovirus encoding rFancc exhibited a repopulating ability that approached wild-type levels. Interestingly, approximately 30% of primary recipients (7 of 22) transplanted with uncorrected Fancc(-/-) cells developed a range of hematopoietic abnormalities including pancytopenia and BM hypoplasia similar to individuals with FA. Hematopoietic abnormalities were detected in only 1 of 22 mice transplanted with Fancc(-/-) cells transduced with a retrovirus encoding rFancc. Moreover, several mice with hematopoietic defects had progenitors that displayed a marked resistance to IFN-gamma, TNF-alpha, and MIP-1alpha compared to both Fancc(-/-) progenitors, which are uniquely hypersensitive to these cytokines, and wild-type progenitors. These data are analogous to studies using progenitors from patients with myelodysplasia and provide functional support for clonal evolution in these mice. Collectively, these data show that gene transfer can enhance HSC repopulating ability and suppresses the tendency for clonal evolution. These studies also reveal potential detrimental effects of ex vivo manipulation for untransduced Fancc(-/-) HSCs.

Haneline LS; Li X; Ciccone SL; Hong P; Yang Y; Broxmeyer HE; Lee SH; Orazi A; Srour EF; Clapp DW

2003-02-01

200

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.

Komaromy, Andras M.; Iwabe, Simone; Chiodo, Vincent A.; Boye, Sanford L.; Hauswirth, William W.; Beltran, William A.; Aguirre, Gustavo D.

2013-01-01

 
 
 
 
201

Inhibition of tumor growth in xenograft nude mice model by recombinant adenovirus-mediated human endostatin gene therapy  

International Nuclear Information System (INIS)

[en] Objective: To investigate the expression efficiency of adenovirus-mediated human endostatin gene (Ad/hEndo) in vitro and in vivo, and to observe its inhibition of tumor growth in xenografted nude mice model. Methods: The expression efficiency of endostatin gene was examined during the infection of Ad/hEndo in nasopharyngeal carcinoma (NPC) CNE-2 cell and human umbilical vein endothelial cells (ECV304) by Western blot and ELISA. The effect on inhibition of growth of NPC CNE-2 xenografted tumors in Balb/c nude mice was observed after administration with Ad/hEndo. The serum endostatin levels were measured by ELISA, and intratumoral microvessel density (MVD) was analyzed. Results: Western blot and ELISA analysis demonstrated high level of endostatin expression in CNE2 and ECV304 cells infected with Ad/ hEndo. The highest concentration of endostatin in supernatant reached 588.34 ng/ml after 72 h of Ad/hEndo infection at a MOI of 20. Ad/hEndo significantly inhibited growth of xenografted CNE-2 (nasopharyngeal carcinoma) tumors with inhibition rate of 46.43% (Ad/ hEndo group versus Ad/LacZ group, t=2.226, P

2003-01-01

202

T-helper cell-mediated proliferation and cytokine responses against recombinant Merkel cell polyomavirus-like particles.  

UK PubMed Central (United Kingdom)

The newly discovered Merkel Cell Polyomavirus (MCPyV) resides in approximately 80% of Merkel cell carcinomas (MCC). Causal role of MCPyV for this rare and aggressive skin cancer is suggested by monoclonal integration and truncation of large T (LT) viral antigen in MCC cells. The mutated MCPyV has recently been found in highly purified leukemic cells from patients with chronic lymphocytic leukemia (CLL), suggesting a pathogenic role also in CLL. About 50-80% of adults display MCPyV-specific antibodies. The humoral immunity does not protect against the development of MCC, as neutralizing MCPyV antibodies occur in higher levels among MCC patients than healthy controls. Impaired T-cell immunity has been linked with aggressive MCC behavior. Therefore, cellular immunity appears to be important in MCPyV infection surveillance. In order to elucidate the role of MCPyV-specific Th-cell immunity, peripheral blood mononuclear cells (PBMC) of healthy adults were stimulated with MCPyV VP1 virus-like particles (VLPs), using human bocavirus (HBoV) VLPs and Candida albicans antigen as positive controls. Proliferation, IFN-?, IL-13 and IL-10 responses were examined in 15 MCPyV-seropositive and 15 seronegative volunteers. With the MCPyV antigen, significantly stronger Th-cell responses were found in MCPyV-seropositive than MCPyV-seronegative subjects, whereas with the control antigens, the responses were statistically similar. The most readily detectable cytokine was IFN-?. The MCPyV antigen tended to induce stronger IFN-? responses than HBoV VLP antigen. Taken together, MCPyV-specific Th-cells elicit vigorous IFN-? responses. IFN-? being a cytokine with major antiviral and tumor suppressing functions, Th-cells are suggested to be important mediators of MCPyV-specific immune surveillance.

Kumar A; Chen T; Pakkanen S; Kantele A; Söderlund-Venermo M; Hedman K; Franssila R

2011-01-01

203

The anti-HBV effect mediated by a novel recombinant eukaryotic expression vector for IFN-alpha.  

UK PubMed Central (United Kingdom)

BACKGROUND: Chronic hepatitis B is a primary cause of liver-related death. Interferon alpha (IFN-alpha) is able to inhibit the replication of hepadnavirus, and the sustained and stable expression of IFN-alpha at appropriate level may be beneficial to HBV clearance. With the development of molecular cloning technology, gene therapy plays a more and more important role in clinical practice. In light of the findings, an attempt to investigate the anti-HBV effects mediated by a eukaryotic expression plasmid (pSecTagB-IFN-alpha) in vitro was carried out. METHODS: HBV positive cell line HepG2.2.15 and its parental cell HepG2 were transfected with pSecTagB-IFN-alpha or empty plasmid by using LipofectamineTM 2000 reagent. The expression levels of IFN-alpha were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA methods. The effects of pSecTagB-IFN-alpha on HBV mRNA, DNA and antigens were analyzed by real-time fluorescence quantitative PCR (qRT-PCR) and ELISA assays. RT-PCR, qRT-PCR and western blot were employed to investigate the influence of pSecTagB-IFN-alpha on IFN-alpha-induced signal pathway. Furthermore, through qRT-PCR and ELISA assays, the suppressive effects of endogenously expressed IFN-alpha and the combination with lamivudine on HBV were also examined. RESULTS: pSecTagB-IFN-alpha could express efficiently in hepatoma cells, and then inhibited HBV replication, characterized by the decrease of HBV S gene (HBs) and HBV C gene (HBc) mRNA, the reduction of HBV DNA load, and the low contents of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). Mechanism research showed that the activation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signal pathway, the up-regulation of IFN-alpha-induced antiviral effectors and double-stranded (ds) RNA sensing receptors by delivering pSecTagB-IFN-alpha, could be responsible for these phenomena. Furthermore, pSecTagB-IFN-alpha vector revealed effectively anti-HBV effect than exogenously added IFN-alpha. Moreover, lamivudine combined with endogenously expressed IFN-alpha exhibited stronger anti-HBV effect than with exogenous IFN-alpha. CONCLUSION: Our results showed that endogenously expressed IFN-alpha can effectively and persistently inhibit HBV replication in HBV infected cells. These observations opened a promising way to design new antiviral genetic engineering drugs based on IFN-alpha.

Yu H; Hou Z; Han Q; Zhang C; Zhang J

2013-08-01

204

MEIOB Targets Single-Strand DNA and Is Necessary for Meiotic Recombination.  

Science.gov (United States)

Meiotic recombination is a mandatory process for sexual reproduction. We identified a protein specifically implicated in meiotic homologous recombination that we named: meiosis specific with OB domain (MEIOB). This protein is conserved among metazoan species and contains single-strand DNA binding sites similar to those of RPA1. Our studies in vitro revealed that both recombinant and endogenous MEIOB can be retained on single-strand DNA. Those in vivo demonstrated the specific expression of Meiob in early meiotic germ cells and the co-localization of MEIOB protein with RPA on chromosome axes. MEIOB localization in Dmc1 (-/-) spermatocytes indicated that it accumulates on resected DNA. Homologous Meiob deletion in mice caused infertility in both sexes, due to a meiotic arrest at a zygotene/pachytene-like stage. DNA double strand break repair and homologous chromosome synapsis were impaired in Meiob (-/-) meiocytes. Interestingly MEIOB appeared to be dispensable for the initial loading of recombinases but was required to maintain a proper number of RAD51 and DMC1 foci beyond the zygotene stage. In light of these findings, we propose that RPA and this new single-strand DNA binding protein MEIOB, are essential to ensure the proper stabilization of recombinases which is required for successful homology search and meiotic recombination. PMID:24068956

Souquet, Benoit; Abby, Emilie; Hervé, Roxane; Finsterbusch, Friederike; Tourpin, Sophie; Le Bouffant, Ronan; Duquenne, Clotilde; Messiaen, Sébastien; Martini, Emmanuelle; Bernardino-Sgherri, Jacqueline; Toth, Attila; Habert, René; Livera, Gabriel

2013-09-19

205

MEIOB Targets Single-Strand DNA and Is Necessary for Meiotic Recombination  

Science.gov (United States)

Meiotic recombination is a mandatory process for sexual reproduction. We identified a protein specifically implicated in meiotic homologous recombination that we named: meiosis specific with OB domain (MEIOB). This protein is conserved among metazoan species and contains single-strand DNA binding sites similar to those of RPA1. Our studies in vitro revealed that both recombinant and endogenous MEIOB can be retained on single-strand DNA. Those in vivo demonstrated the specific expression of Meiob in early meiotic germ cells and the co-localization of MEIOB protein with RPA on chromosome axes. MEIOB localization in Dmc1?/? spermatocytes indicated that it accumulates on resected DNA. Homologous Meiob deletion in mice caused infertility in both sexes, due to a meiotic arrest at a zygotene/pachytene-like stage. DNA double strand break repair and homologous chromosome synapsis were impaired in Meiob?/? meiocytes. Interestingly MEIOB appeared to be dispensable for the initial loading of recombinases but was required to maintain a proper number of RAD51 and DMC1 foci beyond the zygotene stage. In light of these findings, we propose that RPA and this new single-strand DNA binding protein MEIOB, are essential to ensure the proper stabilization of recombinases which is required for successful homology search and meiotic recombination.

Herve, Roxane; Finsterbusch, Friederike; Tourpin, Sophie; Le Bouffant, Ronan; Duquenne, Clotilde; Messiaen, Sebastien; Martini, Emmanuelle; Bernardino-Sgherri, Jacqueline; Toth, Attila; Habert, Rene; Livera, Gabriel

2013-01-01

206

A full-self-consistent RPA description of the 2??? decay  

International Nuclear Information System (INIS)

The RPA treatment of a many body Hamiltonian describing the state of even-even nuclei involved in a 2??? decay is revisited. One shows that renormalizing the Gamow-Teller transition operator by accounting for the ground state correlations requires a similar renormalization for the 1+ dipole density operators which results in activating new boson degrees of freedom. Possible consequences on the Ikeda sum rule and the Gamow-Teller transition amplitude are suggested. A numerical application for a two levels model is presented. (author)

1998-09-05

207

Photoreactions of 16O in self-consistent RPA theory. Pt. 3  

International Nuclear Information System (INIS)

Polarized 16O(?,p0) and 16O(?,n0) reactions below 60 MeV are analyzed in the framework of a self-consistent RPA theory with the Skyrme force Sk3. The calculations include dipole and quadrupole transitions in LWA. Asymmetries, polarizations and analyzing powers are calculated and compared with the available experimental data. Our aim is to show to what extent polarized results give additional information to that extracted from the unpolarized ones, both on the many-body structure of the nucleus and on the nature of the reaction mechanism at intermediate energies. (orig.).

1984-01-01

208

A full self-consistent RPA description of the 2??? decay  

International Nuclear Information System (INIS)

The RPA treatment of a many body Hamiltonian describing the states of even-even nuclei involved in a 2??? decay is revisited. One shows that renormalizing the Gamow-Teller transition operator by accounting for the ground state correlation requires a similar renormalization for the 1+ dipole density operators which results in activating new boson degrees of freedom. Possible consequences on the Ikeda sum rule and the Gamow-Teller transition amplitude are suggested. A numerical application for a two levels model is presented. (authors)

1998-01-01

209

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.).

1998-01-01

210

The Nuclear Scissors Mode by Two Approaches (Wigner Function Moments Versus RPA)  

CERN Multimedia

Two complementary methods to describe the collective motion, RPA and Wigner Function Moments (WFM) method, are compared on an example of a simple model - harmonic oscillator with quadrupole-quadrupole residual interaction. It is shown that they give identical formulae for eigenfrequencies and transition probabilities of all collective excitations of the model including the scissors mode, which is a subject of our especial attention. The normalization factor of the "synthetic" scissors state and its overlap with physical states are calculated analytically. The orthogonality of the spurious state to all physical states is proved rigorously.

Balbutsev, E B

2004-01-01

211

The nuclear scissors mode by two approaches (Wigner function moments versus RPA)  

International Nuclear Information System (INIS)

[en] Two complementary methods to describe the collective motion, RPA and Wigner Function Moments (WFM) method, are compared on an example of a simple model - harmonic oscillator with quadrupole-quadrupole residual interaction. It is shown that they give identical formulae for eigenfrequencies and transition probabilities of all collective excitations of the model including the scissors mode, which is a subject of our especial attention. The normalization factor of the 'synthetic' scissors state and its overlap with physical states are calculated analytically. The orthogonality of the spurious state to all physical states is proved rigorously

2004-01-01

212

The Bogolubov representation of the polaron model and its completely integrable RPA-approximation  

Directory of Open Access Journals (Sweden)

Full Text Available The polaron model in ionic crystal is studied in the Bogolubov representation using a special RPA-approximation. A new exactly solvable approximated polaron model is derived and described in detail. Its free energy at finite temperature is calculated analytically. The polaron free energy in the constant magnetic field at finite temperature is also discussed. Based on the structure of the Bogolubov unitary transformed polaron Hamiltonian there is stated a very important new result: the full polaron model is exactly solvable.

N.N.Bogolubov (jr.); Ya.A. Prykarpatsky; A.A. Ghazaryan

2010-01-01

213

RPA timber assessment update, 1993. Forest Service general technical report (Final)  

Energy Technology Data Exchange (ETDEWEB)

The update reports changes in the nation`s timber resource since the 1989 Resources Planning Act (RPA) timber assessment. The timber resource situation is analyzed to provide projections for future cost and availability of timber products to meet demands. Prospective trends in demands for the supplies of timber and the factors that affect these trends are examined. Changing resource conditions that may lead to policy changed or that may represent opportunities for private or public investment also are identified. Market and resource trends are interpreted to provide an improved basis for managing the resource base.

Haynes, R.W.; Adams, D.M.; Mills, J.R.

1995-03-01

214

Structural and immunological analysis of anthrax recombinant protective antigen adsorbed to aluminum hydroxide adjuvant.  

Science.gov (United States)

New anthrax vaccines currently under development are based on recombinant protective antigen (rPA) and formulated with aluminum adjuvant. Because long-term stability is a desired characteristic of these vaccines, an understanding of the effects of adsorption to aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-Alhydrogel formulations to that of formulations stored for 3 weeks at either room temperature or 37°C in order to assess the changes in rPA structure that might occur upon long-term storage on aluminum adjuvant. Intrinsic fluorescence emission spectra of tryptophan residues indicated that some tertiary structure alterations of rPA occurred during storage on Alhydrogel. Using anti-PA monoclonal antibodies to probe specific regions of the adsorbed rPA molecule, we found that two monoclonal antibodies that recognize epitopes located in domain 1 of PA exhibited greater reactivity to the stored formulations than to freshly prepared formulations. Immunogenicity of rPA-Alhydrogel formulations in mice was assessed by measuring the induction of toxin-neutralizing antibodies, as well as antibodies reactive to 12-mer peptides spanning the length of PA. Mice immunized with freshly prepared formulations developed significantly higher toxin-neutralizing antibody titers than mice immunized with the stored preparations. In contrast, sera from mice immunized with stored preparations exhibited increased reactivity to nine 12-mer peptides corresponding to sequences located throughout the rPA molecule. These results demonstrate that storage of rPA-Alhydrogel formulations can lead to structural alteration of the protein and loss of the ability to elicit toxin-neutralizing antibodies. PMID:22815152

Wagner, Leslie; Verma, Anita; Meade, Bruce D; Reiter, Karine; Narum, David L; Brady, Rebecca A; Little, Stephen F; Burns, Drusilla L

2012-07-18

215

Structural and immunological analysis of anthrax recombinant protective antigen adsorbed to aluminum hydroxide adjuvant.  

UK PubMed Central (United Kingdom)

New anthrax vaccines currently under development are based on recombinant protective antigen (rPA) and formulated with aluminum adjuvant. Because long-term stability is a desired characteristic of these vaccines, an understanding of the effects of adsorption to aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-Alhydrogel formulations to that of formulations stored for 3 weeks at either room temperature or 37°C in order to assess the changes in rPA structure that might occur upon long-term storage on aluminum adjuvant. Intrinsic fluorescence emission spectra of tryptophan residues indicated that some tertiary structure alterations of rPA occurred during storage on Alhydrogel. Using anti-PA monoclonal antibodies to probe specific regions of the adsorbed rPA molecule, we found that two monoclonal antibodies that recognize epitopes located in domain 1 of PA exhibited greater reactivity to the stored formulations than to freshly prepared formulations. Immunogenicity of rPA-Alhydrogel formulations in mice was assessed by measuring the induction of toxin-neutralizing antibodies, as well as antibodies reactive to 12-mer peptides spanning the length of PA. Mice immunized with freshly prepared formulations developed significantly higher toxin-neutralizing antibody titers than mice immunized with the stored preparations. In contrast, sera from mice immunized with stored preparations exhibited increased reactivity to nine 12-mer peptides corresponding to sequences located throughout the rPA molecule. These results demonstrate that storage of rPA-Alhydrogel formulations can lead to structural alteration of the protein and loss of the ability to elicit toxin-neutralizing antibodies.

Wagner L; Verma A; Meade BD; Reiter K; Narum DL; Brady RA; Little SF; Burns DL

2012-09-01

216

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks.  

Science.gov (United States)

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability. PMID:20019063

Bolderson, Emma; Tomimatsu, Nozomi; Richard, Derek J; Boucher, Didier; Kumar, Rakesh; Pandita, Tej K; Burma, Sandeep; Khanna, Kum Kum

2009-12-17

217

Recombinant Programming  

Digital Repository Infrastructure Vision for European Research (DRIVER)

This research report presents a promising new approach to computation called Recombinant Programming. The novelty of our approach is that it separates the program into two layers of computation: the recombination and the interpretation layer. The recombination layer takes sequences as inputs and all...

Pawlak, Renaud; Cuesta, Carlos; Younessi, Houman

218

Photoreactions of 12C, 16O and 40Ca in self-consistent RPA theory. Pt. 1  

International Nuclear Information System (INIS)

The photoreactions of 12C, 16O and 40Ca from particle threshold up to 80 MeV are analysed in the frame of a self-consistent RPA theory with Skyrme interactions. The excitation of the E1 and E2 giant resonances is shown in the energy continuum by referring to photoabsorption (?,p) and (?,n) decay channels. The calculations are performed with a Skyrme III force for its better founded momentum dependence. Correlations between the nuclear dynamics and the properties of the interaction in the spin-spin channel, momentum and density dependence are pointed out. In this connection, an estimate is made of the MEC contribution included in RPA calculations by using Siegert's theorem in the electric transition operator. The E2 decay in (?,p) and (?,n) channels predicted by the RPA is compared with experimental observations of (?,?'), (?,?), (?,n) and (p,?) reactions. (orig.).

1982-01-01

219

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

Energy Technology Data Exchange (ETDEWEB)

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//sup -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/sup -1/ > 0.5 the interaction parameter determined using the RPA theory for q > R/sub g//sup -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 xi and R/sub g/ which is not accounted for by the RPA theory.

Bates, F.S.; Koehler, W.C.; Wignall, G.D.; Fetters, L.J.

1986-12-01

220

RAD52 inactivation is synthetically lethal with deficiencies in BRCA1 and PALB2 in addition to BRCA2 through RAD51-mediated homologous recombination.  

UK PubMed Central (United Kingdom)

Synthetic lethality is an approach to study selective cell killing based on genotype. Previous work in our laboratory has shown that loss of RAD52 is synthetically lethal with BRCA2 deficiency, while exhibiting no impact on cell growth and viability in BRCA2-proficient cells. We now show that this same synthetically lethal relationship is evident in cells with deficiencies in BRCA1 or PALB2, which implicates BRCA1, PALB2 and BRCA2 in an epistatic relationship with one another. When RAD52 was depleted in BRCA1- or PALB2-deficient cells, a severe reduction in plating efficiency was observed, with many abortive attempts at cell division apparent in the double-depleted background. In contrast, when RAD52 was depleted in a BRCA1- or PALB2-wildtype background, a negligible decrease in colony survival was observed. The frequency of ionizing radiation-induced RAD51 foci formation and double-strand break-induced homologous recombination (HR) was decreased by 3- and 10-fold, respectively, when RAD52 was knocked down in BRCA1- or PALB2-depleted cells, with minimal effect in BRCA1- or PALB2-proficient cells. RAD52 function was independent of BRCA1 status, as evidenced by the lack of any defect in RAD52 foci formation in BRCA1-depleted cells. Collectively, these findings suggest that RAD52 is an alternative repair pathway of RAD51-mediated HR, and a target for therapy in cells deficient in the BRCA1-PALB2-BRCA2 repair pathway.

Lok BH; Carley AC; Tchang B; Powell SN

2013-07-01

 
 
 
 
221

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)

[en] 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

222

Skyrme-RPA description of giant resonances in spherical and deformed nuclei  

International Nuclear Information System (INIS)

Ability of the time-dependent density functional theory (TDDFT) with Skyrme forces to describe electric and magnetic resonances (GR) is scrutinized within the separable RPA (SRPA) method recently developed by our group. The method is fully self-consistent and does not need additional parameters. Due to the factorization of the residual interaction, the SRPA drastically reduces the computational effort while keeping the accuracy of involved RPA method. This feature becomes crucial for systematic study of collective dynamics in complex nuclei with their huge configuration space. Both spherical and axially deformed nuclei can be covered. The isovector E1(T=1) and M1 GR is analysed in detail in rare-earth, actinide and superheavy nuclei, including long isotopic chains approaching drip-lines. A special attention is paid to the role of the time-odd current in the Skyrme functional and its influence on the GR properties. We discuss relation of the current with the effective masses and propose a tentative classification of Skyrme forces, based on the description of isovector modes.

2009-01-01

223

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

224

PhaG-mediated synthesis of Poly(3-hydroxyalkanoates) consisting of medium-chain-length constituents from nonrelated carbon sources in recombinant Pseudomonas fragi.  

UK PubMed Central (United Kingdom)

Recently, a new metabolic link between fatty acid de novo biosynthesis and biosynthesis of poly(3-hydroxy-alkanoate) consisting of medium-chain-length constituents (C(6) to C(14)) (PHA(MCL)), catalyzed by the 3-hydroxydecanoyl-[acyl-carrier-protein]:CoA transacylase (PhaG), has been identified in Pseudomonas putida (B. H. A. Rehm, N. Krüger, and A. Steinbüchel, J. Biol. Chem. 273:24044-24051, 1998). To establish this PHA-biosynthetic pathway in a non-PHA-accumulating bacterium, we functionally coexpressed phaC1 (encoding PHA synthase 1) from Pseudomonas aeruginosa and phaG (encoding the transacylase) from P. putida in Pseudomonas fragi. The recombinant strains of P. fragi were cultivated on gluconate as the sole carbon source, and PHA accumulation to about 14% of the total cellular dry weight was achieved. The respective polyester was isolated, and GPC analysis revealed a weight average molar mass of about 130,000 g mol(-1) and a polydispersity of 2.2. The PHA was composed mainly (60 mol%) of 3-hydroxydecanoate. These data strongly suggested that functional expression of phaC1 and phaG established a new pathway for PHA(MCL) biosynthesis from nonrelated carbon sources in P. fragi. When fatty acids were used as the carbon source, no PHA accumulation was observed in PHA synthase-expressing P. fragi, whereas application of the beta-oxidation inhibitor acrylic acid mediated PHA(MCL) accumulation. The substrate for the PHA synthase PhaC1 is therefore presumably directly provided through the enzymatic activity of the transacylase PhaG by the conversion of (R)-3-hydroxydecanoyl-ACP to (R)-3-hydroxydecanoyl-CoA when the organism is cultivated on gluconate. Here we demonstrate for the first time the establishment of PHA(MCL) synthesis from nonrelated carbon sources in a non-PHA-accumulating bacterium, employing fatty acid de novo biosynthesis and the enzymes PhaG (a transacylase) and PhaC1 (a PHA synthase).

Fiedler S; Steinbüchel A; Rehm BH

2000-05-01

225

Spectra of nuclei in the lead region in the framework of the RPA with OBE-G-matrix interactions  

International Nuclear Information System (INIS)

On the base of an existing Computer program for the calculation of particle-hole RPA matrix elements (especially) for finite-range interactions as well for the solution of the particle-hole RPA equations the corresponding matrix elements for two-particle respectively two-hole nuclei were calculated (and tested) and the corresponding RPA equations solved. For the calculation of the nuclear spectra meson-exchange G-matrix interactions were used instead of phenomenological approaches. The former constitute only a part of the effective NN interaction. A direct comparison with the experimental spectra is therefore just as little convenient as the calculation of transition probabilities or an evaluating comparison of TDA and RPA. The results let nevertheless recognize following: (1) The density dependence of the effective NN interaction. (2) For the states of two-particle respectively two-hole nuclei the (?+rho) exchange plays no such dominating role as for unnatural-parity states in double-magic nuclei. (3) An approximation of the missing part of the effective NN interaction by delta-function interactions is not sufficient. (orig.).

1984-01-01

226

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

227

Test of the self-consistent RPA theory in fast particles scattering on nuclei  

International Nuclear Information System (INIS)

Static and transition densities are calculated for ground and low-lying collective states of spherical nuclei 40Ca, 48Ca, 90Zr, 208Pb, as well as positions of these states and the reduced transition probabilities. The calculation is fulfilled self-consistently in framework of the RPA method, based on the Hartree-Fock ground state with the Skyrme S1 and BLV1 interactions. Properties of the obtained static and transition densities are examined in calculations of cross sections of elastic and inelastic scatterings of fast electrons and protons. These cross sections are calculated by means of the phase-shift analysis for electrons and using the Glauber theory for protons. A satisfactory agreement with the experiment is obtained. Both the interactions give, in the whole, near results. In case of 208Pb the BLV1 forces are more preferable.

1979-01-01

228

Momentum and density dependence of the ph interaction in continuum RPA calculations with Skyrme forces  

International Nuclear Information System (INIS)

We analyse the momentum and density dependence of the ph interaction in self-consistent RPA calculations of electromagnetic reactions with Skyrme forces. First, we calculate the Vph00 and Vph01 spin-isospin components in nuclear matter for an SK3 interaction. At the nuclear surface, they have decreasing values with increasing q-values up to momenta q=3 fm-1. As a second point, we show that the predictions of the 12C(e, e') charge response at 400 MeV/c remain practically unchanged when the zero-range quadratic momentum dependence of the SK3 interaction is replaced by the momentum dependence associated with a Yukawa short-ranged force. (orig.).

1988-01-01

229

Photoreactions of 12C, 16O and 40Ca in self consistent RPA theory. Pt. 2  

International Nuclear Information System (INIS)

The (?,p), (?,n) and inverse capture reactions in 12C, 16O and 40Ca below pion threshold have been analysed in the frame of a self-consistent RPA theory with a Skyrme force (Sk3). E1 and E2 transition in LWA are present in the calculation. Thereby, EC linked by gauge invariance to the effective interaction are included in the predicted cross sections. Through an overall comparison with the available data, we attain, in the present context, a good fit of data up to 80 MeV. The observed symmetric behaviour of (?,p) and (?,n) angular distributions is well reproduced. The role of nuclear correlations in the action of EC by gauge invariance is discussed. (orig.).

1984-01-01

230

Thermal Structure and Major Ion Composition of the Venus Ionosphere: First RPA Results from Venus Orbiter.  

UK PubMed Central (United Kingdom)

Thermal plasma quantities measured by, the retarding potential analyzer (RPA) are, together with companion Pioneer Venus measurements, the first in situ measurements of the Venus ionosphere. High ionospheric ion and electron temperatures imply significant solar wind heating of the ionosphere. Comparison of the measured altitude profiles of the dominant ions with an initial modlel indicates that the ionosphere is close to diffusive equilibrium. The ionopause height was observed to vary from 400 to 1000 kilometers in early orbits. The ionospheric particle pressure at the ionopause is apparently balanced at a solar zenith angle of about 70 degrees by the magnetic field pressure with little contribution from energetic solar wind particles. The measured ratio of ionospheric scale height to ionopause radius is consistent with that inferred from previously measured bow shock positions.

Knudsen WC; Spenner K; Whitten RC; Spreiter JR; Miller KL; Novak V

1979-02-01

231

Surface Reengineering of RPA70N Enables Cocrystallization with an Inhibitor of the Replication Protein A Interaction Motif of ATR Interacting Protein.  

UK PubMed Central (United Kingdom)

Replication protein A (RPA) is the primary single-stranded DNA (ssDNA) binding protein in eukaryotes. The N-terminal domain of the RPA70 subunit (RPA70N) interacts via a basic cleft with a wide range of DNA processing proteins, including several that regulate DNA damage response and repair. Small molecule inhibitors that disrupt these protein-protein interactions are therefore of interest as chemical probes of these critical DNA processing pathways and as inhibitors to counter the upregulation of DNA damage response and repair associated with treatment of cancer patients with radiation or DNA-damaging agents. Determination of three-dimensional structures of protein-ligand complexes is an important step for elaboration of small molecule inhibitors. However, although crystal structures of free RPA70N and an RPA70N-peptide fusion construct have been reported, RPA70N-inhibitor complexes have been recalcitrant to crystallization. Analysis of the P61 lattice of RPA70N crystals led us to hypothesize that the ligand-binding surface was occluded. Surface reengineering to alter key crystal lattice contacts led to the design of RPA70N E7R, E100R, and E7R/E100R mutants. These mutants crystallized in a P212121 lattice that clearly had significant solvent channels open to the critical basic cleft. Analysis of X-ray crystal structures, target peptide binding affinities, and (15)N-(1)H heteronuclear single-quantum coherence nuclear magnetic resonance spectra showed that the mutations do not result in perturbations of the RPA70N ligand-binding surface. The success of the design was demonstrated by determining the structure of RPA70N E7R soaked with a ligand discovered in a previously reported molecular fragment screen. A fluorescence anisotropy competition binding assay revealed this compound can inhibit the interaction of RPA70N with the peptide binding motif from the DNA damage response protein ATRIP. The implications of the results are discussed in the context of ongoing efforts to design RPA70N inhibitors.

Feldkamp MD; Frank AO; Kennedy JP; Patrone JD; Vangamudi B; Waterson AG; Fesik SW; Chazin WJ

2013-09-01

232

The effects of the plasmon-LO phonon interaction on the critical densities of RPA approach in a quasi-one-dimensional system  

Scientific Electronic Library Online (English)

Full Text Available Abstract in english In this work we have studied the electron LO phonon interaction on the pair-correlation function g(x) and its dependence on the electronic density for a GaAs-AlGaAs rectangular quantum wire within the random-phase approximation (RPA). We assumed two different values of the wire width. As negative non-physical results are found for lower electronic densities and small interparticle separations in the RPA approach, we have delimited the regions where the RPA approach cannot be used for the calculation of the Q1D electron gas collective excitation.

Machado, P. C. M.; Osório, F. A. P.; Borges, A. N.

2006-06-01

233

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

UK PubMed Central (United Kingdom)

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.

Lauersen KJ; Vanderveer TL; Berger H; Kaluza I; Mussgnug JH; Walker VK; Kruse O

2013-09-01

234

Mean-Field and RPA Approaches to Stable and Unstable Nuclei with Semi-Realistic Interactions  

International Nuclear Information System (INIS)

We have developed semi-realistic NN interactions [1, 2] by modifying the M3Y interaction [3] that was derived from the G-matrix. The modification has been made so that the saturation and the spin-orbit splittings could be reproduced. The new interactions contain finite-range LS and tensor channels, as well as Yukawa-form central channels having reasonable spin and spin-isospin properties. In order to handle such interactions in practical calculations, we have also developed new numerical methods [4-6], in which the Gaussian expansion method [7] is applied. It is noted that these methods have the following advantages: (i) we can efficiently describe the energy-dependent asymptotics of single-particle wave functions at large r, as is typified in arguments on the deformed neutron halo in 40Mg [6], (ii) we can handle various effective interactions, including those having non-locality, and (iii) a single-set of bases is applicable to wide mass range of nuclei and therefore is suitable to systematic calculations. Thereby we can implement Hartree-Fock, Hartree-Fock-Bogolyubov and RPA calculations for stable and unstable nuclei with the semi-realistic interactions. It will be shown first that the new interactions have desired characters for the nuclear matter and for the single- and double-closed nuclei. We shall particularly focus on roles of specific channels of the effective interaction, by studying (a) 'shell evolution' and role of the spin-isospin and the tensor channels [8] in stable and unstable nuclei, and (b) the magnetic response in a fully self-consistent RPA calculation with the tensor force [9]. All these properties seem to be simultaneously and naturally reproduced by the semi-realistic interactions. Thus the semi-realistic interactions are promising in describing various aspects of nuclear structure from stable to drip-line nuclei, in a self-consistent and unified manner. Since they have microscopic origin with minimal modification, we can expect high predictability for unstable nuclei by applying these interactions. Prediction will be given for the neutron drip line for some isotopes and on excited states of several unstable nuclei.(author).

2009-01-01

235

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

236

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

237

Effects of current conservation in continuum RPA-SK3 predictions of photonuclear reactions at intermediate energies  

International Nuclear Information System (INIS)

The incidence of current conservation in (?, p) and (?, n) reactions at intermediate energies has been studied in the frame of the self-consistent RPA-SK3 continuum theory, fulfilling current conservation. Two-body exchange currents, originated from the momentum dependent terms of the Skyrme hamiltonian, are expressed in RPA-SK3 through an isovector convection current carrying an effective nucleon mass mSK(r)?m. The 16O(?,p) and 16O(?,n) angular distributions at energies 60 MeV?E??300 MeV have been calculated and compared with experimental data. Connection is made with the implications of current conservation in nuclear (e,e') charge responses calculated in the same theoretical framework. (orig.).

1991-01-01

238

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

1997-03-01

239

High-efficiency mixing renaturation device beneficial to rPA (radar Performance Analyzer) large-scale renaturation  

UK PubMed Central (United Kingdom)

The invention relates to a high-efficiency mixing renaturation device which is beneficial to rPA large-scale renaturation and is used for adding a denatured protein solution into a renaturation buffer solution. The device comprises a container, a liquid inlet pipe and a propeller, wherein the renaturation buffer solution is loaded in the container the liquid inlet pipe is provided with a plurality of nozzles, one end of the liquid inlet pipe is a liquid outlet end, the other end is a liquid inlet end, and the denatured protein solution is introduced into the liquid inlet end and the propeller is inserted into a position below the liquid level of the renaturation buffer solution. Therefore, the invention can effectively inhabit the agglutination reaction of an rPA inclusion body when in renaturation and markedly improve the renaturation ratio of target protein.

YUFENG QIN; JINHUA YOU; YUANXING ZHANG; XIANGSHAN ZHOU

240

ORC is necessary at the interphase-to-mitosis transition to recruit cdc2 kinase and disassemble RPA foci.  

UK PubMed Central (United Kingdom)

The origin-recognition complex (ORC) has an essential role in defining DNA replication origins and in chromosome segregation. Recent studies in Drosophila orc2 mutants, and in human cells depleted of ORC2, have suggested that this factor is also implicated in mitotic chromosome assembly. We asked whether ORC was required for M phase chromosome assembly independently of its function in DNA replication. We performed depletion assays and reconstitution experiments in Xenopus egg extracts, in conditions of M phase chromosome assembly coupled or uncoupled from DNA replication. We show that, although ORC is dispensable for mitotic chromosome condensation, it is necessary at the interphase-mitosis transition for proper mitotic chromosome assembly to occur in a reaction not strictly dependent on DNA replication. This function involves the recruitment to chromatin of cdc2 kinase and the chromatin disassembly of interphasic replication protein A (RPA) foci. Furthermore, we show that mutations of RPA at the cdc2 kinase site prevents RPA dissociation from chromatin and impairs mitotic chromosome assembly without affecting DNA replication. Our results support the conclusion that in addition to its role in the assembly of prereplication complexes (pre-RCs), at the G1-S transition, ORC is also required for their disassembly at mitotic entry.

Cuvier O; Lutzmann M; Méchali M

2006-03-01

 
 
 
 
241

Prognostic factors in brain metastases: should patients be selected for aggressive treatment according to recursive partitioning analysis (RPA) classes?  

International Nuclear Information System (INIS)

Purpose: To determine whether or not Radiation Therapy Oncology Group (RTOG) recursive partitioning analysis (RPA) derived prognostic classes for patients with brain metastases are generally applicable and can be recommended as rational strategy for patient selection for future clinical trials. Inclusion of time to non-CNS death as additional endpoint besides death from any cause might result in further valuable information, as survival limitation due to uncontrolled extracranial disease can be explored. Methods: We performed a retrospective analysis of prognostic factors for survival and time to non-CNS death in 528 patients treated at a single institution with radiotherapy or surgery plus radiotherapy for brain metastases. For this purpose, patients were divided into groups with Karnofsky performance status (KPS) 0.05 for RPA class II versus III). However, it was 8.5 months in RPA class II patients with controlled primary tumor, which was found to be the only prognostic factor for time to non-CNS death in patients with KPS ?70%. In patients with KPS

2000-01-15

242

On the use of Skyrme forces in self-consistent RPA calculations  

International Nuclear Information System (INIS)

Self-consistent random-phase (RPA) calculations including the continuum are presented using Skyrme forces. The density-dependent interpretation of the interaction is favoured as it does not violate the spin stability. A possible density dependence of the momentum-dependent S- and P-interaction is taken into account, which allows one to vary the incompressibility K and the effective mass m*/m independently. It is shown by analytic relations that these two quantities are the only degrees of freedom left in the parameterization of this Skyrme force, if the ground-state properties shall be reproduced, except for a still open degree of freedom in the spin exchange parameterization. The Landau parameters are discussed as a function of these degrees of freedom in order to find the best possible particle-hole interaction. Continuum calculations of the 1-, 2+ and 3- states in 16O are presented and compared with discretized continuum calculations. It is found that the existing Skyrme forces do not show enough attraction and in addition cause relatively large isospin impurities, in 16O as well as in 208Pb. The influence of large configuration spaces is discussed. A systematic search for an interaction with a stronger particle-hole interaction is presented which seems to favour interactions with a high effective mass, but a low compression modulus. (Auth.).

1977-01-01

243

TFD extension of a self-consistent RPA to finite temperatures  

International Nuclear Information System (INIS)

The self-consistent RPA (SCRPA) developed by Schuck and coauthors is extended to finite temperatures. The corresponding equations are derived by using the formalism of thermofield dynamics. The intrinsic energy of a system is calculated as the expectation value of the Hamiltonian with respect to a T-dependent thermal vacuum state for a thermal-phonon operator. A nonvanishing number of thermal quasiparticles in the vacuum state are assumed. By virtue of the assumption, the thermal Hartree-Fock (HF) equations appear to be coupled to the equations of motion for phonon variables. The thermal occupation numbers are also calculated in a consistent way with the energies of the HF quasiparticles. The approximation is applied to the two-level Lipkin model. Advantages of the thermal SCRPA (TSCRPA) are most obvious at temperatures near the phase-transition point. In the TSCRPA, the phase transition occurs at lower T than in other approximations. Moreover, within the TSCRPA, a statistical behavior of the Lipkin model is described with an appropriate accuracy at any T even if the HF transformation parameter is kept fixed at a value corresponding to the 'spherical' phase of the HF field.

2001-01-01

244

Native signal peptide of human ERp57 disulfide isomerase mediates secretion of active native recombinant ERp57 protein in yeast Saccharomyces cerevisiae.  

UK PubMed Central (United Kingdom)

Human ERp57 protein is disulfide isomerase, facilitating proper folding of glycoprotein precursors in the concert with ER lectin chaperones calreticulin and calnexin. Growing amount of data also associates ERp57 with many different functions in subcellular locations outside the ER. Analysis of protein functions requires substantial amounts of correctly folded, biologically active protein, and in this study we introduce yeast Saccharomyces cerevisiae as a perfect host for production of human ERp57. Our data suggest that native signal peptide of human ERp57 protein is recognized and correctly processed in the yeast cells, which leads to protein secretion. Secreted recombinant ERp57 protein possesses native amino acid sequence and is biologically active. Moreover, secretion allows simple one-step purification of recombinant ERp57 protein with the yields reaching up to 10mg/L.

?iplys E; Žitkus E; Slibinskas R

2013-06-01

245

Native signal peptide of human ERp57 disulfide isomerase mediates secretion of active native recombinant ERp57 protein in yeast Saccharomyces cerevisiae.  

Science.gov (United States)

Human ERp57 protein is disulfide isomerase, facilitating proper folding of glycoprotein precursors in the concert with ER lectin chaperones calreticulin and calnexin. Growing amount of data also associates ERp57 with many different functions in subcellular locations outside the ER. Analysis of protein functions requires substantial amounts of correctly folded, biologically active protein, and in this study we introduce yeast Saccharomyces cerevisiae as a perfect host for production of human ERp57. Our data suggest that native signal peptide of human ERp57 protein is recognized and correctly processed in the yeast cells, which leads to protein secretion. Secreted recombinant ERp57 protein possesses native amino acid sequence and is biologically active. Moreover, secretion allows simple one-step purification of recombinant ERp57 protein with the yields reaching up to 10mg/L. PMID:23528814

?iplys, Evaldas; Žitkus, Eimantas; Slibinskas, Rimantas

2013-03-23

246

Differences between recombinant PTTH and crude brain extracts in cAMP-mediated ecdysteroid secretion from the prothoracic glands of the silkworm, Bombyx mori.  

Science.gov (United States)

The ability of recombinant prothoracicotropic hormone (rPTTH) or crude brain extract (cBRAIN) of Bombyx mori to stimulate ecdysteroid secretion from prothoracic glands (PGs) was investigated throughout the fifth instar and the first day of the pupal stage. Crude brain extracts could stimulate much higher ecdysteroid secretion than rPTTH during a 2h incubation. Recombinant PTTH did not increase the level of glandular cyclic AMP, except on days 4 and 5 of the fifth instar. Glandular cAMP levels were increased by cBRAIN from day 0 until day 5 of the fifth instar with the highest increase on day 3. On this day, rPTTH could not stimulate any increase of ecdysteroid secretion from the PGs during a 30min incubation. On the contrary, PGs incubated with cBRAIN for 30min showed increased secretory activity. Furthermore, on day 3 and in the absence of extracellular Ca(2+), rPTTH did not increase the glandular cAMP levels but cBRAIN did. Recombinant PTTH-stimulated ecdysteroid secretion from day 3 PGs was dependent on extracellular Ca(2+) in a dose-dependent manner. However, cBRAIN could stimulate ecdysteroid secretion even in the absence of extracellular Ca(2+). Taken together, the results of these experiments suggest the presence of a previously unknown cerebral prothoracicotropic factor that can stimulate glandular cAMP levels and ecdysteroid secretion from the PGs of Bombyx mori. PMID:12770324

Dedos, S G.; Fugo, H; Nagata, S; Takamiya, M; Kataoka, H

1999-05-01

247

Differences between recombinant PTTH and crude brain extracts in cAMP-mediated ecdysteroid secretion from the prothoracic glands of the silkworm, Bombyx mori.  

UK PubMed Central (United Kingdom)

The ability of recombinant prothoracicotropic hormone (rPTTH) or crude brain extract (cBRAIN) of Bombyx mori to stimulate ecdysteroid secretion from prothoracic glands (PGs) was investigated throughout the fifth instar and the first day of the pupal stage. Crude brain extracts could stimulate much higher ecdysteroid secretion than rPTTH during a 2h incubation. Recombinant PTTH did not increase the level of glandular cyclic AMP, except on days 4 and 5 of the fifth instar. Glandular cAMP levels were increased by cBRAIN from day 0 until day 5 of the fifth instar with the highest increase on day 3. On this day, rPTTH could not stimulate any increase of ecdysteroid secretion from the PGs during a 30min incubation. On the contrary, PGs incubated with cBRAIN for 30min showed increased secretory activity. Furthermore, on day 3 and in the absence of extracellular Ca(2+), rPTTH did not increase the glandular cAMP levels but cBRAIN did. Recombinant PTTH-stimulated ecdysteroid secretion from day 3 PGs was dependent on extracellular Ca(2+) in a dose-dependent manner. However, cBRAIN could stimulate ecdysteroid secretion even in the absence of extracellular Ca(2+). Taken together, the results of these experiments suggest the presence of a previously unknown cerebral prothoracicotropic factor that can stimulate glandular cAMP levels and ecdysteroid secretion from the PGs of Bombyx mori.

Dedos SG; Fugo H; Nagata S; Takamiya M; Kataoka H

1999-05-01

248

Killing Effect of Ad5/F35-APE1 siRNA Recombinant Adenovirus in Combination with Hematoporphrphyrin Derivative-Mediated Photodynamic Therapy on Human Nonsmall Cell Lung Cancer  

Science.gov (United States)

The main goal of this work is to investigate the killing effects and molecular mechanism of photodynamic therapy (PDT) mediated by the Ad5/F35-APE1 siRNA recombinant adenovirus in combination with a hematoporphrphyrin derivative (HpD) in the A549 human lung adenocarcinoma cell line in vitro to provide a theoretical reference for treating lung cancer by HpD-PDT. By using the technologies of MTT, flow cytometry, ELISA, and western blot, we observed that the proliferation inhibition and apoptosis of the A549 cells were significantly higher than the control group (P < 0.05) after HpD-PDT was performed. The inhibitory efficiency is dependent on the HpD concentration and laser intensity dose. The inhibitory effect on the proliferation of A549 cells of Ad5/F35-APE1 siRNA is more significant after combining with PDT, as indicated by a significant elevation of the intracellular ROS level and the expression of inflammatory factors (P < 0.05). The HpD-PDT-induced expression of the APE1 protein reached the peak after 24 h in A549 cells. The inhibition of APE1 expression in A549 cells was most significant after 48 hours of infection by Ad5/F35-APE1 siRNA recombinant adenovirus (10?MOI). In conclusion, the Ad5/F35-APE1 siRNA recombinant adenovirus could efficiently inhibit the HpD-PDT-induced APE1 expression hence could significantly enhance the killing effect of HpD-PDT in lung cancer cells.

Xia, Lei; Guan, Wei; Wang, Dong; Zhang, Yun-Song; Zeng, Lin-Li; Li, Zeng-Peng; Wang, Ge; Yang, Zhen-Zhou

2013-01-01

249

Killing effect of Ad5/F35-APE1 siRNA recombinant adenovirus in combination with hematoporphrphyrin derivative-mediated photodynamic therapy on human nonsmall cell lung cancer.  

UK PubMed Central (United Kingdom)

The main goal of this work is to investigate the killing effects and molecular mechanism of photodynamic therapy (PDT) mediated by the Ad5/F35-APE1 siRNA recombinant adenovirus in combination with a hematoporphrphyrin derivative (HpD) in the A549 human lung adenocarcinoma cell line in vitro to provide a theoretical reference for treating lung cancer by HpD-PDT. By using the technologies of MTT, flow cytometry, ELISA, and western blot, we observed that the proliferation inhibition and apoptosis of the A549 cells were significantly higher than the control group (P < 0.05) after HpD-PDT was performed. The inhibitory efficiency is dependent on the HpD concentration and laser intensity dose. The inhibitory effect on the proliferation of A549 cells of Ad5/F35-APE1 siRNA is more significant after combining with PDT, as indicated by a significant elevation of the intracellular ROS level and the expression of inflammatory factors (P < 0.05). The HpD-PDT-induced expression of the APE1 protein reached the peak after 24 h in A549 cells. The inhibition of APE1 expression in A549 cells was most significant after 48 hours of infection by Ad5/F35-APE1 siRNA recombinant adenovirus (10?MOI). In conclusion, the Ad5/F35-APE1 siRNA recombinant adenovirus could efficiently inhibit the HpD-PDT-induced APE1 expression hence could significantly enhance the killing effect of HpD-PDT in lung cancer cells.

Xia L; Guan W; Wang D; Zhang YS; Zeng LL; Li ZP; Wang G; Yang ZZ

2013-01-01

250

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

2012-10-15

251

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

UK PubMed Central (United Kingdom)

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.

Nag JK; Shrivastava N; Gupta J; Misra-Bhattacharya S

2013-01-01

252

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

2013-05-07

253

LIM Protein Ajuba Participates in the Repression of the ATR-Mediated DNA Damage Response.  

UK PubMed Central (United Kingdom)

LIM proteins constitute a superfamily characterized by the presence of a LIM domain, known to be involved in protein-protein interactions. Our previous work has implicated members of the Zyxin family of LIM proteins, namely TRIP6 and LPP, in the repression of the DNA damage response (DDR) at telomeres. Here, we describe a role for Ajuba, a closely related LIM molecule, in repressing the ATR-mediated DDR. We found that depletion of Ajuba led to apparent delays in the cell cycle, accompanied with increased Rb phosphorylation, Chk1 phosphorylation, induction of p53, and cell death. Ajuba could be found in a complex with replication protein A (RPA), and its depletion led to RPA phosphorylation, known to be an early event in ATR activation. We propose that Ajuba protects against unscheduled ATR signaling by preventing inappropriate RPA phosphorylation.

Kalan S; Matveyenko A; Loayza D

2013-01-01

254

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

International Nuclear Information System (INIS)

[en] 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)

2011-01-01

255

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  

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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.

Andersson Monica; Warolén Malin; Nilsson Joakim; Selander Martin; Sterky Catharina; Bergdahl Katrin; Sörving Christina; James Stephen R; Doverskog Magnus

2007-01-01

256

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

257

TP53 and RPA3 gene variations were associated with risk of glioma in a Chinese Han population.  

UK PubMed Central (United Kingdom)

Recent advances in human genetic studies have opened new avenues for the identification of susceptibility genes for many complex genetic disorders, especially in the field of rare cancers such as glioma. Glioma is one of the least understood human tumors and the etiology for glioma is barely known. Hundreds of single-nucleotide polymorphisms (SNPs) are found to be related to the risk of glioma in previous studies. This study is committed to investigate the role of heredity in this disorder. To examine and validate how common variants contribute to glioma susceptibility in the Han Chinese population, we evaluated 12 tagging SNPs in a case-control study in the Chinese Han population from Xi'an city of China (301 cases and 302 controls). Overall, two protective alleles and one risk allele for glioma were found by genetic model analyses. In dominant model, the allele "T" of rs6947203 in the RPA3 gene acts as a protective allele [odds ratio (OR), 0.59; 95% confidence interval (CI), 0.22-0.90; p=0.014]. In recessive model, the allele "C" of rs1042522 in the TP53 gene acts as a risk allele (OR, 1.65; 95% CI, 1.05-2.59; p=0.0314). In additive model, the allele "G" of rs4140805 in the RPA3 gene (OR, 0.73; 95% CI, 0.53-0.99; p=0.0437) and the allele "T" of rs6947203 in the RPA3 gene (OR, 0.62; 95% CI, 0.42-0.92; p=0.0177) both act as protective alleles. We also observed a haplotype of "CC" in the TP53 gene with an increased risk of 34% of developing glioma (p=0.0306). Our results, combined with previous studies, ascertain the potential role of the TP53 gene to glioma onset.

Jin T; Zhang J; Li G; Li S; Yang B; Chen C; Cai L

2013-04-01

258

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

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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 < 70%; class II: others). Results: Median number of brain metastases was six (four to 50). Most patients (69%) had extracranial metastases as well. Criteria of RPA Class I (II) were met in 4% (41%), whereas 56% had KPS < 70% and thus were grouped into class III (Tables 1 and 2). Complete or partial remission of brain metastases was found in 46% of patients who underwent computed tomography. Median survival was 4 months, 1-year survival rate 15%. Only age was a borderline significant prognostic factor in univariate analysis ({<=} 50 years vs > 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.)

Nieder, C.; Andratschke, N.; Grosu, A.L.; Molls, M. [Dept. of Radiotherapy and Radiologic Oncology, Klinikum rechts der Isar, Technical Univ. of Munich (Germany)

2003-01-01

259

Self consistent R.P.A. calculation with the finite range and density dependent interaction 'D 1'  

International Nuclear Information System (INIS)

Preliminary results are presented of R.P.A. calculation on 160 with the particle-hole interaction derived from the interaction D 1. The particle-hole interaction is defined as the second derivative of the effective interaction with respect to the density. The density dependent effective interaction of finite range 'D1' is phenomenological in the sense that its relation with realistic nucleon-nucleon interactions was left out. However to prevent unrealistic admixture of spin and isospin terms, each of its components was checked on nuclear matter saturation properties as calculated with more fundamental approaches. (Auth.).

1975-02-24

260

Rapamycin inhibition of baculovirus recombinant (BVr) ribosomal protein S6 kinase (S6K1) is mediated by an event other than phosphorylation  

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Full Text Available Abstract Background Ribosomal protein S6 kinase 1(S6K1) is an evolutionary conserved kinase that is activated in response to growth factors and viral stimuli to influence cellular growth and proliferation. This downstream effector of target of rapamycin (TOR) signaling cascade is known to be directly activated by TOR- kinase mediated hydrophobic motif (HM) phosphorylation at Threonine 412 (T412). Selective loss of this phosphorylation by inactivation of TOR kinase or activation/recruitment of a phosphatase has accordingly been implicated in mediating inhibition by rapamycin. Findings We present evidence that baculovirus driven expression of S6K1 in insect cells (Sf9) fails to activate the enzyme and instead renders it modestly active representing 4-6 folds less activity than its fully active mammalian counterpart. Contrary to the contention that viral infection activates TOR signaling pathway, we report that BVr enzyme fails to exhibit putative TOR dependent phosphorylation at the HM and the resultant phosphorylation at the activation loop (AL) of the enzyme, correlating with the level of activity observed. Surprisingly, the BVr enzyme continued to exhibit sensitivity to rapamycin that remained unaffected by mutations compromised for TOR phosphorylation (T412A) or deletions compromised for TOR binding (?NH 2-46/?CT104). Conclusions These data together with the ability of the BVr enzyme to resist inactivation by phosphatases indicate that inhibition by rapamycin is not mediated by any phosphorylation event in general and TOR dependent phosphorylation in particular.

Beigh Mushtaq A; Showkat Mehvish; ul Hussain Mahboob; Latoo Shafat A; Majeed Sheikh T; Andrabi Khurshid I

2012-01-01

 
 
 
 
261

Thermal aggregation of recombinant protective antigen: aggregate morphology and growth rate.  

UK PubMed Central (United Kingdom)

The thermal aggregation of the biopharmaceutical protein recombinant protective antigen (rPA) has been explored, and the associated kinetics and thermodynamic parameters have been extracted using optical and environmental scanning electron microscopies (ESEMs) and ultraviolet light scattering spectroscopy (UV-LSS). Visual observations and turbidity measurements provided an overall picture of the aggregation process, suggesting a two-step mechanism. Microscopy was used to examine the structure of aggregates, revealing an open morphology formed by the clustering of the microscopic aggregate particles. UV-LSS was used and developed to elucidate the growth rate of these particles, which formed in the first stage of the aggregation process. Their growth rate is observed to be high initially, before falling to converge on a final size that correlates with the ESEM data. The results suggest that the particle growth rate is limited by rPA monomer concentration, and by obtaining data over a range of incubation temperatures, an approach was developed to model the aggregation kinetics and extract the rate constants and the temperature dependence of aggregation. In doing so, we quantified the susceptibility of rPA aggregation under different temperature and environmental conditions and moreover demonstrated a novel use of UV spectrometry to monitor the particle aggregation quantitatively, in situ, in a nondestructive and time-resolved manner.

Belton DJ; Miller AF

2013-01-01

262

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 h...

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

263

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 h...

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

264

Resistance to respiratory syncytial virus (RSV) challenge induced by infection with a vaccinia virus recombinant expressing the RSV M2 protein (Vac-M2) is mediated by CD8+ T cells, while that induced by Vac-F or Vac-G recombinants is mediated by antibodies.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

It was previously demonstrated that the vaccinia virus recombinants expressing the respiratory syncytial virus (RSV) F, G, or M2 (also designated as 22K) protein (Vac-F, Vac-G, or Vac-M2, respectively) induced almost complete resistance to RSV challenge in BALB/c mice. In the present study, we sough...

Connors, M; Kulkarni, A B; Collins, P L; Firestone, C Y; Holmes, K L; Morse, H C; Murphy, B R

265

Effects of recombinant adenovirus-mediated expression of IL-2 and IL-12 in human B lymphoma cells on co-cultured PBMC  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Modulation of the immune system by genetically modified lymphoma cell vaccines is of potential therapeutic value in the treatment of B cell lymphoma. However, the anti-tumor effect of any single immunogene transfer has so far been limited. Combination treatment of recombinant IL-2 and IL-12 has been reported to be synergistic for inducing anti-tumor responses in solid tumors but the potential of IL-2/IL-12 gene modified B cell lymphoma cells has not been explored yet. Methods Using three different human B cell lymphoma cell lines and primary samples from patients with B cell neoplasms, expression levels of the coxsackie B-adenovirus receptor (CAR) and alpha (v) integrins were analyzed by fluorescence-activated cell sorter (FACS). Adenoviral transduction efficiencies were determined by GFP expression analysis and IL-2 and IL-12 cytokine production was quantified by enzyme-linked immunosorbent (ELISA) assays. Proliferative activities of peripheral blood mononuclear cells (PBMC) stimulated with either cytokine derived from supernatants of transduced lymphoma cells were measured by cell proliferation (MTT) assays. An EuTDA cytotoxicity assay was used to compare cytotoxic activities of IL-2 and/or IL-12 stimulated PBMC against unmodified lymphoma cells. Results We found that B cell lymphoma cell lines could be transduced with much higher efficiency than primary tumor samples, which appeared to correlate with the expression of CAR. Adenoviral-expressed IL-2 and IL-12 similarly led to dose-dependent increases in proliferation rates of PBMC obtained from healthy donors. IL-2 and/or IL-12 transduced lymphoma cells were co-cultured with PBMC, which were assayed for their cytolytic activity against unmodified lymphoma cells. We found that IL-2 stimulated PBMC elicited a significant anti-tumor effect but not the combined effect of IL-2/IL-12 or IL-12 alone. Conclusion This study demonstrates that the generation of recombinant adenovirus modified lymphoma cell vaccines based on lymphoma cell lines expressing IL-2 and IL-12 cytokine genes is technically feasible, induces increases in proliferation rates and cytotoxic activity of co-cultured PBMC, and warrants further development for the treatment of lymphoma patients in the future.

Ebert Oliver; Wilbert Dorothee; Buttgereit Peter; Ziske Carsten; Flieger Dimitri; Schmidt-Wolf Ingo GH

2004-01-01

266

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.

Towne Chris; Pertin Marie; Beggah Ahmed T; Aebischer Patrick; Decosterd Isabelle

2009-01-01

267

Three distinct envelope domains, variably present in subgroup B feline leukemia virus recombinants, mediate Pit1 and Pit2 receptor recognition.  

UK PubMed Central (United Kingdom)

Subgroup B feline leukemia viruses (FeLV-Bs) evolve from subgroup A FeLV (FeLV-A) by recombining with portions of endogenous FeLV envelope sequences in the cat genome. The replication properties of FeLV-B are distinct from those of FeLV-A; FeLV-B infects many nonfeline cell lines and recognizes the human Pit1 (HuPit1) receptor, whereas FeLV-A infects primarily feline cells, using a distinct but as yet undefined receptor. Here, we demonstrate that some FeLV-Bs can also use human Pit2 (HuPit2) and hamster Pit2 (HaPit2) for entry. By making viruses that contain chimeric surface (SU) envelope proteins from FeLV-A and FeLV-B, and testing their infectivity, we have defined genetic determinants that confer host range and specific receptor recognition. HuPit1 receptor recognition determinants localize to the N-terminal region of the FeLV-B SU, amino acids 83 to 116, encompassing the N-terminal portion of variable region A (VRA). While this 34-amino-acid domain of the FeLV-B VRA is sufficient for infection of some cells (feline, canine, and human), amino acids 146 to 249 of FeLV-B, which include variable region B (VRB), were required for efficient infection in other cell types (hamster, bovine, and rat). Chimeras encoding FeLV-B VRA and VRB also infected cells expressing HaPit2 and HuPit2 receptors more efficiently than chimeras encoding only the VRA of FeLV-B, suggesting that VRB provides a secondary determinant that is both cell and receptor specific. However, viruses containing additional FeLV-B sequences in the C terminus of SU could not recognize HuPit2, implying that there is a determinant beyond VRB that negatively affects HuPit2 interactions. Thus, Pit2 recognition may drive selection for the generation of specific FeLV-B recombinants, offering an explanation for the two major classes of FeLV-B that have been observed in vivo. Furthermore, the finding that some FeLV-Bs can use both Pit1 and Pit2 may explain previous observations that FeLV-B and GALV, which primarily uses Pit1, display nonreciprocal interference on many cell types.

Boomer S; Eiden M; Burns CC; Overbaugh J

1997-11-01

268

Three distinct envelope domains, variably present in subgroup B feline leukemia virus recombinants, mediate Pit1 and Pit2 receptor recognition.  

Science.gov (United States)

Subgroup B feline leukemia viruses (FeLV-Bs) evolve from subgroup A FeLV (FeLV-A) by recombining with portions of endogenous FeLV envelope sequences in the cat genome. The replication properties of FeLV-B are distinct from those of FeLV-A; FeLV-B infects many nonfeline cell lines and recognizes the human Pit1 (HuPit1) receptor, whereas FeLV-A infects primarily feline cells, using a distinct but as yet undefined receptor. Here, we demonstrate that some FeLV-Bs can also use human Pit2 (HuPit2) and hamster Pit2 (HaPit2) for entry. By making viruses that contain chimeric surface (SU) envelope proteins from FeLV-A and FeLV-B, and testing their infectivity, we have defined genetic determinants that confer host range and specific receptor recognition. HuPit1 receptor recognition determinants localize to the N-terminal region of the FeLV-B SU, amino acids 83 to 116, encompassing the N-terminal portion of variable region A (VRA). While this 34-amino-acid domain of the FeLV-B VRA is sufficient for infection of some cells (feline, canine, and human), amino acids 146 to 249 of FeLV-B, which include variable region B (VRB), were required for efficient infection in other cell types (hamster, bovine, and rat). Chimeras encoding FeLV-B VRA and VRB also infected cells expressing HaPit2 and HuPit2 receptors more efficiently than chimeras encoding only the VRA of FeLV-B, suggesting that VRB provides a secondary determinant that is both cell and receptor specific. However, viruses containing additional FeLV-B sequences in the C terminus of SU could not recognize HuPit2, implying that there is a determinant beyond VRB that negatively affects HuPit2 interactions. Thus, Pit2 recognition may drive selection for the generation of specific FeLV-B recombinants, offering an explanation for the two major classes of FeLV-B that have been observed in vivo. Furthermore, the finding that some FeLV-Bs can use both Pit1 and Pit2 may explain previous observations that FeLV-B and GALV, which primarily uses Pit1, display nonreciprocal interference on many cell types. PMID:9343161

Boomer, S; Eiden, M; Burns, C C; Overbaugh, J

1997-11-01

269

Continuum RPA correlations in quasielastic electron scattering from 12C decay on (e,e'p) and (e,e'n) reaction channels  

International Nuclear Information System (INIS)

We investigate the role of RPA correlations in lognitudinal and transverse response functions for inelastic electron scattering from 12C at momentum transfers ranging between 200 MeV/c and 550 MeV/c. We refer to a continuum self-consistent RPA theory with a SK3 interaction. Electromagnetic operators are taken for uncorrelated nucleons. The partial response on (e,e'p) and (e,e'n) reaction channels is also calculated. Since energy-weighted sum rules are conserved, we can control the effect of the Hartree-Fock non-locality expressed by the SK3 nucleon effective mass as well as the action of creation and descruction of 1p1h pairs on the true ground state induced by the RPA residual interaction. (orig.).

1984-01-01

270

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-?) 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-? 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

271

[Anti-HBV effect of nucleotide analogues on mouse model of chronic HBV infection mediated by recombinant adeno-associated virus 8].  

UK PubMed Central (United Kingdom)

We evaluated the anti-HBV effects of nucleotide analogues, Entecavir (ETV) and Lamivudine (LAM) targeting mouse model of HBV persistent infection with recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome (rAAV8-1.3HBV). Ninety percent (27 of 30 mice) of rAAVS-treated mice were chosen as mouse model. Four groups were orally administrated with different doses of ETV (1 mg/(kgd) or 0.1 mg/(kgd)) and LAM (500 mg/(kgd) or 100 mg/(kgd)) once a day for 10 days. The other two groups were set as normal saline treated and untreated control. We detected the levels of HBV DNA, HBeAg and HBsAg in sera at different time. Results indicate that HBV DNA level decreased significantly (P < 0.05) in drug-treated groups compared with normal saline group after drug administration. Fifteen days after the drug withdrawal, HBV DNA level rebounded back obviously (P < 0.05) in groups with low doses of ETV and LAM. However, there was no apparent change of HBeAg and HBsAg in the whole process among all groups. These results showed that our model could reflect the anti-viral effect of nucleotide analogues. This model can be a useful and convenient tool for anti-HBV drug discovery.

Wang G; Wang G; Dong X; Tian W; Yuchi J; Wei G; Meng H; Wu X

2013-01-01

272

Mycobacterium tuberculosis antigen 85B and ESAT-6 expressed as a recombinant fusion protein in Mycobacterium smegmatis elicits cell-mediated immune response in a murine vaccination model.  

Science.gov (United States)

In this study, we investigated the potential molecular and immunological differences of a recombinant fusion protein (Hybrid-1), comprising of the immunodominant antigens Ag85B and ESAT-6 from Mycobacterium tuberculosis, derived from two different expression systems, namely Mycobacterium smegmatis and Escherichia coli. The fusion protein was successfully expressed and purified from both bacterial hosts and analyzed for any host-dependent post-translational modifications that might affect the immunogenicity of the protein. We investigated the immunogenicity of Hybrid-1 expressed in the two host species in a murine vaccination model, together with a reference standard Hybrid-1 (expressed in E. coli) from the Statens Serum Institut. No evidence of any post-translation modification was found in the M. smegmatis-derived Hybrid-1 fusion protein, nor were there any significant differences in the T-cell responses obtained to the three antigens analyzed. In conclusion, the Hybrid-1 fusion protein was successfully expressed in a homologous expression system using M. smegmatis and this system is worth considering as a primary source for vaccination trials, as it provided protein of excellent yield, stability and free from lipopolysaccharide. PMID:23333882

Tsolaki, Anthony G; Nagy, Judit; Leiva, Sergio; Kishore, Uday; Rosenkrands, Ida; Robertson, Brian D

2013-01-16

273

Mycobacterium tuberculosis antigen 85B and ESAT-6 expressed as a recombinant fusion protein in Mycobacterium smegmatis elicits cell-mediated immune response in a murine vaccination model.  

UK PubMed Central (United Kingdom)

In this study, we investigated the potential molecular and immunological differences of a recombinant fusion protein (Hybrid-1), comprising of the immunodominant antigens Ag85B and ESAT-6 from Mycobacterium tuberculosis, derived from two different expression systems, namely Mycobacterium smegmatis and Escherichia coli. The fusion protein was successfully expressed and purified from both bacterial hosts and analyzed for any host-dependent post-translational modifications that might affect the immunogenicity of the protein. We investigated the immunogenicity of Hybrid-1 expressed in the two host species in a murine vaccination model, together with a reference standard Hybrid-1 (expressed in E. coli) from the Statens Serum Institut. No evidence of any post-translation modification was found in the M. smegmatis-derived Hybrid-1 fusion protein, nor were there any significant differences in the T-cell responses obtained to the three antigens analyzed. In conclusion, the Hybrid-1 fusion protein was successfully expressed in a homologous expression system using M. smegmatis and this system is worth considering as a primary source for vaccination trials, as it provided protein of excellent yield, stability and free from lipopolysaccharide.

Tsolaki AG; Nagy J; Leiva S; Kishore U; Rosenkrands I; Robertson BD

2013-07-01

274

DNA RECOMBINATION IN EUCARYOTIC CELLS BY THE BACTERIOPHAGE PHIC31 RECOMBINATION SYSTEM",  

Science.gov (United States)

This invention provides methods for obtaining specific and stable integration of nucleic acids into eukaryotic cells. The invention makes use of site-specific recombination systems that use prokaryotic recombinase polypeptides, such as the ph:C31 integrase, that can mediate recombination between th...

275

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

276

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; M. Mauz; J. Bange

2013-01-01

277

Systemic treatment after whole-brain radiotherapy may improve survival in RPA class II/III breast cancer patients with brain metastasis.  

Science.gov (United States)

Whole brain radiotherapy (WBRT) is the most widely used treatment for brain metastasis (BM), especially for patients with multiple intracranial lesions. The purpose of this study was to examine the efficacy of systemic treatments following WBRT in breast cancer patients with BM who had different clinical characteristics, based on the classification of the Radiation Therapy Oncology Group recursive partitioning analysis (RPA) and the breast cancer-specific Graded Prognostic Assessment (Breast-GPA). One hundred and one breast cancer patients with BM treated between 2006 and 2010 were analyzed. The median interval between breast cancer diagnosis and identification of BM in the triple-negative patients was shorter than in the luminal A subtype (26 vs. 36 months, respectively; P = 0.021). Univariate analysis indicated that age at BM diagnosis, Karnofsky performance status/recursive partitioning analysis (KPS/RPA) classes, number of BMs, primary tumor control, extracranial metastases and systemic treatment following WBRT were significant prognostic factors for overall survival (OS) (P KPS/RPA classes and systemic treatments following WBRT remained the significant prognostic factors for OS. For RPA class I, the median survival with and without systemic treatments following WBRT was 25 and 22 months, respectively (P = 0.819), while for RPA class II/III systemic treatments significantly improved OS from 7 and 2 months to 11 and 5 months, respectively (P < 0.05). Our results suggested that triple-negative patients had a shorter interval between initial diagnosis and the development of BM than luminal A patients. Systemic treatments following WBRT improved the survival of RPA class II/III patients. PMID:23743596

Zhang, Qian; Chen, Jian; Yu, Xiaoli; Ma, Jinli; Cai, Gang; Yang, Zhaozhi; Cao, Lu; Chen, Xingxing; Guo, Xiaomao; Chen, Jiayi

2013-06-07

278

Systemic treatment after whole-brain radiotherapy may improve survival in RPA class II/III breast cancer patients with brain metastasis.  

UK PubMed Central (United Kingdom)

Whole brain radiotherapy (WBRT) is the most widely used treatment for brain metastasis (BM), especially for patients with multiple intracranial lesions. The purpose of this study was to examine the efficacy of systemic treatments following WBRT in breast cancer patients with BM who had different clinical characteristics, based on the classification of the Radiation Therapy Oncology Group recursive partitioning analysis (RPA) and the breast cancer-specific Graded Prognostic Assessment (Breast-GPA). One hundred and one breast cancer patients with BM treated between 2006 and 2010 were analyzed. The median interval between breast cancer diagnosis and identification of BM in the triple-negative patients was shorter than in the luminal A subtype (26 vs. 36 months, respectively; P = 0.021). Univariate analysis indicated that age at BM diagnosis, Karnofsky performance status/recursive partitioning analysis (KPS/RPA) classes, number of BMs, primary tumor control, extracranial metastases and systemic treatment following WBRT were significant prognostic factors for overall survival (OS) (P < 0.05). Multivariate analysis revealed that KPS/RPA classes and systemic treatments following WBRT remained the significant prognostic factors for OS. For RPA class I, the median survival with and without systemic treatments following WBRT was 25 and 22 months, respectively (P = 0.819), while for RPA class II/III systemic treatments significantly improved OS from 7 and 2 months to 11 and 5 months, respectively (P < 0.05). Our results suggested that triple-negative patients had a shorter interval between initial diagnosis and the development of BM than luminal A patients. Systemic treatments following WBRT improved the survival of RPA class II/III patients.

Zhang Q; Chen J; Yu X; Ma J; Cai G; Yang Z; Cao L; Chen X; Guo X; Chen J

2013-09-01

279

Pig gene knockout by rAAV-mediated homologous recombination: comparison of BRCA1 gene knockout efficiency in Yucatan and Gottingen fibroblasts with slightly different target sequences.  

UK PubMed Central (United Kingdom)

In this study, we compared the gene targeting efficiencies of two rAAV-BRCA1 KO targeting constructs in Yucatan and Göttingen minipig fibroblasts. The homology arms of the constructs consisted exclusively of exonic sequences amplified by PCR from Yucatan genomic DNA. The sequences were identical to those of the reference porcine genome of a Duroc sow (Ensembl Susscrofa 9) and the BRCA1 gene of the Landrace breed (NCBI acc. no. AB271921). Surprisingly, we found that the very efficient gene targeting observed for Yucatan fibroblasts (35% targeting efficiency) was completely absent using either of the two constructs in Göttingen fibroblasts. Sequencing of the relevant BRCA1 exon 11 region (~2 kb) in the Göttingen minipig revealed three single nucleotide differences in the sequence targeted by the left homology arm of the construct (0.3% of the bases) and three or seven in the two right homology regions (0.3 or 0.7% of the bases, respectively). Construction of a novel rAAV-BRCA1 targeting vector based on the Göttingen genomic DNA sequence re-established gene targeting although the efficiency was somewhat lower than that observed for Yucatan fibroblasts. These BRCA1 KO Göttingen fibroblast clones have been used as nuclear donor cells for somatic cell nuclear transfer to generate a Göttingen BRCA1 KO pig model as previously done with the Yucatan breed. The present study illustrates that even a few mismatches present in the homology arms of an efficient rAAV-targeting construct can completely abolish gene targeting by homologous recombination emphasizing the importance of using isogenic DNA even for creating targeting constructs consisting of exon sequences only.

Luo Y; Bolund L; Sřrensen CB

2012-06-01

280

Pig gene knockout by rAAV-mediated homologous recombination: comparison of BRCA1 gene knockout efficiency in Yucatan and Göttingen fibroblasts with slightly different target sequences  

DEFF Research Database (Denmark)

In this study, we compared the gene targeting efficiencies of two rAAV-BRCA1 KO targeting constructs in Yucatan and Göttingen minipig fibroblasts. The homology arms of the constructs consisted exclusively of exonic sequences amplified by PCR from Yucatan genomic DNA. The sequences were identical to those of the reference porcine genome of a Duroc sow (Ensembl Susscrofa 9) and the BRCA1 gene of the Landrace breed (NCBI acc. no. AB271921). Surprisingly, we found that the very efficient gene targeting observed for Yucatan fibroblasts (35% targeting efficiency) was completely absent using either of the two constructs in Göttingen fibroblasts. Sequencing of the relevant BRCA1 exon 11 region (~2 kb) in the Göttingen minipig revealed three single nucleotide differences in the sequence targeted by the left homology arm of the construct (0.3% of the bases) and three or seven in the two right homology regions (0.3 or 0.7% of the bases, respectively). Construction of a novel rAAV-BRCA1 targeting vector based on the Göttingen genomic DNA sequence re-established gene targeting although the efficiency was somewhat lower than that observed for Yucatan fibroblasts. These BRCA1 KO Göttingen fibroblast clones have been used as nuclear donor cells for somatic cell nuclear transfer to generate a Göttingen BRCA1 KO pig model as previously done with the Yucatan breed. The present study illustrates that even a few mismatches present in the homology arms of an efficient rAAV-targeting construct can completely abolish gene targeting by homologous recombination emphasizing the importance of using isogenic DNA even for creating targeting constructs consisting of exon sequences only.

Luo, Yonglun; Bolund, Lars

2012-01-01

 
 
 
 
281

Transport of human recombinant brain-derived neurotrophic factor (BDNF) through the rat blood-brain barrier in vivo using vector-mediated peptide drug delivery.  

Science.gov (United States)

The blood-brain barrier (BBB) transport of brain-derived neurotrophic factor (BDNF) in anesthetized rats was examined in the present studies using vector-mediated peptide drug delivery. Following tritiation, the BDNF was biotinylated via a disulfide linker and was coupled to a covalent conjugate of neutral avidin (NLA), which binds the biotinylated peptide with a high affinity, and the murine OX26 monoclonal antibody to the rat transferrin receptor. Owing to the abundance of transferrin receptors on brain capillary endothelium, the OX26 monoclonal antibody undergoes receptor-mediated transcytosis through the BBB, and the NLA-OX26 conjugate transports biotinylated peptide therapeutics through the BBB. The present studies show that while unconjugated BDNF was not transported through the BBB in vivo, the conjugation of biotinylated BDNF to the NLA-OX26 vector resulted in a marked increase in the brain delivery of BDNF, as defined by measurements of the percentage of the injected dose (ID) delivered per gram of brain. Although BDNF was not transported through the BBB in vivo, this cationic peptide was avidly bound by isolated human brain capillaries via a low-affinity, high-capacity system that was inhibited by protamine and by serum protein binding of BDNF. In conclusion, these studies show that the delivery of unconjugated BDNF to brain is nil owing to the combined effects of negligible BBB transport and rapid systemic clearance of intravenous administered BDNF. The brain delivery of BDNF may be augmented by conjugation of BDNF to BBB drug delivery vectors, such as the NLA-OX26 conjugate. PMID:8058646

Pardridge, W M; Kang, Y S; Buciak, J L

1994-05-01

282

Transport of human recombinant brain-derived neurotrophic factor (BDNF) through the rat blood-brain barrier in vivo using vector-mediated peptide drug delivery.  

UK PubMed Central (United Kingdom)

The blood-brain barrier (BBB) transport of brain-derived neurotrophic factor (BDNF) in anesthetized rats was examined in the present studies using vector-mediated peptide drug delivery. Following tritiation, the BDNF was biotinylated via a disulfide linker and was coupled to a covalent conjugate of neutral avidin (NLA), which binds the biotinylated peptide with a high affinity, and the murine OX26 monoclonal antibody to the rat transferrin receptor. Owing to the abundance of transferrin receptors on brain capillary endothelium, the OX26 monoclonal antibody undergoes receptor-mediated transcytosis through the BBB, and the NLA-OX26 conjugate transports biotinylated peptide therapeutics through the BBB. The present studies show that while unconjugated BDNF was not transported through the BBB in vivo, the conjugation of biotinylated BDNF to the NLA-OX26 vector resulted in a marked increase in the brain delivery of BDNF, as defined by measurements of the percentage of the injected dose (ID) delivered per gram of brain. Although BDNF was not transported through the BBB in vivo, this cationic peptide was avidly bound by isolated human brain capillaries via a low-affinity, high-capacity system that was inhibited by protamine and by serum protein binding of BDNF. In conclusion, these studies show that the delivery of unconjugated BDNF to brain is nil owing to the combined effects of negligible BBB transport and rapid systemic clearance of intravenous administered BDNF. The brain delivery of BDNF may be augmented by conjugation of BDNF to BBB drug delivery vectors, such as the NLA-OX26 conjugate.

Pardridge WM; Kang YS; Buciak JL

1994-05-01

283

Immunoglobulin class switch recombination deficiencies.  

UK PubMed Central (United Kingdom)

Maturation of the secondary antibody repertoire is generated by means of class switch recombination and somatic hypermutation. The molecular mechanisms underlying these important processes have long remained obscure. Inherited defects in class switch recombination variably associated to defects in somatic hypermutation are a group of genetically heterogeneous diseases, the characterization of which has allowed recognition that T-B cell interaction (resulting in CD40-mediated signaling), intrinsic B cell mechanisms, and complex DNA repair machinery are involved in class switch recombination and somatic hypermutation. Elucidation of the molecular defects underlying these disorders has been essential to better understand the molecular basis of immunoglobulin diversification and has offered the opportunity to define the clinical spectrum of these diseases and to prompt more accurate diagnostic and therapeutic approaches.

Kracker S; Gardes P; Mazerolles F; Durandy A

2010-05-01

284

Recursive partitioning analysis (RPA) of prognostic factors in three radiation therapy oncology group (RTOG) brain metastases trials  

International Nuclear Information System (INIS)

Purpose/Objective: Promising results from new approaches such as radiosurgery or stereotactic radiosurgery 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 RTOG brain metastases studies was considered necessary to fully evaluate the benefit of these new interventions. Materials and Methods: 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 pre-treatment characteristics and three treatment-related variables were analyzed. Results: Many factors such as total dose (? 52 Gy vs

1995-01-01

285

Degradation of RPA 202248 [U-14C-phenyl]alpha(-(cyclopropylcarbonyl)-2-(methylsulfonyl)-beta-oxo-4-(trifluromethyl)benzenepropanenitrile), the primary degradation product of isoxaflutole, in an outdoor aquatic microcosm system.  

UK PubMed Central (United Kingdom)

Isoxaflutole, the active ingredient in BALANCE WDG and BALANCE PRO corn herbicides and a co-formulant with the herbicide flufenacet in the product EPIC, is readily degraded in soil and water to RPA 202248 alpha(-(cyclopropylcarbonyl)-2-(methyvlsulfonyl)-beta-oxo-4-(trifluromethyl)benzenepropanenitrile). Because RPA 202248 is responsible at the molecular level for isoxaflutole's herbicidal activity it is important to understand the environmental behavior of the degradation product. Laboratory studies suggest that RPA 202248 is stable to hydrolysis and photolysis in aqueous systems and hence poses a possible environmental concern. As part of a program of work towards understanding the actual field situation, an outdoor microcosm study was carried out. Over the course of the 29-day study, residues remained predominantly in the aqueous phase. A slow but steady degradation of RPA 202248 was observed leading to the formation of RPA 203328 (2-methylsulfonyl-4-trifluoromethylbenzoic acid), which has no herbicidal activity. The half-life of RPA 202248 was calculated to be 103 days. These findings indicate that aqueous degradation should be considered as a potential route of dissipation when assessing the fate of RPA 202248 in large scale impounded water bodies, such as ponds, lakes, or reservoirs in the Mid-West Corn Belt.

Rupprecht JK; Liu A; Kelly I; Allen R

2004-01-01

286

Degradation of RPA 202248 [U-14C-phenyl]alpha(-(cyclopropylcarbonyl)-2-(methylsulfonyl)-beta-oxo-4-(trifluromethyl)benzenepropanenitrile), the primary degradation product of isoxaflutole, in an outdoor aquatic microcosm system.  

Science.gov (United States)

Isoxaflutole, the active ingredient in BALANCE WDG and BALANCE PRO corn herbicides and a co-formulant with the herbicide flufenacet in the product EPIC, is readily degraded in soil and water to RPA 202248 alpha(-(cyclopropylcarbonyl)-2-(methyvlsulfonyl)-beta-oxo-4-(trifluromethyl)benzenepropanenitrile). Because RPA 202248 is responsible at the molecular level for isoxaflutole's herbicidal activity it is important to understand the environmental behavior of the degradation product. Laboratory studies suggest that RPA 202248 is stable to hydrolysis and photolysis in aqueous systems and hence poses a possible environmental concern. As part of a program of work towards understanding the actual field situation, an outdoor microcosm study was carried out. Over the course of the 29-day study, residues remained predominantly in the aqueous phase. A slow but steady degradation of RPA 202248 was observed leading to the formation of RPA 203328 (2-methylsulfonyl-4-trifluoromethylbenzoic acid), which has no herbicidal activity. The half-life of RPA 202248 was calculated to be 103 days. These findings indicate that aqueous degradation should be considered as a potential route of dissipation when assessing the fate of RPA 202248 in large scale impounded water bodies, such as ponds, lakes, or reservoirs in the Mid-West Corn Belt. PMID:15620081

Rupprecht, J Kent; Liu, Anne; Kelly, Iain; Allen, Richard

2004-01-01

287

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

2011-01-01

288

Generation of Modified Pestiviruses by Targeted Recombination  

DEFF Research Database (Denmark)

Infectious cDNA clones are a prerequisite for directed genetic manipulation of pestivirus RNA genomes. We have developed a novel strategy to facilitate manipulation and rescue of modified pestiviruses from infectious cDNA clones based on bacterial artificial chromosomes (BACs). The strategy involves targeted modification of viral cDNA genomes, cloned within BACs, by Red/ET recombination-mediated mutagenesis in E.coli DH10B cells. Using recombination-mediated mutagenesis for the targeted design, the work can be expedited and focused in principal on any sequence within the viral genome and hence is not limited to the use of internal restriction sites. Rescue of modified pestiviruses can be obtained by electroporation of cell cultures with full-length RNA transcripts in vitro transcribed from the recombined BAC clones. We have used this approach to generate a series of new pestivirus BACs modified within different genomic regions and infectious pestiviruses have been rescued from several of these new constructs,demonstrating that recombination-mediated mutagenesis of pestivirus BACs provides a useful tool for expediting the construction of recombinant pestiviruses.

Rasmussen, Thomas Bruun; Friis, Martin Barfred

289

Prognostic factors derived from recursive partition analysis (RPA) of radiation therapy oncology group (RTOG) brain metastases trials applied to surgically resected and irradiated brain metastatic cases  

International Nuclear Information System (INIS)

Purpose: (a) To identify the prognostic factors that determine survival after surgical resection and irradiation of tumors metastatic to brain. (b) To determine if the prognostic factors used in the recursive partition analysis (RPA) of brain metastases cases from Radiation Therapy Oncology Group (RTOG) studies into three distinct survival classes is applicable to surgically resected and irradiated patients. Method: The medical records of 125 patients who had surgical resection and radiotherapy for brain metastases from 1985 to 1997 were reviewed. The patients' disease and treatment related factors were analyzed to identify factors that independently determine survival after diagnosis of brain metastasis. The patients were also grouped into three classes using the RPA-derived prognostic parameters which are: age, performance status, state of the primary disease, and presence or absence of extracranial metastases. Class 1: patients ? 65 years of age, Karnofsky performance status (KPS) of ?70, with controlled primary disease and no extracranial metastases; Class 3: patients with KPS

290

Soft octupole vibrations on superdeformed states in nuclei around 40Ca suggested by Skyrme-HF and self-consistent RPA calculations  

International Nuclear Information System (INIS)

We present the results of fully self-consistent RPA calculation for low-frequency negative-parity modes built on superdeformed states in the 40Ca region. The RPA calculation was carried out using the mixed representation on the three-dimensional Cartesian mesh in a box. The SD shell structure provides a very favorable situation for octupole shape fluctuations, and we show that the coherent excitation of protons and neutrons play an important role in the emergence of strongly collective octupole vibrations built on the SD states in the N=Z nuclei, 32S, 36Ar, 40Ca and 44Ti. In particular, the calculation suggests that a low-frequency, strongly collective K?=1- octupole vibration appears on the SD state in 40Ca. (orig.)

2005-01-01

291

Monitoring and Evaluation of Smolt Migration in the Columbia Basin Volume VIII : Comparison of the RPA Testing Rules, Technical Report 2002.  

Energy Technology Data Exchange (ETDEWEB)

The 2000 FCRPS Biological Opinion (BO) suggested two statistical hypothesis tests to assess the RPA compliance by the years 2005 and 2008. With the decision rules proposed in the BO, Skalski and Ngouenet (2001) developed a compliance framework based on classical t-tests and used Monte-Carlo simulations to calculate power curves. Unfortunately, the two-sample t tests proposed in the BO only have moderate-to-low probability of correctly assessing the true status of the smolt survival recovery. We have developed a superior two-phase regression statistical model for testing the RPA compliance. The two-phase regression model improves the statistical power over the standard two-sample t-tests. In addition, the two-phase regression model has a higher probability of correctly assessing the true status of the smolt survival recovery. These classical statistical power curve approaches do not incorporate prior knowledge into the decision process. Therefore, we propose to examine Bayesian methods that complement classical statistics in situations where uncertainty must be taken into account. The Bayesian analysis will incorporate scientific/biological knowledge/expertise to thoroughly assess the RPA compliance in 2005 and 2008.

Skalski, John; Ngouenet, Roger

2002-08-01

292

Study of the muon number violating (?-,e-) conversion in a nucleus by using quasi-particle RPA  

International Nuclear Information System (INIS)

The exotic (?-, e-) conversion reaction for the 48Ti nucleus is studied in the framework of the quasi-particle random phase approximation (QRPA). For the non-coherent processes the relevant total rate is calculated by summing over partial rates for all the possible intermediate states constructed in the above approximation. For the coherent process the contribution is obtained by using an uncorrelated BCS vacuum. In order to check the validity of closure approximation, which is almost unavoidable in shell-model calculations, we also evaluate the total (?-, e-) conversion rates by QRPA sum-rules by first explicitly calculating a suitable mean excitation energy of the nucleus. The influence of the ground state correlations to the (?-, e-) conversion matrix elements is estimated by using a correlated RPA vacuum. The fraction of the transition rate of the coherent process for each of these methods is calculated and the results are compared to those found previously by using shell-model closure approximation. (orig.).

1994-01-01

293

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; Andreas Vilcinskas

2013-01-01

294

Poliovirus RNA recombination.  

UK PubMed Central (United Kingdom)

We are developing an in vitro system for poliovirus RNA recombination. In this system, two mutant RNAs are replicated with poliovirus RNA-dependent RNA polymerase. Recombination will produce RNAs containing neither mutation and will be the only progeny RNAs that are infectious. We will use this system to determine what proteins and reaction conditions are required for recombination and to study the details of the mechanism of recombination.

Pata J; Kirkegaard K

1991-01-01

295

Recombineering homologous recombination constructs in Drosophila.  

UK PubMed Central (United Kingdom)

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Carreira-Rosario A; Scoggin S; Shalaby NA; Williams ND; Hiesinger PR; Buszczak M

2013-01-01

296

Use of a promoter trap system in Bacillus anthracis and Bacillus subtilis for the development of recombinant protective antigen-based vaccines.  

UK PubMed Central (United Kingdom)

We have recently reported Bacillus anthracis attenuated live vaccine strains efficiently expressing recombinant protective antigen (rPA) and have shown a direct correlation between the level of rPA secreted by these cells and efficacy (S. Cohen, I. Mendelson, Z. Altboum, D. Kobiler, E. Elhanany, T. Bino, M. Leitner, I. Inbar, H. Rosenberg, Y. Gozes, R. Barak, M. Fisher, C. Kronman, B. Velan, and A. Shafferman, Infect. Immun. 68:4549-4558, 2000). To isolate more potent Bacillus promoters for a further increase in the production of rPA, we developed a promoter trap system based on various gfp reporter genes adapted for use in both Bacillus subtilis and B. anthracis backgrounds. Accordingly, a B. anthracis library of 6,000 clones harboring plasmids with chromosomal B. anthracis DNA fragments inserted upstream from gfpuv was constructed. Based on fluorescence intensity, 57 clones carrying potentially strong promoters were identified, some of which were DNA sequenced. The most potent B. anthracis promoter identified (Pntr; 271 bp) was 500 times more potent than the native pagA promoter and 70 times more potent than the alpha-amylase promoter (Pamy). This very potent promoter was tested along with the other promoters (which are three, six, and eight times more potent than Pamy) for the ability to drive expression of rPA in either B. subtilis or B. anthracis. The number of cell-associated pre-PA molecules in B. anthracis was found to correlate well with the strength of the promoter. However, there appeared to be an upper limit to the amount of mature PA secreted into the medium, which did not exceed that driven by Pamy. Furthermore, the rPA constructs fused to the very potent promoters proved to be deleterious to the bacterial hosts and consequently led to genetic instability of the PA expression plasmid. Immunization with attenuated B. anthracis expressing rPA under the control of promoters more potent than Pamy was less efficient in eliciting anti-PA antibodies than that attained with Pamy. The results are consistent with the notion that overexpression of PA leads to severe secretion stress and have practical implications for the design of second-generation rPA-based vaccines.

Gat O; Inbar I; Aloni-Grinstein R; Zahavy E; Kronman C; Mendelson I; Cohen S; Velan B; Shafferman A

2003-02-01

297

Manipulating Mitotic Recombination in the Zebrafish Embryo Through RecQ Helicases  

Digital Repository Infrastructure Vision for European Research (DRIVER)

RecQ DNA helicases resolve Rad-51-mediated recombination and suppress aberrant homologous recombination. RecQ gene loss is associated with cancer susceptibility and increased mitotic recombination. We have developed an in vivo assay based on a zebrafish pigment mutant for suppression of RecQ activit...

Xie, Jing; Bessling, Seneca L.; Cooper, Timothy K.; Dietz, Harry C.; McCallion, Andrew S.; Fisher, Shannon

298

Recombination and Genetic Diversity  

Scientific Electronic Library Online (English)

Full Text Available Abstract in english In this paper we present a spatial stochastic model for genetic recombination, that answers if diversity is preserved in an infinite population of recombinat-ing individuals distributed spatially. We show that, for finite times, recombination may maintain all the various potential different types, but when time grows infinitely, the diversity of individuals extinguishes off. So under the model premisses, recombination and spatial localization alone are not enough to expla (more) in diversity in a population. Further we discuss an application of the model to a controversy regarding the diversity of "Major Histocompatibility Complex" (MHC).

Coutinho, T. C.; Silva, T.T.da; Toledo, G.L.

2012-12-01

299

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

International Nuclear Information System (INIS)

The efficient implementation of electronic structure methods is essential for first principles modeling of molecules and solids. We present here a particularly efficient common framework for methods beyond semilocal density-functional theory (DFT), including Hartree-Fock (HF), hybrid density functionals, random-phase approximation (RPA), second-order Mřller-Plesset perturbation theory (MP2) and the GW method. This computational framework allows us to use compact and accurate numeric atom-centered orbitals (NAOs), popular in many implementations of semilocal DFT, as basis functions. The essence of our framework is to employ the ‘resolution of identity (RI)’ technique to facilitate the treatment of both the two-electron Coulomb repulsion integrals (required in all these approaches) and the linear density-response function (required for RPA and GW). This is possible because these quantities can be expressed in terms of the products of single-particle basis functions, which can in turn be expanded in a set of auxiliary basis functions (ABFs). The construction of ABFs lies at the heart of the RI technique, and we propose here a simple prescription for constructing ABFs 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 accuracy of our RI implementation for Gaussian and NAO basis functions, as well as the convergence behavior of our NAO basis sets for the above-mentioned methods. Benchmark results are presented for the ionization energies of 50 selected atoms and molecules from the G2 ion test set obtained with the GW and MP2 self-energy methods, and the G2-I atomization energies as well as the S22 molecular interaction energies obtained with the RPA method. (paper)

2012-01-01

300

Calculation of the RPA response function of nuclei to quasi-elastic electron scattering with a density-dependent NN interaction; Calcul des fonctions de reponse R.P.A. des noyaux en diffusion quasi-elastique d`electrons avec une interactio N-N dependant de la densite  

Energy Technology Data Exchange (ETDEWEB)

So far, the non-relativistic longitudinal and transverse functions in electron quasi-elastic scattering on the nuclei failed in reproducing satisfactorily the existent experimental data. The calculations including relativistic RPA correlations utilize until now the relativistic Hartree approximation to describe the nuclear matter. But, this provides an incompressibility module two times higher than its experimental value what is an important drawback for the calculation of realistic relativistic RPA correlations. Hence, we have determined the RPA response functions of nuclei by utilising a description of the relativistic nuclear matter leading to an incompressibility module in agreement with the empirical value. To do that we have utilized an interaction in the relativistic Hartree approximation in which we have determined the coupling constants {sigma}-N and {omega}-N as a function of the density in order to reproduce the saturation curve obtained by a Dirac-Brueckner calculation. The results which we have obtained show that the longitudinal response function and the Coulomb sum generally overestimated when one utilizes the pure relativistic Hartree approximation, are here in good agreement with the experimental data for several nuclei

Caillon, J-C.; Labarsouque, J. [Centre d`Etudes Nucleaires, Bordeaux-1 Univ., 33 Gradignan (France)

1997-06-01

 
 
 
 
301

Genetic recombination. [Escherichia coli  

Energy Technology Data Exchange (ETDEWEB)

The molecular pathways of gene recombination are explored and compared in studies of the model organisms, Escherichia coli and phase lambda. In the discussion of data from these studies it seems that recombination varies with the genetic idiosyncrasies of the organism and may also vary within a single organism.

Stahl, F.W.

1987-02-01

302

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  

Energy Technology Data Exchange (ETDEWEB)

Leukotriene (LT)C/sub 4/-D/sub 4/, prostaglandin (PG)E/sub 2/, thromboxane B/sub 2/ (TXB), and 6-ketoprostaglandin F/sub 1/..cap alpha.. (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/sub 4/-D/sub 4/ after phenidone and AA-861 treatment, in PGE/sub 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.

Metcalf, L.E.; Korach, E.S.; Carnathan, G.W.; Shulruff, F.

1986-03-05

303

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

UK PubMed Central (United Kingdom)

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 AM; Hustedt N; Shimada K; Pike BL; Vogel M; Amsler P; Rubin SM; van Leeuwen F; Guénolé A; van Attikum H; Thomä NH; Gasser SM

2012-09-01

304

Randomized Phase II Trial of High-Dose Melatonin and Radiation Therapy for RPA Class 2 Patients With Brain Metastases (RTOG 0119)  

International Nuclear Information System (INIS)

Purpose: To determine if high-dose melatonin for Radiation Therapy Oncology Group (RTOG) recursive partitioning analysis (RPA) Class 2 patients with brain metastases improved survival over historical controls, and to determine if the time of day melatonin was given affected its toxicity or efficacy. RTOG 0119 was a phase II randomized trial for this group of patients. Methods and Materials: RTOG RPA Class 2 patients with brain metastases were randomized to 20 mg of melatonin, given either in the morning (8-9 AM) or in the evening (8-9 PM). All patients received radiation therapy (30 Gy in 10 fractions) in the afternoon. Melatonin was continued until neurologic deterioration or death. The primary endpoint was overall survival time. Neurologic deterioration, as reflected by the Mini-Mental Status Examination, was also measured. Results: Neither of the randomized groups had survival distributions that differed significantly from the historic controls of patients treated with whole-brain radiotherapy. The median survivals of the morning and evening melatonin treatments were 3.4 and 2.8 months, while the RTOG historical control survival was 4.1 months. Conclusions: High-dose melatonin did not show any beneficial effect in this group of patients.

2007-07-01

305

Integrity of the AID serine-38 phosphorylation site is critical for class switch recombination and somatic hypermutation in mice.  

UK PubMed Central (United Kingdom)

Activation-induced cytidine deaminase (AID) is a single-stranded (ss) DNA-specific cytidine deaminase that initiates Ig heavy chain (IgH) class switch recombination (CSR) and Ig somatic hypermutation (SHM) by deaminating cytidines within, respectively, IgH switch (S) regions and Ig variable region (V) exons. AID that is phosphorylated on serine residue 38 interacts with replication protein A (RPA), a ssDNA binding protein, to promote deamination of transcribed double-stranded DNA in vitro, which, along with other evidence, suggests that AID may similarly gain access to transcribed S regions and V exons in vivo. However, the physiological role of AID phosphorylation at serine residue 38 (S38), and even the requirement for the S38 residue, with respect to CSR or SHM has been debated. To address this issue, we used gene targeting to generate an endogenous mouse AID locus that produces AID in which S38 is substituted with alanine (AID(S38A)), a mutant form of AID that retains similar catalytic activity on ssDNA as WT AID (AID(WT)). B cells homozygous for the AID(S38A) mutation show substantially impaired CSR and SHM, correlating with inability of AID(S38A) to interact with endogenous RPA. Moreover, mice haploinsufficient for AID(S38A) have even more severely impaired CSR when compared with mice haploinsufficient for AID(WT), with CSR levels reduced to nearly background levels. These results unequivocally demonstrate that integrity of the AID S38 phosphorylation site is required for normal CSR and SHM in mice and strongly support a role for AID phosphorylation at S38 and RPA interaction in regulating CSR and SHM.

Cheng HL; Vuong BQ; Basu U; Franklin A; Schwer B; Astarita J; Phan RT; Datta A; Manis J; Alt FW; Chaudhuri J

2009-02-01

306

Integrity of the AID serine-38 phosphorylation site is critical for class switch recombination and somatic hypermutation in mice.  

Science.gov (United States)

Activation-induced cytidine deaminase (AID) is a single-stranded (ss) DNA-specific cytidine deaminase that initiates Ig heavy chain (IgH) class switch recombination (CSR) and Ig somatic hypermutation (SHM) by deaminating cytidines within, respectively, IgH switch (S) regions and Ig variable region (V) exons. AID that is phosphorylated on serine residue 38 interacts with replication protein A (RPA), a ssDNA binding protein, to promote deamination of transcribed double-stranded DNA in vitro, which, along with other evidence, suggests that AID may similarly gain access to transcribed S regions and V exons in vivo. However, the physiological role of AID phosphorylation at serine residue 38 (S38), and even the requirement for the S38 residue, with respect to CSR or SHM has been debated. To address this issue, we used gene targeting to generate an endogenous mouse AID locus that produces AID in which S38 is substituted with alanine (AID(S38A)), a mutant form of AID that retains similar catalytic activity on ssDNA as WT AID (AID(WT)). B cells homozygous for the AID(S38A) mutation show substantially impaired CSR and SHM, correlating with inability of AID(S38A) to interact with endogenous RPA. Moreover, mice haploinsufficient for AID(S38A) have even more severely impaired CSR when compared with mice haploinsufficient for AID(WT), with CSR levels reduced to nearly background levels. These results unequivocally demonstrate that integrity of the AID S38 phosphorylation site is required for normal CSR and SHM in mice and strongly support a role for AID phosphorylation at S38 and RPA interaction in regulating CSR and SHM. PMID:19196992

Cheng, Hwei-Ling; Vuong, Bao Q; Basu, Uttiya; Franklin, Andrew; Schwer, Bjoern; Astarita, Jillian; Phan, Ryan T; Datta, Abhishek; Manis, John; Alt, Frederick W; Chaudhuri, Jayanta

2009-02-05

307

Hepatitis B Vaccine (Recombinant)  

Science.gov (United States)

... Hepatitis B Vaccine (Recombinant). -. Products. Engerix-B; Recombivax HB. -. Contact FDA. (800) 835-4709. (301) 827-1800. ... More results from www.fda.gov/biologicsbloodvaccines/vaccines/approvedproducts

308

Surface recombination in semiconductors  

Energy Technology Data Exchange (ETDEWEB)

We propose two general criteria for a surface defect state to act as an efficient, nonradiative recombination center. The first is that the thermal ionization energy should not deviate from the mid-gap energy by more than the relaxation energy of the defect, In this case the activation energy for the recombination is given by the barrier for the capture of the first carrier, whereas the second carrier is captured athermally. The second citerion is related to the position of the average dangling bond energy relative to the band edges. If, as in the cases of InP or InAs, it is located close to a band edge, a low surface recombination velocity is expected. However a much faster recombination is predicated and experimentally observed in the materials with the average dangling bond energy located close to the mid-gap. The relevance of these criteria for the novel wide-gap optoelectronic materials is discussed.

Langer, J.M. [Polish Academy of Sciences, Warsaw (Poland). Inst. of Physics; Walukiewicz, W. [Lawrence Berkeley Lab., CA (United States)

1995-07-01

309

Dielectronic recombination in Li+  

International Nuclear Information System (INIS)

[en] Dielectronic recombination has been investigated for ground-state Li+(1s2) and metastable Li+(1s2s) ions. Absolute recombination rates for the convoluted sum of these configurations are obtained and compared with theoretical calculations. Also, the effect of the space charge on the energy of the observed resonances is investigated as well as the effect of trapped residual ions in reducing the effect of the space charge. copyright 1998 The American Physical Society

1998-01-01

310

Activated recombinant adenovirus proteinases  

Energy Technology Data Exchange (ETDEWEB)

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

Anderson, Carl W. (Stony Brook, NY); Mangel, Walter F. (Shoreham, NY)

1999-08-10

311

Activated recombinant adenovirus proteinases  

Science.gov (United States)

This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

Anderson, C.W.; Mangel, W.F.

1999-08-10

312

Engineering Chinese hamster ovary (CHO) cells for producing recombinant proteins with simple glycoforms by zinc-finger nuclease (ZFN)-mediated gene knockout of mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (Mgat1).  

Science.gov (United States)

While complex N-linked glycoforms are often desired in biotherapeutic protein production, proteins with simple, homogeneous glycan structure have implications for X-ray crystallography and for recombinant therapeutics targeted to the mannose receptor of antigen presenting cells. Mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (Mgat1, also called GnTI) adds N-acetylglucosamine to the Man5GlcNAc2 (Man5) N-glycan structure as part of complex N-glycan synthesis. Here, we report the use of zinc-finger nuclease (ZFN) genome editing technology to create Mgat1 disrupted Chinese hamster ovary (CHO) cell lines. These cell lines allow for the production of recombinant proteins with Man5 as the predominant N-linked glycosylation species. This method provides advantages over previously reported methods to create Mgat1-deficient cell lines. The use of ZFN-based genome editing eliminates potential regulatory concerns associated with random chemical mutagenesis, while retaining the robust growth and productivity characteristics of the parental cell lines. These Mgat1 disrupted cell lines may be used to produce mannose receptor-targeted therapeutic proteins. Cell line generation work can be performed in both Mgat1 disrupted and wild-type host cell lines to conduct X-ray crystallography studies of protein therapeutics in the same cell line used for production. PMID:23777858

Sealover, Natalie R; Davis, Angela M; Brooks, Jeanne K; George, Henry J; Kayser, Kevin J; Lin, Nan

2013-06-15

313

Engineering Chinese hamster ovary (CHO) cells for producing recombinant proteins with simple glycoforms by zinc-finger nuclease (ZFN)-mediated gene knockout of mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (Mgat1).  

UK PubMed Central (United Kingdom)

While complex N-linked glycoforms are often desired in biotherapeutic protein production, proteins with simple, homogeneous glycan structure have implications for X-ray crystallography and for recombinant therapeutics targeted to the mannose receptor of antigen presenting cells. Mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase (Mgat1, also called GnTI) adds N-acetylglucosamine to the Man5GlcNAc2 (Man5) N-glycan structure as part of complex N-glycan synthesis. Here, we report the use of zinc-finger nuclease (ZFN) genome editing technology to create Mgat1 disrupted Chinese hamster ovary (CHO) cell lines. These cell lines allow for the production of recombinant proteins with Man5 as the predominant N-linked glycosylation species. This method provides advantages over previously reported methods to create Mgat1-deficient cell lines. The use of ZFN-based genome editing eliminates potential regulatory concerns associated with random chemical mutagenesis, while retaining the robust growth and productivity characteristics of the parental cell lines. These Mgat1 disrupted cell lines may be used to produce mannose receptor-targeted therapeutic proteins. Cell line generation work can be performed in both Mgat1 disrupted and wild-type host cell lines to conduct X-ray crystallography studies of protein therapeutics in the same cell line used for production.

Sealover NR; Davis AM; Brooks JK; George HJ; Kayser KJ; Lin N

2013-08-01

314

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)C4-D4, prostaglandin (PG)E2, thromboxane B2 (TXB), and 6-ketoprostaglandin F1? (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 4-D4 after phenidone and AA-861 treatment, in PGE2 and 6-KP after phenidone and indomethacin treatment and in TXB after dazoxiben and indomethacin treatment. Dazoxiben significantly (p

1986-03-05

315

Regulation of Meiotic Recombination  

Energy Technology Data Exchange (ETDEWEB)

Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system for assaying recombination using tetrad analysis in a higher eukaryotic system (6). This system enabled the measurement of the frequency and distribution of recombination events at a genome wide level in wild type Arabidopsis (7), construction of genetic linkage maps which include positions for each centromere (8), and modeling of the strength and pattern of interference (9). This proposal extends the use of tetrad analysis in Arabidopsis by using it as the basis for assessing the phenotypes of mutants in genes important for recombination and the regulation of crossover interference and performing a novel genetic screen. In addition to broadening our knowledge of a classic genetic problem - the regulation of recombination by crossover interference - this proposal also provides broader impact by: generating pedagogical tools for use in hands-on classroom experience with genetics, building interdisciplinary collegial partnerships, and creating a platform for participation by junior scientists from underrepresented groups. There are three specific aims: (1) Isolate mutants in Arabidopsis MUS81 homologs using T-DNA and TILLING (2) Characterize recombination levels and interference in mus81 mutants (3) Execute a novel genetic screen, based on tetrad analysis, for genes that regulate meiotic recombination

Gregory p. Copenhaver

2011-11-09

316

Monitoring DNA recombination initiated by HO endonuclease.  

UK PubMed Central (United Kingdom)

DNA double-strand breaks (DSBs) have proven to be very potent initiators of recombination in yeast and other organisms. A single, site-specific DSB initiates homologous DNA repair events such as gene conversion, break-induced replication, and single-strand annealing, as well as nonhomologous end joining, microhomology-mediated end joining, and new telomere addition. When repair is either delayed or prevented, a single DSB can trigger checkpoint-mediated cell cycle arrest. In budding yeast, expressing the HO endonuclease under the control of a galactose-inducible promoter has been instrumental in the study of these processes by providing us a way to synchronously induce a DSB at a unique site in vivo. We describe how the HO endonuclease has been used to study the recombination events in mating-type (MAT) switching. Southern blots provide an overview of the process by allowing one to examine the formation of the DSB, DNA degradation at the break, and formation of the product. Denaturing gels and slot blots as well as PCR have provided important tools to follow the progression of resection in wild-type and mutant cells. PCR has also been important in allowing us to follow the kinetics of certain recombination intermediates such as the initiation of repair DNA synthesis or the removal of nonhomologous Y sequences during MAT switching. Finally chromatin immunoprecipitation has been used to follow the recruitment of key proteins to the DSB and in subsequent steps in DSB repair.

Sugawara N; Haber JE

2012-01-01

317

Cre-loxP-Mediated Recombination between the SIL and SCL Genes Leads to a Block in T-Cell Development at the CD4-CD8- to CD4+CD8+ Transition1  

Science.gov (United States)

In the most common form of stem cell leukemia (SCL) gene rearrangement, an interstitial deletion of 82 kb brings SCL under the control of regulatory elements that normally govern expression of the ubiquitously expressed SCL interrupting locus (SIL) gene, which is located directly upstream of SCL. To investigate the effect of this fusion in a mouse model, a bacterial artificial chromosome (BAC) clone containing both human SIL and SCL genes was isolated, and loxP sites were inserted into intron 1 of both the SIL and SCL genes, corresponding to the sites at which recombination occurs in human T-cell acute lymphocytic leukemia patients. This BAC clone was used to generate transgenic SILloxloxSCL mice. These transgenic mice were subsequently bred to Lck-Cre mice that express the Cre recombinase specifically in the thymus. The BAC transgene was recombined between the two loxP sites in over 50% of the thymocytes from SILloxloxSCL/Cre double-transgenic mice, bringing the SCL gene under the direct control of SIL regulatory elements. Aberrant SCL gene expression in the thymus was verified by reverse transcription-polymerase chain reaction. Using FACS analysis, we found that mice carrying both SILloxloxSCL and Cre transgenes have increased CD4-/CD8- thymocytes compared with transgene-negative mice. In the spleen, these transgenic mice show a marked reduction in the number of mature CD4+ or CD8+ cells. These results demonstrate that conditional activation of SCL under control of SIL regulatory elements can impair normal T-cell development.

Yue Cheng; Zhenhua Zhang; Slape, Christopher; Aplan, Peter D.

2007-01-01

318

Recombination Phenotypes of Escherichia coli greA Mutants  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background The elongation factor GreA binds to RNA polymerase and modulates transcriptional pausing. Some recent research suggests that the primary role of GreA may not be to regulate gene expression, but rather, to promote the progression of replication forks which collide with RNA polymerase, and which might otherwise collapse. Replication fork collapse is known to generate dsDNA breaks, which can be recombinogenic. It follows that GreA malfunction could have consequences affecting homologous recombination. Results Escherichia coli mutants bearing substitutions of the active site acidic residues of the transcription elongation factor GreA, D41N and E44K, were isolated as suppressors of growth inhibition by a toxic variant of the bacteriophage lambda Red-beta recombination protein. These mutants, as well as a D41A greA mutant and a greA deletion, were tested for proficiency in recombination events. The mutations were found to increase the efficiency of RecA-RecBCD-mediated and RecA-Red-mediated recombination, which are replication-independent, and to decrease the efficiency of replication-dependent Red-mediated recombination. Conclusion These observations provide new evidence for a role of GreA in resolving conflicts between replication and transcription.

Poteete Anthony R

2011-01-01

319

Recombinant protein production by in vivo polymer inclusion display.  

UK PubMed Central (United Kingdom)

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.

Grage K; Peters V; Rehm BH

2011-09-01

320

Recombinant protein production by in vivo polymer inclusion display.  

Science.gov (United States)

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. PMID:21803888

Grage, Katrin; Peters, Verena; Rehm, Bernd H A

2011-07-29

 
 
 
 
321

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

322

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

323

The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. Methods We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. Results The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene expression in the mice livers, which did not produce the therapeutic effects on alteration the lipid levels or the inhibition of atherosclerosis development. In contrast, the ribozyme RB15 RNA mediated by scAAV2-TTR-RB15 vector was expressed immediately at day-1 after transduction in HepG2 cells. The apoB mRNA levels were decreased 47% (p = 0.001), compared to the control vector scAAV2-TTR-RB15-mutant. Conclusion This study provided evidence that the rAAV2 single-strand vector mediated a prolonged but not efficient transduction in mouse liver. However, the scAAV2 double-strand vector mediated a rapid and efficient gene expression in liver cells. This strategy using scAAV2 vectors represents a better approach to express small molecules such as ribozyme.

Zhong Shumei; Sun Shihua; Teng Ba-Bie

2004-01-01

324

Nonconservative recombination in Escherichia coli.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Homologous recombination between two duplex DNA molecules might result in two duplex DNA molecules (conservative) or, alternatively, it might result in only one recombinant duplex DNA molecule (nonconservative). Here we present evidence that the mode of homologous recombination is nonconservative in...

Takahashi, N K; Yamamoto, K; Kitamura, Y; Luo, S Q; Yoshikura, H; Kobayashi, I

325

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.

Veronika Altmannová; Peter Kolesár; Lumír Krej?í

2012-01-01

326

Recombination device. Rekombinationsvorrichtung  

Energy Technology Data Exchange (ETDEWEB)

Because of the different compositions of battery gases, recombination is usually incomplete and the electrolyte liquid is lost. According to the invention, this is prevented by converting excess hydrogen with oxygen sucked in from the air. The incoming gas pipe on the filling duct of the battery is provided with an internal gas intake pipe (diameter about 2 mm) and an external transition pipe. This transition pipe has suction holes for air, so that the incoming pipe works like a Bunsen burner. The gas mixture is taken inside an opening funnel to the hydrophobic catalyst via a gas labyrinth. The ballast gases can escape through the holes, the water condenses on the inside of the recombinator case and flows back into the cell via the transition pipe. It passes a porous body between the transition and incoming gas pipes, which blocks the way for the gas flowing upwards.

Winsel, A.; Ledjeff, K.; Bopp, B.U.

1981-10-01

327

Site directed recombination  

Energy Technology Data Exchange (ETDEWEB)

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

Jurka, Jerzy W. (Los Altos, CA)

1997-01-01

328

Relativistic dielectronic recombination theory  

Energy Technology Data Exchange (ETDEWEB)

Dielectronic recombination (DR) is an inverse Auger process in which a free electron is captured by a recombining ion to form a doubly excited autoionizing state. The subsequent decay of the autoionizing state to a stabilized bound state by emitting photons completes the recombination process. DR is an important recombination process for high temperature plasmas. It can affect the ionization balance and level kinetics of the hot plasmas. In addition, the dielectronic satellite lines observed in the emission spectra are frequently used as plasmas diagnostic tools. In the past decade, intense theoretical and experimental studies on the DR process have been carried out. Most of the earlier theoretical calculations on the DR rate coefficients were done either by using a term average approximation or in LS coupling without including the effects of relativity and configuration interaction. The early experimental investigations were concentrated on few times ionized low-Z ions. Recently, the development of electron beam ion trap (EBIT), electron beam ion source (EBIS) and heavy ion storage ring has become possible to produce very highly-charged heavy ions (e.g. U{sup 82+} and Xe{sup 53+})and to study the interaction between electrons and these ions. For highly-charged heavy ions, one excepts that the nonrelativistic method would be inadequate and a relativistic treatment is necessary. To meet this challenge we have developed a relativistic package based on the multiconfiguration Dirac-Fock method and have carried out systematic relativistic calculations of DR cross sections and rate coefficients and resonant transfer and excitation cross sections in ion-atom collisions. In this paper, we will briefly discuss the relativistic calculations of atomic structure and transition rates and will focus for attention on the effects of relativity and intermediate coupling on the DR cross sections and rate coefficients.

Chen, Mau Hsiung

1991-11-01

329

Dielectronic recombination theory  

International Nuclear Information System (INIS)

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

1991-01-01

330

Schematic microscopic approach to the description of M1 transitions between mixed-symmetry and fully symmetric collective states in ?-soft nuclei based on RPA-IBM boson mapping  

Science.gov (United States)

A schematic microscopic method is developed to calculate the M1 transition probabilities between the mixed-symmetry and the fully symmetric states in ?-soft nuclei. The method is based on the random-phase approximation-interacting boson model (RPA-IBM) boson mapping of the most collective isoscalar boson. All other boson modes with higher excitation energies, including the mixed-symmetry boson, are described in the framework of RPA. As an example the M1 transition probabilities are calculated for the 124-134Xe isotopes and compared with the experimental data. The results agree well with the data for the ratio B(M1;1ms+?22+)/B(M1;1ms+?01+). However, the calculated ratio B(M1;2ms+?21+)/B(M1;1ms+?01+) shows a significantly weaker dependence on the mass number than the experimental data.

Jolos, R. V.; Pietralla, N.; Shirikova, N. Yu.; Voronov, V. V.

2011-07-01

331

Schematic microscopic approach to the description of M1 transitions between mixed-symmetry and fully symmetric collective states in ?-soft nuclei based on RPA-IBM boson mapping  

International Nuclear Information System (INIS)

A schematic microscopic method is developed to calculate the M1 transition probabilities between the mixed-symmetry and the fully symmetric states in ?-soft nuclei. The method is based on the random-phase approximation-interacting boson model (RPA-IBM) boson mapping of the most collective isoscalar boson. All other boson modes with higher excitation energies, including the mixed-symmetry boson, are described in the framework of RPA. As an example the M1 transition probabilities are calculated for the 124-134Xe isotopes and compared with the experimental data. The results agree well with the data for the ratio B(M1;1ms+?22+)/B(M1;1ms+?01+). However, the calculated ratio B(M1;2ms+?21+)/B(M1;1ms+?01+) shows a significantly weaker dependence on the mass number than the experimental data.

2011-01-01

332

Immunization of BALB/c mice with recombinant simian virus 40 large tumor antigen induces antibody-dependent cell-mediated cytotoxicity against simian virus 40-transformed cells. An antibody-based mechanism for tumor immunity.  

UK PubMed Central (United Kingdom)

In this report, we describe an immune mechanism that seems to play a role in tumor immunity in BALB/c mice challenged with SV40-transformed cells. This immune mechanism was induced by immunization of mice with rSV40 large tumor Ag (T-Ag). After rSV40 T-Ag immunization, BALB/c mice were protected from a lethal tumor challenge with syngeneic SV40-transformed cells (mKSA). Specifically, we examined CTL activity, NK activity, complement-dependent cytotoxicity, and Ab-dependent cell-mediated cytotoxicity (ADCC) in rSV40 T-Ag-immunized mice that were protected from a subsequent lethal tumor challenge. Immune splenocytes from surviving animals that were subsequently primed in vitro with rSV40 T-Ag demonstrated little to no SV40 T-Ag-specific CTL activity. In addition, natural immunity, as assessed by NK lytic activity, was comparable to that of unimmunized animals. Similarly, minimal significant complement-dependent cytotoxicity of radiolabeled mKSA cells was demonstrated by SV40 T-Ag-specific serum Abs. However, SV40 T-Ag-specific Abs using peritoneal exudate cells as effectors, exhibited significant levels of ADCC against radiolabeled mKSA target cells. Together, these studies indicate that rSV40 T-Ag immunization elicited ADCC that may represent a potential humoral immune mechanism involved in the protection of BALB/c mice from a lethal challenge with syngeneic SV40-transformed cells.

Bright RK; Shearer MH; Kennedy RC

1994-09-01

333

Dielectronic recombination theory  

Energy Technology Data Exchange (ETDEWEB)

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

LaGattuta, K.J.

1991-01-01

334

Dielectronic recombination theory  

Energy Technology Data Exchange (ETDEWEB)

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

LaGattuta, K.J.

1991-12-31

335

Superhelical duplex destabilization and the recombination position effect.  

UK PubMed Central (United Kingdom)

The susceptibility to recombination of a plasmid inserted into a chromosome varies with its genomic position. This recombination position effect is known to correlate with the average G+C content of the flanking sequences. Here we propose that this effect could be mediated by changes in the susceptibility to superhelical duplex destabilization that would occur. We use standard nonparametric statistical tests, regression analysis and principal component analysis to identify statistically significant differences in the destabilization profiles calculated for the plasmid in different contexts, and correlate the results with their measured recombination rates. We show that the flanking sequences significantly affect the free energy of denaturation at specific sites interior to the plasmid. These changes correlate well with experimentally measured variations of the recombination rates within the plasmid. This correlation of recombination rate with superhelical destabilization properties of the inserted plasmid DNA is stronger than that with average G+C content of the flanking sequences. This model suggests a possible mechanism by which flanking sequence base composition, which is not itself a context-dependent attribute, can affect recombination rates at positions within the plasmid.

Sershen CL; Mell JC; Madden SM; Benham CJ

2011-01-01

336

The construction of a new mobile recombineering system of pYM--Red  

UK PubMed Central (United Kingdom)

Recombineering is a new developed genetic engineering technology in the past few years. A new recombineering system named pYM-Red was constructed by gap-repair, that is a technology called in vivo cloning. Linear PCR fragments that amplified from low copy plasmid pACYC184 were used as gene targeting vector. The length of subcloned DNA sequence including Red gene and a series regulatory sequences are about 6.7 kb. The biology function of Red gene in pYM-Red was tested by gene replacement (galk<>kan) of W3110 chromosome. Factors that effect recombination efficiency were precisely confirmed. When induced 10 min at 42 ? and using 300 ng linear DNA fragment as targeting molecules, the efficiency of pYM-Red mediated recombination can reach one positive recombination clone per four thousands electroporation survived cells, it is 5--6 folds higher than pBR322-Red and pKD46 recombination system.

Yu Mei; Zhou Jianguang; Chen Wei; Li Shanhu; Huang Cuifen

2005-01-01

337

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; Denisa Mitrut

2012-01-01

338

Expression of recombinant antibodies.  

UK PubMed Central (United Kingdom)

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

Frenzel A; Hust M; Schirrmann T

2013-01-01

339

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.

Kang Bum; You Hong; Bandyopadhyay Sarmistha; Agrawal Nalini; Melchert Russell B; Basnakian Alexei G; Liu Yong; Hermonat Paul L

2009-01-01

340

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

 
 
 
 
341

The recombination of genetic material  

Energy Technology Data Exchange (ETDEWEB)

Genetic recombination is the major mechanism by which new arrangements of genetic elements are produced in all living organisms, from the simplest bacterial viruses to humans. This volume presents an overview of the types of recombination found in prokaryotes and eukaryotes.

Low, K.B.

1988-01-01

342

Genetic recombination and molecular evolution  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Reduced rates of genetic recombination are often associated with reduced genetic variability and levels of adaptation. Several different evolutionary processes, collectively known as Hill–Robertson (HR) effects, have been proposed as causes of these correlates of recombination. Here, we use DNA sequ...

Gordo, I.; Charlesworth, B.; Kaiser, V.B.; Betancourt, A.J.

343

Cell cycle-dependent formation of Cdc45-Claspin complexes in human cells is compromized by UV-mediated DNA damage.  

UK PubMed Central (United Kingdom)

UNLABELLED: The replication factor Cdc45 has essential functions in the initiation and elongation steps of eukaryotic DNA replication and plays an important role in the intra-S-phase checkpoint. Its interactions with other replication proteins during the cell cycle and after intra-S-phase checkpoint activation are only partially characterized. In the present study, we show that the C terminal part of Cdc45 may mediate its interactions with Claspin. The interactions of human Cdc45 with the three replication factors Claspin, replication protein A and DNA polymerase ? are maximal during the S phase. Following UVC-induced DNA damage, Cdc45-Claspin complex formation is reduced, whereas the binding of Cdc45 to replication protein A is not affected. We also show that treatment of cells with UCN-01 and phosphatidylinositol 3-kinase-like kinase inhibitors does not rescue the UV-induced destabilization of Cdc45-Claspin interactions, suggesting that the loss of the interaction between Cdc45 and Claspin occurs upstream of ataxia telangiectasia and Rad 3-related activation in the intra-S-phase checkpoint. STRUCTURED DIGITAL ABSTRACT: Clapsin physically interacts with Cdc45 by anti bait coimmunoprecipitation (View interaction) Cdc45 physically interacts with RPA32, Clapsin and p125 Pol delta by pull down (View interaction) Cdc45 physically interacts with RPA32 by pull down (View interaction) Cdc45 physically interacts with Clapsin by pull down (View interaction) Cdc45 physically interacts with Clapsin and RPA32 by pull down (View interaction) RPA32 physically interacts with Cdc45 by anti bait coimmunoprecipitation (View interaction).

Broderick R; Rainey MD; Santocanale C; Nasheuer HP

2013-10-01

344

Dielectronic recombination for Li{sup +} ions  

Energy Technology Data Exchange (ETDEWEB)

Dielectronic recombination (DR) was investigated for Li{sup +} + e{sup {minus}} collisions using the electron cooler at the Indiana University Cyclotron Facility (IUCF). For Li{sup +} (1s{sup 2}) ions, DR is expected to occur for relative energies E between 50-60 eV, and for metastable Li{sup +} (1s2s) DR (involving 2s {r_arrow} 2p transitions) should occur for E{sup rel} < 1 eV. Also, for E{sub rel}, near zero, radiative recombination (RR) (inverse photoelectric effect) is expected. This work is an extension of previous work at IUCF for He{sup +} ions. These light He{sup +} and Li{sup +} ions pose stringent tests of DR theory because the electron-electron interaction, which mediates DR, is stronger compared to electron-nucleus interactions than it is for heavier ions. Additionally, the electron coupling in two-electron Li{sup +} gives rise to angular momentum configurations different from those in He{sup +}. In recent measurements at IUCF, the authors have observed DR for both ground-state and metastable Li{sup +} ions; analysis is currently in progress.

Zavodszky, P.A. [Western Michigan Univ., Kalamazoo, MI (United States)

1994-12-31

345

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

International Nuclear Information System (INIS)

[en] 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

346

Site-specific recombination at dif by Haemophilus influenzae XerC.  

UK PubMed Central (United Kingdom)

Xer site-specific recombination at the Escherichia coli chromosomal site dif converts chromosomal dimers to monomers, thereby allowing chromosome segregation during cell division. dif is located in the replication terminus region and binds the E. coli site-specific recombinases EcoXerC and EcoXerD. The Haemophilus influenzae Xer homologues, HinXerC and HinXerD, bind E. coli dif and exchange strands of dif Holliday junctions in vitro. Supercoiled dif sites are not recombined by EcoXerC and EcoXerD in vitro, possibly as a consequence of a regulatory process, which ensures that in vivo recombination at dif is confined to cells that can initiate cell division and contain dimeric chromosomes. In contrast, the combined action of HinXerC and EcoXerD supports in vitro recombination between supercoiled dif sites, thereby overcoming the barrier to dif recombination exhibited by EcoXerC and EcoXerD. The recombination products are catenated and knotted molecules, consistent with recombination occurring with synaptic complexes that have entrapped variable numbers of negative supercoils. Use of catalytically inactive recombinases provides support for a recombination pathway in which HinXerC-mediated strand exchange between directly repeated duplex dif sites generates a Holliday junction intermediate that is resolved by EcoXerD to catenated products. These can undergo a second recombination reaction to generate odd-noded knots.

Neilson L; Blakely G; Sherratt DJ

1999-02-01

347

Comparisons of the RPA, SCRPA, Tamm--Dancoff, and full CI methods by analysis of their transition density matrices, oscillator strengths, and energy moments of oscillator strengths for the electric dipole transitions from the ground state of the B+ ion (frozen K-shell model)  

International Nuclear Information System (INIS)

The RPA, SCRPA, Tamm-Dancoff and full CI methods are compared by analyzing their transition density matrices, oscillator strengths, and energy moments of oscillator strengths for the 1S/sub ground/--1P/sub odd/ transitions of the 4-electron B+ ion in the frozen K-shell approximation. It is found that the RPA gives transition density matrices that are aligned nearly as well as possible along those of the full CI, but have vector lengths that are significantly too long. The corresponding transition energies are significantly too small. These errors compensate to give oscillator strengths for the dominant transition that, for all forms of the oscillator strength, are within 1.6 percent of the corresponding full CI values. The SCRPA gives better transition density matrices than the RPA, but poorer oscillator strengths. The Tamm-Dancoff approximation gives very good values for the mixed length--velocity form of the oscillator strength. The RPA gives a static electric dipole polarizability that is nearly 20 percent larger than that of the full CI. The SCRPA gives a value 15 percent smaller than (and the Tamm-Dancoff approximation gives a mixed length--velocity value that is 11 percent larger than) that of the full CI. Other energy moments of oscillator strengths are also reported. Certain other approximations related to the RPA and the SCRPA are reported as well

1978-01-01

348

Recombinant hormones in osteoporosis.  

UK PubMed Central (United Kingdom)

INTRODUCTION: For the last 10 years, bone anabolic therapy with the recombinant human parathyroid hormone (rhPTH) analogue, teriparatide (rhPTH[1 - 34]), or full-length rhPTH(1 - 84) has been an option in the treatment of osteoporosis. Both drugs are given as a daily subcutaneous injection. In the USA, only teriparatide is marketed. AREAS COVERED: Mechanisms of action by which rhPTH induces bone anabolic effects includes changes in bone remodeling, geometry and mineral density. Data from randomized controlled trials on anti-fracture efficacy are reviewed as well as results from a number of recent studies on administration less than once-a-day or intermittent-/cyclic-therapy. Treatment effects are compared with those of anti-resorptive agents. EXPERT OPINION: In terms of anti-fracture efficacy, treatment with rhPTH is not superior to treatment with potent anti-resorptive agents. However, while the process by which osteoporosis emerges is arrested in response to anti-resorptives, rhPTH acts as a bone anabolic with reversal of the process. Although this mechanism of action seems favorable, the use of rhPTH is limited by a much higher cost than that of anti-resorptive agents. As long as a superior anti-fracture efficacy has not been proven, rhPTH should be confined to patients with severe spinal osteoporosis, including patients in whom treatment with an anti-resorptive has failed.

Rejnmark L

2013-08-01

349

Testing for recombinant erythropoietin.  

UK PubMed Central (United Kingdom)

Erythropoietin (Epo) is a glycoprotein hormone that promotes the production of red blood cells. Recombinant human Epo (rhEpo) is illicitly used to improve performance in endurance sports. Doping in sports is discouraged by the screening of athletes for rhEpo. Both direct tests (indicating the presence of exogeneous Epo isoforms) and indirect tests (indicating hematological changes induced by exogenous Epo administration) can be used for Epo detection. At present, the test adopted by the World Anti Doping Agency is based on a combination of isoelectric focusing and double immunoblotting, and distinguishes between endogenous and rhEpo. However, the adopted monoclonal anti-Epo antibodies are not monospecific. Therefore, the test can occasionally lead to the false-positive detection of rhEpo (epoetin-beta) in post-exercise, protein-rich urine, or in case of contamination of the sample with microorganisms. An improved preanalytical care may counteract a lot of these problems. Adaptation of the criteria may be helpful to further refine direct Epo testing. Indirect tests have the disadvantage that they require blood instead of urine samples, but they can be applied to detect a broader range of performance improving techniques which are illicitly used in sports.

Delanghe JR; Bollen M; Beullens M

2008-03-01

350

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

2009-01-01

351

Recombination system for storage batteries  

Energy Technology Data Exchange (ETDEWEB)

A recombination system for catalytic oxidation of hydrogen in storage battery gases includes a gas supply duct which makes it possible for the combustible gas flowing through it to aspirate from the ambient the necessary combustion air, following the principle of a bunsen burner, and to entrain it to the recombination catalyst. In case of over-supply of gas, an acid separator positioned in the gas supply pipe counteracts the gas aspiration by means of its flow impedance and thereby makes the recombination system safe from overload. It can also be connected following a conventional recombiner, thereby increasing its effectiveness, by receiving the excess hydrogen from same and reacting it with the aid of the air aspiration.

Bopp, B.; Ledjeff, K.; Winsel, A.

1983-03-29

352

Recombination device for storage batteries  

Energy Technology Data Exchange (ETDEWEB)

A recombination device including a gas-tight enclosure connected to receive he discharge gases from a rechargeable storage battery. Catalytic material for the recombination of hydrogen and oxygen to form water is supported within the enclosure. The enclosure is sealed from the atmosphere by a liquid seal including two vertical chambers interconnected with an inverted U-shaped overflow tube. The first chamber is connected at its upper portion to the enclosure and the second chamber communicates at its upper portion with the atmosphere. If the pressure within the enclosure differs as overpressure or vacuum by more than the liquid level, the liquid is forced into one of the two chambers and the overpressure is vented or the vacuum is relieved. The recombination device also includes means for returning recombined liquid to the battery and for absorbing metal hydrides.

Kraft, Helmut (Liederbach, DE); Ledjeff, Konstantin (Bad Krozingen, DE)

1985-01-01

353

Recombinant DNA means and method  

Energy Technology Data Exchange (ETDEWEB)

This patent describes a transformed living cell selected from the group consisting of fungi, yeast and bacteria, and containing genetic material derived from recombinant DNA material and coding for bovine rennin.

Alford, B.L.; Mao, J.I.; Moir, D.T.; Taunton-Rigby, A.; Vovis, G.F.

1987-05-19

354

Hydrogen recombiner development at AECL  

Energy Technology Data Exchange (ETDEWEB)

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 carbonmonoxide, 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)

Dewit, W.A.; Koroll, G.W.; Loesel Sitar, J. [Atomic Energy of Canada Limited, Pinawa, Manitoba (Canada); Graham, W.R.C. [Atomic Energy of Canada Limited, Chalk River, Ontario (Canada)

1997-03-01

355

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

356

Plasmid recombination in Haemophilus influenzae  

Energy Technology Data Exchange (ETDEWEB)

DNA recombination in exponential phase and competent Haemophilus influenzae was measured by an electron microscopic assay that relies on the conversion of plasmid RSF0885 monomers into multimeric forms. Dimer circles were present at a frequency of 2% in plasmid preparations from competent Rd (wild-type) cells; multimers were present at a frequency of 0.2% in preparations from exponential phase cells. Thus, plasmid recombination was stimulated in competent cells. Multimer formation occurred efficiently in cells of the transformation defective mutant rec2, implying that the rec2 gene product is not required for plasmid recombination. However, the absence of multimer plasmids in preparations from competent cells of the transformation defective mutant rec1 suggests that the rec1 gene product is required. Digestion of purified plasmids with restriction endonuclease PvuII, which makes a single cut in the monomer, revealed the presence of recombination intermediates composed of two linear plasmids joined to form two pairs of arms resembling the Greek letter chi. Length measurements of these arms taken from a population of recombination intermediates gave evidence that the plasmids were joined at sites of homology. The distributions of individual DNA strands, at the intersections of the four arms, could be resolved in some recombination intermediates and were of two types. The first type of junction appeared as a single-stranded arm appended to each corner. The second type of junction consisted of a single strand of DNA linking the two linear plasmids at a site of homology. The single-stranded linker was frequently situated at the edge of a short gap on one of the plasmids in the pair. The fine structures of the recombinational joints have been interpreted in terms of previously proposed models of recombination.

McCarthy, D.

1982-01-01

357

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

358

Ethanol production by recombinant hosts  

Energy Technology Data Exchange (ETDEWEB)

Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

Fowler, David E. (Gainesville, FL); Horton, Philip G. (Gainesville, FL); Ben-Bassat, Arie (Gainesville, FL)

1996-01-01

359

Ethanol production by recombinant hosts  

Energy Technology Data Exchange (ETDEWEB)

Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

Ingram, Lonnie O. (Gainesville, FL); Beall, David S. (Gainesville, FL); Burchhardt, Gerhard F. H. (Gainesville, FL); Guimaraes, Walter V. (Vicosa, BR); Ohta, Kazuyoshi (Miyazaki, JP); Wood, Brent E. (Gainesville, FL); Shanmugam, Keelnatham T. (Gainesville, FL)

1995-01-01

360

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

 
 
 
 
361

Investigations for designing catalytic recombiners  

International Nuclear Information System (INIS)

[en] In case of a severe accident in pressurised water reactors (PWR) a high amount of hydrogen up to about 20,000 m3 might be generated and released into the containments. The mixture consisting of hydrogen and oxygen may either burn or detonate, if ignited. In case of detonation the generated shock wave may endanger the components of the plant or the plant itself. Consequently, effective removal of hydrogen is required. The fact that hydrogen and oxygen react exo-thermally on catalytically acting surfaces already at low temperatures generating steam and heat is made use of in catalytic recombiners. They consist of substrates coated with catalyst (mainly platinum or palladium) which are arranged inside a casing. Being passively acting measures, recombiners do not need any additional energy supply. Experimental investigations on catalytic hydrogen recombination are conducted at FZJ (Forschungszentrum Juelich) using three test facilities. The results yield insight in the development potential of contemporary recombiner systems as well as of innovative systems. Detailed investigations on a recombiner section show strong temperature gradients over the surface of a catalytically coated sample. Dependent on the flow velocity, ignition temperature may be reached at the leading edge already at an inlet hydrogen concentration of about 5 vol.-%. The thermal strain of the substrate leads to considerable detachment of catalyst particles probably causing unintended ignition of the flammable mixture. Temperature peaks can be prevented effectively by leaving the first part of the plate uncoated. In order to avoid overheating of the catalyst elements of a recombiner even at high hydrogen concentrations a modular system of porous substrates is proposed. The metallic substrates are coated with platinum at low catalyst densities thus limiting the activity of the single specimen. A modular arrangement of these elements provides high recombination rates over a large hydrogen concentration range without igniting the mixture

2001-01-01

362

Recombination drives vertebrate genome contraction.  

UK PubMed Central (United Kingdom)

Selective and/or neutral processes may govern variation in DNA content and, ultimately, genome size. The observation in several organisms of a negative correlation between recombination rate and intron size could be compatible with a neutral model in which recombination is mutagenic for length changes. We used whole-genome data on small insertions and deletions within transposable elements from chicken and zebra finch to demonstrate clear links between recombination rate and a number of attributes of reduced DNA content. Recombination rate was negatively correlated with the length of introns, transposable elements, and intergenic spacer and with the rate of short insertions. Importantly, it was positively correlated with gene density, the rate of short deletions, the deletion bias, and the net change in sequence length. All these observations point at a pattern of more condensed genome structure in regions of high recombination. Based on the observed rates of small insertions and deletions and assuming that these rates are representative for the whole genome, we estimate that the genome of the most recent common ancestor of birds and lizards has lost nearly 20% of its DNA content up until the present. Expansion of transposable elements can counteract the effect of deletions in an equilibrium mutation model; however, since the activity of transposable elements has been low in the avian lineage, the deletion bias is likely to have had a significant effect on genome size evolution in dinosaurs and birds, contributing to the maintenance of a small genome. We also demonstrate that most of the observed correlations between recombination rate and genome contraction parameters are seen in the human genome, including for segregating indel polymorphisms. Our data are compatible with a neutral model in which recombination drives vertebrate genome size evolution and gives no direct support for a role of natural selection in this process.

Nam K; Ellegren H

2012-01-01

363

The Slx5-Slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination.  

DEFF Research Database (Denmark)

Recombination is important for repairing DNA lesions, yet it can also lead to genomic rearrangements. This process must be regulated, and recently, sumoylation-mediated mechanisms were found to inhibit Rad51-dependent recombination. Here, we report that the absence of the Slx5-Slx8 complex, a newly identified player in the SUMO (small ubiquitin-like modifier) pathway, led to increased Rad51-dependent and Rad51-independent recombination. The increases were most striking during S phase, suggesting an accumulation of DNA lesions during replication. Consistent with this view, Slx8 protein localized to replication centers. In addition, like SUMO E2 mutants, slx8Delta mutants exhibited clonal lethality, which was due to the overamplification of 2 microm, an extrachromosomal plasmid. Interestingly, in both SUMO E2 and slx8Delta mutants, clonal lethality was rescued by deleting genes required for Rad51-independent recombination but not those involved in Rad51-dependent events. These results suggest that sumoylation negatively regulates Rad51-independent recombination, and indeed, the Slx5-Slx8 complex affected the sumoylation of several enzymes involved in early steps of Rad51-independent recombination. We propose that, during replication, the Slx5-Slx8 complex helps prevent DNA lesions that are acted upon by recombination. In addition, the complex inhibits Rad51-independent recombination via modulating the sumoylation of DNA repair proteins. Udgivelsesdato: 2007-Sep

Burgess, Rebecca C; Rahman, Sadia

2007-01-01

364

PROGENITORS OF RECOMBINING SUPERNOVA REMNANTS  

Energy Technology Data Exchange (ETDEWEB)

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

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

2012-05-01

365

Recombinant snake venom prothrombin activators.  

UK PubMed Central (United Kingdom)

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.

Lövgren A

2013-05-01

366

Recombinant exon-encoded resilins for elastomeric biomaterials.  

UK PubMed Central (United Kingdom)

Resilin is an elastomeric protein found in specialized regions of the cuticle of most insects, providing outstanding material properties including high resilience and fatigue lifetime for insect flight and jumping needs. Two exons (1 and 3) from the resilin gene in Drosophila melanogaster were cloned and the encoded proteins expressed as soluble products in Escherichia coli. A heat and salt precipitation method was used for efficient purification of the recombinant proteins. The proteins were solution cast from water and formed into rubber-like biomaterials via horseradish peroxidase-mediated cross-linking. Comparative studies of the two proteins expressed from the two different exons were investigated by Fourier Transform Infrared Spectroscopy (FTIR) and Circular Dichrosim (CD) for structural features. Little structural organization was found, suggesting structural order was not induced by the enzyme-mediated di-tyrosine cross-links. Atomic Force Microscopy (AFM) was used to study the elastomeric properties of the uncross-linked and cross-linked proteins. The protein from exon 1 exhibited 90% resilience in comparison to 63% for the protein from exon 3, and therefore may be the more critical domain for functional materials to mimic native resilin. Further, the cross-linking of the recombinant exon 1 via the citrate-modified photo-Fenton reaction was explored as an alternative di-tyrosine mediated polymerization method and resulted in both highly elastic and adhesive materials. The citrate-modified photo-Fenton system may be suitable for in vivo applications of resilin biomaterials.

Qin G; Rivkin A; Lapidot S; Hu X; Preis I; Arinus SB; Dgany O; Shoseyov O; Kaplan DL

2011-12-01

367

Recombinant Holotoxoid Vaccine against Botulism?  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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 ...

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

368

Recombining helium--neon plasma  

Energy Technology Data Exchange (ETDEWEB)

Recombining helium-neon plasmas are studied theoretically for free electron densities N/sub e/ = 10/sup 13/--10/sup 17/ cm/sup -3/, electron temperatures T/sub e/ = 0.05--0.50 eV, and heavy particle densities N<10/sup 18/ cm/sup -3/. The populations of the helium and neon levels are calculated in the steady sink approximation. It is shown that pure neon plasmas are not well suited as laser media. However, a large stable Ne(4s)-Ne(3p) population inversion can be achieved in a recombining neon--helium plasma. Optimum concentration ratios in the ranges N/sub Ne//N/sub He/< or =10/sup -3/ and N/sub Ne//N/sub e/ = 10/sup 1/--10/sup 3/ are determined, and simple formulas are derived for the Ne(4s) level populations. Inversion is shown to occur both in a recombining He--Ne plasma and when a recombining pure helium plasma is mixed with neon atoms in the ground state.

Belyaev, A.K.

1985-03-01

369

Homologous recombination of exogenous DNA fragments with genomic DNA in somatic cells of mice.  

Science.gov (United States)

We compared liposomes and empty viral capsids for their use as vehicles for DNA transfer into cells and animals. DNA binding capacity was high for liposomes, but DNase I protection of DNA bound to liposomes was only moderate in comparison to DNA incorporated into viral capsids. Cellular uptake of radiolabeled and physiologically active DNA was also compared. For animal studies we chose an endogenous retroposon as target gene. To identify recombinational events we replaced a part of this gene with an artificial sequence not present in the mouse genome. The recombination rate for DNA fragments transfected in Polyoma capsids in live mice was higher than for liposome mediated transfection. Homologous recombination could be observed for both DNA transfer methods, mediated by positively charged liposomes (DOTMA) and by empty Polyoma viral capsids. PMID:1652361

Gareis, M; Harrer, P; Bertling, W M

1991-01-01

370

Xer site-specific recombination. DNA strand rejoining by recombinase XerC.  

UK PubMed Central (United Kingdom)

Xer site-specific recombination functions in the stable maintenance of circular replicons in Escherichia coli. Each of two related recombinase proteins, XerC and XerD, cleaves a specific pair of DNA strands, exchanges them, and rejoins them to the partner DNA molecule during a complete recombination reaction. The rejoining activity of recombinase XerC has been analyzed using isolated covalent XerC-DNA complexes resulting from DNA cleavage reactions upon Holliday junction substrates. These covalent protein-DNA complexes are competent in the rejoining reaction, demonstrating that covalently bound XerC can catalyze strand rejoining in the absence of other proteins. This contrasts with a recombinase-mediated cleavage reaction, which requires the presence of both recombinases, the recombinase mediating catalysis at any given time requiring activation by the partner recombinase. In a recombining nucleoprotein complex, both cleavage and rejoining can occur prior to dissociation of the complex.

Grainge I; Sherratt DJ

1999-03-01

371

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

372

Rapid host adaptation by extensive recombination  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Experimental investigations into virus recombination can provide valuable insights into the biochemical mechanisms and the evolutionary value of this fundamental biological process. Here, we describe an experimental scheme for studying recombination that should be applicable to any recombinogenic vi...

van der Walt, Eric; Rybicki, Edward P.; Varsani, Arvind; Polston, J. E.; Billharz, Rosalind; Donaldson, Lara; Monjane, Adérito L.

373

Glycoengineering approach to half-life extension of recombinant biotherapeutics.  

UK PubMed Central (United Kingdom)

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.

Chen C; Constantinou A; Chester KA; Vyas B; Canis K; Haslam SM; Dell A; Epenetos AA; Deonarain MP

2012-08-01

374

Single-crossover recombination and ancestral recombination trees.  

Science.gov (United States)

We consider the Wright-Fisher model for a population of [Formula: see text] individuals, each identified with a sequence of a finite number of sites, and single-crossover recombination between them. We trace back the ancestry of single individuals from the present population. In the [Formula: see text] limit without rescaling of parameters or time, this ancestral process is described by a random tree, whose branching events correspond to the splitting of the sequence due to recombination. With the help of a decomposition of the trees into subtrees, we calculate the probabilities of the topologies of the ancestral trees. At the same time, these probabilities lead to a semi-explicit solution of the deterministic single-crossover equation. The latter is a discrete-time dynamical system that emerges from the Wright-Fisher model via a law of large numbers and has been waiting for a solution for many decades. PMID:23564407

Baake, Ellen; von Wangenheim, Ute

2013-04-01

375

Single-crossover recombination and ancestral recombination trees.  

UK PubMed Central (United Kingdom)

We consider the Wright-Fisher model for a population of [Formula: see text] individuals, each identified with a sequence of a finite number of sites, and single-crossover recombination between them. We trace back the ancestry of single individuals from the present population. In the [Formula: see text] limit without rescaling of parameters or time, this ancestral process is described by a random tree, whose branching events correspond to the splitting of the sequence due to recombination. With the help of a decomposition of the trees into subtrees, we calculate the probabilities of the topologies of the ancestral trees. At the same time, these probabilities lead to a semi-explicit solution of the deterministic single-crossover equation. The latter is a discrete-time dynamical system that emerges from the Wright-Fisher model via a law of large numbers and has been waiting for a solution for many decades.

Baake E; von Wangenheim U

2013-04-01

376

Dielectronic recombination in laser generated plasmas  

International Nuclear Information System (INIS)

Dielectronic recombination coefficients have been computed for hydrogenic ions from HeII to FeXVI over a range of conditions typical of laser generated plasma. The results are displayed in a set a graphs together with the corresponding collisional radiative recombination coefficients. A comparison of these results indicates plasma conditions where dielectronic recombination is a significant process. (author).

1976-01-01

377

Tissue recombination techniques for mouse embryonic mammary glands.  

UK PubMed Central (United Kingdom)

This review gives detailed technical protocols for dissection of embryonic mammary rudiments and preparation of tissue recombinants composed of embryonic mouse mammary mesenchyme and epithelium. This experimental protocol was used in several seminal experiments that have greatly increased our understanding of embryonic mammary gland development, including the finding that mammary mesenchyme induces and specifies mammary epithelial identity. Analysis of mesenchymal-epithelial interactions has facilitated identification of molecular mediators of cell-cell interactions, in particular the tissue-specific roles of genes expressed in mesenchyme and epithelium during embryonic development.

Cunha GR

2013-06-01

378

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

379

Recombinant vector and eukaryotic host transformed thereby  

Energy Technology Data Exchange (ETDEWEB)

A recombinant plasmid is described comprising: a segment from a first plasmid which is not a lymphotrophic herpes virus segment and which facilitates the replication of the recombinant plasmid in a prokaryotic host; a segment from a lymphotrophic herpes virus which is linked to the first plasmid segment such that is a capable of assisting in maintaining the recombinant plasmid as a plasmid if the recombinant plasmid is inserted into a eukaryotic host that has been transformed by the lymphotrophic herpes virus; and a foreign eukaryotic gene component linked as part of the recombinant plasmid.

Sugden, W.M.

1987-08-11

380

The recombination of argon ion and electron  

International Nuclear Information System (INIS)

There are many uncertain matters about the recombination process on which the decay of a plasma is dependent. Especially, the studies for the recombination phenomena of the high pressure plasma have not been done. In view of the engineering applications of the high pressure plasma, we measured experimentally the recombination coefficient of the atmospheric pressure plasma investigating the decrease of the charged particle density in the very weakly ionized argon-plasma flow. It was found from these experiments that the volume recombination coefficient is the order of 10-8 cm3sec-1 and the recombination on the metal surface is fully catalytic. (auth.)

1975-01-01

 
 
 
 
381

Recombination lasing in heliumlike silicon  

Energy Technology Data Exchange (ETDEWEB)

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.

Apruzese, J.P.; Kepple, P.C.; Davis, J. (Plasma Radiation Branch, Plasma Physics Div., Naval Research Lab., Washington, DC (US)); Pender, J. (Berkeley Research Associates, Inc., Springfield, VA (US))

1988-10-01

382

Recombination lasing in heliumlike silicon  

International Nuclear Information System (INIS)

[en] 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

383

Mechanisms of sister chromatid recombination  

International Nuclear Information System (INIS)

Studies using T948 as a model system have been carried out aimed at elucidating the mechanism of sister chromatid recombination (SCR). Characterization of U.V. light- and x-ray-induced SCR, the relationiship between SCR induction and DNA repair using rad mutations, and the relationship between SCR induction and the time of cell division using cdc mutations are presented. It has been supposed that SCR is induced at the phase of S-G2 following DNA replication, that postreplication break of DNA strands is strongly involved in the induction of SCR, and that induction type of SCR, i.e., conversion type or recombination type, is dependent upon the type of molecular damage of DNA. (Namekawa, K.)

1985-01-01