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RPA Accumulation during Class Switch Recombination Represents 5?–3? DNA-End Resection during the S–G2/M Phase of the Cell Cycle  

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Full Text Available Activation-induced cytidine deaminase (AID promotes chromosomal translocations by inducing DNA double-strand breaks (DSBs at immunoglobulin (Ig genes and oncogenes in the G1 phase. RPA is a single-stranded DNA (ssDNA-binding protein that associates with resected DSBs in the S phase and facilitates the assembly of factors involved in homologous repair (HR, such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage, but its role in Ig gene recombination remains unclear. Here, we demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, recombination-activating genes (RAG, or other nucleases. Small amounts of RPA are deposited at AID targets in G1 in an ATM-dependent manner. In contrast, recruitment in the S–G2/M phase is extensive, ATM independent, and associated with Rad51 accumulation. In the S–G2/M phase, RPA increases in nonhomologous-end-joining-deficient lymphocytes, where there is more extensive DNA-end resection. Thus, most RPA recruitment during class switch recombination represents salvage of unrepaired breaks by homology-based pathways during the S–G2/M phase of the cell cycle.

Arito Yamane

2013-01-01

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

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

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

2014-04-01

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Molecular anatomy of the recombination mediator function of Saccharomyces cerevisiae Rad52  

DEFF Research Database (Denmark)

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

Seong, C.; Sehorn, M.G.

2008-01-01

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Cellular Functions of Human RPA1: MULTIPLE ROLES OF DOMAINS IN REPLICATION, REPAIR, AND CHECKPOINTS*  

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In eukaryotes, the single strand DNA (ssDNA)-binding protein, replication protein A (RPA), is essential for DNA replication, repair, and recombination. RPA is composed of the following three subunits: RPA1, RPA2, and RPA3. The RPA1 subunit contains four structurally related domains and is responsible for high affinity ssDNA binding. This study uses a depletion/replacement strategy in human cells to reveal the contributions of each domain to RPA cellular functions. Muta...

Haring, Stuart J.; Mason, Aaron C.; Binz, Sara K.; Wold, Marc S.

2008-01-01

5

RPA Antagonizes Microhomology-Mediated Repair of DNA Double-Strand Breaks  

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Microhomology-mediated end joining (MMEJ) is a Ku and Ligase IV independent mechanism for repair of DNA double-strand breaks, which contributes to chromosome rearrangements. Here we used a chromosomal end-joining assay to determine the genetic requirements for MMEJ in Saccharomyces cerevisiae. We found that end resection influences the ability to expose microhomologies; however, it is not rate limiting for MMEJ in wild-type cells. The frequency of MMEJ increased by up to 350-fold in rfa1 hypo...

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

2014-01-01

6

Cep164 is a mediator protein required for the maintenance of genomic stability through modulation of MDC1, RPA, and CHK1.  

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The activation of the ataxia telangiectasia mutated (ATM) and ATM/Rad3-related (ATR) kinases triggers a diverse cellular response including the initiation of DNA damage-induced cell cycle checkpoints. Mediator of DNA Damage Checkpoint protein, MDC1, and H2AX are chromatin remodeling factors required for the recruitment of DNA repair proteins to the DNA damage sites. We identified a novel mediator protein, Cep164 (KIAA1052), that interacts with both ATR and ATM. Cep164 is phosphorylated upon replication stress, ultraviolet radiation (UV), and ionizing radiation (IR). Ser186 of Cep164 is phosphorylated by ATR/ATM in vitro and in vivo. The phosphorylation of Ser186 is not affected by RPA knockdown but is severely hampered by MDC1 knockdown. siRNA-mediated silencing of Cep164 significantly reduces DNA damage-induced phosphorylation of RPA, H2AX, MDC1, CHK2, and CHK1, but not NBS1. Analyses of Cep164 knockdown cells demonstrate a critical role of Cep164 in G2/M checkpoint and nuclear divisions. These findings reveal that Cep164 is a key player in the DNA damage-activated signaling cascade. PMID:18283122

Sivasubramaniam, Sudhakar; Sun, Xuemin; Pan, Yen-Ru; Wang, Shaohui; Lee, Eva Y-H P

2008-03-01

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

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

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

2014-01-23

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Visualization of recombination-mediated damage bypass by template switching.  

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Template switching (TS) mediates damage bypass via a recombination-related mechanism involving PCNA polyubiquitination and polymerase ?-dependent DNA synthesis. Using two-dimensional gel electrophoresis and EM, here we characterize TS intermediates arising in Saccharomyces cerevisiae at a defined chromosome locus, identifying five major families of intermediates. Single-stranded DNA gaps of 150-200 nt, and not DNA ends, initiate TS by strand invasion. This causes reannealing of the parental strands and exposure of the nondamaged newly synthesized chromatid, which serves as a replication template for the other blocked nascent strand. Structures resembling double Holliday junctions, postulated to be central double-strand break-repair intermediates but so far visualized only in meiosis, mediate late stages of TS before being processed to hemicatenanes. Our results reveal the DNA transitions accounting for recombination-mediated DNA-damage tolerance in mitotic cells and replication under conditions of genotoxic stress. PMID:25195051

Giannattasio, Michele; Zwicky, Katharina; Follonier, Cindy; Foiani, Marco; Lopes, Massimo; Branzei, Dana

2014-10-01

9

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

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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

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ATR Prohibits Replication Catastrophe by Preventing Global Exhaustion of RPA  

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ATR, activated by replication stress, protects replication forks locally and suppresses origin firing globally. Here, we show that these functions of ATR are mechanistically coupled. Although initially stable, stalled forks in ATR-deficient cells undergo nucleus-wide breakage after unscheduled origin firing generates an excess of single-stranded DNA that exhausts the nuclear pool of RPA. Partial reduction of RPA accelerated fork breakage, and forced elevation of RPA was sufficient to delay such "replication catastrophe" even in the absence of ATR activity. Conversely, unscheduled origin firing induced breakage of stalled forks even in cells with active ATR. Thus, ATR-mediated suppression of dormant origins shields active forks against irreversible breakage via preventing exhaustion of nuclear RPA. This study elucidates how replicating genomes avoid destabilizing DNA damage. Because cancer cells commonly feature intrinsically high replication stress, this study also provides a molecular rationale for their hypersensitivity to ATR inhibitors.

Toledo Lazaro, Luis Ignacio; Altmeyer, Matthias

2013-01-01

11

Activity of the Rhodopseudomonas palustris p-Coumaroyl-Homoserine Lactone-Responsive Transcription Factor RpaR ? †  

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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 (His6-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

2011-01-01

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Cep164 is a mediator protein required for the maintenance of genomic stability through modulation of MDC1, RPA, and CHK1  

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The activation of the ataxia telangiectasia mutated (ATM) and ATM/Rad3-related (ATR) kinases triggers a diverse cellular response including the initiation of DNA damage-induced cell cycle checkpoints. Mediator of DNA Damage Checkpoint protein, MDC1, and H2AX are chromatin remodeling factors required for the recruitment of DNA repair proteins to the DNA damage sites. We identified a novel mediator protein, Cep164 (KIAA1052), that interacts with both ATR and ATM. Cep164 is phosphorylated upon r...

Sivasubramaniam, Sudhakar; Sun, Xuemin; Pan, Yen-ru; Wang, Shaohui; Lee, Eva Y. -h P.

2008-01-01

13

RPA Systems Opportunities Final Report - ARCHIVE  

Commercial strategy – proposals on how RPA should navigate the impending .... \\immediate next steps to provide a platform for the RPA to move forward. .... The \\change from a „task-based? approach to processing claims to one based on ...

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Cre-Mediated Recombination in Rhombic Lip Derivatives  

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To study the development of the cerebellum, we generated a transgenicmouse line Tg(m?6-cre)B1LFR that expresses CRE recombinase under the GABAA receptor ?6 subunit promoter. In this line, recombination of an R26R reporter allele occurred postnatally in granule cells of the cerebellum and dorsal cochlear nucleus, as well as in a subset of precerebellar nuclei in the brainstem. All neurons in which recombination occurred originated during embryogenesis from the rhombic lip. This might be expl...

Fu?nfschilling, Ursula; Reichardt, Louis F.

2002-01-01

15

Recombinant adenovirus-mediated overexpression of 3?-hydroxysteroid-?24 reductase.  

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3?-Hydroxysteroid-?24 reductase (DHCR24) is a multifunctional enzyme that localizes to the endoplasmic reticulum and has neuroprotective and cholesterol-synthesizing activities. DHCR24 overexpression confers neuroprotection against apoptosis caused by amyloid ? deposition. The present study aimed to construct two recombinant adenoviruses driving DHCR24 expression specifically in neurons. Two SYN1 promoter DNA fragments were obtained from human (h) and rat (r). Recombinant Ad-r(h)SYN1-DHCR24 was transfected into AD-293, N2A (mouse neuroblastoma), and MIN6 (mouse pancreatic carcinoma) cells. Western blot analysis showed DHCR24 was specially expressed in 293 and N2A cells, but no specific band was found in MIN6 cells. This demonstrates that the recombinant adenoviruses successfully express DHCR24, and no expression is observed in non-neuronal cells. TUNEL assay results showed apoptosis was inhibited in adenovirus-transfected neurons. Detecting reactive oxygen species by immunofluorescence, we found that adenovirus transfection inhibits apoptosis through scavenging excess reactive oxygen species. Our findings show that the recombinant DHCR24 adenoviruses induce neuron-specific DHCR24 expression, and thereby lay the foundation for further studies on DHCR24 gene therapy for Alzheimer's disease. PMID:25206847

Lu, Xiuli; Jia, Dan; Zhao, Chenguang; Wang, Weiqi; Liu, Ting; Chen, Shuchao; Quan, Xiaoping; Sun, Deliang; Gao, Bing

2014-03-01

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

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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.

Kagale Sateesh

2008-11-01

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Purified human BRCA2 stimulates RAD51-mediated recombination  

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

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

2010-01-01

18

Sustained retrotransposition is mediated by nucleotide deletions and interelement recombinations.  

Science.gov (United States)

The term "C-value paradox" was coined by C. A. Thomas, Jr. in 1971 [Thomas CA (1971) Ann Rev Genetics 5:237-256] to describe the initially puzzling lack of correlation between an organism's genome size and its morphological complexity. Polyploidy and the expansion of repetitive DNA, primarily transposable elements, are two mechanisms that have since been found to account for this differential. While the inactivation of retrotransposons by methylation and their removal from the genome by illegitimate recombination have been well documented, the cause of the apparently periodic bursts of retrotranposon expansion is as yet unknown. We show that the expansion of the CRM1 retrotransposon subfamily in the ancient allotetraploid crop plant corn is linked to the repeated formation of novel recombinant elements derived from two parental retrotransposon genotypes, which may have been brought together during the hybridization of two sympatric species that make up the present day corn genome, thus revealing a unique mechanism linking polyploidy and retrotransposition. PMID:18832157

Sharma, Anupma; Schneider, Kevin L; Presting, Gernot G

2008-10-01

19

Crossover recombination mediated by HIM-18/SLX4-associated nucleases.  

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Meiosis is a specialized cell division program that results in the formation of haploid gametes (i.e., sperm and eggs) from diploid parental cells, and is essential for all sexually reproducing organisms. Crossover formation, the reciprocal exchange of genetic information during recombination, is critical for accurate meiotic chromosome segregation. Misregulation of crossover formation leads to genomic instability and aneuploidy (cells with the incorrect number of chromosomes), resulting in tumorigenesis, birth defects, miscarriages, and infertility in humans. Recently, a shuriken/Swiss army knife-like multi-nuclease complex has been implicated in processing various types of DNA repair intermediates. However, how these nucleases coordinate their functions during repair remained unclear. Our studies in C. elegans revealed genetic redundancies between these nucleases for meiotic crossover formation and that they promote distinct crossover control at different chromosome regions. Specifically, XPF-1 acts redundantly with both MUS-81 and SLX-1 to resolve Holliday junction recombination intermediates into crossover products at designated future crossover sites on chromosome arms. In contrast, SLX-1 is required for suppression of crossovers at the center region of chromosomes. Altogether, our studies have shed light on the interplay between structure-specific endonucleases and uncovered their ability to exert either positive or negative meiotic crossover control on a chromosome region-specific basis. PMID:25057454

Saito, Takamune T; Colaiácovo, Monica P

2014-01-01

20

Homologous recombinational repair factors are recruited and loaded onto the viral DNA genome in Epstein-Barr virus replication compartments.  

Science.gov (United States)

Homologous recombination is an important biological process that facilitates genome rearrangement and repair of DNA double-strand breaks (DSBs). The induction of Epstein-Barr virus (EBV) lytic replication induces ataxia telangiectasia-mutated (ATM)-dependent DNA damage checkpoint signaling, leading to the clustering of phosphorylated ATM and Mre11/Rad50/Nbs1 (MRN) complexes to sites of viral genome synthesis in nuclei. Here we report that homologous recombinational repair (HRR) factors such as replication protein A (RPA), Rad51, and Rad52 as well as MRN complexes are recruited and loaded onto the newly synthesized viral genome in replication compartments. The 32-kDa subunit of RPA is extensively phosphorylated at sites in accordance with those with ATM. The hyperphosphorylation of RPA32 causes a change in RPA conformation, resulting in a switch from the catalysis of DNA replication to the participation in DNA repair. The levels of Rad51 and phosphorylated RPA were found to increase with the progression of viral productive replication, while that of Rad52 proved constant. Furthermore, biochemical fractionation revealed increases in levels of DNA-bound forms of these HRRs. Bromodeoxyuridine-labeled chromatin immunoprecipitation and PCR analyses confirmed the loading of RPA, Rad 51, Rad52, and Mre11 onto newly synthesized viral DNA, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling analysis demonstrated DSBs in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that homologous recombination and/or repair of viral DNA genome might occur, coupled with DNA replication to facilitate viral genome synthesis. PMID:19386720

Kudoh, Ayumi; Iwahori, Satoko; Sato, Yoshitaka; Nakayama, Sanae; Isomura, Hiroki; Murata, Takayuki; Tsurumi, Tatsuya

2009-07-01

 
 
 
 
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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

2012-01-01

22

Overexpressed of RAD51 suppresses recombination defects: a possible mechanism to reverse genomic instability  

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RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early step in HRR is the formation of single-stranded DNA (ss-DNA) coated by RPA (a ss-DNA binding protein). Displacement of RPA by RAD51 is highly regulated and facilitated by a number of different proteins known as the 'recombination mediators'. To assist these recombination mediators, a second group of proteins also is required and we are defining these proteins here as 'recombination co-mediators'. Defects in either recombination mediators or comediators, including BRCA1 and BRCA2, lead to impaired HRR that can genetically be complemented for (i.e. suppressed) by overexpression of RAD51. Defects in HRR have long been known to contribute to genomic instability leading to tumor development. Since genomic instability also slows cell growth, precancerous cells presumably require genomic restabilization to gain a growth advantage. RAD51 is overexpressed in many tumors, and therefore, we hypothesize that the complementing ability of elevated levels of RAD51 in tumors with initial HRR defects limits genomic instability during carcinogenic progression. Of particular interest, this model may also help explain the high frequency of TP53 mutations in human cancers, since wild-type p53 represses RAD51.

Schild, David; Wiese, Claudia

2009-10-15

23

Multiple Regulation of Rad51-Mediated Homologous Recombination by Fission Yeast Fbh1  

Science.gov (United States)

Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCFFbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role in vivo, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange in vitro after strand exchange had been initiated. PMID:25165823

Tsutsui, Yasuhiro; Kurokawa, Yumiko; Ito, Kentaro; Siddique, Md. Shahjahan P.; Kawano, Yumiko; Yamao, Fumiaki; Iwasaki, Hiroshi

2014-01-01

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A Stabilized Respiratory Syncytial Virus Reverse Genetics System Amenable to Recombination Mediated Mutagenesis  

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We describe the first example of combining bacterial artificial chromosome (BAC) recombination-mediated mutagenesis with reverse genetics for a negative strand RNA virus. A BAC-based respiratory syncytial virus (RSV) rescue system was established. An important advantage of this system is that RSV antigenomic cDNA was stabilized in the BAC vector. The RSV genotype chosen was A2-line19F, a chimeric strain previously shown to recapitulate in mice key features of RSV pathogenesis. We recovered tw...

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

2012-01-01

25

Visualization of recombination–mediated damage-bypass by template switching  

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Template switching (TS) mediates damage-bypass via a recombination-related mechanism involving PCNA polyubiquitylation and Polymerase ?-dependent DNA synthesis. Using two-dimensional gel electrophoresis and electron microscopy, here we characterize TS intermediates arising in Saccharomyces cerevisiae at a defined chromosome locus, identifying five major families of intermediates. Single-stranded DNA gaps in the range of 150-200 nucleotides, and not DNA ends, initiate TS by strand invasion. T...

Giannattasio, Michele; Zwicky, Katharina; Follonier, Cindy; Foiani, Marco; Lopes, Massimo; Branzei, Dana

2014-01-01

26

Enzymatic properties of the Caenorhabditis elegans Dna2 endonuclease/helicase and a species-specific interaction between RPA and Dna2  

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In both budding and fission yeasts, a null mutation of the DNA2 gene is lethal. In contrast, a null mutation of Caenorhabditis elegans dna2+ causes a delayed lethality, allowing survival of some mutant C.elegans adults to F2 generation. In order to understand reasons for this difference in requirement of Dna2 between these organisms, we examined the enzymatic properties of the recombinant C.elegans Dna2 (CeDna2) and its interaction with replication-protein A (RPA) from various sources. Like budding yeast Dna2, CeDna2 possesses DNA-dependent ATPase, helicase and endonuclease activities. The specific activities of both ATPase and endonuclease activities of the CeDna2 were considerably higher than the yeast Dna2 (?10- and 20-fold, respectively). CeDna2 endonuclease efficiently degraded a short 5? single-stranded DNA tail (<10 nt) that was hardly cleaved by ScDna2. Both endonuclease and helicase activities of CeDna2 were stimulated by CeRPA, but not by human or yeast RPA, demonstrating a species-specific interaction between Dna2 and RPA. These and other enzymatic properties of CeDna2 described in this paper may shed light on the observation that C.elegans is less stringently dependent on Dna2 for its viability than Saccharomyces cerevisiae. We propose that flaps generated by DNA polymerase ?-mediated displacement DNA synthesis are mostly short in C.elegans eukaryotes, and hence less dependent on Dna2 for viability. PMID:15745997

Kim, Do-Hyung; Lee, Kyoung-Hwa; Kim, Jeong-Hoon; Ryu, Gi-Hyuck; Bae, Sung-Ho; Lee, Byung-Chul; Moon, Kyeong-Yeop; Byun, Si-Myung; Koo, Hyeon-Sook; Seo, Yeon-Soo

2005-01-01

27

Radiation protection authorized persons (RPA) in Germany  

International Nuclear Information System (INIS)

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

28

Homologous-recombination-deficient tumours are dependent on Pol?-mediated repair.  

Science.gov (United States)

Large-scale genomic studies have shown that half of epithelial ovarian cancers (EOCs) have alterations in genes regulating homologous recombination (HR) repair. Loss of HR accounts for the genomic instability of EOCs and for their cellular hyper-dependence on alternative poly-ADP ribose polymerase (PARP)-mediated DNA repair mechanisms. Previous studies have implicated the DNA polymerase ? (Pol? also known as POLQ, encoded by POLQ) in a pathway required for the repair of DNA double-strand breaks, referred to as the error-prone microhomology-mediated end-joining (MMEJ) pathway. Whether Pol? interacts with canonical DNA repair pathways to prevent genomic instability remains unknown. Here we report an inverse correlation between HR activity and Pol? expression in EOCs. Knockdown of Pol? in HR-proficient cells upregulates HR activity and RAD51 nucleofilament assembly, while knockdown of Pol? in HR-deficient EOCs enhances cell death. Consistent with these results, genetic inactivation of an HR gene (Fancd2) and Polq in mice results in embryonic lethality. Moreover, Pol? contains RAD51 binding motifs and it blocks RAD51-mediated recombination. Our results reveal a synthetic lethal relationship between the HR pathway and Pol?-mediated repair in EOCs, and identify Pol? as a novel druggable target for cancer therapy. PMID:25642963

Ceccaldi, Raphael; Liu, Jessica C; Amunugama, Ravindra; Hajdu, Ildiko; Primack, Benjamin; Petalcorin, Mark I R; O'Connor, Kevin W; Konstantinopoulos, Panagiotis A; Elledge, Stephen J; Boulton, Simon J; Yusufzai, Timur; D'Andrea, Alan D

2015-02-12

29

Efficient homologous recombination-mediated genome engineering in zebrafish using TALE nucleases.  

Science.gov (United States)

Custom-designed nucleases afford a powerful reverse genetic tool for direct gene disruption and genome modification in vivo. Among various applications of the nucleases, homologous recombination (HR)-mediated genome editing is particularly useful for inserting heterologous DNA fragments, such as GFP, into a specific genomic locus in a sequence-specific fashion. However, precise HR-mediated genome editing is still technically challenging in zebrafish. Here, we establish a GFP reporter system for measuring the frequency of HR events in live zebrafish embryos. By co-injecting a TALE nuclease and GFP reporter targeting constructs with homology arms of different size, we defined the length of homology arms that increases the recombination efficiency. In addition, we found that the configuration of the targeting construct can be a crucial parameter in determining the efficiency of HR-mediated genome engineering. Implementing these modifications improved the efficiency of zebrafish knock-in generation, with over 10% of the injected F0 animals transmitting gene-targeting events through their germline. We generated two HR-mediated insertion alleles of sox2 and gfap loci that express either superfolder GFP (sfGFP) or tandem dimeric Tomato (tdTomato) in a spatiotemporal pattern that mirrors the endogenous loci. This efficient strategy provides new opportunities not only to monitor expression of endogenous genes and proteins and follow specific cell types in vivo, but it also paves the way for other sophisticated genetic manipulations of the zebrafish genome. PMID:25249466

Shin, Jimann; Chen, Jiakun; Solnica-Krezel, Lilianna

2014-10-01

30

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

Science.gov (United States)

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

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

2012-12-01

31

BRG1 promotes the repair of DNA double-strand breaks by facilitating the replacement of RPA with RAD51.  

Science.gov (United States)

DNA double-strand breaks (DSBs) are a type of lethal DNA damage. The repair of DSBs requires tight coordination between the factors modulating chromatin structure and the DNA repair machinery. BRG1, the ATPase subunit of the chromatin remodelling complex Switch/Sucrose non-fermentable (SWI/SNF), is often linked to tumorigenesis and genome instability, and its role in DSB repair remains largely unclear. In the present study, we show that BRG1 is recruited to DSB sites and enhances DSB repair. Using DR-GFP and EJ5-GFP reporter systems, we demonstrate that BRG1 facilitates homologous recombination repair rather than nonhomologous end-joining (NHEJ) repair. Moreover, the BRG1-RAD52 complex mediates the replacement of RPA with RAD51 on single-stranded DNA (ssDNA) to initiate DNA strand invasion. Loss of BRG1 results in a failure of RAD51 loading onto ssDNA, abnormal homologous recombination repair and enhanced DSB-induced lethality. Our present study provides a mechanistic insight into how BRG1, which is known to be involved in chromatin remodelling, plays a substantial role in the homologous recombination repair pathway in mammalian cells. PMID:25395584

Qi, Wenjing; Wang, Ruoxi; Chen, Hongyu; Wang, Xiaolin; Xiao, Ting; Boldogh, Istvan; Ba, Xueqing; Han, Liping; Zeng, Xianlu

2015-01-15

32

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

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

2006-01-01

33

Intramolecular recombination of chloroplast genome mediated by short direct-repeat sequences in wheat species.  

Science.gov (United States)

Structural alterations of the chloroplast genome tend to occur at "hot spots" on the physical map. To clarify the mechanism of mutation of chloroplast genome structure in higher plants, we determined the nucleotide sequence of the hot-spot region of chloroplast DNAs related to length mutations (deletions/insertions) in Triticum (wheat) and Aegilops. From a comparison of this region in wheat with the corresponding region of tobacco or liverwort, it is evident that one of the open reading frames in tobacco (ORF512) has been replaced in wheat by the rpl23 gene, which is a member of the ribosomal protein gene operon. In the deleted positions and in the original genome of Triticum and Aegilops, consensus sequences forming short direct repeats were found, indicating that these deletions were a result of intramolecular recombination mediated by these short direct-repeat sequences. By two independent recombination events in the Aegilops crassa type of chloroplast genome, which is shared by Triticum monococcum, Ae. bicornis, Ae. sharonensis, Ae. comosa, and Ae. mutica, the novel chloroplast DNA sequences of T. aestivum and Ae. squarrosa were generated. This finding indicates the existence of illegitimate recombination in the chloroplast genome and presents a mechanism for producing genetic diversity of that genome. PMID:3186748

Ogihara, Y; Terachi, T; Sasakuma, T

1988-11-01

34

Homeologous plastid DNA transformation in tobacco is mediated by multiple recombination events.  

Science.gov (United States)

Efficient plastid transformation has been achieved in Nicotiana tabacum using cloned plastid DNA of Solanum nigrum carrying mutations conferring spectinomycin and streptomycin resistance. The use of the incompletely homologous (homeologous) Solanum plastid DNA as donor resulted in a Nicotiana plastid transformation frequency comparable with that of other experiments where completely homologous plastid DNA was introduced. Physical mapping and nucleotide sequence analysis of the targeted plastid DNA region in the transformants demonstrated efficient site-specific integration of the 7.8-kb Solanum plastid DNA and the exclusion of the vector DNA. The integration of the cloned Solanum plastid DNA into the Nicotiana plastid genome involved multiple recombination events as revealed by the presence of discontinuous tracts of Solanum-specific sequences that were interspersed between Nicotiana-specific markers. Marked position effects resulted in very frequent cointegration of the nonselected peripheral donor markers located adjacent to the vector DNA. Data presented here on the efficiency and features of homeologous plastid DNA recombination are consistent with the existence of an active RecA-mediated, but a diminished mismatch, recombination/repair system in higher-plant plastids. PMID:10388829

Kavanagh, T A; Thanh, N D; Lao, N T; McGrath, N; Peter, S O; Horváth, E M; Dix, P J; Medgyesy, P

1999-07-01

35

Recombinant adenoassociated virus 2/5-mediated gene transfer is reduced in the aged rat midbrain.  

Science.gov (United States)

Clinical trials are examining the efficacy of viral vector-mediated gene delivery for treating Parkinson's disease. Although viral vector strategies have been successful in preclinical studies, to date clinical trials have disappointed. This may be because of the fact that preclinical studies fail to account for aging. Aging is the single greatest risk factor for developing Parkinson's disease and age alters cellular processes utilized by viral vectors. We hypothesized that the aged brain would be relatively resistant to transduction when compared with the young adult. We examined recombinant adeno-associated virus 2/5-mediated green fluorescent protein (rAAV2/5 GFP) expression in the young adult and aged rat nigrostriatal system. GFP overexpression was produced in both age groups. However, following rAAV2/5 GFP injection to the substantia nigra aged rats displayed 40%-60% less GFP protein in the striatum, regardless of rat strain or duration of expression. Furthermore, aged rats exhibited 40% fewer cells expressing GFP and 4-fold less GFP messenger RNA. rAAV2/5-mediated gene transfer is compromised in the aged rat midbrain, with deficiencies in early steps of transduction leading to significantly less messenger RNA and protein expression. PMID:25457558

Polinski, Nicole K; Gombash, Sara E; Manfredsson, Fredric P; Lipton, Jack W; Kemp, Christopher J; Cole-Strauss, Allyson; Kanaan, Nicholas M; Steece-Collier, Kathy; Kuhn, Nathan C; Wohlgenant, Susan L; Sortwell, Caryl E

2015-02-01

36

Entropy and Effective temperature in second RPA dynamics  

International Nuclear Information System (INIS)

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

37

Ionizing radiation-induced phosphorylation of RPA p34 is deficient in ataxia telangiectasia and reduced in aged normal fibroblasts  

International Nuclear Information System (INIS)

Replication protein A (RPA, also called human single stranded DNA binding protein, hSSB) is a trimeric, multifunctional protein complex involved in DNA replication, DNA repair and recombination. Phosphorylation of RPA p34 subunit is observed after exposure of cells to radiation and other DNA damaging agents, which implicates the protein not only in repair but also in the regulation of replication on damaged DNA template. Here, we show that the phosphorylation observed in RPA p34 after exposure to ionizing radiation, X- or ?-rays, is reduced and occurs later in primary fibroblasts from patients suffering from ataxia telangiectasia (AT), as compared to normal fibroblasts. We also show that in primary normal human fibroblasts, radiation-induced phosphorylation of RPA p34 is 'age'-dependent and decreases significantly as cultures senesce. Radiation-induced phosphorylation of RPA p34 is nearly absent in non-cycling cells, while the expression of p21cip1/waf1/sdi1 remains inducible. The results demonstrate a growth-stage and culture-age dependency in radiation-induced RPA p34 phosphorylation, and suggest the operation of a signal transduction pathway that is inactivated in senescing or quiescent fibroblasts and defective in AT cells

38

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

Scientific Electronic Library Online (English)

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

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

39

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

Directory of Open Access Journals (Sweden)

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

Paula A. Roratto

2008-01-01

40

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

International Nuclear Information System (INIS)

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

 
 
 
 
41

Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA  

DEFF Research Database (Denmark)

Background Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. Results A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence. Conclusions These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses.

Rasmussen, Thomas Bruun; Risager, Peter Christian

2013-01-01

42

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.

43

I-SceI-mediated plasmid deletion and intra-molecular recombination in Spiroplasma citri.  

Science.gov (United States)

S. citri wild-type strain GII3 carries six plasmids (pSci1 to -6) that are thought to encode determinants involved in the transmission of the spiroplasma by its leafhopper vector. In this study we report the use of meganuclease I-SceI for plasmid deletion in S. citri. Plasmids pSci1NT-I and pSci6PT-I, pSci1 and pSci6 derivatives that contain the tetM selection marker and a unique I-SceI recognition site were first introduced into S. citri strains 44 (having no plasmid) and GII3 (carrying pSci1-6), respectively. Due to incompatibility of homologous replication regions, propagation of the S. citri GII3 transformant in selective medium resulted in the replacement of the natural pSci6 by pSci6PT-I. The spiroplasmal transformants were further transformed by an oriC plasmid carrying the I-SceI gene under the control of the spiralin gene promoter. In the S. citri 44 transformant, expression of I-SceI resulted in rapid loss of pSciNT-I showing that expression of I-SceI can be used as a counter-selection tool in spiroplasmas. In the case of the S. citri GII3 transformant carrying pSci6PT-I, expression of I-SceI resulted in the deletion of plasmid fragments comprising the I-SceI site and the tetM marker. Delineating the I-SceI generated deletions proved they had occurred though recombination between homologous sequences. To our knowledge this is the first report of I-SceI mediated intra-molecular recombination in mollicutes. PMID:21129414

Breton, Marc; Duret, Sybille; Béven, Laure; Dubrana, Marie-Pierre; Renaudin, Joël

2011-02-01

44

Recombinant adenovirus-mediated gene transfer to genitourinary epithelium in vitro and in vivo.  

Science.gov (United States)

Transitional cell carcinoma (TCC) of the bladder is associated with characterized lesions in dominant and recessive oncogenes. The understanding of the molecular basis of tumorigenesis in these instances makes possible the application of gene therapy strategies for TCC. In this regard, the ability to directly access the epithelium of the genitourinary (GU) tract via the urethra provides a practical means to implement these various gene therapy approaches. We thus explored vector strategies to accomplish direct in vivo transduction of GU epithelium. Initially, three human (HT 1197, HT 1376, T24) and one mouse (MBT-2) TCC cell lines were transduced using a recombinant adenoviral vector expressing the firefly luciferase reporter gene, rAd-CMV-Luc. In these studies, reporter gene expression was found to be significantly elevated above background for all four cell lines. Of note, the TCC cell lines HT 1197 and HT 1376 showed expression levels comparable with the cervical carcinoma cell line HeLa, a cell line previously shown to be highly susceptible to recombinant adenovirus-mediated gene transduction. An in vitro time course for T24 and MBT-2 using rAd-CMV-Luc showed peak expression 1 day after transduction for the T24 line and 3 days after transduction for the MBT-2 line, with detectable levels of expression persisting for at least 7 days. As a next step, human and mouse primary tissue deriving from the GU epithelium were transduced using rAd-CMV-Luc. In this assay, luciferase expression levels significantly above background were observed in both instances.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7621262

Bass, C; Cabrera, G; Elgavish, A; Robert, B; Siegal, G P; Anderson, S C; Maneval, D C; Curiel, D T

1995-06-01

45

Wilms' tumor gene WT1 promotes homologous recombination-mediated DNA damage repair.  

Science.gov (United States)

The Wilms' tumor gene WT1 is overexpressed in leukemia and various types of solid tumors and plays an oncogenic role in these malignancies. Alternative splicing at two sites yields four major isoforms, 17AA(+)KTS(+), 17AA(+)KTS(-), 17AA(-)KTS(+), and 17AA(-)KTS(-), and all the isoforms are expressed in the malignancies. However, among the four isoforms, function of WT1[17AA(-)KTS(+)] isoform still remains undetermined. In the present study, we showed that forced expression of WT1[17AA(-)KTS(+)] isoform significantly inhibited apoptosis by DNA-damaging agents such as Doxorubicin, Mitomycin, Camptothesisn, and Bleomycin in immortalized fibroblast MRC5SV and cervical cancer HeLa cells. Knockdown of Rad51, an essential factor for homologous recombination (HR)-mediated DNA repair canceled the resistance to Doxorubicin induced by WT1[17AA(-)KTS(+)] isoform. GFP recombination assay showed that WT1[17AA(-)KTS(+)] isoform alone promoted HR, but that three other WT1 isoforms did not. WT1[17AA(-)KTS(+)] isoform significantly upregulated the expression of HR genes, XRCC2, Rad51D, and Rad54. Knockdown of XRCC2, Rad51D, and Rad54 inhibited the HR activity and canceled resistance to Doxorubicin in MRC5SV cells with forced expression of WT1[17AA(-)KTS(+)] isoform. Furthermore, chromatin immunoprecipitation (ChIP) assay showed the binding of WT1[17AA(-)KTS(+)] isoform protein to promoters of XRCC2 and Rad51D. Immunohistochemical study showed that Rad54 and XRCC2 proteins were highly expressed in the majority of non-small-cell lung cancer (NSCLC) and gastric cancer, and that expression of these two proteins was significantly correlated with that of WT1 protein in NSCLCs. Our results presented here showed that WT1[17AA(-)KTS(+)] isoform had a function to promote HR-mediated DNA repair. © 2014 Wiley Periodicals, Inc. PMID:25418835

Oji, Yusuke; Tatsumi, Naoya; Kobayashi, Junya; Fukuda, Mari; Ueda, Tazu; Nakano, Eri; Saito, Chisae; Shibata, Syohei; Sumikawa, Mihoko; Fukushima, Hisashi; Saito, Akari; Hojo, Nozomi; Suzuki, Miyu; Hoshikawa, Tomoko; Shimura, Tsutomu; Morii, Eiichi; Oka, Yoshihiro; Hosen, Naoki; Komatsu, Kenshi; Sugiyama, Haruo

2014-11-21

46

Recombination during early herpes simplex virus type 1 infection is mediated by cellular proteins.  

Science.gov (United States)

Homologous recombination was examined in cells infected with herpes simplex virus type I. Circular and linear DNA with directly repeated sequences was introduced as recombination substrates into cells. Recombination was measured either by origin-dependent amplification of recombination products or by recombination-dependent expression of luciferase from a disrupted gene. Homologous recombination in baby hamster kidney cells converted linear DNA to circular templates for DNA replication and luciferase expression in the complete absence of virus. The products of homologous recombination were efficiently amplified by the viral replication apparatus. The efficiency of recombination was dependent on the structure of the substrate as well as the cell type. Linear DNA with the direct repeats at internal positions failed to recombine in Balb/c 3T3 cells and induced p53-dependent apoptosis. In contrast, linear DNA with directly repeated sequences precisely at the ends recombined and replicated in 3T3 cells. Homologous recombination in baby hamster kidney cells did not depend on the position of the repeated sequences. We conclude that homologous recombination is independent of viral gene functions and that it is likely to be carried out by cellular proteins. We suggest that homologous recombination between directly repeated sequences in the linear herpes simplex virus type 1 chromosome may help to avoid p53-dependent apoptosis and to promote viral DNA replication. PMID:11069901

Yao, X D; Elias, P

2001-01-26

47

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...

48

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

Science.gov (United States)

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

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

2006-09-20

49

Self-consistent RPA calculations with Skyrme-type interactions: The skyrme_rpa program  

Science.gov (United States)

Random Phase Approximation (RPA) calculations are nowadays an indispensable tool in nuclear physics studies. We present here a complete version implemented with Skyrme-type interactions, with the spherical symmetry assumption, that can be used in cases where the effects of pairing correlations and of deformation can be ignored. The full self-consistency between the Hartree-Fock mean field and the RPA excitations is enforced, and it is numerically controlled by comparison with energy-weighted sum rules. The main limitations are that charge-exchange excitations and transitions involving spin operators are not included in this version. Program summaryProgram title: skyrme_rpa (v 1.00) Catalogue identifier: AENF_v1_0 Program summary URL: http://cpc.cs.qub.ac.uk/summaries/AENF_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 5531 No. of bytes in distributed program, including test data, etc.: 39435 Distribution format: tar.gz Programming language: FORTRAN-90/95; easily downgradable to FORTRAN-77. Computer: PC with Intel Celeron, Intel Pentium, AMD Athlon and Intel Core Duo processors. Operating system: Linux, Windows. RAM: From 4 MBytes to 150 MBytes, depending on the size of the nucleus and of the model space for RPA. Word size: The code is written with a prevalent use of double precision or REAL(8) variables; this assures 15 significant digits. Classification: 17.24. Nature of problem: Systematic observations of excitation properties in finite nuclear systems can lead to improved knowledge of the nuclear matter equation of state as well as a better understanding of the effective interaction in the medium. This is the case of the nuclear giant resonances and low-lying collective excitations, which can be described as small amplitude collective motions in the framework of the Random Phase Approximation (RPA). This work provides a tool where one starts from an assumed form of nuclear effective interaction (the Skyrme forces) and builds the self-consistent Hartree-Fock mean field of a given nucleus, and then the RPA multipole excitations of that nucleus. Solution method: The Hartree-Fock (HF) equations are solved in a radial mesh, using a Numerov algorithm. The solutions are iterated until self-consistency is achieved (in practice, when the energy eigenvalues are stable within a desired accuracy). In the obtained mean field, unoccupied states necessary for the RPA calculations are found. For all single-particle states, box boundary conditions are assumed. To solve the RPA problem for a given value of total angular momentum and parity J? a coupled basis is constructed and the RPA matrix is diagonalized (protons and neutrons are treated explicitly, and no approximation related to the use of isospin formalism is introduced). The transition amplitudes and transition strengths associated to given external operators are calculated. The HF densities and RPA transition densities are also evaluated. Restrictions: The main restrictions are related to the assumed spherical symmetry and absence of pairing correlations. Running time: The typical running time depends strongly on the nucleus, on the multipolarity, on the choice of the model space and of course on the computer. It can vary from a few minutes to several hours.

Colò, Gianluca; Cao, Ligang; Van Giai, Nguyen; Capelli, Luigi

2013-01-01

50

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

Science.gov (United States)

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

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

2013-12-01

51

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

Directory of Open Access Journals (Sweden)

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

Farley Daniel C

2010-12-01

52

Quark recombination in inclusive spectra  

International Nuclear Information System (INIS)

The reaction A1A2 ? (?/sup +-//K+)X has been analyzed using the quark recombination model where a secondary meson is mediated by combining spectator constituent quarks of the baryons belonging to the incident nucleus with the anti-quarks from the sea, the even taking place at a specific value of the Bjorken variable chi g approx. = 1/3. The inclusive cross-section R(E/d3sigma/dp3) is scaled in terms R(PA2??-x) and/or R(PP? ?-x), the scaling turns out to be a function of the probability functions that one or two constituent quarks will be wounded and also of the geometrical factors, depending on the location of the interaction. The model is moderately successful for low momenta secondary mesons. For high momentum component, the triple Regge mechanism with the ? and nucleon trajectory is invoked, where the elementary cross-sections are convoluted with the momentum distribution of the nucleons, bearing in mind the Pauli principle. The internal fermi motion is important. Finally, a generalized recombination model, depending on the probability distribution functions of up and down quarks is also developed to analyze the inclusive spectra

53

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

54

RPA correction to the optical potential  

International Nuclear Information System (INIS)

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. 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. (authors)

55

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)

56

Extravasation of intravascular fluid mediated by the systemic administration of recombinant interleukin 2.  

Science.gov (United States)

Adoptive immunotherapy with lymphokine-activated killer cells and recombinant interleukin 2 (IL 2) can produce significant reduction of visceral metastases in tumor-bearing mice and, as shown recently, in humans with disseminated cancer. Because further dose escalations of IL 2 have been prevented by the development of a vascular leak syndrome (VLS) in both mice and humans, we investigated this VLS in mice undergoing the systemic administration of high-dose IL 2. A model for quantitating capillary permeability was used in which 125I-bovine serum albumin was injected i.v., and 2 hr later, tissues were counted in a gamma analyzer. A permeability index (PI) was calculated by dividing the mean counts per minute (cpm) of tissues from IL 2-treated mice by those from control animals. The injection of IL 2 produced increases in vascular permeability that were most pronounced in the thymus, spleen, lungs, liver, and kidneys (PI = 18.0, 10.0, 9.7, 6.7, and 6.3, respectively, on day 6). The development of the VLS was highly dependent on the number of days of IL 2 treatment (for example, the lungs contained 638, 1382, 3350, and 6187 cpm after 0, 1, 3, and 6 days of IL 2, respectively). Moreover, the degree of the VLS was directly related to the dose of IL 2 administered. Measurement of the wet and dry weights of lungs from IL 2-treated mice demonstrated that IL 2 produced a dramatic increase in their water weight (from 0.10 g at base line to 0.22 g after 200,000 U of IL 2 for 6 days). The injection of the IL 2 excipient failed to induce capillary leakage in tissues. Immunosuppression of mice by pretreatment irradiation (500 rad) or by injection of cyclophosphamide or by concurrent use of cortisone acetate markedly reduced or eliminated the development of the VLS. Similarly, the VLS was not observed in nude mice receiving IL 2. Thus, the administration of IL 2 produces a dose-limiting VLS that may be mediated, directly or indirectly, by host lymphoid elements. PMID:3528289

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

1986-09-01

57

Blocking the B7-H4 pathway with novel recombinant antibodies enhances T cell-mediated antitumor responses.  

Science.gov (United States)

B7-H4 inhibits T-cell activation and is widely expressed by solid neoplasms. We have recently demonstrated that the expression of B7-H4 on the surface of malignant cells in vivo is inducible, and that novel anti-B7-H4 recombinant antibodies can reverse the inhibition of tumor-specific T cells. Thus, antibodies targeting the B7-H4 pathways may extend the survival of cancer patients by restoring T cell-mediated antitumor responses. PMID:24083083

Dangaj, Denarda; Scholler, Nathalie

2013-08-01

58

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

59

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

Directory of Open Access Journals (Sweden)

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

Vassetzky Yegor S

2008-12-01

60

Various applications of TALEN- and CRISPR/Cas9-mediated homologous recombination to modify the Drosophila genome  

Science.gov (United States)

ABSTRACT Modifying the genomes of many organisms is becoming as easy as manipulating DNA in test tubes, which is made possible by two recently developed techniques based on either the customizable DNA binding protein, TALEN, or the CRISPR/Cas9 system. Here, we describe a series of efficient applications derived from these two technologies, in combination with various homologous donor DNA plasmids, to manipulate the Drosophila genome: (1) to precisely generate genomic deletions; (2) to make genomic replacement of a DNA fragment at single nucleotide resolution; and (3) to generate precise insertions to tag target proteins for tracing their endogenous expressions. For more convenient genomic manipulations, we established an easy-to-screen platform by knocking in a white marker through homologous recombination. Further, we provided a strategy to remove the unwanted duplications generated during the “ends-in” recombination process. Our results also indicate that TALEN and CRISPR/Cas9 had comparable efficiency in mediating genomic modifications through HDR (homology-directed repair); either TALEN or the CRISPR/Cas9 system could efficiently mediate in vivo replacement of DNA fragments of up to 5?kb in Drosophila, providing an ideal genetic tool for functional annotations of the Drosophila genome. PMID:24659249

Yu, Zhongsheng; Chen, Hanqing; Liu, Jiyong; Zhang, Hongtao; Yan, Yan; Zhu, Nannan; Guo, Yawen; Yang, Bo; Chang, Yan; Dai, Fei; Liang, Xuehong; Chen, Yixu; Shen, Yan; Deng, Wu-Min; Chen, Jianming; Zhang, Bo; Li, Changqing; Jiao, Renjie

2014-01-01

 
 
 
 
61

Spurious modes in extended RPA theories  

International Nuclear Information System (INIS)

The necessary conditions that the spurious state associated with the translational motion and its double-phonon state have zero excitation energy in Extended RPA (ERPA) theories which include both one-body and two-body amplitudes are investigated using the small-amplitude limit of the time-dependent density-matrix theory (STDDM). STDDM provides us with a quite general form of ERPA, as compared with other similar theories, in the sense that all components of one-body and two-body amplitudes are taken into account. Two conditions are found necessary to guarantee the above property of the single and double spurious states: The first is that no truncation in the single-particle space should be made. This condition is necessary for the closure relation to be used and is common for the single and double spurious states. The second depends on the mode. For the single spurious state all components of the one-body amplitudes must be included, and for the double spurious state all components of one-body and two-body amplitudes have to be included. It is also shown that the Kohn theorem and the continuity equations for transition densities and currents hold under the same conditions as the spurious states. ERPA theories formulated using the Hartree-Fock ground state have a non-hermiticity problem. A method for formulating ERPA with hermiticity is also proposed using the time-dependent density-matrix formalism. (orig.)

62

FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori  

Science.gov (United States)

A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they h...

63

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)

64

RPA Correlations and Nuclear Densities in Relativistic Mean Field Approach  

CERN Document Server

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.

Van Giai, N; Meng, J; Giai, Nguyen Van; Liang, Haozhao; Meng, Jie

2007-01-01

65

Recombinant gamma interferon causes neutrophil migration mediated by the release of a macrophage neutrophil chemotactic factor.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A dose-dependent neutrophil migration was observed following the injection of purified (Hu IFN-gamma) or recombinant (rIFN-gamma) human gamma interferon into rat peritoneal cavities. This finding contrasts with their inability to cause chemotaxis in vitro in the Boyden chamber. Neutrophil migration into peritoneal cavities and subcutaneous air pouches induced by both preparations of interferon was abolished by pretreatment of the animals with dexamethasone. IFN-gamma-induced neutrophil migrat...

Ribeiro, R. A.; Cunha, F. Q.; Ferreira, S. H.

1990-01-01

66

Hapten Mediated Display and Pairing of Recombinant Antibodies Accelerates Assay Assembly for Biothreat Countermeasures  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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 s...

Sherwood, Laura J.; Andrew Hayhurst

2012-01-01

67

Recombinant Vesicular Stomatitis Virus Vector Mediates Postexposure Protection against Sudan Ebola Hemorrhagic Fever in Nonhuman Primates?  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Recombinant vesicular stomatitis virus (VSV) vectors expressing homologous filoviral glycoproteins can completely protect rhesus monkeys against Marburg virus when administered after exposure and can partially protect macaques after challenge with Zaire ebolavirus. Here, we administered a VSV vector expressing the Sudan ebolavirus (SEBOV) glycoprotein to four rhesus macaques shortly after exposure to SEBOV. All four animals survived SEBOV challenge, while a control animal that received a nons...

Geisbert, Thomas W.; Daddario-dicaprio, Kathleen M.; Williams, Kinola J. N.; Geisbert, Joan B.; Leung, Anders; Feldmann, Friederike; Hensley, Lisa E.; Feldmann, Heinz; Jones, Steven M.

2008-01-01

68

Transgenic or plant expression vector-mediated recombination of Plum Pox Virus.  

Science.gov (United States)

Different mutants of an infectious full-length clone (p35PPV-NAT) of Plum pox virus (PPV) were constructed: three mutants with mutations of the assembly motifs RQ and DF in the coat protein gene (CP) and two CP chimeras with exchanges in the CP core region of Zucchini yellow mosaic virus and Potato virus Y. The assembly mutants were restricted to single infected cells, whereas the PPV chimeras were able to produce systemic infections in Nicotiana benthamiana plants. After passages in different transgenic N. benthamiana plants expressing the PPV CP gene with a complete (plant line 4.30.45.) or partially deleted 3'-nontranslated region (3'-NTR) (plant line 17.27. 4.), characterization of the viral progeny of all mutants revealed restoration of wild-type virus by recombination with the transgenic CP RNA only in the presence of the complete 3'-NTR (4.30.45.). Reconstitution of wild-type virus was also observed following cobombardment of different assembly-defective p35PPV-NAT together with a movement-defective plant expression vector of Potato virus X expressing the intact PPV-NAT CP gene transiently in nontransgenic N. benthamiana plants. Finally, a chimeric recombinant virus was detected after cobombardment of defective p35PPV-NAT with a plant expression vector-derived CP gene from the sour cherry isolate of PPV (PPV-SoC). This chimeric virus has been established by a double recombination event between the CP-defective PPV mutant and the intact PPV-SoC CP gene. These results demonstrate that viral sequences can be tested for recombination events without the necessity for producing transgenic plants. PMID:10906199

Varrelmann, M; Palkovics, L; Maiss, E

2000-08-01

69

Resolution of Dicentric Chromosomes by Ty-Mediated Recombination in Yeast  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We have integrated a plasmid containing a yeast centromere, CEN5, into the HIS4 region of chromosome III by transformation. Of the three transformant colonies examined, none contained a dicentric chromosome, but all contained a rearranged chromosome III. In one transformant, rearrangement occurred by homologous recombination between two Ty elements; one on the left arm and the other on the right arm of chromosome III. This event produced a ring chromosome (ring chromosome III) of about 60 kb...

Surosky, Richard T.; Tye, Bik-kwoon

1985-01-01

70

Recombinant murine erythropoietin receptor expressed in avian erythroid progenitors mediates terminal erythroid differentiation in vitro  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The biological activity of the recombinant murine erythropoietin receptor (muEpoR) has so far been ascertained only in nonerythroid, established cell lines ectopically expressing the exogenous receptor. Here we show that the regulation of proliferation and differentiation by the muEpoR can be studied in chicken erythroid cells capable of terminal differentiation. The cloned muEpoR was introduced into primary and immortalized chicken erythroblast clones transformed by conditional oncogenes, us...

Steinlein, P.; Deiner, E.; Leutz, A.; Beug, H.

1994-01-01

71

Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells.  

Science.gov (United States)

In this study we describe optimization of polyethylenimine (PEI)-mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell-specific production. The optimal conditions for transient transfection of suspension-adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG4 monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 microg 10(6) cells(-1) mL(-1) at a PEI nitrogen:DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin-like growth factor (LR3-IGF) and a reduction in culture temperature to 32 degrees C were found to increase product titer 2- and 3-fold, respectively. However, mild hypothermia and LR3-IGF acted synergistically to increase product titer 11-fold. Although increased product titer in the presence of LR3-IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post-transfection increased both the integral of viable cell concentration (IVC) and cell-specific Mab production rate. For cultures maintained at 32 degrees C in the presence of LR3-IGF, IVC and qMab were increased 4- and 2.5-fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 degrees C in the presence of LR3-IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3-fold increase in accumulated Mab titer from approximately 13 to approximately 39 mg L(-1). Under these conditions, Mab glycosylation at Asn297 remained essentially constant and similar to that of the same Mab produced by stably transfected GS-CHO cells. From these data we suggest that the efficiency of transient production processes (protein output per rDNA input) can be significantly improved using a combination of mild hypothermia and growth factor(s) to yield an extended "activated hypothermic synthesis". PMID:16739959

Galbraith, Douglas J; Tait, Andrew S; Racher, Andrew J; Birch, John R; James, David C

2006-01-01

72

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

Decottignies, Anabelle

2007-01-01

73

Characterization of Alu and recombination-associated motifs mediating a large homozygous SPG7 gene rearrangement causing hereditary spastic paraplegia.  

Science.gov (United States)

Spastic paraplegia type 7 (SPG7) is one of the most common forms of autosomal recessive hereditary spastic paraplegia (AR-HSP). Although over 77 different mutations have been identified in SPG7 patients, only 9 gross deletions have been reported with only a few of them being fully characterized. Here, we present a detailed description of a large homozygous intragenic SPG7 gene rearrangement involving a 5144-base pair (bp) genomic loss (c. 1450-446_1779?+?746 delinsAAAGTGCT) encompassing exons 11 to 13, identified in a Spanish AR-HSP family. Analysis of the deletion junction sequences revealed that the 5' breakpoint of this SPG7 gene deletion was located within highly homologous Alu sequences where the 3' breakpoint appears to be flanked by the core crossover hotspot instigator (chi)-like sequence (GCTGG). Furthermore, an 8-bp (AAAGTTGCT) conserved sequence at the breakpoint junction was identified, suggesting that the most likely mechanism for the occurrence of this rearrangement is by Alu microhomology and chi-like recombination-associated motif-mediated multiple exon deletion. Our results are consistent with non-allelic homologous recombination and non-homologous end joining in deletion mutagenesis for the generation of rearrangements. This study provides more evidence associating repeated elements as a genetic mechanism underlying neurodegenerative disorders, highlighting their importance in human diseases. PMID:25398481

López, Eva; Casasnovas, Carlos; Giménez, Javier; Matilla-Dueñas, Antoni; Sánchez, Ivelisse; Volpini, Víctor

2014-11-16

74

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

DEFF Research Database (Denmark)

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

Bonde, Mads; Klausen, Michael S.

2014-01-01

75

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

Science.gov (United States)

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

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

2014-07-01

76

Site-specific gene integration in rice genome mediated by the FLP-FRT recombination system.  

Science.gov (United States)

Plant transformation based on random integration of foreign DNA often generates complex integration structures. Precision in the integration process is necessary to ensure the formation of full-length, single-copy integration. Site-specific recombination systems are versatile tools for precise genomic manipulations such as DNA excision, inversion or integration. The yeast FLP-FRT recombination system has been widely used for DNA excision in higher plants. Here, we report the use of FLP-FRT system for efficient targeting of foreign gene into the engineered genomic site in rice. The transgene vector containing a pair of directly oriented FRT sites was introduced by particle bombardment into the cells containing the target locus. FLP activity generated by the co-bombarded FLP gene efficiently separated the transgene construct from the vector-backbone and integrated the backbone-free construct into the target site. Strong FLP activity, derived from the enhanced FLP protein, FLPe, was important for the successful site-specific integration (SSI). The majority of the transgenic events contained a precise integration and expressed the transgene. Interestingly, each transgenic event lacked the co-bombarded FLPe gene, suggesting reversion of the integration structure in the presence of the constitutive FLPe expression. Progeny of the precise transgenic lines inherited the stable SSI locus and expressed the transgene. This work demonstrates the application of FLP-FRT system for site-specific gene integration in plants using rice as a model. PMID:21083801

Nandy, Soumen; Srivastava, Vibha

2011-08-01

77

The RPA description of the quantum damped harmonic oscillator  

International Nuclear Information System (INIS)

We have developed a microscopic, time reversible model which behaves like a damped harmonic oscillator for times short compared to the system's Poincare time. The model assumes an RPA description of initial collective states within a restricted subspace, then traces their time evolution when an additional subspace (the reservoir) is coupled to the restricted subspace. The assumption that this time evolution is also governed by RPA dynamics is utilized. The evolution of packets initialized as the shifted eigenstates of a undamped oscillator of arbitrary frequency (different from the frequency of the collective phonon) allows the explicit study of the expectation values and the fluctuations in the collective coordinates. In all cases, the fluctuations decay to their zero point values. Due to the decay of the fluctuations, the energy loss rate deviates from the classical result. The RPA description contrasts with both the Albrecht and the Kostin descriptions. (orig.)

78

Wigner Function Moments versus RPA in a simple model  

Science.gov (United States)

Two complementary methods to describe the collective motion, RPA and Wigner Function Moments (WFM) method, are compared using an example of a simple model—harmonic oscillator with quadrupole-quadrupole residual interaction. It is shown that they give identical formulas for eigenfrequencies and transition probabilities of all collective excitations of the model, including the scissors mode, which is a subject of our special attention. The exact relation between the variables of the two methods and the respective dynamical equations is established. The transformation of the RPA spectrum into the WFM one is explained.

Balbutsev, E. B.; Schuck, P.

2006-12-01

79

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 a.sflk-1 gene therapy might be a feasible approach for inhibiting the development of corneal neovascularization

80

Developing protocols for recombinant adeno-associated virus-mediated gene therapy in space.  

Science.gov (United States)

With the advent of the era of International Space Station (ISS) and Mars exploration, it is important more than ever to develop means to cure genetic and acquired diseases, which include cancer and AIDS, for these diseases hamper human activities. Thus, our ultimate goal is to develop protocols for gene therapy, which are suitable to humans on the earth as well as in space. Specifically, we are trying to cure the hemoglobinopathies, beta-thalassemia (Cooley's anemia) and sickle cell anemia, by gene therapy. These well-characterized molecular diseases serve as models for developing ex vivo gene therapy, which would apply to other disorders as well. For example, the procedure may become directly relevant to treating astronauts for space-anemia, immune suppression and bone marrow derived tumors, e.g. leukemia. The adeno-associated virus serotype 2 (AAV2) is a non-pathogenic human parvovirus with broad host-range and tissue specificity. Exploiting these characteristics we have been developing protocols for recombinant AAV2 (rAAV)-based gene therapy. With the rAAV constructs and hematopoietic stem cell (HSC) culture systems in hand, we are currently attempting to cure the mouse model of beta-thalassemia [C57BL/6- Hbbth/Hbbth, Hb(d-minor)] by HSC transplantation (HST) as well as by gene therapy. This paper describes the current status of our rAAV-gene therapy research. PMID:12697549

Ohi, S

2000-07-01

 
 
 
 
81

Effects of recombinant activated factor VII on thrombin-mediated feedback activation of coagulation.  

Science.gov (United States)

Thrombin is a key hemostatic enzyme, which propagates its own generation by activating factors V, VIII, and XI. Sustained thrombin generation also activates thrombin-activatable fibrinolysis inhibitor (TAFI), which stabilizes fibrin clot against fibrinolysis. Recombinant activated factor VII (rFVIIa) is considered a novel hemostatic intervention for refractory bleeding, but rebleeding episodes related to fibrinolysis still occur. The present study aimed to investigate the antifibrinolytic effects of rFVIIa in relation to thrombin generation. Using thrombelastography, the effects of rFVIIa on thrombin-activated fibrin formation and on fibrinolysis induced by tissue plasminogen activator were evaluated in various factor-deficient plasma samples. A Thrombinoscope was used to quantitate thrombin generation. Thrombin increased antifibrinolytic activity in a concentration-dependent manner as demonstrated by a longer clot lysis time. In plasma deficient in factors V, VIII, IX, X, or XI, clot lysis occurred early (factor-XI-deficient plasma. A normal clot lysis time was observed in factor-XIII-deficient or dual antithrombin/factor-VIII-deficient plasma. Inhibition of TAFI increased the rate of fibrinolysis. Thrombin generation was delayed or decreased in single factor-deficient plasma except for factor XIII deficiency. After rFVIIa addition, the peak thrombin generation reached over 100 nmol/l in factor-XI-deficient plasma, but not in plasma deficient in factors V, VIII, IX, or X. Thrombin generation and subsequent activation of TAFI were important for clot stability. We conclude that rFVIIa therapy does not compensate for increased susceptibility to fibrinolysis due to lack of factor(s) necessary for the formation of tenase and prothrombinase. PMID:18277134

Taketomi, Taro; Szlam, Fania; Bader, Stephen O; Sheppard, Chelsea A; Levy, Jerrold H; Tanaka, Kenichi A

2008-03-01

82

Recombinase Polymerase Amplification (RPA) of CaMV-35S Promoter and nos Terminator for Rapid Detection of Genetically Modified Crops  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that enables the amplification of DNA within 30 min at a constant temperature of 37–42 °C by simulating in vivo DNA recombination. In this study, based on the regulatory sequence of the cauliflower mosaic virus 35S (CaMV-35S) promoter and the Agrobacterium tumefaciens nopaline synthase gene (nos) terminator, which are widely incorporated in genetically modified (GM) crops, we designe...

Chao Xu; Liang Li; Wujun Jin; Yusong Wan

2014-01-01

83

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

DEFF Research Database (Denmark)

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

Hagen, Lars; Kavli, Bodil

2008-01-01

84

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

Directory of Open Access Journals (Sweden)

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

2010-04-01

85

TCreERT2, a Transgenic Mouse Line for Temporal Control of Cre-Mediated Recombination in Lineages Emerging from the Primitive Streak or Tail Bud  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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...

Anderson, Matthew J.; Naiche, L. A.; Wilson, Catherine P.; Elder, Cindy; Swing, Deborah A.; Lewandoski, Mark

2013-01-01

86

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) with the RPA high-affinity ssDNA-binding domains (RPA70AB). Heteronuclear NMR chemical shift mapping revealed that Tag-OBD binds to RPA70AB at a site distal from the ssDNA-binding sites and that RPA7...

Jiang, Xiaohua; Klimovich, Vitaly; Arunkumar, Alphonse I.; Hysinger, Erik B.; Wang, Yingda; Ott, Robert D.; Guler, Gulfem D.; Weiner, Brian; Chazin, Walter J.; Fanning, Ellen

2006-01-01

87

Recombinase Polymerase Amplification (RPA of CaMV-35S Promoter and nos Terminator for Rapid Detection of Genetically Modified Crops  

Directory of Open Access Journals (Sweden)

Full Text Available Recombinase polymerase amplification (RPA is a novel isothermal DNA amplification and detection technology that enables the amplification of DNA within 30 min at a constant temperature of 37–42 °C by simulating in vivo DNA recombination. In this study, based on the regulatory sequence of the cauliflower mosaic virus 35S (CaMV-35S promoter and the Agrobacterium tumefaciens nopaline synthase gene (nos terminator, which are widely incorporated in genetically modified (GM crops, we designed two sets of RPA primers and established a real-time RPA detection method for GM crop screening and detection. This method could reliably detect as few as 100 copies of the target molecule in a sample within 15–25 min. Furthermore, the real-time RPA detection method was successfully used to amplify and detect DNA from samples of four major GM crops (maize, rice, cotton, and soybean. With this novel amplification method, the test time was significantly shortened and the reaction process was simplified; thus, this method represents an effective approach to the rapid detection of GM crops.

Chao Xu

2014-10-01

88

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

Directory of Open Access Journals (Sweden)

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.

Hope Ian A

2010-03-01

89

E1 strength in light nuclei: Skyrme RPA analysis  

International Nuclear Information System (INIS)

The giant dipole resonance (GDR) in N = 28 isotones (48Ca, 50Ti, 52Cr, 54Fe) is analyzed in the framework of the Skyrme random-phase-approximation (RPA). Three Skyrme forces, SkM*, SLy6 and SV-bas, are used. The effects beyond RPA are simulated by the double folding procedure. We show that dipole strength exhibits a large collective shift, which testifies to a strong impact of the residual interaction and signals on considerable anharmonic effects. In 52Cr, a significant pairing impact is found. For exception of 50Ti, an acceptable agreement with the experiment data is obtained, which justifies the ability of Skyrme forces to describe GDR in light nuclei. (author)

90

Generalized Scalar Multiplication Secure against SPA, DPA, and RPA  

Science.gov (United States)

In the execution on a smart card, elliptic curve cryptosystems have to be secure against side channel attacks such as the simple power analysis (SPA), the differential power analysis (DPA), and the refined power analysis (RPA), and so on. MMM-algorithm proposed by Mamiya, Miyaji, and Morimoto is a scalar multiplication algorithm secure against SPA, DPA, and RPA, which can decrease the computational complexity by increasing the size of a pre-computed table. However, it provides only 4 different cases of pre-computed tables. From the practical point of view, a wider range of time-memory tradeoffs is usually desired. This paper generalizes MMM-algorithm to improve the flexibility of tables as well as the computational complexity. Our improved algorithm is secure, efficient and flexible for the storage size.

Miyaji, Atsuko

91

Restoration of broken symmetries in Self-Consistent RPA  

Digital Repository Infrastructure Vision for European Research (DRIVER)

It is shown that the Self-Consistent RPA (SCRPA) approach allows in a very natural way to restore symmetries, spontaneously broken on the mean field level. This is achieved via the introduction of a second Lagrange multiplier which constrains the variance of the symmetry operator to zero. This important feature of SCRPA, here pointed out for the first time, is illustrated employing a simplified model of the nuclear superfluidity.

Rabhi, A.; Schuck, P.; Bennaceur, R.; Chanfray, G.; Dukelsky, J.

2001-01-01

92

Giant resonances using realistic interactions and second RPA  

International Nuclear Information System (INIS)

The Unitary Correlation Operator Method (UCOM) considers explicitly the short-range correlations induced in nuclei by the nucleon-nucleon (NN) interaction and provides a way to derive a universal, phase-shift equivalent effective NN potential starting from a realistic one. The correlated potential can then be used within standard many-body methods and tractable Hilbert spaces. Recent applications have shown that first-order RPA with a two-body UCOM potential can not, in general, reproduce 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. In this work we employ a UCOM interaction in Second RPA (SRPA) calculations of GRs. We find that the inclusion of second-order configurations - which effectively dress the underlying single-particle states with self-energy insertions - produces sizable corrections. These appear essential for a realistic description of GRs when using the UCOM. We argue that effects of higher than second order should be negligible. Therefore, UCOM-SRPA emerges as a promising tool for consistent calculations of GRs in closed-shell nuclei. This is an interesting development, since SRPA can accommodate more physics than RPA (e.g., fragmentation). Remaining discrepancies due to missing three-body terms and self-consistency issues of the model are discussed

93

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

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

2012-01-01

94

Microscopic description of hot nuclei: the SPA+RPA approach  

International Nuclear Information System (INIS)

We discuss the static path plus random phase approximation (SPA+RPA) to the partition function of warm finite nuclei. The method, derived from the auxiliary field path integral representation of the partition function, takes into account large amplitude statistical fluctuations around the mean field, and is able to provide an accurate evaluation of thermodynamics properties within finite configuration spaces. We present some recent improvements which include the treatment of repulsive terms in the interaction, the exact implementation in canonical and in restricted grand canonical ensembles with fixed number parity, and the evaluation of response and strength functions

95

Microscopic description of hot nuclei: the SPA+RPA approach  

CERN Document Server

We discuss the static path plus random phase approximation (SPA+RPA) to the partition function of warm finite nuclei. The method, derived from the auxiliary field path integral representation of the partition function, takes into account large amplitude statistical fluctuations around the mean field, and is able to provide an accurate evaluation of thermodynamics properties within finite configuration spaces. We present some recent improvements which include the treatment of repulsive terms in the interaction, the exact implementation in canonical and in restricted grand canonical ensembles with fixed number parity, and the evaluation of response and strength functions.

Rossignoli, R; Ring, P

1999-01-01

96

RPA sum rules for giant resonances at finite temperature  

International Nuclear Information System (INIS)

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

97

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

Science.gov (United States)

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) with the RPA high-affinity ssDNA-binding domains (RPA70AB). Heteronuclear NMR chemical shift mapping revealed that Tag-OBD binds to RPA70AB at a site distal from the ssDNA-binding sites and that RPA70AB, Tag-OBD, and an 8-nucleotide ssDNA form a stable ternary complex. Intact RPA and Tag also interact stably in the presence of an 8-mer, but Tag dissociates from the complex when RPA binds to longer oligonucleotides. Together, our results imply that an allosteric change in RPA quaternary structure completes the loading reaction. A mechanistic model is proposed in which the ternary complex is a key intermediate that directly couples origin DNA unwinding to RPA loading on emerging ssDNA. PMID:17110927

Jiang, Xiaohua; Klimovich, Vitaly; Arunkumar, Alphonse I; Hysinger, Erik B; Wang, Yingda; Ott, Robert D; Guler, Gulfem D; Weiner, Brian; Chazin, Walter J; Fanning, Ellen

2006-11-29

98

Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling  

Science.gov (United States)

ATR kinase is a critical upstream regulator of the checkpoint response to various forms of DNA damage. Previous studies have shown that ATR is recruited via its binding partner ATR-interacting protein (ATRIP) to replication protein A (RPA)-covered single-stranded DNA (RPA-ssDNA) generated at sites of DNA damage where ATR is then activated by TopBP1 to phosphorylate downstream targets including the Chk1 signal transducing kinase. However, this critical feature of the human ATR-initiated DNA damage checkpoint signaling has not been demonstrated in a defined system. Here we describe an in vitro checkpoint system in which RPA-ssDNA and TopBP1 are essential for phosphorylation of Chk1 by the purified ATR-ATRIP complex. Checkpoint defective RPA mutants fail to activate ATR kinase in this system, supporting the conclusion that this system is a faithful representation of the in vivo reaction. Interestingly, we find that an alternative form of RPA (aRPA), which does not support DNA replication, can substitute for the checkpoint function of RPA in vitro, thus revealing a potential role for aRPA in the activation of ATR kinase. We also find that TopBP1 is recruited to RPA-ssDNA in a manner dependent on ATRIP and that the N terminus of TopBP1 is required for efficient recruitment and activation of ATR kinase. PMID:20616048

Choi, Jun-Hyuk; Lindsey-Boltz, Laura A.; Kemp, Michael; Mason, Aaron C.; Wold, Marc S.; Sancar, Aziz

2010-01-01

99

Rpa4, a homolog of the 34-kilodalton subunit of the replication protein A complex.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Replication protein A (RPA) is a complex of three polypeptides of 70, 34, and 13 kDa isolated from diverse eukaryotes. The complex is a single-stranded DNA-binding protein essential for simian virus 40-based DNA replication in vitro and for viability in the yeast Saccharomyces cerevisiae. We have identified a new 30-kDa human protein which interacts with the 70- and 13-kDa subunits of RPA, with a yeast two-hybrid/interaction trap method. This protein, Rpa4, has 47% identity with Rpa2, the 34-...

Keshav, K. F.; Chen, C.; Dutta, A.

1995-01-01

100

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

Science.gov (United States)

Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells. PMID:22761925

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

2012-01-01

 
 
 
 
101

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

102

Activity of the Rhodopseudomonas palustris p-Coumaroyl-Homoserine Lactone-Responsive Transcription Factor RpaR ? †  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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 severa...

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

2011-01-01

103

Regulatory Functions of the N-terminal Domain of the 70-kDa Subunit of Replication Protein A (RPA)*  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Replication protein A (RPA) is the major single-stranded DNA-binding protein in eukaryotes. RPA is composed of three subunits of 70, 32, and 14 kDa. The N-terminal domain of the 70-kDa subunit (RPA70) has weak DNA binding activity, interacts with proteins, and is involved in cellular DNA damage response. To define the mechanism by which this domain regulates RPA function, we analyzed the function of RPA forms containing a deletion of the N terminus of RPA70 and mutatio...

Binz, Sara K.; Wold, Marc S.

2008-01-01

104

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

Science.gov (United States)

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

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

2014-02-01

105

Transfer of the symbiotic plasmid of Rhizobium etli CFN42 requires cointegration with p42a, which may be mediated by site-specific recombination.  

Science.gov (United States)

Plasmid p42a from Rhizobium etli CFN42 is self-transmissible and indispensable for conjugative transfer of the symbiotic plasmid (pSym). Most pSym transconjugants also inherit p42a. pSym transconjugants that lack p42a always contain recombinant pSyms, which we designated RpSyms*. RpSyms* do not contain some pSym segments and instead have p42a sequences, including the replication and transfer regions. These novel recombinant plasmids are compatible with wild-type pSym, incompatible with p42a, and self-transmissible. The symbiotic features of derivatives simultaneously containing a wild-type pSym and an RpSym* were analyzed. Structural analysis of 10 RpSyms* showed that 7 shared one of the two pSym-p42a junctions. Sequencing of this common junction revealed a 53-bp region that was 90% identical in pSym and p42a, including a 5-bp central region flanked by 9- to 11-bp inverted repeats reminiscent of bacterial and phage attachment sites. A gene encoding an integrase-like protein (intA) was localized downstream of the attachment site on p42a. Mutation or the absence of intA abolished pSym transfer from a recA mutant donor. Complementation with the wild-type intA gene restored transfer of pSym. We propose that pSym-p42a cointegration is required for pSym transfer; cointegration may be achieved either through homologous recombination among large reiterated sequences or through IntA-mediated site-specific recombination between the attachment sites. Cointegrates formed through the site-specific system but resolved through RecA-dependent recombination or vice versa generate RpSyms*. A site-specific recombination system for plasmid cointegration is a novel feature of these large plasmids and implies that there is unique regulation which affects the distribution of pSym in nature due to the role of the cointegrate in conjugative transfer. PMID:15516565

Brom, Susana; Girard, Lourdes; Tun-Garrido, Cristina; García-de los Santos, Alejandro; Bustos, Patricia; González, Víctor; Romero, David

2004-11-01

106

Enzymatic properties of the Caenorhabditis elegans Dna2 endonuclease/helicase and a species-specific interaction between RPA and Dna2  

Digital Repository Infrastructure Vision for European Research (DRIVER)

In both budding and fission yeasts, a null mutation of the DNA2 gene is lethal. In contrast, a null mutation of Caenorhabditis elegans dna2+ causes a delayed lethality, allowing survival of some mutant C.elegans adults to F2 generation. In order to understand reasons for this difference in requirement of Dna2 between these organisms, we examined the enzymatic properties of the recombinant C.elegans Dna2 (CeDna2) and its interaction with replication-protein A (RPA) from various sources. Like b...

Kim, Do-hyung; Lee, Kyoung-hwa; Kim, Jeong-hoon; Ryu, Gi-hyuck; Bae, Sung-ho; Lee, Byung-chul; Moon, Kyeong-yeop; Byun, Si-myung; Koo, Hyeon-sook; Seo, Yeon-soo

2005-01-01

107

H2O2 regulates recombinant Ca2+ channel alpha1C subunits but does not mediate their sensitivity to acute hypoxia.  

Science.gov (United States)

Acute hypoxic inhibition of the pore-forming alpha(1C) subunit of the L-type Ca(2+) channel mediates hypoxic arterial vasodilatation, a physiological response which matches tissue O(2) demand and supply in the systemic vasculature. In numerous O(2)-sensing cell types, reactive O(2) species (ROS) have been proposed as mediators linking lowered O(2) levels with the appropriate cellular response. In this study, we examined the roles of H(2)O(2) and NADPH oxidase as mediators of hypoxic inhibition of recombinant alpha(1C) subunits. Human cardiac L-type Ca(2+) channel alpha(1C) subunits were stably expressed in HEK 293 cells. Ca(2+) currents were recorded using the whole-cell configuration of the patch-clamp technique. Bath application of 100microM H(2)O(2) significantly enhanced depolarisation-evoked Ca(2+) currents in a voltage-dependent manner, while dialysis with 1000Uml(-1) catalase reduced these currents. In the presence of catalase, hypoxic inhibition of Ca(2+) currents was not significantly different compared to non-dialysed controls. The NADPH oxidase inhibitors diphenylene iodonium (10microM) and phenylarsine oxide (5microM) were without effect on either basal Ca(2+) currents or responses to hypoxia. Thus, endogenous production of H(2)O(2) regulates the alpha(1C) subunit. However, neither suppression of H(2)O(2) levels nor inhibition of NADPH oxidase is involved in O(2)-dependent regulation of the Ca(2+) channel. PMID:15110764

Hudasek, Kristin; Brown, Stephen T; Fearon, Ian M

2004-05-21

108

Solving the RPA eigenvalue equation in real-space  

Energy Technology Data Exchange (ETDEWEB)

We present a computational method to solve the RPA eigenvalue equation employing a uniform grid representation in three-dimensional Cartesian coordinates. The conjugate gradient method is used for this purpose as an interactive method for a generalized eigenvalue problem. No construction of unoccupied orbitals is required in the procedure. We expect this method to be useful for systems lacking spatial symmetry to calculate accurate eigenvalues and transition matrix elements of a few low-lying excitations. Some applications are presented to demonstrate the feasibility of the method, considering the simplified mean-field model as an example of a nuclear physics system and the electronic excitations in molecules with time-dependent density functional theory as an example of an electronic system. (author)

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

2002-12-01

109

Thermal nuclear pairing within the self-consistent quasiparticle RPA  

CERN Document Server

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

Dang, N Dinh

2010-01-01

110

Inter-chromosomal recombination of Mll and Af9 genes mediated by cre-loxP in mouse development.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Chromosomal translocations are crucial events in the aetiology of many leukaemias, lymphomas and sarcomas, resulting in enforced oncogene expression or the creation of novel fusion genes. The study of the biological outcome of such events ideally requires recapitulation of the tissue specificity and timing of the chromosomal translocation itself. We have used the Cre-loxP system of phage P1 to induce de novo Mll–Af9 chromosomal recombination during mouse development. loxP sites were introdu...

Collins, Ec; Pannell, R.; Simpson, Em; Forster, A.; Rabbitts, Th

2000-01-01

111

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

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

Feng, Zhihui; Zhang, Junran

2012-01-01

112

PTEN microdeletions in T-cell acute lymphoblastic leukemia are caused by illegitimate RAG-mediated recombination events.  

Science.gov (United States)

Phosphatase and tensin homolog (PTEN)-inactivating mutations and/or deletions are an independent risk factor for relapse of T-cell acute lymphoblastic leukemia (T-ALL) patients treated on Dutch Childhood Oncology Group or German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia protocols. Some monoallelic mutated or PTEN wild-type patients lack PTEN protein, implying that additional PTEN inactivation mechanisms exist. We show that PTEN is inactivated by small deletions affecting a few exons in 8% of pediatric T-ALL patients. These microdeletions were clonal in 3% and subclonal in 5% of patients. Conserved deletion breakpoints are flanked by cryptic recombination signal sequences (cRSSs) and frequently have non-template-derived nucleotides inserted in between breakpoints, pointing to an illegitimate RAG recombination-driven activity. Identified cRSSs drive RAG-dependent recombination in a reporter system as efficiently as bona fide RSSs that flank gene segments of the T-cell receptor locus. Remarkably, equivalent microdeletions were detected in thymocytes of healthy individuals. Microdeletions strongly associate with the TALLMO subtype characterized by TAL1 or LMO2 rearrangements. Primary and secondary xenotransplantation of TAL1-rearranged leukemia allowed development of leukemic subclones with newly acquired PTEN microdeletions. Ongoing RAG activity may therefore actively contribute to the acquisition of preleukemic hits, clonal diversification, and disease progression. PMID:24904117

Mendes, Rui D; Sarmento, Leonor M; Canté-Barrett, Kirsten; Zuurbier, Linda; Buijs-Gladdines, Jessica G C A M; Póvoa, Vanda; Smits, Willem K; Abecasis, Miguel; Yunes, J Andres; Sonneveld, Edwin; Horstmann, Martin A; Pieters, Rob; Barata, João T; Meijerink, Jules P P

2014-07-24

113

A Vector System for ABC Transporter-Mediated Secretion and Purification of Recombinant Proteins in Pseudomonas Species.  

Science.gov (United States)

Pseudomonas fluorescens is an efficient platform for recombinant protein production. P. fluorescens has an ABC transporter secreting endogenous thermostable lipase (TliA) and protease, which can be exploited to transport recombinant proteins across the cell membrane. In this study, the expression vector pDART was constructed by inserting tliDEF, genes encoding the ABC transporter, along with the construct of the lipase ABC transporter recognition domain (LARD), into pDSK519, a widely used shuttle vector. When the gene for the target protein was inserted into the vector, the C-terminally fused LARD allowed it to be secreted through the ABC transporter into the extracellular medium. After secretion of the fused target protein, the LARD containing a hydrophobic C terminus enabled its purification through hydrophobic interaction chromatography (HIC) using a methyl-Sepharose column. Alkaline phosphatase (AP) and green fluorescent protein (GFP) were used to validate the expression, export, and purification of target proteins by the pDART system. Both proteins were secreted into the extracellular medium in P. fluorescens. In particular, AP was secreted in several Pseudomonas species with its enzymatic activity in extracellular media. Furthermore, purification of the target protein using HIC yielded some degree of AP and GFP purification, where AP was purified to almost a single product. The pDART system will provide greater convenience for the secretory production and purification of recombinant proteins in Gram-negative bacteria, such as Pseudomonas species. PMID:25548043

Ryu, Jaewook; Lee, Ukjin; Park, Jiye; Yoo, Do-Hyun; Ahn, Jung Hoon

2015-03-01

114

Small Plasmids Harboring qnrB19: a Model for Plasmid Evolution Mediated by Site-Specific Recombination at oriT and Xer Sites  

Science.gov (United States)

Plasmids pPAB19-1, pPAB19-2, pPAB19-3, and pPAB19-4, isolated from Salmonella and Escherichia coli clinical strains from hospitals in Argentina, were completely sequenced. These plasmids include the qnrB19 gene and are 2,699, 3,082, 2,989, and 2,702 nucleotides long, respectively, and they share extensive homology among themselves and with other previously described small qnrB19-harboring plasmids. The genetic environment of qnrB19 in all four plasmids is identical to that in these other plasmids and in transposons such as Tn2012, Tn5387, and Tn5387-like. Nucleotide sequence comparisons among these and previously described plasmids showed a variable region characterized by being flanked by an oriT locus and a Xer recombination site. We propose that this arrangement could play a role in the evolution of plasmids and present a model for DNA swapping between plasmid molecules mediated by site-specific recombination events at oriT and a Xer target site. PMID:22290975

Tran, Tung; Andres, Patricia; Petroni, Alejandro; Soler-Bistué, Alfonso; Albornoz, Ezequiel; Zorreguieta, Angeles; Reyes-Lamothe, Rodrigo; Sherratt, David J.; Corso, Alejandra

2012-01-01

115

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

Science.gov (United States)

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

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

2013-05-01

116

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

International Nuclear Information System (INIS)

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

117

Passive immunity to yersiniae mediated by anti-recombinant V antigen and protein A-V antigen fusion peptide.  

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LcrV (V antigen), a known unstable 37.3-kDa monomeric peptide encoded on the ca. 70-kb Lcr plasmid of Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica, has been implicated as a regulator of the low-calcium response, virulence factor, and protective antigen. In this study, lcrV of Y. pestis was cloned into protease-deficient Escherichia coli BL21. The resulting recombinant V antigen underwent marked degradation from the C-terminal end during purification, yielding majo...

Motin, V. L.; Nakajima, R.; Smirnov, G. B.; Brubaker, R. R.

1994-01-01

118

Atmospheric-pressure plasma jet induces DNA double-strand breaks that require a Rad51-mediated homologous recombination for repair in Saccharomyces cerevisiae.  

Science.gov (United States)

Non-thermal plasma generated under atmospheric pressure produces a mixture of chemically reactive molecules and has been developed for a number of biomedical applications. Recently, plasma jet has been proposed as novel cancer therapies based on the observation that free radicals generated by plasma jet induce mitochondria-mediated apoptotic cell death. We show here that air plasma jet induces DNA double-strand breaks (DSBs) in yeast chromosomes leading to genomic instability and loss of viability, which are alleviated by Rad51, the yeast homolog of Escherichiacoli RecA recombinase, through DNA damage repair by a homologous recombination (HR) process. Hypersensitivity of rad51 mutant to air plasma was not restored by antioxidant treatment unlike sod1 mutant that was highly sensitive to reactive oxygen species (ROS) challenge, suggesting that plasma jet induces DSB-mediated cell death independent of ROS generation. These results may provide a new insight into the mechanism of air plasma jet-induced cell death. PMID:25086216

Lee, Yoonna; Kim, Kangil; Kang, Kyu-Tae; Lee, Jong-Soo; Yang, Sang Sik; Chung, Woo-Hyun

2014-10-15

119

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

Science.gov (United States)

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

Decottignies, Anabelle

2007-07-01

120

Perfluorochemical (PFC) liquid enhances recombinant adenovirus vector-mediated viral interleukin-10 (AdvIL-10) expression in rodent lung  

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Abstract Adenovirus and cationic liposome mediated transfer of Interleukin-10 (IL-10), a potent anti-inflammatory cytokine, has been shown to decrease pro-inflammatory cytokine levels and overall lung inflammation in models of lung transplantation and injury. Limitations to current approaches of IL-10 gene therapy include poor vector delivery methods and pro-inflammatory properties of human IL-10 under certain conditions. We hypothesize that using perfluorochemical (PFC) liquid to d...

Zimmerman Jerry J; Bonneau Laura L; Li John T; Weiss Daniel J

2007-01-01

 
 
 
 
121

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 < .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

122

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

123

Correlation between frequency of non-allelic homologous recombination and homology properties: evidence from homology-mediated CNV mutations in the human genome.  

Science.gov (United States)

Non-allelic homologous recombination (NAHR) is one of the key mechanisms of DNA rearrangement. NAHR occurring between direct homologous repeats can generate genomic copy number variation (CNV) and make significant contributions to both genome evolution and human diseases such as cancer. Intriguingly, previous observations on the rare CNVs at certain genomic disorder loci suggested that NAHR frequency could be dependent on homology properties. However, such a correlation remains unclear at the other NAHR-mediated CNV loci, especially the common CNVs in human populations. Different from the rare CNVs associated with genomic disorders, it is challenging to identify de novo NAHR events at common CNV loci. Therefore, our previously proposed statistic M was employed in estimating relative mutation rate for the NAHR-mediated CNVs in human populations. By utilizing generalized regression neural network and principal component analysis in studying 4330 CNVs ascertained in 3 HapMap populations, we identified the CNVs mediated by NAHR between paired segmental duplications (SDs) and further revealed the correlations between SD properties and NAHR probability. SD length and inter-SD distance were shown to make major contributions to the occurrence of NAHR, whereas chromosomal position and sequence similarity of paired SDs are also involved in NAHR. An integrated effect of SD properties on NAHR frequency was revealed for the common CNVs in human populations. These observations can be well explained by ectopic synapsis in NAHR together with our proposed model of chromosomal compression/extension/looping (CCEL) for homology mis-pairing. Our findings showed the important roles of SDs in NAHR and human genomic evolution. PMID:25324539

Peng, Zhen; Zhou, Weichen; Fu, Wenqing; Du, Renqian; Jin, Li; Zhang, Feng

2014-10-16

124

An open-shell RPA by means of seniority and reduced isospin projections  

International Nuclear Information System (INIS)

A real-particle RPA formalism for open-shell nuclei is presented which can be applied to both isoscalar vibration and isospin-splitting isovector vibration. In order to keep boson approximation meaningful even under the existence of partially-occupied orbits, one-body operator for description of normal mode is modified by means of projection operators which project onto wave functions with stretched seniority, defined in the isospin formalism, and with specific reduced isospin. Validity of boson approximation ensures bermiticity of RPA equations of motion and clarifies association with quasi-particle RPA. Nuclear excitation arising from the rearrangement of nucleons in the same partially-occupied orbit can be taken into account, while it is impossible in the RPA by Rowe who adopted one-body operators themselves for description of normal mode. (author)

125

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

International Nuclear Information System (INIS)

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<0.05) and 49.70% (Ad/ hEndo group versus DMEM group, t=2.254, P<0.05), respectively. In the study group, serum levels of endostatin in treated group were much higher than that of control groups for 3- and 7- day term. The intratumoral MVD also decreased significantly in the treated tumors (9.95±2.20 versus 18.54±1.80, t=7.158, P< 0.05). Conclusion: Adenovirus-mediated h0.05). Conclusion: Adenovirus-mediated human endostatin gene had obtained high level of expression in vitro and in vivo, and significantly inhibited the angiogenesis and growth of CNE-2 xenografted tumors in nude mice

126

Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Here we describe a triple transgenic mouse system, which combines the tissue specificity of any Cre-transgenic line with the inducibility of the reverse tetracycline transactivator (rtTA)/tetracycline-responsive element (tet-O)-driven transgenes. To ensure reliable rtTA expression in a broad range of cell types, we have targeted the rtTA transgene into the ROSA26 locus. The rtTA expression, however, is conditional to a Cre recombinase-mediated excision of a STOP region from the ROSA26 locus. ...

Belteki, Gusztav; Haigh, Jody; Kabacs, Nikolett; Haigh, Katharina; Sison, Karen; Costantini, Frank; Whitsett, Jeff; Quaggin, Susan E.; Nagy, Andras

2005-01-01

127

Reactive oxygen species regulate DNA copy number in isolated yeast mitochondria by triggering recombination-mediated replication  

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Mitochondrial DNA (mtDNA) encodes proteins that are essential for cellular ATP production. Reactive oxygen species (ROS) are respiratory byproducts that damage mtDNA and other cellular components. In Saccharomyces cerevisiae, the oxidized base excision-repair enzyme Ntg1 introduces a double-stranded break (DSB) at the mtDNA replication origin ori5; this DSB initiates the rolling-circle mtDNA replication mediated by the homologous DNA pairing protein Mhr1. Thus, ROS may play a role in the regu...

Hori, Akiko; Yoshida, Minoru; Shibata, Takehiko; Ling, Feng

2009-01-01

128

Recombinant aequorin as a reporter for receptor-mediated changes of intracellular Ca2+ -levels in Drosophila S2 cells.  

Science.gov (United States)

The bioluminescent Ca(2+)-sensitive reporter protein, aequorin, was employed to develop an insect cell-based functional assay system for monitoring receptor-mediated changes of intracellular Ca(2)(+)-concentrations. Drosophila Schneider 2 (S2) cells were genetically engineered to stably express both apoaequorin and the insect tachykinin-related peptide receptor, STKR. Lom-TK III, an STKR agonist, was shown to elicit concentration-dependent bioluminescent responses in these S2-STKR-Aeq cells. The EC(50) value for the calcium effect detected by means of aequorin appeared to be nearly identical to the one that was measured by means of Fura-2, a fluorescent Ca(2)(+)-indicator. In addition, this aequorin-based method was also utilised to study receptor antagonists. Experimental analysis of the effects exerted by spantide I, II and III, three potent substance P antagonists, on Lom-TK III-stimulated S2-STKR-Aeq cells showed that these compounds antagonise STKR-mediated responses in a concentration-dependent manner. The rank order of inhibitory potencies was spantide III > spantide II > spantide I. PMID:12488971

Torfs, Herbert; Poels, Jeroen; Detheux, Michel; Dupriez, Vincent; Van Loy, Tom; Vercammen, Linda; Vassart, Gilbert; Parmentier, Marc; Vanden Broeck, Jozef

2002-04-01

129

A comparison of the phenotypic and genetic stability of recombinant Trichoderma spp. generated by protoplast- and Agrobacterium-mediated transformation.  

Science.gov (United States)

Four different Trichoderma strains, T. harzianum CECT 2413, T. asperellum T53, T. atroviride T11 and T. longibrachiatum T52, which represent three of the four sections contained in this genus, were transformed by two different techniques: a protocol based on the isolation of protoplasts and a protocol based on Agrobacterium-mediated transformation. Both methods were set up using hygromycin B or phleomycin resistance as the selection markers. Using these techniques, we obtained phenotypically stable transformants of these four different strains. The highest transformation efficiencies were obtained with the T. longibrachiatum T52 strain: 65-70 transformants/microg DNA when transformed with the plasmid pAN7-1 (hygromycin B resistance) and 280 transformants/107 spores when the Agrobacterium-mediated transformation was performed with the plasmid pUR5750 (hygromycin B resistance). Overall, the genetic analysis of the transformants showed that some of the strains integrated and maintained the transforming DNA in their genome throughout the entire transformation and selection process. In other cases, the integrated DNA was lost. PMID:16953173

Cardoza, Rosa Elena; Vizcaino, Juan Antonio; Hermosa, Maria Rosa; Monte, Enrique; Gutiérrez, Santiago

2006-08-01

130

The lactoferrin receptor may mediate the reduction of eosinophils in the duodenum of pigs consuming milk containing recombinant human lactoferrin.  

Science.gov (United States)

Lactoferrin is part of the immune system and multiple tissues including the gastrointestinal (GI) tract, liver, and lung contain receptors for lactoferrin. Lactoferrin has many functions, including antimicrobial, immunomodulatory, and iron binding. Additionally, lactoferrin inhibits the migration of eosinophils, which are constitutively present in the GI tract, and increase during inflammation. Lactoferrin suppresses eosinophil infiltration into the lungs and eosinophil migration in -vitro. Healthy pigs have a large population of eosinophils in their small intestine and like humans, pigs have small intestinal lactoferrin receptors (LFR); thus, pigs were chosen to investigate the effects of consumption of milk containing recombinant human lactoferrin (rhLF-milk) on small intestinal eosinophils and expression of eosinophilic cytokines. In addition, LFR localization was analyzed in duodenum and circulating eosinophils to determine if the LFR could play a role in lactoferrin's ability to inhibit eosinophil migration. In the duodenum there were significantly fewer eosinophils/unit area in pigs fed rhLF-milk compared to pigs fed control milk (p = 0.025); this was not seen in the ileum (p = 0.669). In the duodenum, no differences were observed in expression of the LFR, or any eosinophil migratory cytokines, and the amount of LFR protein was not different (p = 0.386). Immunohistochemistry (IHC) showed that within the duodenum the LFR localized on the brush border of villi, crypts, and within the lamina propria. Circulating eosinophils also contained LFRs, which may be a mechanism allowing lactoferrin to directly inhibit eosinophil migration. PMID:25085595

Cooper, Caitlin; Nonnecke, Eric; Lönnerdal, Bo; Murray, James

2014-10-01

131

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

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

MetteChristensen

2010-01-01

132

Cyanamide Potentiates the Ethanol-Induced Impairment of Receptor-Mediated Endocytosis in a Recombinant Hepatic Cell Line Expressing Alcohol Dehydrogenase Activity  

Science.gov (United States)

Ethanol administration has been shown to alter receptor-mediated endocytosis in the liver. We have developed a recombinant hepatic cell line stably transfected with murine alcohol dehydrogenase cDNA to serve as an in vitro model to investigate these ethanol-induced impairments. In the present study, transfected cells were maintained in the absence or presence of 25?mM ethanol for 7 days, and alterations in endocytosis by the asialoglycoprotein receptor were determined. The role of acetaldehyde in this dysfunction was also examined by inclusion of the aldehyde dehydrogenase inhibitor, cyanamide. Our results showed that ethanol metabolism impaired internalization of asialoorosomucoid, a ligand for the asialoglycoprotein receptor. The addition of cyanamide potentiated the ethanol-induced defect in internalization and also impaired degradation of the ligand in the presence of ethanol. These results indicate that the ethanol-induced impairment in endocytosis is exacerbated by the inhibition of aldehyde dehydrogenase, suggesting the involvement of acetaldehyde in this dysfunction. PMID:22518324

Clemens, Dahn L.; Tuma, Dean J.; Casey, Carol A.

2012-01-01

133

The amino-terminal fragment of human urokinase directs a recombinant chimeric toxin to target cells: internalization is toxin mediated.  

Science.gov (United States)

In contrast to two-chain urokinase (uPA), a chemical conjugate between uPA and native saporin (a cytotoxic plant seed ribosome-inactivating protein) did not require plasminogen activator inhibitors to be internalized. To dissect this pathway, we constructed a chimera consisting of the amino-terminal fragment (ATF) of human urokinase fused to a saporin isoform (SAP-3). The chimeric ATF-SAP toxin was expressed in Escherichia coli, purified, and characterized for its ribosome-inactivating activity. Besides being a potent inhibitor of protein synthesis in cell-free assays, ATF-SAP was specifically cytotoxic toward cells expressing human uPAR. Competition experiments indicated that both the human uPAR and the LDL-related receptor protein are involved in mediating the cell killing ability of ATF-SAP. We conclude that neither plasminogen activator inhibitors nor the catalytic moiety of urokinase are necessary to initiate these internalization pathways. Thus, saporin may play a role similar to plasminogen activator inhibitors in its ability to trigger internalization of uPAR-bound ligands through endocytic receptors. PMID:9367352

Fabbrini, M S; Carpani, D; Bello-Rivero, I; Soria, M R

1997-11-01

134

Phosphorylation and cellular function of the human Rpa2 N-terminus in the budding yeast Saccharomyces cerevisiae.  

Science.gov (United States)

Maintenance of genome integrity is critical for proper cell growth. This occurs through accurate DNA replication and repair of DNA lesions. A key factor involved in both DNA replication and the DNA damage response is the heterotrimeric single-stranded DNA (ssDNA) binding complex Replication Protein A (RPA). Although the RPA complex appears to be structurally conserved throughout eukaryotes, the primary amino acid sequence of each subunit can vary considerably. Examination of sequence differences along with the functional interchangeability of orthologous RPA subunits or regions could provide insight into important regions and their functions. This might also allow for study in simpler systems. We determined that substitution of yeast Replication Factor A (RFA) with human RPA does not support yeast cell viability. Exchange of a single yeast RFA subunit with the corresponding human RPA subunit does not function due to lack of inter-species subunit interactions. Substitution of yeast Rfa2 with domains/regions of human Rpa2 important for Rpa2 function (i.e., the N-terminus and the loop 3-4 region) supports viability in yeast cells, and hybrid proteins containing human Rpa2 N-terminal phospho-mutations result in similar DNA damage phenotypes to analogous yeast Rfa2 N-terminal phospho-mutants. Finally, the human Rpa2 N-terminus (NT) fused to yeast Rfa2 is phosphorylated in a manner similar to human Rpa2 in human cells, indicating that conserved kinases recognize the human domain in yeast. The implication is that budding yeast represents a potential model system for studying not only human Rpa2 N-terminal phosphorylation, but also phosphorylation of Rpa2 N-termini from other eukaryotic organisms. PMID:25499885

Ghospurkar, Padmaja L; Wilson, Timothy M; Liu, Shengqin; Herauf, Anna; Steffes, Jenna; Mueller, Erica N; Oakley, Gregory G; Haring, Stuart J

2015-02-01

135

Tensor hypercontracted ppRPA: Reducing the cost of the particle-particle random phase approximation from O(r 6) to O(r 4)  

Science.gov (United States)

In recent years, interest in the random-phase approximation (RPA) has grown rapidly. At the same time, tensor hypercontraction has emerged as an intriguing method to reduce the computational cost of electronic structure algorithms. In this paper, we combine the particle-particle random phase approximation with tensor hypercontraction to produce the tensor-hypercontracted particle-particle RPA (THC-ppRPA) algorithm. Unlike previous implementations of ppRPA which scale as O(r6), the THC-ppRPA algorithm scales asymptotically as only O(r4), albeit with a much larger prefactor than the traditional algorithm. We apply THC-ppRPA to several model systems and show that it yields the same results as traditional ppRPA to within mH accuracy. Our method opens the door to the development of post-Kohn Sham functionals based on ppRPA without the excessive asymptotic cost of traditional ppRPA implementations.

Shenvi, Neil; van Aggelen, Helen; Yang, Yang; Yang, Weitao

2014-07-01

136

Tensor hypercontracted ppRPA: Reducing the cost of the particle-particle random phase approximation from O(r?6) to O(r?4)  

International Nuclear Information System (INIS)

In recent years, interest in the random-phase approximation (RPA) has grown rapidly. At the same time, tensor hypercontraction has emerged as an intriguing method to reduce the computational cost of electronic structure algorithms. In this paper, we combine the particle-particle random phase approximation with tensor hypercontraction to produce the tensor-hypercontracted particle-particle RPA (THC-ppRPA) algorithm. Unlike previous implementations of ppRPA which scale as O(r6), the THC-ppRPA algorithm scales asymptotically as only O(r4), albeit with a much larger prefactor than the traditional algorithm. We apply THC-ppRPA to several model systems and show that it yields the same results as traditional ppRPA to within mH accuracy. Our method opens the door to the development of post-Kohn Sham functionals based on ppRPA without the excessive asymptotic cost of traditional ppRPA implementations

137

Mutations in the BRCT binding site of BRCA1 result in hyper-recombination.  

Science.gov (United States)

We introduced a K1702M mutation in the BRCA1 BRCT domain known to prevent the binding of proteins harboring pS-X-X-F motifs such as Abraxas-RAP80, BRIP1, and CtIP. Surprisingly, rather than impairing homologous recombination repair (HRR), expression of K1702M resulted in hyper-recombination coinciding with an accumulation of cells in S-G2 and no effect on nonhomologous end-joining. These cells also showed increased RAD51 and RPA nuclear staining. More pronounced effects were seen with a naturally occurring BRCT mutant (M1775R) that also produced elevated levels of ssDNA, in part co-localizing with RPA, in line with excessive DNA resection. M1775R induced unusual, thread-like promyelocytic leukemia (PML) nuclear bodies and clustered RPA foci rather than the typical juxtaposed RPA-PML foci seen with wild-type BRCA1. Interestingly, K1702M hyper-recombination diminished with a second mutation in the BRCA1 RING domain (I26A) known to reduce BRCA1 ubiquitin-ligase activity. Thesein vitro findings correlated with elevated nuclear RAD51 and RPA staining of breast cancer tissue from a patient with the M1775R mutation. Altogether, the disruption of BRCA1 (BRCT)-pS-X-X-F protein binding results in ubiquitination-dependent hyper-recombination via excessive DNA resection and the appearance of atypical PML-NBs. Thus, certain BRCA1 mutations that cause hyper-recombination instead of reduced DSB repair might lead to breast cancer. PMID:21666281

Dever, Seth M; Golding, Sarah E; Rosenberg, Elizabeth; Adams, Bret R; Idowu, Michael O; Quillin, John M; Valerie, Nicholas; Xu, Bo; Povirk, Lawrence F; Valerie, Kristoffer

2011-05-01

138

Mutations in the BRCT binding site of BRCA1 result in hyper-recombination  

Science.gov (United States)

We introduced a K1702M mutation in the BRCA1 BRCT domain known to prevent the binding of proteins harboring pS-X-X-F motifs such as Abraxas-RAP80, BRIP1, and CtIP. Surprisingly, rather than impairing homologous recombination repair (HRR), expression of K1702M resulted in hyper-recombination coinciding with an accumulation of cells in S-G2 and no effect on nonhomologous end-joining. These cells also showed increased RAD51 and RPA nuclear staining. More pronounced effects were seen with a naturally occurring BRCT mutant (M1775R) that also produced elevated levels of ssDNA, in part co-localizing with RPA, in line with excessive DNA resection. M1775R induced unusual, thread-like promyelocytic leukemia (PML) nuclear bodies and clustered RPA foci rather than the typical juxtaposed RPA-PML foci seen with wild-type BRCA1. Interestingly, K1702M hyper-recombination diminished with a second mutation in the BRCA1 RING domain (I26A) known to reduce BRCA1 ubiquitin-ligase activity. These in vitro findings correlated with elevated nuclear RAD51 and RPA staining of breast cancer tissue from a patient with the M1775R mutation. Altogether, the disruption of BRCA1 (BRCT)-pS-X-X-F protein binding results in ubiquitination-dependent hyper-recombination via excessive DNA resection and the appearance of atypical PML-NBs. Thus, certain BRCA1 mutations that cause hyper-recombination instead of reduced DSB repair might lead to breast cancer. PMID:21666281

Dever, Seth M.; Golding, Sarah E.; Rosenberg, Elizabeth; Adams, Bret R.; Idowu, Michael O.; Quillin, John M.; Valerie, Nicholas; Xu, Bo; Povirk, Lawrence F.; Valerie, Kristoffer

2011-01-01

139

RPA assists HSF1 access to nucleosomal DNA by recruiting histone chaperone FACT.  

Science.gov (United States)

Transcription factor access to regulatory elements is prevented by the nucleosome. Heat shock factor 1 (HSF1) is a winged helix transcription factor that plays roles in control and stressed conditions by gaining access to target elements, but mechanisms of HSF1 access are not well known in mammalian cells. Here, we show the physical interaction between the wing motif of human HSF1 and replication protein A (RPA), which is involved in DNA metabolism. Depletion of RPA1 abolishes HSF1 access to the promoter of HSP70 in unstressed condition and delays its rapid activation in response to heat shock. The HSF1-RPA complex leads to preloading of RNA polymerase II and opens the chromatin structure by recruiting a histone chaperone, FACT. Furthermore, this interaction is required for melanoma cell proliferation. These results provide a mechanism of constitutive HSF1 access to nucleosomal DNA, which is important for both basal and inducible gene expression. PMID:22940245

Fujimoto, Mitsuaki; Takaki, Eiichi; Takii, Ryosuke; Tan, Ke; Prakasam, Ramachandran; Hayashida, Naoki; Iemura, Shun-ichiro; Natsume, Tohru; Nakai, Akira

2012-10-26

140

Benchmarking van der Waals functionals with noncontact RPA calculations on graphene-Ag(111)  

Science.gov (United States)

We have benchmarked long range behavior of seven different van der Waals functionals comparing them with our ACF-RPA correlation calculations for graphene on a Ag(111) system. Correlation given by the second version of van der Waals density functional vdW-DF2 agrees remarkably well with our random phase approximation (RPA) calculation in the long range region. In the intermediate and shorter range regions combining vdW-DF2 correlation with proper exchange functional becomes important. We compared the results of the van der Waals functionals in this region to the previous RPA calculations and to some extent to experimental observations, and calculated that the combined vdW-DF2(C09x) or rev-vdW-DF2 functionals show satisfactory behavior.

Lon?ari?, Ivor; Despoja, Vito

2014-08-01

 
 
 
 
141

The RPA and the restoration of translation symmetry of rotating nucleus hamiltonian  

International Nuclear Information System (INIS)

The translation symmetry of Hamiltonian of rotating nucleus and its relation to the solutions of equations of motion in random phase approximation RPA are investigated. The requirement of restoration of translation symmetry of rotating nucleus Hamiltonian broken by deformed average nucleon field is used for construction of the residual interactions leading to the appearance of the vibrational states near the nucleus yrast line. With these residual interactions the equations of motion in the framework of RPA are solved. The method of ex-- traction of the spurious modes, connected with nucleus center of ass motion in lab. system, from the RPA solutions is proposed. The formulae for ener ies and structure of the odd parity vibrational states near the yrast line are found

142

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.  

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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 ...

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

1995-01-01

143

The Calliphora rpa mutant lacks the PDZ domain-assembled INAD signalling complex.  

Science.gov (United States)

The visual transduction cascade of fly photoreceptors is a G protein-coupled phospholipase C-signalling pathway which is assembled into a supramolecular signalling complex by the PDZ (postsynaptic density protein-95, discs large, Z0-1) domain protein INAD (inactivation no afterpotential D). The norpA-encoded phospholipase Cbeta, the light-activated transient receptor potential (TRP) Ca2+ channel and an eye-specific protein kinase C are bound to INAD and together form the core of the signalling complex. In the present study we show that the Calliphora rpa mutant, which has previously been hypothesized to represent an equivalent of Drosophila norpA mutants, has normal amounts of norpA mRNA but fails to express inaD mRNA. Electrophysiological recordings from the eyes of the rpa mutant reveal that the electroretinogram is reduced (about 12% of wild type) but not completely absent, and that it exhibits markedly prolonged deactivation kinetics. Furthermore, rpa mutants display a slow, light-dependent degeneration of the photoreceptor cells. With respect to the INAD signalling complex, the rpa mutant is similar to the Drosophila inaD null mutant: not only INAD itself, but also the other core components of the INAD signalling complex, are reduced or absent in photoreceptor membranes of rpa flies. Residual TRP is localized throughout the plasma membrane of the photoreceptor cell, rather than being restricted to the microvillar photoreceptor membrane. [35S]methionine-labelling of newly synthesized retinal proteins reveals that TRP is synthesized in the rpa mutant at wild-type level, but is transported to or incorporated into the microvillar photoreceptor membrane at a much lower rate. We thus suggest, that the formation of the INAD signalling complex is required for specifically targeting its components to the photoreceptor membrane. PMID:11069586

Huber, A; Belusic, G; Da Silva, N; Bähner, M; Gerdon, G; Draslar, K; Paulsen, R

2000-11-01

144

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

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

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

1990-09-01

145

Muscle-specific cell ablation conditional upon Cre-mediated DNA recombination in transgenic mice leads to massive spinal and cranial motoneuron loss.  

Science.gov (United States)

We describe here a binary transgenic system based on Cre-mediated DNA recombination for genetic cell ablation in mice that enabled us to obtain skeletal muscle-deficient embryos by mating two phenotypically normal transgenic lines. In those embryos, skeletal muscles are eliminated as a consequence of the expression of the gene encoding the diphtheria toxin A fragment. Cell ablation occurs gradually beginning approximately on embryonic day (E) 12.5, and by E18-5 almost all skeletal muscle is absent. Analysis of the consequences of muscle cell ablation revealed that almost all spinal motoneurons are lost by E18.5, providing strong evidence that survival of spinal motoneurons during embryogenesis is dependent on signals from their target tissue, skeletal muscle, and that trophic signals produced by nonmuscle sources are sufficient to support survival of no more than 10% of embryonic spinal motoneurons in the absence of muscle-derived signals. There was also substantial loss of cranial (hypoglossal and facial) motoneurons in the muscle-deficient embryos, thus indicating that cranial motoneuron survival is also dependent on trophic signals produced by their target tissue. Although spinal motoneurons are a major target of spinal interneurons, the loss of motoneurons did not affect interneuron survival. Muscle-deficient embryos had a cleft palate and abnormalities of the lower jaw, raising the possibility that they might serve as a mouse model for the human disorder, Robin sequence. The data reported here demonstrate the utility of a binary transgenic system for obtaining mouse embryos in which a specific cell population has been ablated, so that its role in embryonic development can be studied. PMID:9630749

Grieshammer, U; Lewandoski, M; Prevette, D; Oppenheim, R W; Martin, G R

1998-05-15

146

IPPP - a program for the RPA calculation of transmission mechanics of spin-spin coupling constants  

International Nuclear Information System (INIS)

The IPPP program calculates NMR spin-spin coupling constants in molecular systems, based on a standard INDO computation, within the RPA (or CHF) approximation solved by the polarization propagator technique. The transmission mechanisms are analyzed by the inner projection of the propagator matrix. (orig.)

147

G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding.  

Science.gov (United States)

Human telomeres terminate with a single-stranded 3' G overhang, which can be recognized as a DNA damage site by replication protein A (RPA). The protection of telomeres (POT1)/POT1-interacting protein 1 (TPP1) heterodimer binds specifically to single-stranded telomeric DNA (ssTEL) and protects G overhangs against RPA binding. The G overhang spontaneously folds into various G-quadruplex (GQ) conformations. It remains unclear whether GQ formation affects the ability of POT1/TPP1 to compete against RPA to access ssTEL. Using single-molecule Förster resonance energy transfer, we showed that POT1 stably loads to a minimal DNA sequence adjacent to a folded GQ. At 150 mM K(+), POT1 loading unfolds the antiparallel GQ, as the parallel conformation remains folded. POT1/TPP1 loading blocks RPA's access to both folded and unfolded telomeres by two orders of magnitude. This protection is not observed at 150 mM Na(+), in which ssTEL forms only a less-stable antiparallel GQ. These results suggest that GQ formation of telomeric overhangs may contribute to suppression of DNA damage signals. PMID:24516170

Ray, Sujay; Bandaria, Jigar N; Qureshi, Mohammad H; Yildiz, Ahmet; Balci, Hamza

2014-02-25

148

Second RPA dynamics at finite temperature: time-evolutions of dynamical operators  

International Nuclear Information System (INIS)

Time-evolutions of dynamical operators, in particular the generalized density matrix comprising both diagonal and off-diagonal elements, are investigated within the framework of second RPA dynamics at finite temperature. The calculation of the density matrix previously carried out through the appliance of the second RPA master equation by retaining only the slowly oscillating coupling terms is extended to include in the interaction Hamiltonian both the rapidly and slowly oscillating coupling terms. The extended second RPA master equation, thereby formulated without making use of the so-called resonant approximation, is analytically solved and a closed expression for the generalized density matrix is extracted. We provide illustrative examples of the generalized density matrix for various specific initial conditions. We turn particularly our attention to the Poisson distribution type of initial condition for which we deduce specifically a particular form of the density matrix from the solution of the Fokker-Planck equation for the coherent state representation. The relation of the Fokker-Planck equation to the second RPA master equation and its properties are briefly discussed. The oversight incurred in the time-evolution of operators by the resonant approximation is elucidated. The first and second moments of collective coordinates are also computed in relation to the expectation value of various dynamical operators involved in the extended master equation

149

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

International Nuclear Information System (INIS)

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

150

Increased vascular permeability in organs mediated by the systemic administration of lymphokine-activated killer cells and recombinant interleukin-2 in mice.  

Science.gov (United States)

Significant tumor regressions in mice with established carcinomas, sarcomas, and melanomas and in humans with advanced cancers have been observed following immunotherapy with lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (IL-2). However, dose escalations of LAK cells and IL-2 have been prevented by the development of a vascular leak syndrome (VLS). Although IL-2 alone can produce this VLS, we investigated the role of transferred LAK cells, generated by the incubation of syngeneic splenocytes in IL-2, in mediating this phenomenon. We used a murine model to quantitate the vascular leak by measuring the extravasation of iv injected 125I-bovine serum albumin. A permeability index (PI) was calculated by dividing the mean cpm of tissues from treated mice by those from control animals. The systemic transfer of LAK cells and IL-2 produced a significantly greater extravasation of albumin in the lungs, liver, and kidneys than after Hanks' balanced salt solution, IL-2, or LAK cells alone (in the lungs, for example, PI = 4.7, 1.4, and 1.6 after LAK cells and IL-2, LAK cells alone, and IL-2 alone, respectively). To eliminate the contribution to the leak by host lymphocytes, we irradiated mice before cell transfer. As compared to controls, LAK cells and IL-2 resulted in higher extravasation in the lungs, liver, kidneys, and spleen. However, a similar vascular leak was not observed in the lungs, liver, and kidneys after treatment with IL-2 plus fresh or cultured (without IL-2) splenocytes. Moreover, the combination of IL-2 excipient and LAK cells or IL-2 and irradiated LAK cells did not produce a fluid leak. The development of the VLS by LAK cells was directly related to the dose of concurrently administered IL-2 and the number of injected cells. Depletion of Thy 1.2-positive lymphocytes using antibody and complement prior to generating the LAK cells used in adoptive transfer did not abrogate the VLS in any of the organs tested. Similarly, depletion of L3T4 and Lyt-2 positive cells in vivo using monoclonal antibodies prior to harvesting spleens for generation of LAK cells also had no impact on the VLS. In contrast, in vitro treatment of LAK precursor cells with antibody to asialo-GM-1 plus complement completely eliminated the VLS when the depleted cells were cultured in IL-2 and subsequently transferred.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:3258039

Ettinghausen, S E; Puri, R K; Rosenberg, S A

1988-04-01

151

Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination  

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During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schiz...

Lorenz, Alexander; Mehats, Alize?e; Osman, Fekret; Whitby, Matthew C.

2014-01-01

152

Function of Rad51 paralogs in eukaryotic homologous recombinational repair  

International Nuclear Information System (INIS)

Full text: Homologous recombinational repair (HRR) is an important mechanism for maintaining genetic integrity and cancer prevention by accurately repair of DNA double strand breaks induced by environmental insults or occurred in DNA replication. A critical step in HRR is the polymerization of Rad51 on single stranded DNA to form nuclear protein filaments, the later conduct DNA strand paring and exchange between homologous strands. A number of proteins, including replication protein A (RPA), Rad52 and Rad51 paralogs, are suggested to modulate or facilitate the process of Rad51 filament formation. Five Rad51 paralogs, namely XRCC2, XRCC3, Rad51B, Rad51C and Rad51D have been identified in eucaryotic cells. These proteins show distant protein sequence identity to Rad51, to yeast Rad51 paralogs (Rad55 and Rad57) and to each other. Hamster or chicken mutants of Rad51 paralogs exhibit hypersensitivity to a variety of DNA damaging agents, especially cross-linking agents, and are defective in assembly of Rad51 onto HRR site after DNA damage. Recent data from our and other labs showed that Rad51 paralogs constitute two distinct complexes in cell extracts, one contains XRCC2, Rad51B, Rad51C and Rad51D, and the other contains Rad51C and XRCC3. Rad51C is involved in both complexes. Our results also showed that XRCC3-Rad51C complex interacts with Rad51 in vivo. Furthermore, overexpression of Rad52 can partially suppress the hypersensitivity of XRCC2 mutant irs1 to ionizing radiatiy of XRCC2 mutant irs1 to ionizing radiation and corrected the defects in Rad51 focus formation. These results suggest that XRCC2 and other Rad51 paralogs play a mediator function to Rad51 in the early stage of HRR

153

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

CERN Document Server

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

154

The RPA — Optimising a processor array architecture for implementation using VLSI  

Science.gov (United States)

The RPA project aims at developing a general purpose array architecture exhibiting a wide range of perceived user parallelism. It comprises an array of efficient single-bit-slice cells, which can be configured in any number to form the processing elements of a processor array. Any number of cells in any connected path may form a processing element, although for algorithm design and routing consideration, closed loops are the most practical topology. This paper outlines the architecture of this cell, which is considerably more efficient than the ICL DAP or GEC GRID for many word oriented operations. This paper also highlights the problems faced in implementing this form of architecture in silicon. The RPA architecture is therefore discussed in relation to the constraints imposed by this implementation and also in relation to the efficient realisation of multi-mode arithmetic.

Jesshope, C. R.

1985-07-01

155

Transfer of the Symbiotic Plasmid of Rhizobium etli CFN42 Requires Cointegration with p42a, Which May Be Mediated by Site-Specific Recombination  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Plasmid p42a from Rhizobium etli CFN42 is self-transmissible and indispensable for conjugative transfer of the symbiotic plasmid (pSym). Most pSym transconjugants also inherit p42a. pSym transconjugants that lack p42a always contain recombinant pSyms, which we designated RpSyms*. RpSyms* do not contain some pSym segments and instead have p42a sequences, including the replication and transfer regions. These novel recombinant plasmids are compatible with wild-type pSym, incompatible with p42a, ...

Brom, Susana; Girard, Lourdes; Tun-garrido, Cristina; Garci?a-de Los Santos, Alejandro; Bustos, Patricia; Gonza?lez, Vi?ctor; Romero, David

2004-01-01

156

RPA spin-isospin nuclear response in the deep inelastic region  

International Nuclear Information System (INIS)

The spin-isospin volume responses of a finite nucleus are evaluated in the RPA frame, utilizing a harmonic oscillator basis. Particular emphasis is given to the mixing between the longitudinal and transverse couplings, which arise at the nuclear surface. We show that it reduces somewhat the contrast between the two spin responses. We compare the calculated transverse response with the experimental one extracted from deep inelastic electron scattering

157

The Bogolubov Representation of the Polaron Model and Its Completely Integrable RPA-Approximation  

International Nuclear Information System (INIS)

The polaron model in ionic crystal is studied in the N. 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 N. Bogolubov unitary transformed polaron Hamiltonian a very important new result is stated: the full polaron model is exactly solvable. (author)

158

Mean-Field and RPA Approaches to Stable and Unstable Nucleiwith Semi-Realistic NN Interaction  

Science.gov (United States)

The semi-realistic NN interactionshave been applied to nuclear structure studies in the mean-field and RPA framework. It has been clarified that the tensor force plays crucial roles in M1 strengths in ^{208}Pb and E2 strengths to 2^+_1 in the Sn isotopes, as well as in Z- or N-dependence of the shell structure. The semi-realistic effective NN interactions provide us with a promising tool to describe nuclear properties on microscopic basis.

Nakada, H.

159

Quantum interference terms in nonmesonic weak decay of $\\Lambda$-hypernuclei within a RPA formalism  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Single and double coincidence nucleon spectra in the $\\Lambda$-hypernuclei weak decay are evaluated and discussed using a microscopic formalism. Nuclear matter is employed together with the local density approximation which allows us to analyze the $^{12}_{\\Lambda}C$ hypernucleus non-mesonic weak decay. Final state interactions (FSI) are included via the first order (in the nuclear residual interaction) terms to the RPA, where the strong residual interaction is modelled by a...

Bauer, E.

2007-01-01

160

The nature of excess electrons in anatase and rutile from hybrid DFT and RPA.  

Science.gov (United States)

The behavior of excess electrons in undoped and defect free bulk anatase and rutile TiO2 has been investigated by state-of-the-art electronic structure methods including hybrid density functional theory (DFT) and the random phase approximation (RPA). Consistent with experiment, charge trapping and polaron formation is observed in both anatase and rutile. The difference in the anisotropic shape of the polarons is characterized, confirming for anatase the large polaron picture. For anatase, where polaron formation energies are small, charge trapping is observed also with standard hybrid functionals, provided the simulation cell is sufficiently large (864 atoms) to accommodate the lattice relaxation. Even though hybrid orbitals are required as a starting point for RPA in this system, the obtained polaron formation energies are relatively insensitive to the amount of Hartree-Fock exchange employed. The difference in trapping energy between rutile and anatase can be obtained accurately with both hybrid functionals and RPA. Computed activation energies for polaron hopping and delocalization clearly show that anatase and rutile might have different charge transport mechanisms. In rutile, only hopping is likely, whereas in anatase hopping and delocalization are competing. Delocalization will result in conduction-band-like and thus enhanced transport. Anisotropic conduction, in agreement with experimental data, is observed, and results from the tendency to delocalize in the [001] direction in rutile and the (001) plane in anatase. For future work, our calculations serve as a benchmark and suggest RPA on top on hybrid orbitals (PBE0 with 30% Hartree-Fock exchange), as a suitable method to study the rich chemistry and physics of TiO2. PMID:25360624

Spreafico, Clelia; VandeVondele, Joost

2014-12-21

 
 
 
 
161

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

Two complementary methods to describe the collective motion, RPA and Wigner function moments 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 here is the subject of our special attention. The exact relation between the variables o...

Balbutsev, E. B.; Schuck, P.

2005-01-01

162

MASC - a small Remotely Piloted Aircraft (RPA) for wind energy research  

Science.gov (United States)

Originally designed for atmospheric boundary layer research, the MASC (Multipurpose Airborne Sensor Carrier) RPA (Remotely Piloted Aircraft, also known as Unmanned Aerial Vehicle, UAV) is capable of making in-situ measurements of temperature, humidity and wind in high resolution and precision. The autopilot system ROCS (Research Onboard Computer System) enables the aircraft to fly pre-defined routes between waypoints at constant altitude and airspeed. The system manages to operate in wind speeds up to 15 m s-1 safely. It is shown that a MASC can fly as close as one rotor diameter upstream and downstream of running wind turbines at these wind speeds and take valuable data of incoming flow and wake. The flexible operation of an RPA at the size of a MASC can be a major advantage of the system compared to tower measurements and remote sensing in wind energy research. In the project "Lidar Complex" comparisons of RPA measurements with lidar systems and tower measurements are carried out at two different test sites. First results, including turbulence and wake measurements, from a campaign in autumn 2013 are presented.

Wildmann, N.; Hofsäß, M.; Weimer, F.; Joos, A.; Bange, J.

2014-05-01

163

Deep-sequencing identification of the genomic targets of the cytidine deaminase AID and its cofactor RPA in B lymphocytes  

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The cytidine deaminase AID hypermutates immunoglobulin genes but can also target oncogenes, leading to tumorigenesis. The extent of AID’s promiscuity and its predilection for immunoglobulin genes are unknown. We report here that AID interacted broadly with promoter-proximal sequences associated with stalled polymerases and chromatin-activating marks. In contrast, genomic occupancy of replication protein A (RPA), an AID cofactor, was restricted to immunoglobulin genes. The recruitment of RPA...

Yamane, Arito; Resch, Wolfgang; Kuo, Nan; Kuchen, Stefan; Li, Zhiyu; Sun, Hong-wei; Robbiani, Davide F.; Mcbride, Kevin; Nussenzweig, Michel C.; Casellas, Rafael

2011-01-01

164

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

Science.gov (United States)

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

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

2013-11-01

165

A Limited Number of Transducible Hepatocytes Restricts a Wide-Range Linear Vector Dose Response in Recombinant Adeno-Associated Virus-Mediated Liver Transduction  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Recombinant adeno-associated virus (rAAV) vectors are promising vehicles for achieving stable liver transduction in vivo. However, the mechanisms of liver transduction are not fully understood, and furthermore, the relationships between rAAV dose and levels of transgene expression, total number of hepatocytes transduced, and proportion of integrated vector genomes have not been well established. To begin to elucidate the liver transduction dose response with rAAV vectors, we injected mice wit...

Nakai, Hiroyuki; Thomas, Clare E.; Storm, Theresa A.; Fuess, Sally; Powell, Sharon; Wright, J. Fraser; Kay, Mark A.

2002-01-01

166

Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination.  

Science.gov (United States)

During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs. A common attribute of these protein complexes is an ability to stabilize the Rad51/Dmc1 nucleoprotein filament, and we propose that it is this property that imposes constraints on which enzymes gain access to the recombination intermediate, thereby controlling the manner in which it is processed and resolved. PMID:25414342

Lorenz, Alexander; Mehats, Alizée; Osman, Fekret; Whitby, Matthew C

2014-12-16

167

Cosmological Recombination  

Science.gov (United States)

In this thesis we focus on studying the physics of cosmological recombination and how the details of recombination affect the Cosmic Microwave Background (CMB) anisotropies. We present a detailed calculation of the spectral line distortions on the CMB spectrum arising from the Lyman-alpha and the lowest two-photon transitions in the recombination of hydrogen (H), and the corresponding lines from helium (He). The peak of these distortions mainly comes from the Lyman-alpha transition and occurs at about 170 microns, which is the Wien part of the CMB. The major theoretical limitation for extracting cosmological parameters from the CMB sky lies in the precision with which we can calculate the cosmological recombination process. With this motivation, we perform a multi-level calculation of the recombination of H and He with the addition of the spin-forbidden transition for neutral helium (He I), plus the higher order two-photon transitions for H and among singlet states of He I. We find that the inclusion of the spin-forbidden transition results in more than a percent change in the ionization fraction, while the other transitions give much smaller effects. Last we modify RECFAST by introducing one more parameter to reproduce recent numerical results for the speed-up of helium recombination. Together with the existing hydrogen `fudge factor', we vary these two parameters to account for the remaining dominant uncertainties in cosmological recombination. By using a Markov Chain Monte Carlo method with Planck forecast data, we find that we need to determine the parameters to better than 10% for He I and 1% for H, in order to obtain negligible effects on the cosmological parameters.

Wong, Wan Yan

2008-11-01

168

DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis. Results Sequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the Vibrio proteolyticus 5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into Escherichia coli, the chimeric RNA (3×pen aRNA was expressed constitutively from E. coli rrnB P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid-solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse. Conclusions The feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs in vivo using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use in clinical and biomedical research.

Willson Richard C

2010-12-01

169

Cosmological Recombination  

CERN Document Server

In this thesis we focus on studying the physics of cosmological recombination and how the details of recombination affect the Cosmic Microwave Background (CMB) anisotropies. We present a detailed calculation of the spectral line distortions on the CMB spectrum arising from the Lyman-alpha and the lowest two-photon transitions in the recombination of hydrogen (H), and the corresponding lines from helium (He). The peak of these distortions mainly comes from the Lyman-alpha transition and occurs at about 170 microns, which is the Wien part of the CMB. The major theoretical limitation for extracting cosmological parameters from the CMB sky lies in the precision with which we can calculate the cosmological recombination process. With this motivation, we perform a multi-level calculation of the recombination of H and He with the addition of the spin-forbidden transition for neutral helium (He I), plus the higher order two-photon transitions for H and among singlet states of He I. We find that the inclusion of the s...

Wong, Wan Yan

2008-01-01

170

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

Science.gov (United States)

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

Wildmann, Norman; Bange, Jens

2014-05-01

171

RPA Review  

Degree of inherent control in addressing these drivers. 55. 3.4. Looking ..... still \\has to process, in most other respects it appears to be flying blind, leading us to \\question ..... Notifiable Pest Quarantine inspections for FERA (formerly PHSI). \\2000.

172

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.

2010-01-01

173

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

Science.gov (United States)

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

Wildmann, Norman; Bange, Jens

2014-05-01

174

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

International Nuclear Information System (INIS)

Two complementary methods to describe the collective motion, RPA and Wigner-function-moments 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

175

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

CERN Document Server

Two complementary methods to describe the collective motion, RPA and Wigner function moments 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 here is the subject of our special attention. The exact relation between the variables of the two methods and the respective dynamical equations is established. 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

2005-01-01

176

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

CERN Document Server

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

177

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

International Nuclear Information System (INIS)

Two complementary methods to describe the collective motion, the RPA and the method of Wigner function moments, are compared using a simple model as an example--a harmonic oscillator with quadrupole-quadrupole residual interaction. It is shown that they give identical formulas for eigenfrequencies and transition probabilities of all collective excitations of the model, including the scissors mode, which is a subject of our special 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

178

Recombinant adeno-associated virus (rAAV)-mediated expression of a human gamma-globin gene in human progenitor-derived erythroid cells.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Effective gene therapy for the severe hemoglobin (Hb) disorders, sickle-cell anemia and thalassemia, will require an efficient method to transfer, integrate, and express a globin gene in primary erythroid cells. To evaluate recombinant adeno-associated virus (rAAV) for this purpose, we constructed a rAAV vector encoding a human gamma-globin gene (pJM24/vHS432A gamma). Its 4725-nucleotide genome consists of two 180-bp AAV inverted terminal repeats flanking the core elements of hypersensitive s...

Miller, J. L.; Donahue, R. E.; Sellers, S. E.; Samulski, R. J.; Young, N. S.; Nienhuis, A. W.

1994-01-01

179

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

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

2009-01-01

180

SETD2-Dependent Histone H3K36 Trimethylation Is Required for Homologous Recombination Repair and Genome Stability  

Directory of Open Access Journals (Sweden)

Full Text Available Modulating chromatin through histone methylation orchestrates numerous cellular processes. SETD2-dependent trimethylation of histone H3K36 is associated with active transcription. Here, we define a role for H3K36 trimethylation in homologous recombination (HR repair in human cells. We find that depleting SETD2 generates a mutation signature resembling RAD51 depletion at I-SceI-induced DNA double-strand break (DSB sites, with significantly increased deletions arising through microhomology-mediated end-joining. We establish a presynaptic role for SETD2 methyltransferase in HR, where it facilitates the recruitment of C-terminal binding protein interacting protein (CtIP and promotes DSB resection, allowing Replication Protein A (RPA and RAD51 binding to DNA damage sites. Furthermore, reducing H3K36me3 levels by overexpressing KDM4A/JMJD2A, an oncogene and H3K36me3/2 demethylase, or an H3.3K36M transgene also reduces HR repair events. We propose that error-free HR repair within H3K36me3-decorated transcriptionally active genomic regions promotes cell homeostasis. Moreover, these findings provide insights as to why oncogenic mutations cluster within the H3K36me3 axis.

Sophia X. Pfister

2014-06-01

 
 
 
 
181

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

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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 cell...

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

2013-01-01

182

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

Directory of Open Access Journals (Sweden)

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

2012-03-01

183

On the role of anti-bound states in the RPA description of the giant monopole resonance  

International Nuclear Information System (INIS)

The limit of the applicability of the resonant Random Phase Approximation (RPA) method is tested by calculating escape widths in the giant monopole resonance of 16O and comparing them to the results of a time dependent Hartree-Fock calculation. Though the widths of the narrow s-wave component agree reasonably well, the broad p-wave component shows large disagreement, which cannot be cured by complementing the basis with anti-bound states in the RPA calculation. (author) 18 refs.; 3 tabs

184

Sex, Not Genotype, Determines Recombination Levels in Mice  

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Recombination, the precise physical breakage and rejoining of DNA between homologous chromosomes, plays a central role in mediating the orderly segregation of meiotic chromosomes in most eukaryotes. Despite its importance, the factors that control the number and placement of recombination events within a cell remain poorly defined. The rate of recombination exhibits remarkable species specificity, and, within a species, recombination is affected by the physical size of the chromosome, chromos...

Lynn, Audrey; Schrump, Stefanie; Cherry, Jonathan; Hassold, Terry; Hunt, Patricia

2005-01-01

185

A functional recT gene for recombineering of Clostridium.  

Science.gov (United States)

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

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

2014-03-10

186

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

187

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

Directory of Open Access Journals (Sweden)

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

Beggah Ahmed T

2009-09-01

188

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

189

A Semi-Classical, Microscopic Model for Nuclear Collective Rotation Plus RPA  

International Nuclear Information System (INIS)

Collective rotation and vibration of deformed nuclei are described semiclassically but microscopically by first transforming the time-dependent Schrodinger equation to a rotating frame, while preserving time-reversal invariance, and then applying a variational method. The rotating-frame axes are chosen to coincide with the principal axes of the expectation of an arbitrary, symmetric second-rank tensor operator ?. It is shown that the equations derived for the rotational and vibrational motions decouple completely due to the rotational invariance of the Hamiltonian and diagonality of the expectation of ? in the rotating frame. The equations describing the vibration reduce to those of the RPA. The equation describing the rotation generalizes that of the conventional cranking model (CM). The predicted rotation moment of inertia is shown to reduce to that of the CM for special types of particle interactions

190

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.)

191

Antitumor therapy mediated by 5-fluorocytosine and a recombinant fusion protein containing TSG-6 hyaluronan binding domain and yeast cytosine deaminase.  

Science.gov (United States)

Matrix attachment therapy (MAT) is an enzyme prodrug strategy that targets hyaluronan in the tumor extracellular matrix to deliver a prodrug converting enzyme near the tumor cells. A recombinant fusion protein containing the hyaluronan binding domain of TSG-6 (Link) and yeast cytosine deaminase (CD) with an N-terminal His(x6) tag was constructed to test MAT on the C26 colon adenocarcinoma in Balb/c mice that were given 5-fluorocytosine (5-FC) in the drinking water. LinkCD was expressed in Escherichia coli and purified by metal-chelation affinity chromatography. The purified LinkCD fusion protein exhibits a K(m) of 0.33 mM and V(max) of 15 microM/min/microg for the conversion of 5-FC to 5-fluorouracil (5-FU). The duration of the enzyme activity for LinkCD was longer than that of CD enzyme at 37 degrees C: the fusion protein retained 20% of its initial enzyme activity after 24 h, and 12% after 48 h. The LinkCD fusion protein can bind to a hyaluronan oligomer (12-mer) at a K(D) of 55 microM at pH 7.4 and a K(D) of 5.32 microM at pH 6.0 measured using surface plasmon resonance (SPR). To evaluate the antitumor effect of LinkCD/5-FC combination therapy in vivo, mice received intratumoral injections of LinkCD on days 11 and 14 after C26 tumor implantation and the drinking water containing 10 mg/mL of 5-FC starting on day 11. To examine if the Link domain by itself was able to reduce tumor growth, we included treatment groups that received LinkCD without 5-FC and Link-mtCD (a functional mutant that lacks cytosine deaminase activity) with 5-FC. Animals that received LinkCD/5-FC treatment showed significant tumor size reduction and increased survival compared to the CD/5-FC treatment group. Treatment groups that were unable to produce 5-FU had no effect on the tumor growth despite receiving the fusion protein that contained the Link domain. The results indicate that a treatment regime consisting of a fusion protein containing the Link domain, the active CD enzyme, and the prodrug 5-FC is sufficient to produce an antitumor effect. Thus, the LinkCD fusion protein is an alternative to antibody-directed prodrug enzyme therapy (ADEPT) approaches for cancer treatment. PMID:19265397

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

2009-01-01

192

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

International Nuclear Information System (INIS)

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

193

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 SciELO Brazil | Language: English 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 no [...] n-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.

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

2006-06-01

194

Efficient repair of all types of single-base mismatches in recombination intermediates in Chinese hamster ovary cells. Competition between long-patch and G-T glycosylase-mediated repair of G-T mismatches.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Repair of all 12 single-base mismatches in recombination intermediates was investigated in Chinese hamster ovary cells. Extrachromosomal recombination was stimulated by double-strand breaks in regions of shared homology. Recombination was predicted to occur via single-strand annealing, yielding heteroduplex DNA (hDNA) with a single mismatch. Nicks were expected on opposite strands flanking hDNA, equidistant from the mismatch. Unlike studies of covalently closed artificial hDNA substrates, all...

Bill, C. A.; Duran, W. A.; Miselis, N. R.; Nickoloff, J. A.

1998-01-01

195

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

CERN Document Server

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

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

2008-01-01

196

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

197

Optimization of transfection mediated by calcium phosphate for plasmid rAAV-LacZ (recombinant adeno-associated virus-beta-galactosidase reporter gene) production in suspension-cultured HEK-293 (human embryonic kidney 293) cells.  

Science.gov (United States)

rAAV (recombinant adeno-associated virus) has become a very useful gene-delivery vector for gene therapy. However, it is very difficult to generate rAAV using triple transfection on a commercial scale, owing to its low productivity and inconveniently adhesive nature of its culture. An optimal suspension-culture transfection procedure was developed for rAAV-LacZ production in suspended HEK-293 cells mediated by calcium phosphate (lacZ, a reporter gene, codes for beta-galactosidase). The study showed that cytotoxicity of transfection complexes and cell aggregation in suspension culture were two key factors affecting high suspension-culture transfection efficiency. Cytotoxicity of transfection complexes was influenced effectively by mixture of Ca(2+) and plasmid DNA when their concentrations were decreased from 300 to 150 mM and from 3.0 to 1.5 microg/ml respectively, as manifested by a relatively higher cell viability after suspension-culture transfection. Moreover, the transfection efficiency was still less than 15%. In addition, we explored the disruption of cell aggregation and the control of transfection-complex size with 2.0 mM EGTA treatment for 30 min before transfection and the addition of 100 mM Mg(2+) during transfection respectively, procedures which enhanced transfection efficiency significantly, owing to more contact and endocytosis between cells and transfection complexes. Finally, the high transfection level and rAAV-LacZ titre achieved under optimized suspension-culture transfection conditions, namely 40% and 5 x 10(11) v.g. (vector genomes)/60 ml of medium respectively, is promising for the technique's application in the large-scale production of rAAV. PMID:17052245

Feng, Lei; Guo, Meijin; Zhang, Shuxiang; Chu, Ju; Zhuang, Yingping; Zhang, Siliang

2007-02-01

198

Recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in Staphylococcus aureus  

Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (?4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates. © 2014 Hill-Cawthorne et al.

Hill-Cawthorne, Grant A.

2014-06-27

199

Recombinant Technology and Probiotics  

Directory of Open Access Journals (Sweden)

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

Icy D’Silva

2011-09-01

200

Recombinant Technology and Probiotics  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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 molecule...

Icy D’Silva

2011-01-01

 
 
 
 
201

Reconstitution of recombination-associated DNA synthesis with human proteins  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

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

2013-01-01

202

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

203

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

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

2003-01-01

204

Single-stranded heteroduplex intermediates in ? Red homologous recombination  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background The Red proteins of lambda phage mediate probably the simplest and most efficient homologous recombination reactions yet described. However the mechanism of dsDNA recombination remains undefined. Results Here we show that the Red proteins can act via full length single stranded intermediates to establish single stranded heteroduplexes at the replication fork. We created asymmetrically digestible dsDNA substrates by exploiting the fact that Red? exonuclease activity requires a 5' phosphorylated end, or is blocked by phosphothioates. Using these substrates, we found that the most efficient configuration for dsDNA recombination occurred when the strand that can prime Okazaki-like synthesis contained both homology regions on the same ssDNA molecule. Furthermore, we show that Red recombination requires replication of the target molecule. Conclusions Hence we propose a new model for dsDNA recombination, termed 'beta' recombination, based on the formation of ssDNA heteroduplexes at the replication fork. Implications of the model were tested using (i an in situ assay for recombination, which showed that recombination generated mixed wild type and recombinant colonies; and (ii the predicted asymmetries of the homology arms, which showed that recombination is more sensitive to non-homologies attached to 5' than 3' ends. Whereas beta recombination can generate deletions in target BACs of at least 50 kb at about the same efficiency as small deletions, the converse event of insertion is very sensitive to increasing size. Insertions up to 3 kb are most efficiently achieved using beta recombination, however at greater sizes, an alternative Red-mediated mechanism(s appears to be equally efficient. These findings define a new intermediate in homologous recombination, which also has practical implications for recombineering with the Red proteins.

Zhang Youming

2010-07-01

205

DNA rearrangement mediated by inverted repeats.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Inverted repeats of DNA are widespread in the genomes of eukaryotes and prokaryotes and can mediate genome rearrangement. We studied rearrangement mediated by plasmid-borne inverted repeats in Escherichia coli. We show that inverted repeats can mediate an efficient and recA-independent recombination event. Surprisingly, the product of this recombination is not that of simple inversion between the inverted repeats, but almost exclusively an unusual head-to-head dimer with complex DNA rearrange...

Bi, X.; Liu, L. F.

1996-01-01

206

Therapeutic Recombinant Monoclonal Antibodies  

Science.gov (United States)

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

Bakhtiar, Ray

2012-01-01

207

Mean-field and RPA approaches to stable and unstable nuclei with semi-realistic NN interactions  

Science.gov (United States)

Important roles of the NN interaction in the shell evolution, which have been pointed out by Otsuka and his collaborators, can be investigated by the self-consistent mean-field approaches with the semi-realistic interactions. We show that the shell evolution in the Z = 50 and Z = 20 nuclei is appropriately described by the semi-realistic interactions that include the realistic tensor force. The M1 strengths in 208Pb are also reproduced by the semi-realistic interactions in the HF+RPA, which are another manifestation of the tensor force in nuclear structure.

Nakada, H.

2013-07-01

208

Range-separated approach to the RPA correlation applied to van der Waals bond and to diffusion of defects  

Science.gov (United States)

The Random Phase Approximation (RPA) is a promising approximation to the exchange-correlation energy of Density Functional Theory (DFT), since it contains the van der Waals (vdW) interaction and yields a potential with the correct band gap. However, its calculation is computationally very demanding. We apply a range separation concept [1] to RPA and demonstrate how it drastically speeds up the calculations without loss of accuracy. The scheme is succesfully applied to a layered system subjected to weak vdW attraction and to address the controversy of the self-diffusion in silicon [2]. We calculate the formation and migration energies of self-interstitials and vacancies taking into account atomic relaxations. The obtained activation energies deviate significantly from the earlier calculations that were affected by the band gap problem and challenge some of the experimental interpretations [3]: the diffusion of vacancies and interstitials have almost the same activation energy.[4pt] [1] J. Toulouse, F. Colonna, and A. Savin, Phys. Rev. A 70, 062505 (2004).[0pt] [2] F. Bruneval, Phys. Rev. Lett. 108, 256403 (2012).[0pt] [3] H. Bracht, E. E. Haller, and R. Clark-Phelps, Phys. Rev. Lett. 81, 393 (1998).

Bruneval, Fabien

2013-03-01

209

Static correlation and electron localization in molecular dimers from the self-consistent RPA and GW approximation  

CERN Document Server

We investigate static correlation and delocalization errors in the self-consistent GW and random-phase approximation (RPA) by studying molecular dissociation of the H_2 and LiH molecules. Although both approximations are diagrammatically identical, the non-locality and frequency dependence of the GW self-energy crucially influence the different energy contributions to the total energy as compared to the use of a static local potential in the RPA. The latter leads to significantly larger correlation energies which allows for a better description of static correlation at intermediate bond distances. The substantial error found in GW is further analyzed by comparing spin-restricted and spin-unrestricted calculations. At large but finite nuclear separation their difference gives an estimate of the so-called fractional spin error normally determined only in the dissociation limit. Furthermore, a calculation of the dipole moment of the LiH molecule at dissociation reveals a large delocalization error in GW making t...

Hellgren, Maria; Rohr, Daniel R; Ren, Xinguo; Rubio, Angel; Scheffler, Matthias; Rinke, Patrick

2014-01-01

210

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

Directory of Open Access Journals (Sweden)

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

N. Wildmann

2013-08-01

211

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

212

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

Directory of Open Access Journals (Sweden)

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

N. Wildmann

2013-03-01

213

Fundamental study of recombination and recombineering in Escherichia coli  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Recombination and recombineering systems have been used in Escherichia coli to recombinant DNA sequences. With endonuclease and DNA lipase the bacterial plasmid and target DNA fragment can bind together and recombinant for a new DNA sequences. Red Proteins have been used in recombineering system to perform the function as the enzymes in recombination system, and faster and easier than the other way of recombinant new DNA sequences in E.coli. In this report we get to know the pr...

Sun, Xiaohang; Huang, Yang

2008-01-01

214

Recombinant factor IX.  

Science.gov (United States)

Despite the introduction of recombinant preparations of factor VIII and recombinant factor VII and VIIa, patients with other forms of hemophilia, especially hemophilia B, have remained at increased risk for blood borne viruses because of a lack of clinically utilizable preparations of recombinant factor IX. This report describes the state of current tests with a recently licensed preparation of recombinant factor IX, BeneFix, from Genetics Institute. Structurally, functionally, and therapeutically, recombinant factor IX is comparable to monoclonal plasma-derived factor IX. The only observed difference between recombinant and plasma factor IX is the recovery in pharmacokinetic studies where recombinant factor IX recovery was approximately 72% that of a plasma factor IX product. This difference is attributed to be due to minor differences in the post-translational modification of recombinant factor IX compared to plasma. These studies demonstrate that recombinant factor IX is effective in the treatment of hemophilia B and has the safety profile expected from a product prepared by recombinant technology. PMID:9198163

White, G C; Beebe, A; Nielsen, B

1997-07-01

215

Multiprotein complex between the GPI-anchored CyRPA with PfRH5 and PfRipr is crucial for Plasmodium falciparum erythrocyte invasion.  

Science.gov (United States)

Erythrocyte invasion by Plasmodium falciparum merozoites is a highly intricate process in which Plasmodium falciparum reticulocyte binding-like homologous protein 5 (PfRH5) is an indispensable parasite ligand that binds with its erythrocyte receptor, Basigin. PfRH5 is a leading blood-stage vaccine candidate because it exhibits limited polymorphisms and elicits potent strain-transcending parasite neutralizing antibodies. However, the mechanism by which it is anchored to the merozoite surface remains unknown because both PfRH5 and the PfRH5-interacting protein (PfRipr) lack transmembrane domains and GPI anchors. Here we have identified a conserved GPI-linked parasite protein, Cysteine-rich protective antigen (CyRPA) as an interacting partner of PfRH5-PfRipr that tethers the PfRH5/PfRipr/CyRPA multiprotein complex on the merozoite surface. CyRPA was demonstrated to be GPI-linked, localized in the micronemes, and essential for erythrocyte invasion. Specific antibodies against the three proteins successfully detected the intact complex in the parasite and coimmunoprecipitated the three interacting partners. Importantly, full-length CyRPA antibodies displayed potent strain-transcending invasion inhibition, as observed for PfRH5. CyRPA does not bind with erythrocytes, suggesting that its parasite neutralizing antibodies likely block its critical interaction with PfRH5-PfRipr, leading to a blockade of erythrocyte invasion. Further, CyRPA and PfRH5 antibody combinations produced synergistic invasion inhibition, suggesting that simultaneous blockade of the PfRH5-Basigin and PfRH5/PfRipr/CyRPA interactions produced an enhanced inhibitory effect. Our discovery of the critical interactions between PfRH5, PfRipr, and the GPI-anchored CyRPA clearly defines the components of the essential PfRH5 adhesion complex for P. falciparum erythrocyte invasion and offers it as a previously unidentified potent target for antimalarial strategies that could abrogate formation of the crucial multiprotein complex. PMID:25583518

Reddy, K Sony; Amlabu, Emmanuel; Pandey, Alok K; Mitra, Pallabi; Chauhan, Virander S; Gaur, Deepak

2015-01-27

216

Quasispecies and recombination.  

Science.gov (United States)

Recombination is introduced into Eigen's theory of quasispecies evolution. Comparing numerical simulations of the rate equations in the non-recombining and recombining cases show that recombination has a strong effect on the error threshold and, for a wide range of mutation rates, gives rise to two stable fixed points in the dynamics. This bi-stability results in the existence of two error thresholds. However, we prove that, for low mutation rates the bi-stability breaks down and the unique equilibrium distribution is concentrated around the sequence with highest fitness. PMID:16982076

Jacobi, Martin Nilsson; Nordahl, Mats

2006-12-01

217

Recombination and Genetic Diversity  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English 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, b [...] ut when time grows infinitely, the diversity of individuals extinguishes off. So under the model premisses, recombination and spatial localization alone are not enough to explain diversity in a population. Further we discuss an application of the model to a controversy regarding the diversity of "Major Histocompatibility Complex" (MHC).

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

2012-12-01

218

Microfluidic lab-on-a-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification (RPA).  

Science.gov (United States)

For the first time we demonstrate a self-sufficient lab-on-a-foil system for the fully automated analysis of nucleic acids which is based on the recently available isothermal recombinase polymerase amplification (RPA). The system consists of a novel, foil-based centrifugal microfluidic cartridge including prestored liquid and dry reagents, and a commercially available centrifugal analyzer for incubation at 37 degrees C and real-time fluorescence detection. The system was characterized with an assay for the detection of the antibiotic resistance gene mecA of Staphylococcus aureus. The limit of detection was blow-molding and sealed with a self-adhesive tape. The demonstrated system excels existing PCR based lab-on-a-chip platforms in terms of energy efficiency and time-to-result. Applications are suggested in the field of mobile point-of-care analysis, B-detection, or in combination with continuous monitoring systems. PMID:20300675

Lutz, Sascha; Weber, Patrick; Focke, Max; Faltin, Bernd; Hoffmann, Jochen; Müller, Claas; Mark, Daniel; Roth, Günter; Munday, Peter; Armes, Niall; Piepenburg, Olaf; Zengerle, Roland; von Stetten, Felix

2010-04-01

219

Enabling simulation at the fifth rung of DFT: Large scale RPA calculations with excellent time to solution  

Science.gov (United States)

The Random Phase Approximation (RPA), which represents the fifth rung of accuracy in Density Functional Theory (DFT), is made practical for large systems. Energies of condensed phase systems containing thousands of explicitly correlated electrons and 1500 atoms can now be computed in minutes and less than 1 h, respectively. GPU acceleration is employed for dense and sparse linear algebra, while communication is minimized by a judicious data layout. The performance of the algorithms, implemented in the widely used CP2K simulation package, has been investigated on hybrid Cray XC30 and XK7 architectures, up to 16,384 nodes. Our results emphasize the importance of good network performance, in addition to the availability of GPUs and generous on node memory. A new level of predictivity has thus become available for routine application in Monte Carlo and molecular dynamics simulations.

Del Ben, Mauro; Schütt, Ole; Wentz, Tim; Messmer, Peter; Hutter, Jürg; VandeVondele, Joost

2015-02-01

220

Perceived Risk and Efficacy Beliefs as Motivators of Change: Use of the Risk Perception Attitude (RPA) Framework To Understand Health Behaviors.  

Science.gov (United States)

Introduces the risk perception attitude (RPA) framework that categorizes individuals into one of four attitudinal groups: responsive, avoidance, proactive, and indifference. Conducts two studies using college students as subjects. Finds that when risk and efficacy are made salient, people's risk perception guides most of their subsequent actions,…

Rimal, Rajiv N.; Real, Kevin

2003-01-01

 
 
 
 
221

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

International Nuclear Information System (INIS)

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

222

Bocavirus Infection Induces a DNA Damage Response That Facilitates Viral DNA Replication and Mediates Cell Death?  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Minute virus of canines (MVC) is an autonomous parvovirus that replicates efficiently without helper viruses in Walter Reed/3873D (WRD) canine cells. We previously showed that MVC infection induces mitochondrion-mediated apoptosis and G2/M-phase arrest in infected WRD cells. However, the mechanism responsible for these effects has not been established. Here, we report that MVC infection triggers a DNA damage response in infected cells, as evident from phosphorylation of H2AX and RPA32. We dis...

Luo, Yong; Chen, Aaron Yun; Qiu, Jianming

2011-01-01

223

Vaccination of Rhesus Macaques with Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Env V3 Elicits Neutralizing Antibody-Mediated Protection against Simian-Human Immunodeficiency Virus with a Homologous but Not a Heterologous V3 Motif  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Although the correlates of vaccine-induced protection against human immunodeficiency virus type 1 (HIV-1) are not fully known, it is presumed that neutralizing antibodies (NAb) play a role in controlling virus infection. In this study, we examined immune responses elicited in rhesus macaques following vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing an HIV-1 Env V3 antigen (rBCG Env V3). We also determined the effect of vaccination on protection against ch...

Someya, Kenji; Cecilia, Dayaraj; Ami, Yasushi; Nakasone, Tadashi; Matsuo, Kazuhiro; Burda, Sherri; Yamamoto, Hiroshi; Yoshino, Naoto; Kaizu, Masahiko; Ando, Shuji; Okuda, Kenji; Zolla-pazner, Susan; Yamazaki, Shudo; Yamamoto, Naoki; Honda, Mitsuo

2005-01-01

224

MEIOB exhibits single-stranded DNA-binding and exonuclease activities and is essential for meiotic recombination  

Science.gov (United States)

Meiotic recombination enables the reciprocal exchange of genetic material between parental homologous chromosomes and ensures faithful chromosome segregation during meiosis in sexually reproducing organisms. This process relies on the complex interaction of DNA repair factors, and many steps remain poorly understood in mammals. Here we report the identification of MEIOB, a meiosis-specific protein, in a proteomics screen for novel meiotic chromatin-associated proteins in mice. MEIOB contains an OB domain with homology to one of the RPA1 OB folds. MEIOB binds to single-stranded DNA and exhibits 3’ to 5’ exonuclease activity. MEIOB forms a complex with RPA and with SPATA22, and these three proteins colocalize in foci that are associated with meiotic chromosomes. Strikingly, chromatin localization and stability of MEIOB depends on SPATA22 and vice versa. Meiob-null mouse mutant exhibits a failure in meiosis and sterility in both sexes. Our results suggest that MEIOB is required for meiotic recombination and chromosomal synapsis. PMID:24240703

Luo, Mengcheng; Yang, Fang; Leu, N. Adrian; Landaiche, Jessica; Handel, Mary Ann; Benavente, Ricardo; La Salle, Sophie; Wang, P. Jeremy

2013-01-01

225

Enhancement of antibody class switch recombination by the cumulative activity of four separate elements1  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Class switch recombination of antibody isotype is mediated by a recombinational DNA deletion event, and must be robustly upregulated during antigen-driven differentiation of B cells. The enhancer region 3? of the C? gene is important for the upregulation of switch recombination. Using a transgene of the entire heavy chain constant region locus, we now demonstrate that it is the four 3? enhancer elements themselves (a total of 4.7 kb) that are responsible for the upregulation, rather than...

Dunnick, Wesley A.; Shi, Jian; Zerbato, Jennifer M.; Fontaine, Clinton A.; Collins, John T.

2011-01-01

226

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

Science.gov (United States)

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

McInnis, Christine E; Blackwell, Helen E

2014-01-01

227

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

2011-03-01

228

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

229

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

Directory of Open Access Journals (Sweden)

Full Text Available 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 IoxP 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 SILIoxloxSCL 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 IoxP sites in over 50% of the thymocytes from SILIoxloxSCL/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 SILIoxloxSCL and Cre transgenes have increased CD4-/CD8- thymocytes compared with transgenenegative 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

2007-04-01

230

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. PMID:17460775

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

2007-01-01

231

Phenylbutyrate inhibits homologous recombination induced by camptothecin and methyl methanesulfonate  

DEFF Research Database (Denmark)

Homologous recombination is accompanied by extensive changes to chromatin organization at the site of DNA damage. Some of these changes are mediated through acetylation/deacetylation of histones. Here, we show that recombinational repair of DNA damage induced by the anti-cancer drug camptothecin (CPT) and the alkylating agent methyl methanesulfonate (MMS) is blocked by sodium phenylbutyrate (PBA) in the budding yeast Saccharomyces cerevisiae. In particular, PBA suppresses CPT- and MMS-induced genetic recombination as well as DNA double-strand break repair during mating-type interconversion. Treatment with PBA is accompanied by a dramatic reduction in histone H4 lysine 8 acetylation. Live cell imaging of homologous recombination proteins indicates that repair of CPT-induced DNA damage is redirected to a non-recombinogenic pathway in the presence of PBA without loss in cell viability. In contrast, the suppression of MMS-induced recombination by PBA is accompanied by a dramatic loss in cell viability. Taken together, our results demonstrate that PBA inhibits DNA damage-induced homologous recombination likely by mediating changes in chromatin acetylation. Moreover, the combination of PBA with genotoxic agents can lead to different cell fates depending on the type of DNA damage inflicted.

Kaiser, Gitte Schalck; Germann, Susanne Manuela

2011-01-01

232

The theory and computational implementation of quadratically convergent Hartree-Fock orbital optimization methods and their relationship to the time-dependent Hartree-Fock and RPA methods  

International Nuclear Information System (INIS)

The theory of quadratically convergent Hartree-Fock or self-consistent field (QC-SCF) orbital optimization is presented using the language of second quantization. Two methods that are appropriate for the computational implementation of QC-SCF are described: the Newton-Raphson method and an approximate super configuration interaction (CI) approach, both of which can be implemented such that no four-index transformation is necessary. The Newton-Raphson formulation of QC-SCF is shown to be equivalent to solving the frequency-independent coupled perturbation Hartree-Fock equations, and consequently a close relationship exists between QC-SCF and the more general time-dependent coupled perturbation Hartree-Fock (TDCPHF) theory and the related theories of random phase approximation (RPA) and time-dependent Hartree-Fock. Matrix element expressions that are needed for RPA naturally arise in QC-SCF too, and a list of these for open-shell ground state wavefunctions is also given. Computational techniques that are believed to be useful for the solution of the TDCPHF and RPA problems are also briefly discussed

233

Polarity in adenovirus recombination.  

Science.gov (United States)

The distributions of the crossovers necessary to generate ts+ genomes have been examined in a collection of clonally unrelated ts+ recombinants from a set of ts X ts adenovirus crosses. In a cross between two parents that are grossly heterologous between map units 80.2 and 91.5, the distribution of crossovers was significantly skewed toward the left-hand end of the genome, with a declining frequency proceeding rightward. This gradient of recombination was modified by the removal of the right-hand heterology and by the presence of another region of heterology between map units 3.67 and 10.11. In a cross where the ts markers were flanked by both heterologies, no gradient was observed and ts+ recombinants were characterized by a higher rate of supernumerary crossovers. In a cross designed so that one ts marker was internal to two heterologies, crossovers were found disproportionately between the second ts marker and the nearby heterology. In addition, ts+ recombinants formed by crossing over internal to the heterologies again were accompanied by a high frequency of supernumerary crossovers. Finally, ts+ recombinant frequencies in crosses identical except for the presence of either one or two flanking heterologies were markedly lower in the latter case. These data, taken together, suggest that a major pathway of adenovirus recombination initiates at, or near, the molecular termini and is perhaps driven by the displaced single strands produced during DNA replication. Internal initiation, on the other hand, may employ these single strands to form genetic "patches." PMID:6330982

Munz, P L; Young, C S

1984-06-01

234

Mediation Analysis  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Mediating variables are prominent in psychological theory and research. A mediating variable transmits the effect of an independent variable on a dependent variable. Differences between mediating variables and confounders, moderators, and covariates are outlined. Statistical methods to assess mediation and modern comprehensive approaches are described. Future directions for mediation analysis are discussed.

Mackinnon, David P.; Fairchild, Amanda J.; Fritz, Matthew S.

2007-01-01

235

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

236

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 patus 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

237

In the Nicotiana sylvestris CMSII mutant, a recombination-mediated change 5' to the first exon of the mitochondrial nad1 gene is associated with lack of the NADH:ubiquinone oxidoreductase (complex I) NAD1 subunit.  

Science.gov (United States)

We previously reported that the Nicotiana sylvestris CMSII mutant mitochondrial DNA carried a large deletion. Several expressed sequences, most of which are duplicated, and the unique copy of the nad7 gene encoding the NAD7 subunit of the NADH:ubiquinone oxidoreductase complex (complex I) are found in the deletion. Here, we show that the orf87-nad3-nad1/A cotranscription unit transcribed from a unique promoter element in the wild-type, is disrupted in CMSII. Nad3, orf87 and the promoter element are part of the deleted sequence, whilst the nad1/A sequence is present and transcribed from a new promoter brought by the recombination event, as indicated by Northern and primer extension experiments. However, Western analyses of mitochondrial protein fractions and of complex I purified using anti-NAD9 affinity columns, revealed that NAD1 is lacking in CMSII mitochondria. Our results suggest that translation of nad1 transcripts rather than transcription itself could be altered in the mutant. Consequences of lack of this submit belonging the membrane arm of complex I and thought to contain the ubiquinone-binding site, are discussed. PMID:10215845

Gutierres, S; Combettes, B; De Paepe, R; Mirande, M; Lelandais, C; Vedel, F; Chétrit, P

1999-04-01

238

Recombinant hormones in osteoporosis  

DEFF Research Database (Denmark)

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.

Rejnmark, Lars; Rejnmark, Lars

2013-01-01

239

Recombineering linear BACs.  

Science.gov (United States)

Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells. PMID:25239740

Chen, Qingwen; Narayanan, Kumaran

2015-01-01

240

Dissociative recombination; potential curves  

International Nuclear Information System (INIS)

Calculations of potential curves of oxygen, hydrogen and helium molecules responsible for dissociative electron recombination with positive ions of these molecules are presented. The scope of calculations ensuring sufficient accuracy of quantitative results in determination of spectroscopic properties of molecules is presented

 
 
 
 
241

Dissociative recombination of NH+  

Science.gov (United States)

We have experimentally investigated dissociative recombination of NH+ with electrons using a merged ion and electron beam configuration in a storage ring. A fast counting and position sensitive imaging detector enabled us to perform fragment imaging measurements over relative electron-ion collision energies from 0 to 12 eV. The results show unprecedented details on product excitation and on the reaction dynamics.

Yang, B.; Novotný, O.; Krantz, C.; Buhr, H.; Mended, M.; Nordhorn, C.; Geppert, W. D.; Berg, M.; Bing, D.; Domesle, C.; Grussie, F.; Savin, D. W.; Schwalm, D.; Cai, X.; Wolf, A.

2014-04-01

242

Minutes and group memories from all NERBC/USGS-RPA power plant siting task force meetings through October, 1980. Appendix  

International Nuclear Information System (INIS)

The New England River Basins Commission/United States Geological Survey-Resource Planning Analysis Office (NERBC/USGS-RPA) Power Plant Siting Task Force has formerly met seven times between July 1979 and August 1980. At the first meeting on July 13, 1979, the members agreed that there were many problems with the current process of selecting sites for power plants in New England, and that they would work by consensus to find solutions for these problems. At the second meeting on October 19, 1979, NERBC staff presented information on the site selection and approval processes in New England. The Task Force began a preliminary discussion of problems in these processes, and agreed that the initial scope of work of the Task Force would focus on issues in site selection. At the third meeting on January 18, 1980, the Task Force began initial discussions in three areas: imperfections in the site selection process, stakeholders in the site selection process, and principles to guide solutions to the problems in site selection. On March 7, 1980, at the fourth meeting, the Task Force continued discussions on imperfections, stakeholders, and principles. At the fifth meeting on May 2, 1980, the Task Force reached a wide range of agreements on the difficulties encountered in the site selection process and on the principles guiding problem solving in site selection. At the sixth meeting on May 29, 1980, the Task Force focused on solutions to the problems identified at earlier meetingshe problems identified at earlier meetings. Groups of Task Force members constructed eight different scenarios describing alternative power plant siting processes. In July 1980, the Task Force met for the seventh time and refined the eight scenarios, paring them down to five. An attempt was made to develop two scenarios using the common elements from the five. One of these two graphic models was based on government involvement in the site selection process, and the other was based on stakeholder involvement in the process

243

Vaccination with recombinant 4×M2e.HSP70c fusion protein as a universal vaccine candidate enhances both humoral and cell-mediated immune responses and decreases viral shedding against experimental challenge of H9N2 influenza in chickens.  

Science.gov (United States)

As cellular immunity is essential for virus clearance, it is commonly accepted that no adequate cellular immunity is achieved by all available inactivated HA-based influenza vaccines. Thus, an improved influenza vaccine to induce both humoral and cell-mediated immune responses is urgently required to control LPAI H9N2 outbreaks in poultry farms. M2e-based vaccines have been suggested and developed as a new generation of universal vaccine candidate against influenza A infection. Our previous study have shown that a prime-boost administration of recombinant 4×M2e.HSP70c (r4M2e/H70c) fusion protein compared to conventional HA-based influenza vaccines provided full protection against lethal dose of influenza A viruses in mice. In the present study, the immunogenicity and protective efficacy of (r4M2e/H70c) was examined in chickens. The data reported herein show that protection against H9N2 viral challenge was significantly increased in chickens by injection of r4M2e/H70c compared with injection of conventional HA-based influenza vaccine adjuvanted with MF59 or recombinant 4×M2e (r4M2e) without HSP70c. Oropharyngeal and cloacal shedding of the virus was detected in all of the r4M2e/H70c vaccinated birds at 2 days after challenge, but the titer was low and decreased rapidly to reach undetectable levels at 7 days after challenge. Moreover, comparison of protective efficacy against LPAI H9N2 in birds intramuscularly immunized with r4M2e/H70c likely represented the ability of the M2e-based vaccine in providing cross-protection against heterosubtypic H9N2 challenge and also allowed the host immune system to induce HA-homosubtype neutralizing antibody against H9N2 challenge. This protective immunity might be attributed to enhanced cell-mediated immunity, which is interpreted as increased lymphocytes proliferation, increased levels of Th1-type (IFN-?) and Th2-type (IL-4) cytokines production and increased CD4(+) to CD8(+) ratios, resulting from the injection of four tandem repeats of the ectodomain of the conserved influenza matrix protein M2 (4×M2e) genetically fused to C-terminus of Mycobacterium tuberculosis HSP70 (mHSP70c). PMID:25293397

Dabaghian, Mehran; Latify, Ali Mohammad; Tebianian, Majid; Nili, Hassan; Ranjbar, Ali Reza Tevangar; Mirjalili, Ali; Mohammadi, Mashallah; Banihashemi, Reza; Ebrahimi, Seyyed Mahmoud

2014-11-01

244

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

DEFF Research Database (Denmark)

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

Vejrup-Hansen, Rasmus; Mizuno, Ken'Ichi

2011-01-01

245

DNA rearrangement mediated by inverted repeats.  

Science.gov (United States)

Inverted repeats of DNA are widespread in the genomes of eukaryotes and prokaryotes and can mediate genome rearrangement. We studied rearrangement mediated by plasmid-borne inverted repeats in Escherichia coli. We show that inverted repeats can mediate an efficient and recA-independent recombination event. Surprisingly, the product of this recombination is not that of simple inversion between the inverted repeats, but almost exclusively an unusual head-to-head dimer with complex DNA rearrangement. Moreover, this recombination is dramatically reduced by increasing the distance separating the repeats. These results can be readily explained by a model involving reciprocal switching of the leading and lagging strands of DNA replication within the inverted repeats, which leads to the formation of a Holliday junction. Reciprocal strand switching during DNA replication might be a common mechanism for genome rearrangement associated with inverted duplication. PMID:8570641

Bi, X; Liu, L F

1996-01-23

246

SUMO Wrestles with Recombination  

Directory of Open Access Journals (Sweden)

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

Lumír Krej?í

2012-07-01

247

Recombinant vaccines against leptospirosis.  

Science.gov (United States)

Leptospirosis is an important neglected infectious disease that occurs in urban environments, as well as in rural regions worldwide. Rodents, the principal reservoir hosts of pathogenic Leptospira spp., and other infected animals shed the bacteria in their urine. During occupational or even recreational activities, humans that come into direct contact with infected animals or with a contaminated environment, particularly water, are at risk of infection. Prevention of urban leptospirosis is largely dependent on sanitation measures that are often difficult to implement, especially in developing countries. Vaccination with inactivated whole-cell preparations (bacterins) has limited efficacy due to the wide antigenic variation of the pathogen. Intensive efforts towards developing improved recombinant vaccines are ongoing. During the last decade, many reports on the evaluation of recombinant vaccines have been published. Partial success has been obtained with some surface-exposed protein antigens. The combination of protective antigens and new adjuvants or delivery systems may result in the much-needed effective vaccine. PMID:22048111

Dellagostin, Odir A; Grassmann, André A; Hartwig, Daiane D; Félix, Samuel R; da Silva, Éverton F; McBride, Alan J A

2011-11-01

248

Nonradiative recombination in semiconductors  

CERN Document Server

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

Abakumov, VN; Yassievich, IN

1991-01-01

249

Demystified… recombinant antibodies  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Recombinant antibodies are important tools for biomedical research and are increasingly being used as clinical diagnostic/therapeutic reagents. In this article, a background to humanised antibodies is given, together with details of the generation of antibody fragments—for example, single chain Fv fragments. Phage antibody fragments are fast becoming popular and can be generated by simple established methods of affinity enrichment from libraries derived from immune cells. Phage display meth...

Smith, K. A.; Nelson, P. N.; Warren, P.; Astley, S. J.; Murray, P. G.; Greenman, J.

2004-01-01

250

Radiative recombination rate coefficients  

Energy Technology Data Exchange (ETDEWEB)

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

Brinzanescu, O. [Gesellschaft fuer Schwerionenforschung mbH, Darmstadt (Germany); National Inst. for Laser, Plasma and Radiation Physics, Bucharest Magurele (Romania); Stoehlker, T. [Gesellschaft fuer Schwerionenforschung mbH, Darmstadt (Germany); Frankfurt Univ. (Germany). Inst. fuer Kernphysik

2001-07-01

251

Radiative recombination rate coefficients  

Energy Technology Data Exchange (ETDEWEB)

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

Brinzanescu, O. [Gesellschaft fuer Schwerionenforschung mbH, Darmstadt (Germany); National Inst. for Laser, Plasma and Radiation Physics, Bucharest (Romania); Stoehlker, T. [Gesellschaft fuer Schwerionenforschung mbH, Darmstadt (Germany); Frankfurt Univ. (Germany). Inst. fuer Kernphysik

2000-10-01

252

Soluble recombinant influenza vaccines.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Soluble, recombinant forms of influenza A virus haemagglutinin and neuraminidase have been produced in cells of lower eukaryotes, and shown in a mouse model to induce complete protective immunity against a lethal virus challenge. Soluble neuraminidase, produced in a baculovirus system, consisted of tetramers, dimers and monomers. Only the tetramers were enzymatically active. The immunogenicity decreased very considerably in the order tetra > di > mono. Therefore, we fused the head part of the...

Fiers, W.; Neirynck, S.; Deroo, T.; Saelens, X.; Jou, W. M.

2001-01-01

253

Recombinant Influenza Vaccines  

Digital Repository Infrastructure Vision for European Research (DRIVER)

This review covers the problems encountered in the construction and production of new recombinant influenza vaccines. New approaches to the development of influenza vaccines are investigated; they include reverse genetics methods, production of virus-like particles, and DNA- and viral vector-based vaccines. Such approaches as the delivery of foreign genes by DNA- and viral vector-based vaccines can preserve the native structure of antigens. Adenoviral vectors are a promising...

Sedova, E. S.; Shcherbinin, D. N.; Migunov, A. I.; Smirnov, Iu A.; Logunov, D. Iu; Shmarov, M. M.; Tsybalova, L. M.; Naroditskii?, B. S.; Kiselev, O. I.; Gintsburg, A. L.

2012-01-01

254

Hydrogen-oxygen recombiner  

International Nuclear Information System (INIS)

An apparatus for efficiently and safely recombining hydrogen and oxygen gas to form water vapor, the apparatus being particularly adapted for use with a nuclear reactor system in which potentially dangerous hydrogen gas, evolved within the containment vessel during certain postulated accident conditions, can be eliminated. Further, this apparatus also aids in the removal of certain radioactive contaminents from the gases in a containment vessel

255

The recombination epoch revisited  

Science.gov (United States)

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

Krolik, Julian H.

1989-01-01

256

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 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

257

Resonant scattering and recombination of pseudodegenerate WIMPs  

International Nuclear Information System (INIS)

We consider the direct and indirect detection signatures of weakly interacting massive particles (WIMPs) ?0 in kinematic regimes with a heavier, but nearly degenerate, charged state ?±. For small splittings of O(10) MeV, the scattering of WIMPs off nuclei may be dominated by inelastic recombination processes mediated by the formation of (?-N) bound states, leading to a distinct signature for direct detection. These cross sections are bound primarily by limits on the abundance of heavy isotopes, and may be considerably larger than the elastic scattering cross section in more conventional models. If the mass splitting is too large for recombination to occur, there may still be a significant resonant enhancement of loop-induced electromagnetic form factors of the WIMP, which can enhance the elastic scattering cross section. We also discuss how this regime affects the annihilation cross section and indirect detection signatures, and note the possibility of a significant mono-energetic ? signal, mediated by resonant processes near the (?+?-) bound-state threshold.

258

Lipopolysaccharide induced conversion of recombinant prion protein.  

Science.gov (United States)

The conformational conversion of the cellular prion protein (PrP(C)) to the ?-rich infectious isoform PrP(Sc) is considered a critical and central feature in prion pathology. Although PrP(Sc) is the critical component of the infectious agent, as proposed in the "protein-only" prion hypothesis, cellular components have been identified as important cofactors in triggering and enhancing the conversion of PrP(C) to proteinase K resistant PrP(Sc). A number of in vitro systems using various chemical and/or physical agents such as guanidine hydrochloride, urea, SDS, high temperature, and low pH, have been developed that cause PrP(C) conversion, their amplification, and amyloid fibril formation often under non-physiological conditions. In our ongoing efforts to look for endogenous and exogenous chemical mediators that might initiate, influence, or result in the natural conversion of PrP(C) to PrP(Sc), we discovered that lipopolysaccharide (LPS), a component of gram-negative bacterial membranes interacts with recombinant prion proteins and induces conversion to an isoform richer in ? sheet at near physiological conditions as long as the LPS concentration remains above the critical micelle concentration (CMC). More significant was the LPS mediated conversion that was observed even at sub-molar ratios of LPS to recombinant ShPrP (90-232). PMID:24819168

Saleem, Fozia; Bjorndahl, Trent C; Ladner, Carol L; Perez-Pineiro, Rolando; Ametaj, Burim N; Wishart, David S

2014-01-01

259

Human XPC-hHR23B interacts with XPA-RPA in the recognition of triplex-directed psoralen DNA interstrand crosslinks  

Digital Repository Infrastructure Vision for European Research (DRIVER)

DNA interstrand crosslinks (ICLs) represent a severe form of damage that blocks DNA metabolic processes and can lead to cell death or carcinogenesis. The repair of DNA ICLs in mammals is not well characterized. We have reported previously that a key protein complex of nucleotide excision repair (NER), XPA-RPA, recognizes DNA ICLs. We now report the use of triplex technology to direct a site-specific psoralen ICL to a target DNA substrate to determine whether the human global genome NER damage...

Thoma, Brian S.; Wakasugi, Mitsuo; Christensen, Jesper; Reddy, Madhava C.; Vasquez, Karen M.

2005-01-01

260

Transcript-RNA-templated DNA recombination and repair.  

Science.gov (United States)

Homologous recombination is a molecular process that has multiple important roles in DNA metabolism, both for DNA repair and genetic variation in all forms of life. Generally, homologous recombination involves the exchange of genetic information between two identical or nearly identical DNA molecules; however, homologous recombination can also occur between RNA molecules, as shown for RNA viruses. Previous research showed that synthetic RNA oligonucleotides can act as templates for DNA double-strand break (DSB) repair in yeast and human cells, and artificial long RNA templates injected in ciliate cells can guide genomic rearrangements. Here we report that endogenous transcript RNA mediates homologous recombination with chromosomal DNA in yeast Saccharomyces cerevisiae. We developed a system to detect the events of homologous recombination initiated by transcript RNA following the repair of a chromosomal DSB occurring either in a homologous but remote locus, or in the same transcript-generating locus in reverse-transcription-defective yeast strains. We found that RNA-DNA recombination is blocked by ribonucleases H1 and H2. In the presence of H-type ribonucleases, DSB repair proceeds through a complementary DNA intermediate, whereas in their absence, it proceeds directly through RNA. The proximity of the transcript to its chromosomal DNA partner in the same locus facilitates Rad52-driven homologous recombination during DSB repair. We demonstrate that yeast and human Rad52 proteins efficiently catalyse annealing of RNA to a DSB-like DNA end in vitro. Our results reveal a novel mechanism of homologous recombination and DNA repair in which transcript RNA is used as a template for DSB repair. Thus, considering the abundance of RNA transcripts in cells, RNA may have a marked impact on genomic stability and plasticity. PMID:25186730

Keskin, Havva; Shen, Ying; Huang, Fei; Patel, Mikir; Yang, Taehwan; Ashley, Katie; Mazin, Alexander V; Storici, Francesca

2014-11-20

 
 
 
 
261

Did the universe recombine?  

International Nuclear Information System (INIS)

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

262

Excitation energy and angular momentum dependence of nuclear level densities and spin cut-off factor in SPA and SPA + RPA approaches  

International Nuclear Information System (INIS)

We investigate the excitation energy (E*) and angular momentum (J) dependence of nuclear level density and spin cut-off factor (?) within microscopic approaches based on SPA and its extension SPA+RPA representation of the grand partition function for quadrupole-quadrupole interaction model Hamiltonian. For 110Sn, we find that excitation energy dependence of the total level density obtained within these approaches is significantly different. On the other hand, these approaches yield similar behaviour for J-dependence of the level density at fixed values of E*. Values of ?SPA+RPA at low E* are found to be slightly smaller than ?SPA but they tend to become almost the same at higher E* (>30 MeV). We also find that Bethe's formula for fixed-J level density based on the spin cut-off approximation can be used to compute ?(E*,J) near the yrast line provided one uses an appropriate value of the spin cut-off factor. (orig.)

263

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 < 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

264

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

CERN Document Server

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

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

2012-01-01

265

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

266

Nonreplicative RNA Recombination in Poliovirus  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Current models of recombination between viral RNAs are based on replicative template-switch mechanisms. The existence of nonreplicative RNA recombination in poliovirus is demonstrated in the present study by the rescue of viable viruses after cotransfections with different pairs of genomic RNA fragments with suppressed translatable and replicating capacities. Approximately 100 distinct recombinant genomes have been identified. The majority of crossovers occurred between nonhomologous segments...

Gmyl, Anatoly P.; Belousov, Evgeny V.; Maslova, Svetlana V.; Khitrina, Elena V.; Chetverin, Alexander B.; Agol, Vadim I.

1999-01-01

267

Detecting variable (V, diversity (D and joining (J gene segment recombination using a two-colour fluorescence system  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Diversity of immunoglobulins and the T cell antigen receptors is achieved via the recombination activating gene (RAG-mediated rearrangement of variable (V, diversity (D and joining (J gene segments, and this underpins the efficient recognition of a seemingly limitless array of antigens. Analysis of V(DJ recombination activity is typically performed using extrachromosomal recombination substrates that are recovered from transfected cells and selected using bacterial transformation. We have developed a two-colour fluorescence-based system that simplifies detection of both deletion and inversion joining events mediated by RAG proteins. Results This system employs two fluorescent reporter genes that differentially mark unrearranged substrates and those that have undergone RAG-mediated deletion or inversion events. The recombination products bear the hallmarks of true V(DJ recombination and activity can be detected using fluorescence microscopy or flow cytometry. Recombination events can be detected without the need for cytotoxic selection of recombination products and the system allows analysis of recombination activity using substrates integrated into the genome. Conclusions This system will be useful in the analysis and exploitation of the V(DJ recombination machinery and suggests that similar approaches could be used to replace expression of one gene with another during lymphocyte development.

Scott Gina B

2010-03-01

268

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors.  

Science.gov (United States)

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a 'targeting' vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors. PMID:25590226

Reddy, Thimma R; Kelsall, Emma J; Fevat, Léna M S; Munson, Sarah E; Cowley, Shaun M

2015-01-01

269

Hormad1 Mutation Disrupts Synaptonemal Complex Formation, Recombination, and Chromosome Segregation in Mammalian Meiosis  

Science.gov (United States)

Meiosis is unique to germ cells and essential for reproduction. During the first meiotic division, homologous chromosomes pair, recombine, and form chiasmata. The homologues connect via axial elements and numerous transverse filaments to form the synaptonemal complex. The synaptonemal complex is a critical component for chromosome pairing, segregation, and recombination. We previously identified a novel germ cell–specific HORMA domain encoding gene, Hormad1, a member of the synaptonemal complex and a mammalian counterpart to the yeast meiotic HORMA domain protein Hop1. Hormad1 is essential for mammalian gametogenesis as knockout male and female mice are infertile. Hormad1 deficient (Hormad1?/?) testes exhibit meiotic arrest in the early pachytene stage, and synaptonemal complexes cannot be visualized by electron microscopy. Hormad1 deficiency does not affect localization of other synaptonemal complex proteins, SYCP2 and SYCP3, but disrupts homologous chromosome pairing. Double stranded break formation and early recombination events are disrupted in Hormad1?/? testes and ovaries as shown by the drastic decrease in the ?H2AX, DMC1, RAD51, and RPA foci. HORMAD1 co-localizes with ?H2AX to the sex body during pachytene. BRCA1, ATR, and ?H2AX co-localize to the sex body and participate in meiotic sex chromosome inactivation and transcriptional silencing. Hormad1 deficiency abolishes ?H2AX, ATR, and BRCA1 localization to the sex chromosomes and causes transcriptional de-repression on the X chromosome. Unlike testes, Hormad1?/? ovaries have seemingly normal ovarian folliculogenesis after puberty. However, embryos generated from Hormad1?/? oocytes are hyper- and hypodiploid at the 2 cell and 8 cell stage, and they arrest at the blastocyst stage. HORMAD1 is therefore a critical component of the synaptonemal complex that affects synapsis, recombination, and meiotic sex chromosome inactivation and transcriptional silencing. PMID:21079677

Erdin, Serpil Uckac; Yatsenko, Svetlana A.; Kloc, Malgorzata; Yang, Fang; Wang, P. Jeremy; Meistrich, Marvin L.; Rajkovic, Aleksandar

2010-01-01

270

Somatic and Germinal Recombination of a Direct Repeat in Arabidopsis  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

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

1992-01-01

271

Optimal Recombination in Genetic Algorithms  

Digital Repository Infrastructure Vision for European Research (DRIVER)

This paper surveys results on complexity of the optimal recombination problem (ORP), which consists in finding the best possible offspring as a result of a recombination operator in a genetic algorithm, given two parent solutions. We consider efficient reductions of the ORPs, allowing to establish polynomial solvability or NP-hardness of the ORPs, as well as direct proofs of hardness results.

Eremeev, Anton V.; Kovalenko, Julia V.

2013-01-01

272

Dielectronic recombination lines of C+  

International Nuclear Information System (INIS)

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

273

Dielectronic Recombination Lines of C+  

CERN Document Server

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

Sochi, Taha

2012-01-01

274

Recombinant TCR ligand reverses clinical signs and CNS damage of EAE induced by recombinant human MOG1  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Increasing evidence suggests that in addition to T cell dependent effector mechanisms, autoantibodies are also involved in the pathogenesis of MS, including demyelinating antibodies specific for myelin oligodendrocyte glycoprotein (MOG). Our previous studies have demonstrated that recombinant T cell receptor ligands (RTLs) are very effective for treating T cell mediated experimental autoimmune encephalomyelitis (EAE). In order to expand the scope of RTL therapy in MS patients, it was of inter...

Sinha, Sushmita; Subramanian, Sandhya; Emerson-webber, Ashley; Lindner, Maren; Burrows, Gregory G.; Grafe, Marjorie; Linington, Christopher; Vandenbark, Arthur A.; Bernard, Claude C. A.; Offner, Halina

2010-01-01

275

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

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

1991-01-01

276

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

International Nuclear Information System (INIS)

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

277

Recombinant Goat VEGF164 Increases Hair Growth by Painting Process on the Skin of Shaved Mouse  

Digital Repository Infrastructure Vision for European Research (DRIVER)

To detect goat vascular endothelial growth factor (VEGF)-mediated regrowth of hair, full-length VEGF164 cDNA was cloned from Inner Mongolia cashmere goat (Capra hircus) into the pET-his prokaryotic expression vector, and the recombinant plasmid was transferred into E. coli BL21 cells. The expression of recombinant 6×his-gVEGF164 protein was induced by 0.5 mM isopropyl thio-?-D-galactoside at 32°C. Recombinant goat VEGF164 (rgVEGF164) was purified and identi ed by western blot using monoclo...

Bao, Wenlei; Yin, Jianxin; Liang, Yan; Guo, Zhixin; Wang, Yanfeng; Liu, Dongjun; Wang, Xiao; Wang, Zhigang

2014-01-01

278

Magnetic susceptibility of a CuO2 plane in the La2CuO4 system: I. RPA treatment of the Dzyaloshinskii-Moriya Interactions  

CERN Document Server

Motivated by recent experiments on undoped La2CuO4, which found pronounced temperature-dependent anisotropies in the low-field magnetic susceptibility, we have investigated a two-dimensional square lattice of S=1/2 spins that interact via Heisenberg exchange plus the symmetric and anti-symmetric Dzyaloshinskii-Moriya anisotropies. We describe the transition to a state with long-ranged order, and find the spin-wave excitations, with a mean-field theory, linear spin-wave analysis, and using Tyablikov's RPA decoupling scheme. We find the different components of the susceptibility within all of these approximations, both below and above the N'eel temperature, and obtain evidence of strong quantum fluctuations and spin-wave interactions in a broad temperature region near the transition.

Tabunshchyk, K V

2005-01-01

279

Delayed recombination and standard rulers  

International Nuclear Information System (INIS)

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

280

Brca2-Pds5 complexes mobilize persistent meiotic recombination sites to the nuclear envelope.  

Science.gov (United States)

Homologous recombination is required for reciprocal exchange between homologous chromosome arms during meiosis. Only select meiotic recombination events become chromosomal crossovers; the majority of recombination outcomes are noncrossovers. Growing evidence suggests that crossovers are repaired after noncrossovers. Here, I report that persisting recombination sites are mobilized to the nuclear envelope of Drosophila pro-oocytes during mid-pachytene. Their number correlates with the average crossover rate per meiosis. Proteomic and interaction studies reveal that the recombination mediator Brca2 associates with lamin and the cohesion factor Pds5 to secure persistent recombination sites at the nuclear envelope. In Rad51(-/-) females, all persistent DNA breaks are directed to the nuclear envelope. By contrast, a reduction of Pds5 or Brca2 levels abolishes the movement and has a negative impact on crossover rates. The data suggest that persistent meiotic DNA double-strand breaks might correspond to crossovers, which are mobilized to the nuclear envelope for their repair. The identification of Brca2-Pds5 complexes as key mediators of this process provides a first mechanistic explanation for the contribution of lamins and cohesins to meiotic recombination. PMID:25588834

Kusch, Thomas

2015-02-15

 
 
 
 
281

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 < 70. Patients who do not qualify for Class 1 or 3 are grouped as Class 2. The survival of these patients was determined from the time of diagnosis of brain metastases to the time of death. Results: The median survival of the entire group was 9.5 months. The three classes of patients as grouped had median survivals of 14.8, 9.9, and 6.0 months respectively (p = 0.0002). Age of < 65 yearstively (p = 0.0002). Age of < 65 years, KPS of ? 70, controlled primary disease, absence of extracranial metastases, complete surgical resection of the brain lesion(s) were found to be independent prognostic factors for survival; the total dose of radiation was not. Conclusion: Based on the results of this study, the patients and disease characteristics have significant impact on the survival of patients with brain metastases treated with a combination of surgical resection and radiotherapy. These parameters could be used in selecting patients who would benefit most from such treatment

282

Aspergillus: sex and recombination.  

Science.gov (United States)

The genus Aspergillus is one of the most widespread groups of fungi on Earth, comprised of about 300-350 species with very diverse lifestyles. Most species produce asexual propagula (conidia) on conidial heads. Despite their ubiquity, a sexual cycle has not yet been identified for most of the aspergilli. Where sexual reproduction is present, species exhibit either homothallic (self fertile) or heterothallic (obligate outcrossing) breeding systems. A parasexual cycle has also been described in some Aspergillus species. As in other fungi, sexual reproduction is governed by mating-type (MAT) genes, which determine sexual identity and are involved in regulating later stages of sexual development. Previous population genetic studies have indicated that some supposedly asexual aspergilli exhibit evidence of a recombining population structure, suggesting the presence of a cryptic sexual cycle. In addition, genome analyses have revealed networks of genes necessary for sexual reproduction in several Aspergillus species, again consistent with latent sexuality in these fungi. Knowledge of MAT gene presence has then successfully been applied to induce sexual reproduction between MAT1-1 and MAT1-2 isolates of certain supposedly asexual aspergilli. Recent progress in understanding the extent and significance of sexual reproduction is described here, with special emphasis on findings that are relevant to clinically important aspergilli. PMID:25118872

Varga, János; Szigeti, Gyöngyi; Baranyi, Nikolett; Kocsubé, Sándor; O'Gorman, Céline M; Dyer, Paul S

2014-12-01

283

Bipolaron recombination in conjugated polymers.  

Science.gov (United States)

By using the Su-Schrieffer-Heeger model modified to include electron-electron interactions, the Brazovskii-Kirova symmetry breaking term and an external electric field, we investigate the scattering process between a negative and a positive bipolaron in a system composed of two coupled polymer chains. Our results show that the Coulomb interactions do not favor the bipolaron recombination. In the region of weak Coulomb interactions, the two bipolarons recombine into a localized excited state, while in the region of strong Coulomb interactions they can not recombine. Our calculations show that there are mainly four channels for the bipolaron recombination reaction: (1) forming a biexciton, (2) forming an excited negative polaron and a free hole, (3) forming an excited positive polaron and a free electron, (4) forming an exciton, a free electron, and a free hole. The yields for the four channels are also calculated. PMID:21861583

Sun, Zhen; Stafström, Sven

2011-08-21

284

Controlled release from recombinant polymers.  

Science.gov (United States)

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

Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

2014-09-28

285

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

286

Alphavirus vectors as recombinant vaccines  

Digital Repository Infrastructure Vision for European Research (DRIVER)

ALPHAVIRUS VECTORS AS RECOMBINANT VACCINES Peter Berglund Doctoral dissertation from the Microbiology and TumorbiologyCenter Karolinska Institute, Sweden This thesis describes further developments of an expression system based on thealphavirus Semliki Forest virus (SFV), and its potential use as recombinant vaccine. The RNA genome of SFV contains a 3'open reading frame (ORF) encoding the structuralproteins, and a 5' ORF coding for the viral replicase. Transfecti...

Berglund, Peter

1997-01-01

287

Ethanol production by recombinant hosts  

Science.gov (United States)

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

288

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

289

Delayed recombination and cosmic parameters  

International Nuclear Information System (INIS)

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

290

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

291

Tyrosine phosphorylation enhances RAD52-mediated annealing by modulating its DNA binding.  

Science.gov (United States)

RAD52 protein has an important role in homology-directed DNA repair by mediating RAD51 nucleoprotein filament formation on single-stranded DNA (ssDNA) protected by replication protein-A (RPA) and annealing of RPA-coated ssDNA. In human, cellular response to DNA damage includes phosphorylation of RAD52 by c-ABL kinase at tyrosine 104. To address how this phosphorylation modulates RAD52 function, we used an amber suppressor technology to substitute tyrosine 104 with chemically stable phosphotyrosine analogue (p-Carboxymethyl-L-phenylalanine, pCMF). The RAD52(Y104pCMF) retained ssDNA-binding activity characteristic of unmodified RAD52 but showed lower affinity for double-stranded DNA (dsDNA) binding. Single-molecule analyses revealed that RAD52(Y104pCMF) specifically targets and wraps ssDNA. While RAD52(Y104pCMF) is confined to ssDNA region, unmodified RAD52 readily diffuses into dsDNA region. The Y104pCMF substitution also increased the ssDNA annealing rate and allowed overcoming the inhibitory effect of dsDNA. We propose that phosphorylation at Y104 enhances ssDNA annealing activity of RAD52 by attenuating dsDNA binding. Implications of phosphorylation-mediated activation of RAD52 annealing activity are discussed. PMID:21804533

Honda, Masayoshi; Okuno, Yusuke; Yoo, Jungmin; Ha, Taekjip; Spies, Maria

2011-08-17

292

Recombination processes in ionised plasmas  

Science.gov (United States)

The observational analysis of astrophysical plasmas relies on accurate calculations of the atomic processes involved. The recombination spectra of singly ionised oxygen (O il) and carbon (C il) present excellent tools for investigating regions such as planetary nebulae and H II regions. In this thesis, detailed treatments of the recombination processes of both O II and C II are presented. Using the R-matrix solution to the close coupling equations, I present the results of accurate photoionisation calculations. Bound state energy levels are determined and oscillator strengths calculated for both species. Recombination coefficients were evalu ated for low n and 1, for C II in LS-coupling, and 0 II in intermediate coupling, taking particular care to treat resonances effectively. Sample photoionisation cross-sections are presented for both species, and compared to previous work. A complete radiative-cascade model is treated for both species, in order to determine line emissivities under nebular conditions at a wide range of temperatures and densities. Collisional effects are treated for C II, along with, for the first time, the effects of high temperature dielectronic recombination, allowing the modelling of regions of much higher electron temperature than previous work. The O II calculations were performed under intermediate coupling for the first time, allowing the effects of non-statistical popula tions of the parent ion fine-structure levels and dielectronic recombination onto bound states within this fine-structure to be taken into account in line emissivities. Detailed comparison with previous theoretical work was made for both species. The application of the C II and 0 n recombination spectra to determining tempera ture and densities from the observed spectra of a number of ionised nebulae is considered. The potential for using the new recombination spectra as diagnostic tools to solve some of the key problems in the study of ionised nebulae is demonstrated.

Bastin, Robert

293

Investigations for designing catalytic recombiners  

Energy Technology Data Exchange (ETDEWEB)

In case of a severe accident in pressurised water reactors (PWR) a high amount of hydrogen up to about 20,000 m{sup 3} 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.

Broeckerhoff, P.; Reinecke, E.A. [Forschungszentrum Juelich GmbH (Germany). Inst. fuer Sicherheitsforschung und Reaktortechnik

2001-07-01

294

Investigations for designing catalytic recombiners  

International Nuclear Information System (INIS)

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

295

Recombining WMAP: Constraints on ionizing and resonance radiation at recombination  

International Nuclear Information System (INIS)

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

296

Different functions for the domains of the Arabidopsis thaliana RMI1 protein in DNA cross-link repair, somatic and meiotic recombination  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Recombination intermediates, such as double Holliday junctions, can be resolved by nucleases or dissolved by the combined action of a DNA helicase and a topoisomerase. In eukaryotes, dissolution is mediated by the RTR complex consisting of a RecQ helicase, a type IA topoisomerase and the structural protein RecQ-mediated genome instability 1 (RMI1). Throughout eukaryotes, the RTR complex is involved in DNA repair and in the suppression of homologous recombination (HR) in somatic cells. Surpris...

Bonnet, Simone; Knoll, Alexander; Hartung, Frank; Puchta, Holger

2013-01-01

297

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

International Nuclear Information System (INIS)

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

298

Upstream and downstream processing of recombinant IgA.  

Science.gov (United States)

Immunoglobulin A (IgA) is the most abundant antibody class in the human body and has a unique role in mediating immunity. The ever-increasing knowledge about the potential of IgAs has renewed interest in this antibody class for therapeutic use against a variety of infectious and malignant diseases, and as a preventive agent for mucosal pathogens. Despite the considerable therapeutic potential of IgA the exploration thereof has often been hampered due to difficulties in producing and purifying desired quantities. Large amounts of pure IgA will be required for in vivo studies. This work reviews current achievements and bottlenecks in upstream and downstream processing of recombinant IgA from a biotechnological point of view. We also highlight recent accomplishments with diverse expression systems and presents different affinity techniques for the capture of recombinant IgA to compare their purification potential. PMID:25257601

Reinhart, David; Kunert, Renate

2015-02-01

299

Multifaceted regulation of V(D)J recombination  

Science.gov (United States)

V(D)J recombination is responsible for generating an enormous repertoire of immunoglobulins and T cell receptors, therefore it is a centerpiece to the formation of the adaptive immune system. The V(D)J recombination process proceeds through two steps, site-specific cleavage at RSS (Recombination Signal Sequence) site mediated by the RAG recombinase (RAG1/2) and the subsequent imprecise resolution of the DNA ends, which is carried out by the ubiquitous non-homologous end joining pathway (NHEJ). The V(D)J recombination reaction is obliged to be tightly controlled under all circumstances, as it involves generations of DNA double strand breaks, which are considered the most dangerous lesion to a cell. Multifaceted regulatory mechanisms have been evolved to create great diversity of the antigen receptor repertoire while ensuring genome stability. The RAG-mediated cleavage reaction is stringently regulated at both the pre-cleavage stage and the post-cleavage stage. Specifically, RAG1/2 first forms a pre-cleavage complex assembled at the boarder of RSS and coding flank, which ensures the appropriate DNA targeting. Subsequently, this complex initiates site-specific cleavage, generating two types of double stranded DNA breaks, hairpin-ended coding ends (HP-CEs) and blunt signal ends (SEs). After the cleavage, RAG1/2 proteins bind and retain the recombination ends to form post-cleavage complexes (PCC), which collaborates with the NHEJ machinery for appropriate transfer of recombination ends to NHEJ for proper end resolution. However, little is known about the molecular basis of this collaboration, partly attributed to the lack of sensitive assays to reveal the interaction of PCC with HP-CEs. Here, for the first time, by using two complementary fluorescence-based techniques, fluorescence anisotropy and fluorescence resonance energy transfer (FRET), I managed to monitor the RAG1/2-catalyzed cleavage reaction in real time, from the pre-cleavage to the post-cleavage stages. By examining the dynamic fluorescence changes during the RAG-mediated cleavage reactions, and by manipulating the reaction conditions, I was able to characterize some fundamental properties of RAG-DNA interactions before and after cleavage. Firstly, Mg 2+, known as a physiological cofactor at the excision step, also promotes the HP-CEs retention in the RAG complex after cleavage. Secondly, the structure of pre-cleavage complex may affect the subsequent collaborations with NHEJ for end resolution. Thirdly, the non-core region of RAG2 may have differential influences on the PCC retention of HP-CEs and SEs. Furthermore, I also provide the first evidence of RAG1-mediated regulation of RAG2. Our study provides important insights into the multilayered regulatory mechanisms, in modulating recombination events in developing lymphocytes and paves the way for possible development of detection and diagnotic markers for defective recombination events that are often associated immunodeficiency and/or lymphoid malignancy.

Wang, Guannan

300

Inhomogeneous recombinations during cosmic reionization  

CERN Document Server

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

Sobacchi, Emanuele

2014-01-01

 
 
 
 
301

Theoretical studies of dissociative recombination  

Science.gov (United States)

The calculation of dissociative recombination rates and cross sections over a wide temperature range by theoretical quantum chemical techniques is described. Model calculations on electron capture by diatomic ions are reported which illustrate the dependence of the rates and cross sections on electron energy, electron temperature, and vibrational temperature for three model crossings of neutral and ionic potential curves. It is shown that cross sections for recombination to the lowest vibrational level of the ion can vary by several orders of magnitude depending upon the position of the neutral and ionic potential curve crossing within the turning points of the v = 1 vibrational level. A new approach for calculating electron capture widths is reported. Ab initio calculations are described for recombination of O2(+) leading to excited O atoms.

Guberman, S. L.

1985-01-01

302

The Dissociative Recombination of OH(+)  

Science.gov (United States)

Theoretical quantum chemical calculations of the cross sections and rates for the dissociative recombination of the upsilon = 0 level of the ground state of OH(+) show that recombination occurs primarily along the 2 (2)Pi diabatic route. The products are 0((1)D) and a hot H atom with 6.1 eV kinetic energy. The coupling to the resonances is very small and the indirect recombination mechanism plays only a minor role. The recommended value for the rate coefficient is (6.3 +/- 0.7) x 10(exp -9)x (T(e)/1300)(exp -0.48) cu.cm/s for 10 less than T(e) less than 1000 K.

Guberman, Steven L.

1995-01-01

303

Recombination energy of N22+  

International Nuclear Information System (INIS)

The recombination energy of N22+ has been computed using N2+, N2+ and N2 potential curves from the literature. Vibrational overlaps and energies liberated in the various N22+(3?sub(g)-, 1?sub(g)+, 3PIsub(u), 1PIsub(u)) ? N2+(X2?sub(g)+, A2PIsub(u), B2?sub(u)?+, C2?sub(u)+) vibronic transitions have been computed and used as input for determination of the N22+ recombination energy. (orig.)

304

Dissociative recombination of protonated methanol  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The branching ratios of the different reaction pathways and the overall rate coefficients of the dissociative recombination reactions of CH3OH2+ and CD3OD2+ have been measured at the CRYRING storage ring located in Stockholm, Sweden. Analysis of the data yielded the result that formation of methanol or deuterated methanol accounted for only 3 and 6% of the total rate in CH3OH2+ and CD3OD2+, respectively. Dissociative recombination of both isotopomeres mainly involves fragmentation of the C–...

Geppert, Wolf; Hamberg, Mathias; Thomas, Richard D.; O?sterdahl, Fabian; Hellberg, Fredrik; Zhaunerchyk, Vitali; Ehlerding, Anneli; Millar, Tom; Roberts, Helen; Semaniak, Jacek; Af Ugglas, Magnus; Ka?llberg, Anders; Simonsson, Ansgar; Kaminska, Magdalena; Larsson, Mats

2006-01-01

305

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

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

Chisholm, Karen M.; Aubert, Sarah D.; Freese, Krister P.; Zakian, Virginia A.; King, Mary-claire; Welcsh, Piri L.

2012-01-01

306

Horizontal transmissible protection against myxomatosis and rabbit hemorrhagic disease by using a recombinant myxoma virus.  

Science.gov (United States)

We have developed a new strategy for immunization of wild rabbit populations against myxomatosis and rabbit hemorrhagic disease (RHD) that uses recombinant viruses based on a naturally attenuated field strain of myxoma virus (MV). The recombinant viruses expressed the RHDV major capsid protein (VP60) including a linear epitope tag from the transmissible gastroenteritis virus (TGEV) nucleoprotein. Following inoculation, the recombinant viruses induced specific antibody responses against MV, RHDV, and the TGEV tag. Immunization of wild rabbits by the subcutaneous and oral routes conferred protection against virulent RHDV and MV challenges. The recombinant viruses showed a limited horizontal transmission capacity, either by direct contact or in a flea-mediated process, promoting immunization of contact uninoculated animals. PMID:10627521

Bárcena, J; Morales, M; Vázquez, B; Boga, J A; Parra, F; Lucientes, J; Pagès-Manté, A; Sánchez-Vizcaíno, J M; Blasco, R; Torres, J M

2000-02-01

307

Night-time electron temperature troughs in the equatorial topside ionosphere revealed from RPA experiments on the ISS-b satellite  

International Nuclear Information System (INIS)

Night-time electron temperature (Tsub(e)), electron density (Nsub(e)) and the mean ion mass (Msub(i)) of the topside ionosphere at altitudes of about 1100 km have been obtained from the RPA experiments on ISS-b. Global maps of these quantities are derived by adopting the spherical surface harmonic functions fitted to the satellite data by means of a least-square method. The night-time map of Tsub(e) shows a marked equatorial trough which is limited to a 1000 longitudinal sector and a 200 latitudinal extent. The center of the trough is located a few degrees 'summerward' of the dip equator. The longitude of the trough is strongly controlled by the magnetic declination. This longitude changes rapidly around autumnal equinox from the region of maximum westward declination to that of eastward declination, the opposite change occurs around the time of vernal equinox. The maps of Msub(i) and Nsub(e) in low latitudes have features apparently related to the Tsub(e) trough. These features can be explained in terms of field-aligned interhemispheric plasma flow driven by the neutral air wind. (author)

308

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

309

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

Directory of Open Access Journals (Sweden)

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

Garcia-Cruz Raquel

2009-06-01

310

Cre/Lox Recombination in the Lower Urinary Tract  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Tbx18 is a T-Box transcription factor that has specific expression and indispensible function in the lower urinary tract (Airik et al., 2006). Here, we report the generation and characterization of a bacterial artificial chromosome (BAC) transgene expressing Cre under the control of Tbx18 regulatory elements. When crossed to the ROSA26R-lacZ reporter mice, the Tbx18-Cre transgene mediates loxP recombination in the mesenchymal derivatives in the lower urinary tract, especially in the smooth mu...

Wang, Yinqiu; Tripathi, Piyush; Guo, Qiusha; Coussens, Matthew; Ma, Liang; Chen, Feng

2009-01-01

311

Recombination in immunoglobulin gene loci  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

Komisarenko S. V.; Halytskiy V. A.

2009-01-01

312

In utero recombinant adeno-associated virus gene transfer in mice, rats, and primates  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Abstract Background Gene transfer into the amniotic fluid using recombinant adenovirus vectors was shown previously to result in high efficiency transfer of transgenes into the lungs and intestines. Adenovirus mediated in utero gene therapy, however, resulted in expression of the transgene for less than 30 days. Recombinant adenovirus associated viruses (rAAV) have the advantage of maintaining the viral genome in daughter cells thus providing for long-term expression...

Marrero Luis; Dunn Daisy; Larson Janet E; Garrett Deiadra J; Craig, Cohen J.

2003-01-01

313

Recombineering reveals a diverse collection of ribosomal proteins L4 and L22 that confer resistance to macrolide antibiotics  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Mutations in ribosomal proteins L4 and L22 confer resistance to erythromycin and other macrolide antibiotics in a variety of bacteria. L4 and L22 have elongated loops whose tips converge in the peptide exit tunnel near the macrolide binding site, and resistance mutations typically affect residues within these loops. Here, we use bacteriophage ? Red-mediated recombination, or “recombineering”, to uncover new L4 and L22 alleles that confer macrolide resistance in Escherichia coli. We rando...

Diner, Elie J.; Hayes, Christopher S.

2009-01-01

314

The ?0 polarization and the recombination mechanism  

International Nuclear Information System (INIS)

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

315

Bocavirus infection induces a DNA damage response that facilitates viral DNA replication and mediates cell death.  

Science.gov (United States)

Minute virus of canines (MVC) is an autonomous parvovirus that replicates efficiently without helper viruses in Walter Reed/3873D (WRD) canine cells. We previously showed that MVC infection induces mitochondrion-mediated apoptosis and G(2)/M-phase arrest in infected WRD cells. However, the mechanism responsible for these effects has not been established. Here, we report that MVC infection triggers a DNA damage response in infected cells, as evident from phosphorylation of H2AX and RPA32. We discovered that both ATM (ataxia telangiectasia-mutated kinase) and ATR (ATM- and Rad3-related kinase) were phosphorylated in MVC-infected WRD cells and confirmed that ATM activation was responsible for the phosphorylation of H2AX, whereas ATR activation was required for the phosphorylation of RPA32. Both pharmacological inhibition of ATM activation and knockdown of ATM in MVC-infected cells led to a significant reduction in cell death, a moderate correction of cell cycle arrest, and most importantly, a reduction in MVC DNA replication and progeny virus production. Parallel experiments with an ATR-targeted small interfering RNA (siRNA) had no effect. Moreover, we identified that this ATM-mediated cell death is p53 dependent. In addition, we localized the Mre11-Rad50-Nbs1 (MRN) complex, the major mediator as well as a substrate of the ATM-mediated DNA damage response pathway to MVC replication centers during infection, and show that Mre11 knockdown led to a reduction in MVC DNA replication. Our findings are the first to support the notion that an autonomous parvovirus is able to hijack the host DNA damage machinery for its own replication and for the induction of cell death. PMID:21047968

Luo, Yong; Chen, Aaron Yun; Qiu, Jianming

2011-01-01

316

Population inversion in recombining hydrogen plasma  

International Nuclear Information System (INIS)

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

317

Shock and tissue injury induced by recombinant human cachectin.  

Science.gov (United States)

Cachectin (tumor necrosis factor), a protein produced in large quantities by endotoxin-activated macrophages, has been implicated as an important mediator of the lethal effect of endotoxin. Recombinant human cachectin was infused into rats in an effort to determine whether cachectin, by itself, can elicit the derangements of host physiology caused by administration of endotoxin. When administered in quantities similar to those produced endogenously in response to endotoxin, cachectin causes hypotension, metabolic acidosis, hemoconcentration, and death within minutes to hours, as a result of respiratory arrest. Hyperglycemia and hyperkalemia were also observed after infusion. At necropsy, diffuse pulmonary inflammation and hemorrhage were apparent on gross and histopathologic examination, along with ischemic and hemorrhagic lesions of the gastrointestinal tract, and acute renal tubular necrosis. Thus, it appears that a single protein mediator (cachectin) is capable of inducing many of the deleterious effects of endotoxin. PMID:3764421

Tracey, K J; Beutler, B; Lowry, S F; Merryweather, J; Wolpe, S; Milsark, I W; Hariri, R J; Fahey, T J; Zentella, A; Albert, J D

1986-10-24

318

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

Osorio, Yanira; Ghiasi, Homayon

2003-01-01

319

Characterization of recombinantly expressed matrilin VWA domains.  

Science.gov (United States)

VWA domains are the predominant independent folding units within matrilins and mediate protein-protein interactions. Mutations in the matrilin-3 VWA domain cause various skeletal diseases. The analysis of the pathological mechanisms is hampered by the lack of detailed structural information on matrilin VWA domains. Attempts to resolve their structures were hindered by low solubility and a tendency to aggregation. We therefore took a comprehensive approach to improve the recombinant expression of functional matrilin VWA domains to enable X-ray crystallography and nuclear magnetic resonance (NMR) studies. The focus was on expression in Escherichia coli, as this allows incorporation of isotope-labeled amino acids, and on finding conditions that enhance solubility. Indeed, circular dichroism (CD) and NMR measurements indicated a proper folding of the bacterially expressed domains and, interestingly, expression of zebrafish matrilin VWA domains and addition of N-ethylmaleimide yielded the most stable proteins. However, such proteins did still not crystallize and allowed only partial peak assignment in NMR. Moreover, bacterially expressed matrilin VWA domains differ in their solubility and functional properties from the same domains expressed in eukaryotic cells. Structural studies of matrilin VWA domains will depend on the use of eukaryotic expression systems. PMID:25462806

Becker, Ann-Kathrin A; Mikolajek, Halina; Werner, Jörn M; Paulsson, Mats; Wagener, Raimund

2015-03-01

320

Dielectronic Recombination of Argon-Like Ions  

CERN Document Server

We present a theoretical investigation of dielectronic recombination (DR) of Ar-like ions that sheds new light on the behavior of the rate coefficient at low-temperatures where these ions form in photoionized plasmas. We provide results for the total and partial Maxwellian-averaged DR rate coefficients from the initial ground level of K II -- Zn XIII ions. It is expected that these new results will advance the accuracy of the ionization balance for Ar-like M-shell ions and pave the way towards a detailed modeling of astrophysically relevant X-ray absorption features. We utilize the AUTOSTRUCTURE computer code to obtain the accurate core-excitation thresholds in target ions and carry out multiconfiguration Breit-Pauli (MCBP) calculations of the DR cross section in the independent-processes, isolated-resonance, distorted-wave (IPIRDW) approximation. Our results mediate the complete absence of direct DR calculations for certain Ar-like ions and question the reliability of the existing empirical rate formulas, of...

Nikoli?, D; Korista, K T; Badnell, N R

2010-01-01

 
 
 
 
321

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.)

322

Recombination lasing in heliumlike silicon  

International Nuclear Information System (INIS)

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

323

Nondisjunction of chromosome 15: Origin and recombination  

Energy Technology Data Exchange (ETDEWEB)

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

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

1993-09-01

324

Nonhomologous recombination in human cells.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Nonhomologous recombination (NHR) is a major pathway for the repair of chromosomal double-strand breaks in the DNA of somatic cells. In this study, a comparison was made between the nonhomologous end joining of transfected adenovirus DNA fragments in vivo and the ability of purified human proteins to catalyze nonhomologous end joining in vitro. Adenovirus DNA fragments were shown to be efficiently joined in human cells regardless of the structure of the ends. Sequence analysis of these juncti...

Derbyshire, M. K.; Epstein, L. H.; Young, C. S.; Munz, P. L.; Fishel, R.

1994-01-01

325

Recombinant erythropoietin in clinical practice  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The introduction of recombinant human erythropoietin (RHuEPO) has revolutionised the treatment of patients with anaemia of chronic renal disease. Clinical studies have demonstrated that RHuEPO is also useful in various non-uraemic conditions including haematological and oncological disorders, prematurity, HIV infection, and perioperative therapies. Besides highlighting both the historical and functional aspects of RHuEPO, this review discusses the applications of RHuEPO in clinical practice a...

Ng, T.; Marx, G.; Littlewood, T.; Macdougall, I.

2003-01-01

326

Dielectronic recombination in He+ ions  

International Nuclear Information System (INIS)

Dielectronic recombination involving 1s+e-?nln'l' transitions has been investigated for He+ ions. This work was done using the ion storage ring and electron cooler at the Indiana University Cyclotron Facility. Resonant maxima from DR were observed, but the energy resolution was insufficient to identify individual transitions. The magnitude of the measured cross sections appears to be about a factor of two lower than theory. (orig.)

327

Coherent and stochastic contributions of compound resonances in atomic processes: electron recombination, photoionization and scattering  

CERN Document Server

In open-shell atoms and ions, processes such as photoionization, combination (Raman) scattering, electron scattering and recombination, are often mediated by many-electron compound resonances. We show that their interference (neglected in independent-resonance approximation) leads to a coherent contribution, which determines the energy-averaged total cross sections of electron- and photon-induced reactions found from the optical theorem. On the other hand, the partial cross sections (e.g., electron recombination, combination photon scattering) are dominated by the stochastic contributions. Thus, the optical theorem provides a link between the stochastic and coherent contributions of the compound resonances.

Flambaum, V V; Gribakin, G F

2014-01-01

328

Current Trends of HIV Recombination Worldwide  

Science.gov (United States)

One of the major characteristics of HIV-1 is its high genetic variability and extensive heterogeneity. This characteristic is due to its molecular traits, which in turn allows it to vary, recombine, and diversify at a high frequency. As such, it generates complex molecular forms, termed recombinants, which evade the human immune system and so survive. There is no sequence constraint to the recombination pattern as it appears to occur at inter-group (between groups M and O), as well as interand intra-subtype within group M. Rapid emergence and active global transmission of HIV-1 recombinants, known as circulating recombinant forms (CRFs) and unique recombinant forms (URFs), requires urgent attention. To date, 55 CRFs have been reported around the world. The first CRF01_AE originated from Central Africa but spread widely in Asia. The most recent CRF; CRF55_01B is a recombinant form of CRF01_AE and subtype B, although its origin is yet to be publicly disclosed. HIV-1 recombination is an ongoing event and plays an indispensable role in HIV epidemics in different regions. Africa, Asia and South America are identified as recombination hot-spots. They are affected by continual emergence and cocirculation of newly emerging CRFs and URFs, which are now responsible for almost 20% of HIV-1 infections worldwide. Better understanding of recombinants is necessary to determine their biological and molecular attributes. PMID:24470968

Lau, Katherine A.; Wong, Justin J.L.

2013-01-01

329

Current trends of HIV recombination worldwide  

Directory of Open Access Journals (Sweden)

Full Text Available One of the major characteristics of HIV-1 is its high genetic variability and extensive heterogeneity. This characteristic is due to its molecular traits, which in turn allows it to vary, recombine, and diversify at a high frequency. As such, it generates complex molecular forms, termed recombinants, which evade the human immune system and so survive. There is no sequence constraint to the recombination pattern as it appears to occur at inter-group (between groups M and O, as well as inter- and intra-subtype within group M. Rapid emergence and active global transmission of HIV-1 recombinants, known as circulating recombinant forms (CRFs and unique recombinant forms (URFs, requires urgent attention. To date, 55 CRFs have been reported around the world. The first CRF01_AE originated from Central Africa but spread widely in Asia. The most recent CRF; CRF55_01B is a recombinant form of CRF01_AE and subtype B, although its origin is yet to be publicly disclosed. HIV-1 recombination is an ongoing event and plays an indispensable role in HIV epidemics in different regions. Africa, Asia and South America are identified as recombination hot-spots. They are affected by continual emergence and co-circulation of newly emerging CRFs and URFs, which are now responsible for almost 20% of HIV-1 infections worldwide. Better understanding of recombinants is necessary to determine their biological and molecular attributes.

Justin J.L. Wong

2013-06-01

330

Bacteriophage recombination systems and biotechnical applications.  

Science.gov (United States)

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

Nafissi, Nafiseh; Slavcev, Roderick

2014-04-01

331

Heterogeneity in recombinant protein production  

DEFF Research Database (Denmark)

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

Schalén, Martin; Johanson, Ted

2012-01-01

332

Atomic excitation and recombination in external fields  

International Nuclear Information System (INIS)

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

333

Theoretical studies of dissociative recombination  

Science.gov (United States)

In the collision of electrons with molecules and molecular ions, excitation and dissociation are dominated by resonant processes, where the electron becomes temporarily trapped, changing the forces felt by the nuclei. We have carried out calculations on the resonant process leading to dissociative reccombination. We separate the problem into two steps. First, the resonance parameters are obtained from accurate electron scattering calculations using the Complex Kohn variational method. Then these parameters are used as input to the dynamics calculations. We will illustrate the method with the study of dissociative recombination in HF+ CF+ HCO/HCO+ and H2O+.

Roos, J.; Ngassam, V.; Larson, Å.; Orel, A. E.

2009-11-01

334

Recombination fluorescence in ultracold neutral plasmas  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We present the first measurements and simulations of recombination fluorescence in ultracold neutral plasmas. In contrast with previous work, experiment and simulation are in significant disagreement. Comparison with a recombination model suggests that the disagreement could be due to the high energy portion of the electron energy distribution or to large energy changes in electron/Rydberg scattering. Recombination fluorescence opens a new diagnostic window in ultracold plas...

Bergeson, S. D.; Robicheaux, F.

2007-01-01

335

Recombinant DNA production of spider silk proteins.  

Science.gov (United States)

Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. PMID:24119078

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

2013-11-01

336

Consequences of recombination on traditional phylogenetic analysis.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We investigate the shape of a phylogenetic tree reconstructed from sequences evolving under the coalescent with recombination. The motivation is that evolutionary inferences are often made from phylogenetic trees reconstructed from population data even though recombination may well occur (mtDNA or viral sequences) or does occur (nuclear sequences). We investigate the size and direction of biases when a single tree is reconstructed ignoring recombination. Standard software (PHYLIP) was used to...

Schierup, Mh; Hein, J.

2000-01-01

337

Consequences of recombination on traditional phylogenetic analysis.  

DEFF Research Database (Denmark)

We investigate the shape of a phylogenetic tree reconstructed from sequences evolving under the coalescent with recombination. The motivation is that evolutionary inferences are often made from phylogenetic trees reconstructed from population data even though recombination may well occur (mtDNA or viral sequences) or does occur (nuclear sequences). We investigate the size and direction of biases when a single tree is reconstructed ignoring recombination. Standard software (PHYLIP) was used to construct the best phylogenetic tree from sequences simulated under the coalescent with recombination. With recombination present, the length of terminal branches and the total branch length are larger, and the time to the most recent common ancestor smaller, than for a tree reconstructed from sequences evolving with no recombination. The effects are pronounced even for small levels of recombination that may not be immediately detectable in a data set. The phylogenies when recombination is present superficially resemble phylogenies for sequences from an exponentially growing population. However, exponential growth has a different effect on statistics such as Tajima's D. Furthermore, ignoring recombination leads to a large overestimation of the substitution rate heterogeneity and the loss of the molecular clock. These results are discussed in relation to viral and mtDNA data sets. Udgivelsesdato: 2000-Oct

Schierup, M H; Hein, J

2000-01-01

338

Recombinant production of the therapeutic peptide lunasin  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Lunasin is a chemopreventive peptide produced in a number of plant species. It comprises a helical region with homology to a region of chromatin binding proteins, an Arg-Gly-Asp cell adhesion motif and eight aspartic acid residues. In vitro studies indicate that lunasin suppresses chemical and oncogene driven transformation of mammalian cells. We have explored efficient recombinant production of lunasin by exploiting the Clostridium thermocellum CipB cellulose binding domain (CBD as a fusion partner protein. Results We used a pET28 vector to express a CBD-lunasin fusion with a hexahistidine tag and Tobacco Etch Virus protease site, to allow protease-mediated release of native lunasin. Autoinduction in E. coli BL21 (DE3 Star cells achieved expression of 3.35 g/L of CBD-lunasin fusion protein. The final yield of lunasin was 210 mg/L corresponding to 32% of the theoretical yield. Purification by cellulose binding and nickel affinity chromatography were tested with the latter proving more satisfactory. The effects of CBD-lunasin expression on growth and morphology of the E. coli cells were examined by light and electron microscopy revealing an altered morphology in a proportion of cells. Cell division appeared to be inhibited in these cells resulting in elongated, non-septated cells. Conclusions The use of CBD as a fusion partner gave high protein yields by autoinduction, with lunasin release by TEV protease cleavage. With some optimisation this approach could provide a potentially valuable route for production of this therapeutic peptide. Over-expression in the host cells manifest as a cell division defect in a population of the cells, presumably mimicking some aspect of the chemopreventive function observed in mammalian cells.

Kyle Stuart

2012-02-01

339

The Fission Yeast FANCM Ortholog Directs Non-Crossover Recombination During Meiosis  

Science.gov (United States)

The formation of healthy gametes depends on programmed DNA double strand breaks (DSBs), which are each repaired as a crossover (CO) or non-crossover (NCO) from a homologous template. Although most of these DSBs are repaired without giving COs, little is known about the genetic requirements of NCO-specific recombination. We show that Fml1, the Fanconi anemia complementation group M (FANCM)-ortholog of Schizosaccharomyces pombe, directs the formation of NCOs during meiosis in competition with the Mus81-dependent pro-CO pathway. We also define the Rad51/Dmc1-mediator Swi5-Sfr1 as a major determinant in biasing the recombination process in favour of Mus81, to ensure the appropriate amount of COs to guide meiotic chromosome segregation. The conservation of these proteins from yeast to Humans suggests that this interplay may be a general feature of meiotic recombination. PMID:22723423

Lorenz, Alexander; Osman, Fekret; Sun, Weili; Nandi, Saikat; Steinacher, Roland; Whitby, Matthew C.

2012-01-01

340

Reflectometric interference spectroscopy-based immunosensing using immobilized antibody via His-tagged recombinant protein A.  

Science.gov (United States)

The proposed approach demonstrated in this study provides an immunosensing system based on reflectometric interference spectroscopy (RIfS) in combination with an antibody immobilization method using histidine-tagged recombinant protein A. Carboxymethyldextran (CMD) was immobilized on a 3-aminopropyltriethoxysilane-treated a silicon nitride-coated silicon wafer, followed by chelating histidine-tagged recombinant protein A with copper (II) ions. The CMD-layer was found to be advantageous in terms of not only immobilization of histidine-tagged recombinant protein A-mediated an antibody against myoglobin (anti-Myo) but also prevention of non-specific binding of myoglobin. Myoglobin was repeatedly detected, and the apparent detection limit was 0.1 ?g mL(-1). The proposed RIfS-based protein sensing system, in conjunction with the easy preparation of silicon-based inexpensive immunosensing chips, is expected to be applicable for label-free optical detection for other proteins in various fields. PMID:25060725

Choi, Hyung Woo; Sakata, Yasuhiko; Ooya, Tooru; Takeuchi, Toshifumi

2015-02-01

 
 
 
 
341

Identifying the nature of charge recombination in organic solar cells from charge-transfer state electroluminescence  

Energy Technology Data Exchange (ETDEWEB)

Charge-transfer (CT) state electroluminescence is investigated in several polymer:fullerene bulk heterojunction solar cells. The ideality factor of the electroluminescence reveals that the CT emission in polymer:fullerene solar cells originates from free-carrier bimolecular recombination at the donor-acceptor interface, rather than a charge-trap-mediated process. The fingerprint of the presence of nonradiative trap-assisted recombination, a voltage-dependent CT electroluminescence quantum efficiency, is only observed for the P3HT:PCBM system, which is explained by a reduction of the competing bimolecular recombination rate. These results are in agreement with measurements of the illumination-intensity dependence of the open-circuit voltage. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

Wetzelaer, Gert-Jan A.H. [Molecular Electronics, Zernike Institute for Advanced Materials, University of Groningen (Netherlands); Dutch Polymer Institute, Eindhoven (Netherlands); Kuik, Martijn [Molecular Electronics, Zernike Institute for Advanced Materials, University of Groningen (Netherlands); Blom, Paul W.M. [Molecular Electronics, Zernike Institute for Advanced Materials, University of Groningen (Netherlands); TNO/Holst Centre, Eindhoven (Netherlands)

2012-10-15

342

Recombinant Goat VEGF164 Increases Hair Growth by Painting Process on the Skin of Shaved Mouse.  

Science.gov (United States)

To detect goat vascular endothelial growth factor (VEGF)-mediated regrowth of hair, full-length VEGF164 cDNA was cloned from Inner Mongolia cashmere goat (Capra hircus) into the pET-his prokaryotic expression vector, and the recombinant plasmid was transferred into E. coli BL21 cells. The expression of recombinant 6×his-gVEGF164 protein was induced by 0.5 mM isopropyl thio-?-D-galactoside at 32°C. Recombinant goat VEGF164 (rgVEGF164) was purified and identi ed by western blot using monoclonal anti-his and anti-VEGF antibodies. The rgVEGF164 was smeared onto the dorsal area of a shaved mouse, and we noted that hair regrowth in this area was faster than in the control group. Thus, rgVEGF164 increases hair growth in mice. PMID:25178380

Bao, Wenlei; Yin, Jianxin; Liang, Yan; Guo, Zhixin; Wang, Yanfeng; Liu, Dongjun; Wang, Xiao; Wang, Zhigang

2014-09-01

343

Sexual Recombination in Gibberella zeae.  

Science.gov (United States)

ABSTRACT We developed a method for inducing sexual outcrosses in the homothallic Ascomycete fungus Gibberella zeae (anamorph: Fusarium graminearum). Strains were marked with different nitrate nonutilizing (nit) mutations, and vegetative compatibility groups served as additional markers in some crosses. Strains with complementary nit mutations were cocultured on carrot agar plates. Ascospores from individual perithecia were plated on a minimal medium (MM) containing nitrate as the sole nitrogen source. Crosses between different nit mutants segregated in expected ratios (3:1 nit(-):nit(+)) from heterozygous perithecia. Analysis of vegetative compatibility groups of progeny of two crosses indicated two and three vegetative incompatibility (vic) genes segregating, respectively. For rapid testing of sexual recombination between nit mutants, perithecia were inverted over MM to deposit actively discharged ascospores. Development of proto-trophic wild-type colonies was taken as evidence of sexual recombination. Strains of G. zeae group 2 from Japan, Nepal, and South Africa, and from Indiana, Kansas, and Ohio in the United States were sexually interfertile. Four group 1 strains were not interfertile among themselves or with seven group 2 strains. Attempts to cross G. zeae with representatives of F. acuminatum, F. avenaceum, F. culmorum, F. crookwellense, F. oxysporum, and three mating populations of G. fujikuroi were not successful. PMID:18944794

Bowden, R L; Leslie, J F

1999-02-01

344

Rapid Purification of Recombinant Histones  

Science.gov (United States)

The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method “rapid histone purification” (RHP) as it circumvents isolation of inclusion bodies and thereby cuts out the most time-consuming step of traditional purification protocols. Instead of inclusion body isolation, whole cell extracts are prepared under strongly denaturing conditions that directly solubilize inclusion bodies. By ion exchange chromatography, the histones are purified from the extracts. The protocol has been successfully applied to all four canonical Drosophila and human histones. RHP histones and histones that were purified from isolated inclusion bodies had similar purities. The different purification strategies also did not impact the quality of octamers reconstituted from these histones. We expect that the RHP protocol can be readily applied to the purification of canonical histones from other species as well as the numerous histone variants. PMID:25090252

Klinker, Henrike; Becker, Peter B.; Mueller-Planitz, Felix

2014-01-01

345

Charge recombination in undoped cuprates  

Science.gov (United States)

We theoretically analyze the process of charge recombination in the planar Mott-Hubbard insulators with the aim to explain the short picosecond-range lifetimes of photoexcited carriers, experimentally studied via pump-probe experiments on the undoped cuprates. The recombination mechanism consists of two essential ingredients: the formation of a metastable s -type bound holon-doublon pair, i.e., the Mott exciton, and the decay of such an excitonic state via the multimagnon emission. In spite of the large gap that requires many bosons to be emitted, the latter process is fast due to a large exchange scale and strong charge-spin coupling in planar systems. As the starting microscopic model we consider the single-band Hubbard model and then a more realistic three-band model for cuprates, both leading to the same minimal one. The decay rate of the exciton is evaluated numerically via the Fermi golden rule, having consistency also with the direct time-evolution calculation. The decay rate reveals exponential dependence on the ratio of the Mott-Hubbard gap and the exchange coupling, the result qualitatively reproduced also within a toy exciton-boson model.

Lenar?i?, Zala; Prelovšek, Peter

2014-12-01

346

Recombinant protein production in yeasts.  

Science.gov (United States)

Recombinant protein production is a multibillion-dollar market. The development of a new product begins with the choice of a production host. While one single perfect host for every protein does not exist, several expression systems ranging from bacterial hosts to mammalian cells have been established. Among them, yeast cell factories combine the advantages of being single cells, such as fast growth and easy genetic manipulation, as well as eukaryotic features including a secretory pathway leading to correct protein processing and post-translational modifications. In this respect, especially the engineering of yeast glycosylation to produce glycoproteins of human-like glycan structures is of great interest. Additionally, different attempts of cellular engineering as well as the design of different production processes that are leading to improved productivities are presented. With the advent of cheaper next-generation sequencing techniques, systems biotechnology approaches focusing on genome scale analyses will advance and accelerate yeast cell factories and thus recombinant protein production processes in the near future. In this review we summarize advantages and limitations of the main and most promising yeast hosts, including Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha as those presently used in large scale production of heterologous proteins. PMID:22160907

Mattanovich, Diethard; Branduardi, Paola; Dato, Laura; Gasser, Brigitte; Sauer, Michael; Porro, Danilo

2012-01-01

347

Dissociative recombination in planetary ionospheres  

Science.gov (United States)

Ionization in planetary atmospheres can be produced by solar photoionization, photoelectron impact ionization, and, in auroral regions, by impact of precipitating particles. This ionization is lost mainly in dissociative recombination (DR) of molecular ions. Although atomic ions cannot undergo DR, they can be transformed locally through ion-molecule reactions into molecular ions, or they may be transported vertically or horizontally to regions of the atmosphere where such transformations are possible. Because DR reactions tend to be very exothermic, they can be an important source of kinetically or internally excited fragments. In interplanetary thermospheres, the neutral densities decrease exponentially with altitude. Below the homopause (or turbopause), the atmosphere is assumed to be throughly mixed by convection and/or turbulence. Above the homopause, diffusion is the major transport mechanism, and each species is distributed according to its mass, with the logarithmic derivative of the density with repect to altitude given approximately by -1/H, where H = kT/mg is the scale height. In this expression, T is the neutral temperature, g is the local acceleratiion of gravity, and m is the mass of the species. Thus lighter species become relatively more abundant, and heavier species less abundant, as the altitude increases. This variation of the neutral composition can lead to changes in the ion composition; furthermore, as the neutral densities decrease, dissociative recombination becomes more important relative to ion-neutral reactions as a loss mechanism for molecular ions.

Fox, J. L.

1993-01-01

348

Fine-Scale Population Recombination Rates, Hotspots, and Correlates of Recombination in the Medicago truncatula Genome  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Recombination rates vary across the genome and in many species show significant relationships with several genomic features, including distance to the centromere, gene density, and GC content. Studies of fine-scale recombination rates have also revealed that in several species, there are recombination hotspots, that is, short regions with recombination rates 10–100 greater than those in surrounding regions. In this study, we analyzed whole-genome resequence data from 26 accessions of the mo...

Paape, Timothy; Zhou, Peng; Branca, Antoine; Briskine, Roman; Young, Nevin; Tiffin, Peter

2012-01-01

349

Fish vaccine antigens produced or delivered by recombinant DNA technologies.  

Science.gov (United States)

Current efforts to develop vaccines, particularly for aquacultured species, have turned largely to biotechnology because it provides the means to inexpensively produce sufficient quantities of the immunoprotective antigen. These efforts have resulted in several prototype vaccines for fish and the publication of a large number of articles on the subject. However, there are only a few recombinant DNA-based vaccines for aquaculture in the licensing pipeline. Continued funding of research on recombinant DNA vaccines comes from the recognition by industry and government funding agencies that this research can lead to an increased understanding of the mechanisms in protective immunity. This is especially important for fish and shellfish species since our knowledge of the immune mechanisms in these animals is pitifully meagre. This presentation discusses the relative merits of the different recombinant DNA technologies that have been used to produce viral vaccines for fish and the promising approaches that are under consideration to increase the efficacy of these vaccines. There are many approaches to antigen production by recombinant DNA techniques including: (i) the preparation of purified antigenic proteins produced from the cloned viral genes in a variety of vector/host expression systems, (ii) chemical synthesis or the use of fusion vectors to produce peptides corresponding to known epitopes, (iii) defined attenuations, i.e. specific genetic alterations, of live virus vaccines, (iv) the use of live bacterial or viral vectors to deliver resistance genes or viral antigens, (v) anti-idiotype antibodies, and (vi) DNA vaccines where purified plasmid DNA expressing the pathogen gene under a eucaryotic promoter is injected. All of these technologies have been used more or less successfully in the development of vaccines for aquacultured species. However, the requirements for safety, effectiveness, ease of application and low cost/dose restrict their commercial development for aquaculture. The ideal viral vaccine for aquaculture must be effective in preventing death, be inexpensive to produce and license, provide immunity of long duration, and be easily administered. In addition, these vaccines must not only provide protection against the lethal effects of virus infection but prevent the formation of virus persistence. This is especially true for infectious haematopoietic necrosis virus (IHNV) which has been shown to persist in survivors in the presence of high antibody levels. Since resolution of virus persistence is thought to be correlate with cell-mediated immunity, vaccines designed to augment the cell-mediated immunity must be developed for fish. Approaches that are being considered include the use of cytokines in combination with subunit vaccines and the use of specific MHC-I inducer adjuvants with the vaccine. The "tailoring" of vaccine immunogenicity using different combinations of antigen and adjuvant will be presented. PMID:9270855

Leong, J C; Anderson, E; Bootland, L M; Chiou, P W; Johnson, M; Kim, C; Mourich, D; Trobridge, G

1997-01-01

350

PhiC31 integrase induces efficient site-specific recombination in the Capra hircus genome.  

Science.gov (United States)

Streptomyces phage ?C31 integrase induces efficient site-specific recombination capable of integrating exogenous genes at pseudo attP sites in human, mouse, rat, rabbit, sheep, Drosophila, and bovine genomes. However, the ?C31-mediated recombination between attB and the corresponding pseudo attP sites has not been investigated in Capra hircus. Here, we identified eight pseudo attP sites located in the intron or intergenic regions of the C. hircus genome, and demonstrated different levels of foreign gene expression after ?C31 integrase-mediated integration. These pseudo attP sites share similar sequences with each other and with pseudo attP sites in other mammalian genomes, and these are associated with a neighboring consensus motif found in other genomes. The application of the ?C31 integrase system in C. hircus provides a new option for genetic engineering of this economically important goat species. PMID:24754538

Ma, Haiyan; Ma, Qingwen; Lu, Yao; Wang, Juan; Hu, Wei; Gong, Zhijuan; Cai, Linlin; Huang, Ying; Huang, Shu-Zhen; Zeng, Fanyi

2014-08-01

351

Modifiers of (CAG)(n) instability in Machado-Joseph disease (MJD/SCA3) transmissions: an association study with DNA replication, repair and recombination genes.  

Science.gov (United States)

Twelve neurological disorders are caused by gene-specific CAG/CTG repeat expansions that are highly unstable upon transmission to offspring. This intergenerational repeat instability is clinically relevant since disease onset, progression and severity are associated with repeat size. Studies of model organisms revealed the involvement of some DNA replication and repair genes in the process of repeat instability, however, little is known about their role in patients. Here, we used an association study to search for genetic modifiers of (CAG)n instability in 137 parent-child transmissions in Machado-Joseph disease (MJD/SCA3). With the hypothesis that variants in genes involved in DNA replication, repair or recombination might alter the MJD CAG instability patterns, we screened 768 SNPs from 93 of these genes. We found a variant in ERCC6 (rs2228528) associated with an expansion bias of MJD alleles. When using a gene-gene interaction model, the allele combination G-A (rs4140804-rs2972388) of RPA3-CDK7 is also associated with MJD instability in a direction-dependent manner. Interestingly, the transcription-coupled repair factor ERCC6 (aka CSB), the single-strand binding protein RPA, and the CDK7 kinase part of the TFIIH transcription repair complex, have all been linked to transcription-coupled repair. This is the first study performed in patient samples to implicate specific modifiers of CAG instability in humans. In summary, we found variants in three transcription-coupled repair genes associated with the MJD mutation that points to distinct mechanisms of (CAG)n instability. PMID:25026993

Martins, Sandra; Pearson, Christopher E; Coutinho, Paula; Provost, Sylvie; Amorim, António; Dubé, Marie-Pierre; Sequeiros, Jorge; Rouleau, Guy A

2014-10-01

352

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 nventional 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

353

Episomal Persistence of Recombinant Adenoviral Vector Genomes during the Cell Cycle In Vivo  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Previously we showed that recombinant adenoviral helper-dependent (HD) vectors result in long-term transgene expression levels in vivo which slowly declined by 95% over a period of 1 year. In this study, we further establish that this was not predominantly immune mediated. To determine if cell turnover was responsible for the loss of transgene expression, we induced rapid hepatocyte cell cycling in mouse liver, by performing a surgical two-thirds partial hepatectomy. We observed a 55 and 65% ...

Ehrhardt, Anja; Xu, Hui; Kay, Mark A.

2003-01-01

354

The Purified and Recombinant Legionella pneumophila Chaperonin Alters Mitochondrial Trafficking and Microfilament Organization?  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A portion of the total cellular pool of the Legionella pneumophila chaperonin, HtpB, is found on the bacterial cell surface, where it can mediate invasion of nonphagocytic cells. HtpB continues to be abundantly produced and released by internalized L. pneumophila and may thus have postinvasion functions. We used here two functional models (protein-coated beads and expression of recombinant proteins in CHO cells) to investigate the competence of HtpB in mimicking early intracellular traffickin...

Chong, Audrey; Lima, Celia A.; Allan, David S.; Nasrallah, Gheyath K.; Gardun?o, Rafael A.

2009-01-01

355

T Cell Response of Asymptomatic Leishmania chagasi Infected Subjects to Recombinant Leishmania Antigens  

Digital Repository Infrastructure Vision for European Research (DRIVER)

In areas of Leishmania chagasi transmission the ability to control leishmania infection is associated with IFN-g production. In visceral leishmaniasis down-regulation of T cell responses is mediated by interleukin-10 (IL-10). In this study we evaluated the lymphoproliferative response, IFN-g and IL-10 production on lymphocyte cultures stimulated with recombinant leishmania antigens in subjects with asymptomatic L. chagasi infection. There was a statistically significant difference in the lymp...

Sérgio Ricardo Costa; Oliveira Ju?nior, Argemiro D.; Olívia Bacellar; Carvalho, Edgar M.

1999-01-01

356

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

Plasmid DNA substrates were X-irradiated and injected into the nuclei of Xenopus laevis oocytes. After incubation for 20 h, DNA was recovered from the oocytes and analyzed simultaneously for repair and for intermolecular homologous recombination by electrophoresis and bacterial transformation. Oocyte-mediated repair of DNA strand breaks was observed with both methods. Using a repair-deficient mutant Escherichia coli strain and its repair-proficient parent as hosts for the transformation assay...

Sweigert, S. E.; Carroll, D.

1990-01-01

357

Expression of recombinant plasmids in mammalian cells is enhanced by sodium butyrate.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We have studied the effects of sodium butyrate on DNA-mediated gene transfer in an effort to investigate interrelationships between chromatin structure and expression of recombinant plasmids. Our results demonstrate that butyrate affects the early stages of gene activity following DNA uptake at least two levels. First, the number of cells able to express foreign DNA increases from 10% to up to 40%. Second, there is an increase in enhancer-dependent transcription, approximately 30 fold in HeLa...

Gorman, C. M.; Howard, B. H.; Reeves, R.

1983-01-01

358

How well do we understand cosmological recombination?  

Science.gov (United States)

The major theoretical limitation for extracting cosmological parameters from the cosmic microwave background (CMB) sky lies in the precision with which we can calculate the cosmological recombination process. Uncertainty in the details of hydrogen and helium recombination could effectively increase the errors or bias the values of the cosmological parameters derived from the Planck satellite, for example. Here, we modify the cosmological recombination code RECFAST by introducing one more parameter to reproduce the recent numerical results for the speed-up of the helium recombination. Together with the existing hydrogen fudge factor, we vary these two parameters to account for the remaining dominant uncertainties in cosmological recombination. By using the COSMOMC code with Planck forecast data, we find that we need to determine the parameters to better than 10 per cent for HeI and 1 per cent for H, in order to obtain negligible effects on the cosmological parameters. For helium recombination, if the existing studies have calculated the ionization fraction to the 0.1 per cent level by properly including the relevant physical processes, then we already have numerical calculations which are accurate enough for Planck. For hydrogen, setting the fudge factor to speed up low-redshift recombination by 14 per cent appears to be sufficient for Planck. However, more work still needs to be done to carry out comprehensive numerical calculations of all the relevant effects for hydrogen, as well as to check for effects which couple hydrogen and helium recombination through the radiation field.

Wong, Wan Yan; Moss, Adam; Scott, Douglas

2008-05-01

359

RNAi and heterochromatin repress centromeric meiotic recombination  

DEFF Research Database (Denmark)

During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essential in most species for proper homologue segregation. Nevertheless, recombination is repressed specifically in and around the centromeres of chromosomes, apparently because rare centromeric (or pericentromeric) recombination events, when they do occur, can disrupt proper segregation and lead to genetic disabilities, including birth defects. The basis by which centromeric meiotic recombination is repressed has been largely unknown. We report here that, in fission yeast, RNAi functions and Clr4-Rik1 (histone H3 lysine 9 methyltransferase) are required for repression of centromeric recombination. Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis.

Ellermeier, Chad; Higuchi, Emily C

2010-01-01

360

Combined trap for laser stimulated recombination  

International Nuclear Information System (INIS)

We present a combined Paul-Penning trap to study CO2 laser stimulated recombination of protons and electrons. The trap is located in the center of an optical resonator for 11 ?m radiation. In this manner the available laser intensity is enhanced for stimulated recombination to the n=1 state of hydrogen

 
 
 
 
361

Electron-ion recombination at low energy  

International Nuclear Information System (INIS)

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

362

Dissociative recombination of highly symmetric polyatomic ions.  

Science.gov (United States)

A general first-principles theory of dissociative recombination is developed for highly symmetric molecular ions and applied to H(3)O(+) and CH(3)(+), which play an important role in astrophysical, combustion, and laboratory plasma environments. The theoretical cross sections obtained for the dissociative recombination of the two ions are in good agreement with existing experimental data from storage ring experiments. PMID:22324682

Douguet, Nicolas; Orel, Ann E; Greene, Chris H; Kokoouline, Viatcheslav

2012-01-13

363

Dielectronic recombination lines of C{sup +}  

Energy Technology Data Exchange (ETDEWEB)

The present paper presents atomic data generated to investigate the recombination lines of C II in the spectra of planetary nebulae. These data include energies of bound and autoionizing states, oscillator strengths and radiative transition probabilities, autoionization probabilities, and recombination coefficients. The R-matrix method of electron scattering theory was used to describe the C{sup 2+} plus electron system.

Sochi, Taha, E-mail: taha.sochi@kcl.ac.uk; Storey, Peter J.

2013-11-15

364

Quality control of homologous recombination.  

Science.gov (United States)

Exogenous and endogenous genotoxic agents, such as ionizing radiation and numerous chemical agents, cause DNA double-strand breaks (DSBs), which are highly toxic and lead to genomic instability or tumorigenesis if not repaired accurately and efficiently. Cells have over evolutionary time developed certain repair mechanisms in response to DSBs to maintain genomic integrity. Major DSB repair mechanisms include non-homologous end joining and homologous recombination (HR). Using sister homologues as templates, HR is a high-fidelity repair pathway that can rejoin DSBs without introducing mutations. However, HR execution without appropriate guarding may lead to more severe gross genome rearrangements. Here we review current knowledge regarding the factors and mechanisms required for accomplishment of accurate HR. PMID:24858417

Liu, Ting; Huang, Jun

2014-10-01

365

Recombination at silicon dangling bonds  

International Nuclear Information System (INIS)

In the past, pulsed electrically detected magnetic resonance experiments (pEDMR) with silicon dangling bonds (db) in hydrogenated microcrystalline silicon (?c-Si:H) showed that at low temperatures, two db recombination mechanisms exist where electrons are captured (i) by dbs directly (db-dc) or (ii) via band-tail states (tail-db). Here, similar experiments on hydrogenated amorphous silicon (a-Si:H) and crystalline silicon/silicondioxide interfaces (c-Si/SiO2) are presented. They show that at low temperatures, only the db-dc is detectable at dbs in the c-Si/SiO2 interface (Pb centers) while in a-Si:H, only tail-db processes are observed

366

Fundamental Studies of Recombinant Hydrogenases  

Energy Technology Data Exchange (ETDEWEB)

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

Adams, Michael W

2014-01-25

367

Disruption of mouse RAD54 reduces ionizing radiation resistance and homologous recombination.  

Science.gov (United States)

Double-strand DNA break (DSB) repair by homologous recombination occurs through the RAD52 pathway in Saccharomyces cerevisiae. Its biological importance is underscored by the conservation of many RAD52 pathway genes, including RAD54, from fungi to humans. We have analyzed the phenotype of mouse RAD54-/- (mRAD54-/-) cells. Consistent with a DSB repair defect, these cells are sensitive to ionizing radiation, mitomycin C, and methyl methanesulfonate, but not to ultraviolet light. Gene targeting experiments demonstrate that homologous recombination in mRAD54-/- cells is reduced compared to wild-type cells. These results imply that, besides DNA end-joining mediated by DNA-dependent protein kinase, homologous recombination contributes to the repair of DSBs in mammalian cells. Furthermore, we show that mRAD54-/- mice are viable and exhibit apparently normal V(D)J and immunoglobulin class-switch recombination. Thus, mRAD54 is not required for the recombination processes that generate functional immunoglobulin and T cell receptor genes. PMID:9108475

Essers, J; Hendriks, R W; Swagemakers, S M; Troelstra, C; de Wit, J; Bootsma, D; Hoeijmakers, J H; Kanaar, R

1997-04-18

368

The Study of the Inhibition of the Recombinant TACE Prodomain to Endotoxemia in Mice  

Directory of Open Access Journals (Sweden)

Full Text Available Objective: To demonstrate the inhibitory function of the prodomain of tumor necrosis factor-? (TNF-? converting enzyme (TACE on TACE activity and to develop an approach to interfere with inflammation processes. Methods: The cDNA encoding the fulllength ectodomain (T1300 and prodomain (T591 of TACE were amplified by RT-PCR. The expression plasmids (pET-28a (+-T1300 and pET-28a (+-T591 were constructed and transformed into E. coli BL21. After Ni2+-NTA resin affinity chromatography, the recombinant T591 protein was obtained and assayed. In order to detect its inhibiton of TACE activity, the mice in the LPS-induced endotoxemia model group were treated with the recombinant TACE prodomain protein prior to the injection of LPS. Murine peritoneal macrophages were isolated from mice abdominal cavity for FCM and the liver, kidney and lung were removed for traditionally histopathology sectioning. Results: The FCM results showed that the recombinant prodomain protein decreased the release of the sTNF-?, which mediated the accumulation of TNF-? on the surface of macrophage cells. HE staining proved that the recombinant protein can decrease the inflammatory response in internal organs of endotoxaemia mice. Conclusions: The recombinant prodomain of TACE has the ability to inhibit sTNF-? release, which indicates that prodomain is an effective antagonist of TACE and might be useful in the molecular design of anti-inflammatory drugs.

Yuzhen Yang

2009-12-01

369

Cross-species surface display of functional spirochetal lipoproteins by recombinant Borrelia burgdorferi.  

Science.gov (United States)

Surface-exposed lipoproteins of relapsing fever (RF) and Lyme borreliosis Borrelia spirochetes mediate certain interactions of the bacteria with their arthropod and vertebrate hosts. RF spirochetes such as Borrelia hermsii serially evade the host's antibody response by multiphasic antigenic variation of Vsp and Vlp proteins. Furthermore, the expression of Vsp1 and Vsp2 by Borrelia turicatae is associated with neurotropism and higher blood densities, respectively. In contrast to RF Borrelia species, the Lyme borreliosis spirochete Borrelia burgdorferi is amenable to genetic manipulation. To facilitate structure-function analyses of RF surface lipoproteins, we used recombinant plasmids to introduce full-length vsp1 and vsp2 as well as two representative vlp genes into B. burgdorferi cells. Recombinant B. burgdorferi cells constitutively expressed the proteins under the control of the B. burgdorferi flaB promoter. Antibody and protease accessibility assays indicated proper surface exposure and folding. Expression of Vsp1 and Vsp2 conferred glycosaminoglycan binding to recombinant B. burgdorferi cells that was similar to that observed with purified recombinant proteins and B. turicatae expressing native Vsp. These data demonstrate that the lipoprotein modification and export mechanisms in the genus Borrelia are conserved. They also validate the use of recombinant B. burgdorferi in studies of surface lipoprotein structure-function and the biogenesis of spirochete membranes. PMID:14977951

Zückert, Wolfram R; Lloyd, Jill E; Stewart, Philip E; Rosa, Patricia A; Barbour, Alan G

2004-03-01

370

[Recombinant proteasomal alpha-type subunits exhibit endoribonuclease activity].  

Science.gov (United States)

26S proteasome is a multi-subunit protein complex that consists of the regulatory 19S and the catalytic 20S subcomplexes. The major cellular function of the proteasome is protein degradation. It has been found recently that the 20S particle, besides its proteolytic activity, also possesses endoribonuclease activity. The latter is mediated by two alpha-type subunits (alpha1 and alpha5). In this report we have analyzed the remaining alpha-type subunits for their ability to hydrolyze RNA. We found that all of the recombinant subunits tested exhibited endoribonuclease activity which depended on the origin of RNA and the presence of bivalent ions in the reaction. These results indicate that the endoribonuclease activity of proteasomes may play an important role in cellular metabolism of RNA. PMID:21427980

Fedorova, O A; Moiseeva, T N; Mittenberg, A G; Barlev, N A

2010-01-01

371

Rad54: the Swiss Army knife of homologous recombination?  

Science.gov (United States)

Homologous recombination (HR) is a ubiquitous cellular pathway that mediates transfer of genetic information between homologous or near homologous (homeologous) DNA sequences. During meiosis it ensures proper chromosome segregation in the first division. Moreover, HR is critical for the tolerance and repair of DNA damage, as well as in the recovery of stalled and broken replication forks. Together these functions preserve genomic stability and assure high fidelity transmission of the genetic material in the mitotic and meiotic cell divisions. This review will focus on the Rad54 protein, a member of the Snf2-family of SF2 helicases, which translocates on dsDNA but does not display strand displacement activity typical for a helicase. A wealth of genetic, cytological, biochemical and structural data suggests that Rad54 is a core factor of HR, possibly acting at multiple stages during HR in concert with the central homologous pairing protein Rad51. PMID:16935872

Heyer, Wolf-Dietrich; Li, Xuan; Rolfsmeier, Michael; Zhang, Xiao-Ping

2006-01-01

372

Protective efficacy induced by recombinant Clostridium difficile toxin fragments.  

Science.gov (United States)

Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection. PMID:23716610

Leuzzi, Rosanna; Spencer, Janice; Buckley, Anthony; Brettoni, Cecilia; Martinelli, Manuele; Tulli, Lorenza; Marchi, Sara; Luzzi, Enrico; Irvine, June; Candlish, Denise; Veggi, Daniele; Pansegrau, Werner; Fiaschi, Luigi; Savino, Silvana; Swennen, Erwin; Cakici, Osman; Oviedo-Orta, Ernesto; Giraldi, Monica; Baudner, Barbara; D'Urzo, Nunzia; Maione, Domenico; Soriani, Marco; Rappuoli, Rino; Pizza, Mariagrazia; Douce, Gillian R; Scarselli, Maria

2013-08-01

373

Protective Efficacy Induced by Recombinant Clostridium difficile Toxin Fragments  

Science.gov (United States)

Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection. PMID:23716610

Leuzzi, Rosanna; Spencer, Janice; Buckley, Anthony; Brettoni, Cecilia; Martinelli, Manuele; Tulli, Lorenza; Marchi, Sara; Luzzi, Enrico; Irvine, June; Candlish, Denise; Veggi, Daniele; Pansegrau, Werner; Fiaschi, Luigi; Savino, Silvana; Swennen, Erwin; Cakici, Osman; Oviedo-Orta, Ernesto; Giraldi, Monica; Baudner, Barbara; D'Urzo, Nunzia; Maione, Domenico; Soriani, Marco; Rappuoli, Rino; Pizza, Mariagrazia

2013-01-01

374

Recombinant expression and biochemical characterization of sugarcane legumain.  

Science.gov (United States)

Plant legumains, also termed vacuolar processing enzymes (VPEs), are cysteine peptidases that play key roles in plant development, senescence, programmed cell death and defense against pathogens. Despite the increasing number of reports on plant cysteine peptidases, including VPEs, the characterization of sugarcane VPEs and their inhibition by endogenous cystatins have not yet been described. This is the first report of the biochemical characterization of a sugarcane cysteine peptidase. In this work, a recombinant sugarcane legumain was expressed in Pichia pastoris and characterized. Kinetic studies of the recombinant CaneLEG revealed that this enzyme has the main characteristics of VPEs, such as self-activation and activity under acidic pH. CaneLEG activity was strongly inhibited when incubated with sugarcane cystatin 3 (CaneCPI-3). Quantitative analysis of CaneLEG and CaneCPI-3 gene expression indicated a tissue-specific expression pattern for both genes throughout sugarcane growth, with the strong accumulation of CaneLEG transcripts throughout the internode development. Furthermore, the CaneLEG and CaneCPI-3 genes exhibited up-regulation in plantlets treated with abscisic acid (ABA). These results suggest that CaneCPI-3 may be a potential endogenous inhibitor of CaneLEG and these genes may be involved in plant stress response mediated by ABA. Also, the expression analysis provides clues for the putative involvement of CaneLEG and CaneCPI-3 in sugarcane development and phytohormone response. PMID:22721948

Santos-Silva, Ludier K; Soares-Costa, Andrea; Gerald, Lee T S; Meneghin, Silvana P; Henrique-Silva, Flavio

2012-08-01

375

DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange  

Digital Repository Infrastructure Vision for European Research (DRIVER)

RAD51, an essential eukaryotic DNA recombinase, promotes homologous pairing and strand exchange during homologous recombination and the recombinational repair of double strand breaks. Mutations that up- or down-regulate RAD51 gene expression have been identified in several tumors, suggesting that inappropriate expression of the RAD51 activity may cause tumorigenesis. To identify chemical compounds that affect the RAD51 activity, in the present study, we performed the RAD51-mediated strand exc...

Ishida, Takako; Takizawa, Yoshimasa; Kainuma, Takashi; Inoue, Jin; Mikawa, Tsutomu; Shibata, Takehiko; Suzuki, Hidekazu; Tashiro, Satoshi; Kurumizaka, Hitoshi

2009-01-01

376

A MEDIATOR METHYLATION MYSTERY: JMJD1C DEMETHYLATES MDC1 TO REGULATE DNA DAMAGE REPAIR  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Mediator of DNA-Damage Checkpoint 1 (MDC1) has a central role in repair of DNA double-strand breaks (DSBs) by both homologous recombination and nonhomologous end joining, and its function is regulated by post-translational phosphorylation, ubiquitylation, and SUMOylation. In this issue, a new study by Watanabe et al. reveals that methylation of MDC1 is also critical for its function in DSB repair and specifically affects repair through BRCA1-dependent homologous recombination.

Lu, Jian; Matunis, Michael J.

2013-01-01

377

Recombination analysis of Soybean mosaic virus sequences reveals evidence of RNA recombination between distinct pathotypes  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract RNA recombination is one of the two major factors that create RNA genome variability. Assessing its incidence in plant RNA viruses helps understand the formation of new isolates and evaluate the effectiveness of crop protection strategies. To search for recombination in Soybean mosaic virus (SMV, the causal agent of a worldwide seed-borne, aphid-transmitted viral soybean disease, we obtained all full-length genome sequences of SMV as well as partial sequences encoding the N-terminal most (P1 protease and the C-terminal most (capsid protein; CP viral protein. The sequences were analyzed for possible recombination events using a variety of automatic and manual recombination detection and verification approaches. Automatic scanning identified 3, 10, and 17 recombination sites in the P1, CP, and full-length sequences, respectively. Manual analyses confirmed 10 recombination sites in three full-length SMV sequences. To our knowledge, this is the first report of recombination between distinct SMV pathotypes. These data imply that different SMV pathotypes can simultaneously infect a host cell and exchange genetic materials through recombination. The high incidence of SMV recombination suggests that recombination plays an important role in SMV evolution. Obtaining additional full-length sequences will help elucidate this role.

Babu Mohan

2008-11-01

378

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

Science.gov (United States)

The adjuvant effects of cytokines in humoral and cell-mediated immunity to herpes simplex virus type 1 (HSV-1) have been examined in mice using HSV-1 recombinant viruses expressing murine interleukin-2 (IL-2), IL-4, or gamma interferon (IFN-?) gene. Groups of naive BALB/c mice were immunized intraperitoneally with one or three doses of the HSV-1 recombinant viruses expressing IL-2, IL-4, or IFN-? or with parental control virus. Despite similar replication kinetics, these three recombinant viruses elicited different immune responses to HSV-1 on immunization. Immunization with the recombinant virus expressing IL-4 elicited a humoral response of greater magnitude than immunization with the recombinant viruses expressing IL-2 or IFN-? or with parental virus. In contrast, immunization with recombinant virus expressing IL-2 elicited a higher cytotoxic T-cell response than immunization with viruses expressing IL-4 or IFN-?. Stimulation in vitro of splenocytes obtained from the mice immunized with UV-inactivated HSV-1 McKrae resulted in a TH1 pattern of cytokine expression irrespective of the recombinant virus used in the immunization. As observed for the parental virus, both CD4+ and CD8+ T cells contributed equally to the production of IL-2 by the splenocytes of mice immunized with any of the three recombinant viruses. However, the pattern of IFN-? production by CD4+ and CD8+ T cells differed according to the recombinant virus used. After lethal ocular challenge, all immunized mice were protected completely against death and manifestations of eye disease caused by HSV-1, which are typical responses in unimmunized mice. Mice immunized with IL-4-expressing virus cleared the virus from their eyes more rapidly than mice immunized with IL-2- or IFN-?-expressing virus. Taken together, our results suggest that, in contrast to IFN-? which did not exhibit an adjuvant effect, both IL-4 and IL-2 act as adjuvants in immunization with HSV, with IL-4 showing greater efficacy. PMID:12719570

Osorio, Yanira; Ghiasi, Homayon

2003-01-01

379

Quark Recombination in Heavy Ion Collisions  

CERN Document Server

Data on high energy nuclear collisions collected at the Relativistic Heavy Ion Collider over the past decade have provided convincing evidence that hadronization is quite different in hot nuclear environments compared to p+p collisions. In particular, the data suggest that we see traces of quark degrees of freedom in elliptic flow, with the implication that collective flow is generated on the parton level and is transfered to hadrons through a simple recombination step. In this contribution we review the experimental evidence for quark recombination and discuss some recombination models which are used to describe these effects.

Fries, Rainer J

2011-01-01

380

Recombination in narrow-gapped semiconductors  

International Nuclear Information System (INIS)

In narrow-gapped semiconductors of the type Hgsub(1-x)Cdsub(x)Te as well as in lead chalcogenides and their mixed crystals with energy gaps of some tenths of eV, the band-band recombination processes dominate if the samples are sufficiently perfect in their crystal lattices. The relative importance of the radiative or Auger recombination depends on the width of the energy gap and the charge carrier concentration. In the extreme case of very narrow energy gaps plasmon and one-electron recombination occurs additionally

 
 
 
 
381

Chi Enhances Heteroduplex DNA Levels during Recombination  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The major pathway of homologous recombination in Escherichia coli, the RecBCD pathway, is stimulated by Chi sites. To determine whether Chi enhances an early or late step in recombination, we measured formation of heteroduplex DNA (hDNA) in extracts of lambda-infected E. coli. Chi elevated hDNA levels in these extracts, supporting a role for Chi early (before hDNA formation) in recombination. RecA protein and RecBCD enzyme were both necessary for detection of hDNA, indicating that they, too, ...

Holbeck, S. L.; Smith, G. R.

1992-01-01

382

Recombinant vaccine for canine parvovirus in dogs.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

VP2 is the major component of canine parvovirus (CPV) capsids. The VP2-coding gene was engineered to be expressed by a recombinant baculovirus under the control of the polyhedrin promoter. A transfer vector that contains the lacZ gene under the control of the p10 promoter was used in order to facilitate the selection of recombinants. The expressed VP2 was found to be structurally and immunologically indistinguishable from authentic VP2. The recombinant VP2 shows also the capability to self-as...

Lo?pez Turiso, J. A.; Corte?s, E.; Marti?nez, C.; Ruiz Yba?n?ez, R.; Simarro, I.; Vela, C.; Casal, I.

1992-01-01

383

Dissociative Recombination without a Curve Crossing  

Science.gov (United States)

Ab initio calculations show that a curve crossing is not always needed for a high dissociative- recombination cross section. For HeH(+), in which no neutral states cross the ion potential curve, dissociative recombination is driven by the nuclear kinetic-energy operator on adiabatic potential curves. The kinetic-energy derivative operator allows for capture into repulsive curves that are outside of the classical turning points for the nuclear motion. The dominant dissociative route is the C (2)Sigma(+) state leading to H(n = 2) atoms. An analogous mechanism is proposed for the dissociative recombination of H3(+).

Guberman, Steven L.

1994-01-01

384

Dissociative recombination of CH4(+).  

Science.gov (United States)

CH4(+) is an important molecular ion in the astrochemistry of diffuse clouds, dense clouds, cometary comae, and planetary ionospheres. However, the rate of one of the common destruction mechanisms for molecular ions in these regions, dissociative recombination (DR), is somewhat uncertain. Here, we present absolute measurements for the DR of CH4(+) made using the heavy ion storage ring CRYRING in Stockholm, Sweden. From our collision-energy dependent cross-sections, we infer a thermal rate constant of k(Te) = 1.71(±0.02) × 10(–6)(Te/300)(?0.66(±0.02)) cm3 s(–1) over the region of electron temperatures 10 ? Te ? 1000 K. At low collision energies, we have measured the branching fractions of the DR products to be CH4 (0.00 ± 0.00); CH3 + H (0.18 ± 0.03); CH2 + 2H (0.51 ± 0.03); CH2 + H2 (0.06 ± 0.01); CH + H2 + H (0.23 ± 0.01); and CH + 2H2 (0.02 ± 0.01), indicating that two or more C–H bonds are broken in 80% of all collisions. PMID:23651407

Thomas, Richard D; Kashperka, Iryna; Vigren, E; Geppert, Wolf D; Hamberg, Mathias; Larsson, Mats; af Ugglas, Magnus; Zhaunerchyk, Vitali

2013-10-01

385

Rico: An Accurate Cosmological Recombination Code  

CERN Document Server

We present Rico, a code designed to compute the ionization fraction of the Universe during the epoch of hydrogen and helium recombination with an unprecedented combination of speed and accuracy. This is accomplished by training the machine learning code Pico on the calculations of a multi-level cosmological recombination code which self-consistently includes several physical processes that were neglected previously. After training, Rico is used to fit the free electron fraction as a function of the cosmological parameters. While, for example at low redshifts (z<~900), much of the net change in the ionization fraction can be captured by lowering the hydrogen fudge factor in Recfast by about 3%, Rico provides a means of effectively using the accurate ionization history of the full recombination code in the standard cosmological parameter estimation framework without the need to add new or refined fudge factors or functions to a simple recombination model. Within the new approach presented here it is easy to ...

Fendt, W A; Rubiño-Martín, J A; Wandelt, B D

2008-01-01

386

How well do we understand cosmological recombination?  

CERN Document Server

We modify RECFAST by introducing one more parameter to reproduce the recent numerical results for the speed-up of the helium recombination. Together with the hydrogen fudge factor, we vary these two parameters to account for the remaining dominant uncertainties in cosmological recombination. By using the CosmoMC code with Planck forecast data, we find that we need to determine the parameter better than ten per cent for HeI and one per cent for H, in order to obtain correct constraints on the cosmological parameters. For helium recombination, if the existing studies have properly considered the relevant physical processes to calculate the ionization fraction at 0.1 per cent level, then we already have numerical calculations which are accurate for Planck. However, there is still no similar comprehensive numerical calculation for hydrogen recombination, which will be an urgent task.

Wong, Wan Yan; Scott, Douglas

2007-01-01

387

Recombinant Proteins: Hopes for Tissue Engineering  

Directory of Open Access Journals (Sweden)

Full Text Available Proteins constitute a group of key molecules with many applications in tissue engineering. Use of proteins provided from natural sources has several limitations which are overcome by the use of recombinant proteins. So far, the recombinant forms of many proteins with tissue engineering applications have been developed including structural proteins, growth factors and cytokines. This technology has enabled the development of specifically designed proteins such as growth factors with matrix binding domains, and hybrid structural proteins with improved mechanical properties. Recombinant proteins are produced either ex vivo or in vivo, by local gene therapy protocols, and are of medical and economic benefits. Due to the high applicability of recombinant proteins in tissue engineering, it is recommended to include this platform as an infrastructural element in any tissue engineering program.

Sepideh Hamzehlou

2012-06-01

388

Recombinant Proteins: Hopes for Tissue Engineering  

Science.gov (United States)

SUMMARY Proteins constitute a group of key molecules with many applications in tissue engineering. Use of proteins provided from natural sources has several limitations which are overcome by the use of recombinant proteins. So far, the recombinant forms of many proteins with tissue engineering applications have been developed including structural proteins, growth factors and cytokines. This technology has enabled the development of specifically designed proteins such as growth factors with matrix binding domains, and hybrid structural proteins with improved mechanical properties. Recombinant proteins are produced either ex vivo or in vivo, by local gene therapy protocols, and are of medical and economic benefits. Due to the high applicability of recombinant proteins in tissue engineering, it is recommended to include this platform as an infrastructural element in any tissue engineering program. PMID:23678450

Farajollahi, Mohammad Morad; Hamzehlou, Sepideh; Mehdipour, Ahmad; Samadikuchaksaraei, Ali

2012-01-01

389

Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine  

Science.gov (United States)

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

390

Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine  

Science.gov (United States)

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

391

Recombinant Human Papillomavirus (HPV) Bivalent Vaccine  

Science.gov (United States)

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

392

Recombination during expansion of ultracold plasma  

International Nuclear Information System (INIS)

Signals of ultracold plasma are observed by two-photon ionization of laser-cooled caesium atoms in a magneto-optical trap. Recombination of ions and electrons into Rydberg atoms during the expansion of ultracold plasma is investigated by using state-selective field ionization spectroscopy. The dependences of recombination on initial electron temperature (1–70 K) and initial ion density (?1010 cm?3) are investigated. The measured dependence on initial ion density is N1.547+0.004 at a delay time of 5 ?s. The recombination rate rapidly declines as initial electron temperature increases when delay time is increased. The distributions of Rydberg atoms on different values of principal quantum number n, i.e. n = 30–60, at an initial electron temperature of 3.3 K are also investigated. The main experimental results are approximately explained by the three-body recombination theory. (atomic and molecular physics)

393

Constraints from jet calculus on quark recombination  

International Nuclear Information System (INIS)

Within the quantum-chromodynamic jet-calculus formalism, we deduce an equation describing recombination of quarks and antiquarks into mesons within a quark or gluon jet. This equation relates the recombination function R(x1,x2,x) used in current literature to the fragmentation function for producing that same meson out of the parton initiating the jet. We submit currently used recombination functions to our consistency test, taking as input mainly the u-quark fragmentation ''data'' into ?+ mesons. The qq-bar?? recombination functions popular in the literature are consistent with measured fragmentation functions, but they must be supplemented by other contributions to provide the full D?+/sub u/. We also discuss the Q2 dependence of the resulting fragmentation functions

394

Recombination Effects on Supernovae Light-Curves  

CERN Document Server

Supernovae of type IIP are marked by the long plateau seen in their optical light curves. The plateau is believed to be the result of a recombination wave that propagates through the outflowing massive hydrogen envelope. Here, we analytically investigate the transition from a fully ionized envelope to a partially recombined one and its effects on the SN light curve. The motivation is to establish the underlying processes which dominate the evolution at late times when recombination takes place in the envelope, yet early enough so that $^{56}$Ni decay is a negligible source of energy. We assume a simple, yet adequate, hydrodynamic profile of the envelope and study the mechanisms which dominate the energy emission and the observed temperature. We consider the diffusion of photons through the envelope while analyzing the ionization fraction and the coupling between radiation and gas. We find that once recombination starts, the observed temperature decreases slowly in time. However, in a typical red supergiant (R...

Goldfriend, Tomer; Sari, Re'em

2014-01-01

395

A trans-Complementing Recombination Trap Demonstrates a Low Propensity of Flaviviruses for Intermolecular Recombination?  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Intermolecular recombination between the genomes of closely related RNA viruses can result in the emergence of novel strains with altered pathogenic potential and antigenicity. Although recombination between flavivirus genomes has never been demonstrated experimentally, the potential risk of generating undesirable recombinants has nevertheless been a matter of concern and controversy with respect to the development of live flavivirus vaccines. As an experimental system for investigating the a...

Taucher, Christian; Berger, Angelika; Mandl, Christian W.

2010-01-01

396

Recombinant Proteins: Hopes for Tissue Engineering  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Proteins constitute a group of key molecules with many applications in tissue engineering. Use of proteins provided from natural sources has several limitations which are overcome by the use of recombinant proteins. So far, the recombinant forms of many proteins with tissue engineering applications have been developed including structural proteins, growth factors and cytokines. This technology has enabled the development of specifically designed proteins such as growth factors with matrix bin...

Sepideh Hamzehlou; Mohammad Morad Farajollahi; Ahmad Mehdipour; Ali Samadikuchaksaraei

2012-01-01

397

Signals From the Epoch of Cosmological Recombination  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The physical ingredients to describe the epoch of cosmological recombination are amazingly simple and well-understood. This fact allows us to take into account a very large variety of physical processes, still finding potentially measurable consequences for the energy spectrum and temperature anisotropies of the Cosmic Microwave Background (CMB). In this contribution we provide a short historical overview in connection with the cosmological recombination epoch and its connec...

Sunyaev, R. A.; Chluba, J.

2009-01-01

398

Dissociative Recombination of Small Polyatomic Molecules  

Science.gov (United States)

Simplified analytical expressions for dissociative recombination driven by the Jahn-Teller and Renner-Teller interactions are discussed. These expressions allow straightforward predictions of isotope effects, and the results are discussed for both H3+ and HCO+ and their isotopomers. Although the predicted effects are generally in good agreement with experiment, discrepancies indicate areas worthy of more extensive investigation. Some general features of isotope effects in the dissociative recombination of other systems are also discussed.

Pratt, Stephen T.; Jungen, Christian

2011-07-01

399

Engineering recombinant chicken antibodies for improved characteristics  

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Phage libraries are a versatile source of recombinant antibody fragments directed against a wide variety of antigens. Recombinant antibodies have the advantage that they can be engineered to improve their binding or other characteristics. A chicken single chain variable fragment (scFv) phage library was panned against the 16 kDa antigen of Mycobacterium tuberculosis. Three phage displayed antibodies were obtained which bound specifically to the antigen. In soluble scFv format, howev...

Sixholo, Joy

2009-01-01

400

Live recombinant BHV/BRSV vaccine  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

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

1998-01-01

 
 
 
 
401

Recombination-generation currents in degenerate semiconductors  

Science.gov (United States)

The classical Shockley-Read-Hall theory of free carrier recombination and generation via traps is extended to degenerate semiconductors. A concise and simple expression is found which avoids completely the concept of a Fermi level, a concept which is alien to nonequilibrium situations. Assumptions made in deriving the recombination generation current are carefully delineated and are found to be basically identical to those made in the original theory applicable to nondegenerate semiconductors.

Von Roos, O.

1978-01-01

402

Dissociative Recombination of Astrochemically Interesting Ions  

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In this thesis the major work described concerns experimental determination of the dissociative recombination (DR) reaction for several molecular ions of astrochemical interest. DR is the process where an electron recombines with a molecular ion to form an excited neutral that disintegrates into two or more neutral fragments to release the gained excess energy. It is very efficient under cold conditions and therefore ubiquitously occurring in interstellar environments such as dark clouds and ...

Hamberg, Mathias

2010-01-01

403

Electron Recombination with Small Molecular Ions  

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In this thesis I have theoretically studied electron recombination processes with small molecular ions. In these kind of processes resonant states are involved. To calculate the potential energy for these states as a function of internuclear distance, structure calculations and scattering calculations have to be performed. So far I have been studying the ion-pair formation with in electron recombination with H3+. The cross section for this process has been calculated using different kind of m...

Brinne Roos, Johanna

2007-01-01

404

Chromosome breakage and recombination at fragile sites.  

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Chromosomal fragile sites are points on chromosomes that usually appear as nonstaining chromosome or chromatid gaps. It has frequently been suggested that fragile sites may be involved in chromosome breakage and recombination events. We and others have previously shown that fragile sites predispose to intrachromosomal recombination as measured by sister-chromatid exchanges. These findings suggested that fragile site expression often, if not always, is accompanied by DNA strand breakage. In th...

Glover, T. W.; Stein, C. K.

1988-01-01

405

Genistein directly inhibits native and recombinant NMDA receptors.  

Science.gov (United States)

The protein tyrosine kinase (PTK) inhibitor genistein has been widely used to examine potential effects of tyrosine phosphorylation on neurotransmitter function. We report here that genistein inhibits N-methyl-d-aspartate (NMDA) receptors through a direct effect. Whole-cell NMDA-activated current was recorded in native receptors from mouse hippocampal slice culture and rat recombinant NR1aNR2A and NR1aNR2B receptors transiently expressed in HEK293 cells. Extracellular application of genistein and NMDA reversibly inhibited NMDA-activated current. The inhibition of NMDA-activated current by genistein applied externally was not affected when genistein was also pre-equilibrated in the intracellular solution. Daidzein, an analog of genistein that does not block PTK, also inhibited NMDA-activated current. Coapplication of lavendustin A, a specific inhibitor of PTK, had no effect on the NMDA response. Moreover, genistein-induced inhibition of NMDA-activated current displayed concentration- and voltage-dependence. Our results demonstrate that genistein has a direct inhibitory effect on NMDA receptors that is not mediated via inhibition of tyrosine kinase. Thus, other PTK inhibitors may be more suitable for studying involvement of PTKs in NMDA receptor-mediated events. PMID:20303997

Huang, Renqi; Singh, Meharvan; Dillon, Glenn H

2010-06-01

406

Lack of MSH2 involvement differentiates V(D)J recombination from other non-homologous end joining events.  

Science.gov (United States)

V(D)J recombination and class switch recombination are the two DNA rearrangement events used to diversify the mouse and human antibody repertoires. While their double strand breaks (DSBs) are initiated by different mechanisms, both processes use non-homologous end joining (NHEJ) in the repair phase. DNA mismatch repair elements (MSH2/MSH6) have been implicated in the repair of class switch junctions as well as other DNA DSBs that proceed through NHEJ. MSH2 has also been implicated in the regulation of factors such as ATM and the MRN (Mre11, Rad50, Nbs1) complex, which are involved in V(D)J recombination. These findings led us to examine the role of MSH2 in V(D)J repair. Using MSH2-/- and MSH2+/+ mice and cell lines, we show here that all pathways involving MSH2 are dispensable for the generation of an intact pre-immune repertoire by V(D)J recombination. In contrast to switch junctions and other DSBs, the usage of terminal homology in V(D)J junctions is not influenced by MSH2. Thus, whether the repair complex for V(D)J recombination is of a canonical NHEJ type or a separate microhomology-mediated-end joining (MMEJ) type, it does not involve MSH2. This highlights a distinction between the repair of V(D)J recombination and other NHEJ reactions. PMID:16314305

Larijani, Mani; Zaheen, Ahmad; Frieder, Darina; Wang, Yuxun; Wu, Gillian E; Edelmann, Winfried; Martin, Alberto

2005-01-01

407

Recombination hotspots and host susceptibility modulate the adaptive value of recombination during maize streak virus evolution  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Maize streak virus -strain A (MSV-A; Genus Mastrevirus, Family Geminiviridae, the maize-adapted strain of MSV that causes maize streak disease throughout sub-Saharan Africa, probably arose between 100 and 200 years ago via homologous recombination between two MSV strains adapted to wild grasses. MSV recombination experiments and analyses of natural MSV recombination patterns have revealed that this recombination event entailed the exchange of the movement protein - coat protein gene cassette, bounded by the two genomic regions most prone to recombination in mastrevirus genomes; the first surrounding the virion-strand origin of replication, and the second around the interface between the coat protein gene and the short intergenic region. Therefore, aside from the likely adaptive advantages presented by a modular exchange of this cassette, these specific breakpoints may have been largely predetermined by the underlying mechanisms of mastrevirus recombination. To investigate this hypothesis, we constructed artificial, low-fitness, reciprocal chimaeric MSV genomes using alternating genomic segments from two MSV strains; a grass-adapted MSV-B, and a maize-adapted MSV-A. Between them, each pair of reciprocal chimaeric genomes represented all of the genetic material required to reconstruct - via recombination - the highly maize-adapted MSV-A genotype, MSV-MatA. We then co-infected a selection of differentially MSV-resistant maize genotypes with pairs of reciprocal chimaeras to determine the efficiency with which recombination would give rise to high-fitness progeny genomes resembling MSV-MatA. Results Recombinants resembling MSV-MatA invariably arose in all of our experiments. However, the accuracy and efficiency with which the MSV-MatA genotype was recovered across all replicates of each experiment depended on the MSV susceptibility of the maize genotypes used and the precise positions - in relation to known recombination hotspots - of the breakpoints required to re-create MSV-MatA. Although the MSV-sensitive maize genotype gave rise to the greatest variety of recombinants, the measured fitness of each of these recombinants correlated with their similarity to MSV-MatA. Conclusions The mechanistic predispositions of different MSV genomic regions to recombination can strongly influence the accessibility of high-fitness MSV recombinants. The frequency with which the fittest recombinant MSV genomes arise also correlates directly with the escalating selection pressures imposed by increasingly MSV-resistant maize hosts.

Monjane Adérito L

2011-12-01

408

H-Ras regulation of TRAIL death receptor mediated apoptosis  

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TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through the death receptors (DRs) 4 and/or 5 expressed on the cell surface. Multiple clinical trials are underway to evaluate the antitumor activity of recombinant human TRAIL and agonistic antibodies to DR4 or DR5. However, their therapeutic potential is limited by the high frequency of cancer resistance. Here we provide evidence demonstrating the role of H-Ras in TRAIL receptor mediated apoptosis. By analyzing the genome wide m...

Chen, Jun-jie; Bozza, William P.; Di, Xu; Zhang, Yaqin; Hallett, William; Zhang, Baolin

2014-01-01

409

Microhomology-mediated deletion and gene conversion in African trypanosomes  

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Antigenic variation in African trypanosomes is induced by DNA double-strand breaks (DSBs). In these protozoan parasites, DSB repair (DSBR) is dominated by homologous recombination (HR) and microhomology-mediated end joining (MMEJ), while non-homologous end joining (NHEJ) has not been reported. To facilitate the analysis of chromosomal end-joining, we established a system whereby inter-allelic repair by HR is lethal due to loss of an essential gene. Analysis of intrachromosomal end joining in ...

Glover, Lucy; Jun, Junho; Horn, David

2011-01-01

410

Recombinant plants provide a new approach to the production of bacterial polysaccharide for vaccines.  

Science.gov (United States)

Bacterial polysaccharides have numerous clinical or industrial uses. Recombinant plants could offer the possibility of producing bacterial polysaccharides on a large scale and free of contaminating bacterial toxins and antigens. We investigated the feasibility of this proposal by cloning and expressing the gene for the type 3 synthase (cps3S) of Streptococcus pneumoniae in Nicotinia tabacu