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1

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

Science.gov (United States)

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

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

2014-04-01

2

DNA-PK phosphorylation of RPA32 Ser4/Ser8 regulates replication stress checkpoint activation, fork restart, homologous recombination and mitotic catastrophe.  

Science.gov (United States)

Genotoxins and other factors cause replication stress that activate the DNA damage response (DDR), comprising checkpoint and repair systems. The DDR suppresses cancer by promoting genome stability, and it regulates tumor resistance to chemo- and radiotherapy. Three members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, ATM, ATR, and DNA-PK, are important DDR proteins. A key PIKK target is replication protein A (RPA), which binds single-stranded DNA and functions in DNA replication, DNA repair, and checkpoint signaling. An early response to replication stress is ATR activation, which occurs when RPA accumulates on ssDNA. Activated ATR phosphorylates many targets, including the RPA32 subunit of RPA, leading to Chk1 activation and replication arrest. DNA-PK also phosphorylates RPA32 in response to replication stress, and we demonstrate that cells with DNA-PK defects, or lacking RPA32 Ser4/Ser8 targeted by DNA-PK, confer similar phenotypes, including defective replication checkpoint arrest, hyper-recombination, premature replication fork restart, failure to block late origin firing, and increased mitotic catastrophe. We present evidence that hyper-recombination in these mutants is ATM-dependent, but the other defects are ATM-independent. These results indicate that DNA-PK and ATR signaling through RPA32 plays a critical role in promoting genome stability and cell survival in response to replication stress. PMID:24819595

Ashley, Amanda K; Shrivastav, Meena; Nie, Jingyi; Amerin, Courtney; Troksa, Kyle; Glanzer, Jason G; Liu, Shengqin; Opiyo, Stephen O; Dimitrova, Diana D; Le, Phuong; Sishc, Brock; Bailey, Susan M; Oakley, Greg G; Nickoloff, Jac A

2014-09-01

3

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.

Plate, Iben; Mortensen, Uffe Hasbro

2008-01-01

4

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

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

Wang Xuan

2004-01-01

5

Visualization of recombination-mediated damage bypass by template switching.  

Science.gov (United States)

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

6

GENERATION OF RECOMBINANT BACULOVIRUS VIA LIPOSOME MEDIATED TRANSFECTION  

Science.gov (United States)

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

7

Anthrax sub-unit vaccine: the structural consequences of binding rPA83 to Alhydrogel®.  

Science.gov (United States)

An anthrax sub-unit vaccine, comprising recombinant Protective Antigen (rPA83) and aluminium hydroxide adjuvant (Alhydrogel®) is currently being developed. Here, a series of biophysical techniques have been applied to free and adjuvant bound antigen. Limited proteolysis and fluorescence identified no changes in rPA83 tertiary structure following binding to Alhydrogel and the bound rPA83 retained two structurally important calcium ions. For adsorbed rPA83, differential scanning calorimetry revealed a small reduction in unfolding temperature but a large decrease in unfolding enthalpy whilst urea unfolding demonstrated unchanged stability but a loss of co-operativity. Overall, these results demonstrate that interactions between rPA83 and Alhydrogel have a minimal effect on the folded protein structure and suggest that antigen destabilisation is not a primary mechanism of Alhydrogel adjuvancy. This study also shows that informative structural characterisation is possible for adjuvant bound sub-unit vaccines. PMID:21964315

Soliakov, Andrei; Kelly, Ian F; Lakey, Jeremy H; Watkinson, Allan

2012-01-01

8

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

Science.gov (United States)

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

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

2014-03-01

9

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

Science.gov (United States)

The Red recombination system of bacteriophage lambda is widely used for genetic engineering because of its ability to promote recombination between bacterial chromosomes or plasmids and linear DNA species introduced by electroporation. The process is known to be intimately tied to replication, but the cellular functions which participate with Red in this process are largely unknown. Here two such functions are identified: the GrpE-DnaK-DnaJ chaperone system, and DNA polymerase I. Mutations in either function are found to decrease the efficiency of Red recombination. grpE and dnaJ mutations which greatly decrease Red recombination with electroporated DNA species have only small effects on Red-mediated transduction. This recombination event specificity suggests that the involvement of GrpE-DnaJ-DnaK is not simply an effect on Red structure or stability. PMID:23840702

Poteete, Anthony R.

2013-01-01

10

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

Directory of Open Access Journals (Sweden)

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

Kagale Sateesh

2008-11-01

11

Measurements of DNA-loop formation via Cre-mediated recombination.  

Science.gov (United States)

The Cre-recombination system has become an important tool for genetic manipulation of higher organisms and a model for site-specific DNA-recombination mechanisms employed by the ?-Int superfamily of recombinases. We report a novel quantitative approach for characterizing the probability of DNA-loop formation in solution using time-dependent ensemble Förster resonance energy transfer measurements of intra- and inter-molecular Cre-recombination kinetics. Our method uses an innovative technique for incorporating multiple covalent modifications at specific sites in covalently closed DNA. Because the mechanism of Cre recombinase does not conform to a simple kinetic scheme, we employ numerical methods to extract rate constants for fundamental steps that pertain to Cre-mediated loop closure. Cre recombination does not require accessory proteins, DNA supercoiling or particular metal-ion cofactors and is thus a highly flexible system for quantitatively analyzing DNA-loop formation in vitro and in vivo. PMID:22589415

Shoura, Massa J; Vetcher, Alexandre A; Giovan, Stefan M; Bardai, Farah; Bharadwaj, Anusha; Kesinger, Matthew R; Levene, Stephen D

2012-08-01

12

Evidence that the normal route of replication-allowed Red-mediated recombination involves double-chain ends.  

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Recombination mediated by the Red pathway of bacteriophage lambda is focused towards sites of double-chain cuts. Double-chain ends created either by type II restriction enzymes acting at unmodified recognition sites or by lambda's packaging enzyme, terminase, acting at cos are utilized in a manner similar to the double-chain break repair pathway of recombination in yeast. When lambda is allowed to recombine during replicative growth, spontaneous recombination is approximately evenly distribut...

Thaler, D. S.; Stahl, M. M.; Stahl, F. W.

1987-01-01

13

Insertion sequence inversions mediated by ectopic recombination between terminal inverted repeats.  

Science.gov (United States)

Transposable elements are widely distributed and diverse in both eukaryotes and prokaryotes, as exemplified by DNA transposons. As a result, they represent a considerable source of genomic variation, for example through ectopic (i.e. non-allelic homologous) recombination events between transposable element copies, resulting in genomic rearrangements. Ectopic recombination may also take place between homologous sequences located within transposable element sequences. DNA transposons are typically bounded by terminal inverted repeats (TIRs). Ectopic recombination between TIRs is expected to result in DNA transposon inversions. However, such inversions have barely been documented. In this study, we report natural inversions of the most common prokaryotic DNA transposons: insertion sequences (IS). We identified natural TIR-TIR recombination-mediated inversions in 9% of IS insertion loci investigated in Wolbachia bacteria, which suggests that recombination between IS TIRs may be a quite common, albeit largely overlooked, source of genomic diversity in bacteria. We suggest that inversions may impede IS survival and proliferation in the host genome by altering transpositional activity. They may also alter genomic instability by modulating the outcome of ectopic recombination events between IS copies in various orientations. This study represents the first report of TIR-TIR recombination within bacterial IS elements and it thereby uncovers a novel mechanism of structural variation for this class of prokaryotic transposable elements. PMID:21187977

Ling, Alison; Cordaux, Richard

2010-01-01

14

Visualization of recombination-mediated damage-bypass by template switching  

Science.gov (United States)

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. This causes re-annealing of the parental strands and exposure of the non-damaged newly synthesized chromatid as template for replication by the other blocked nascent strand. Structures resembling double Holliday Junctions, postulated to be central double-strand break repair intermediates, but so far only visualized 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 for replication under conditions of genotoxic stress. PMID:25195051

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

2014-01-01

15

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.)

16

Recombination-mediated genetic engineering of Plasmodium berghei DNA.  

Science.gov (United States)

DNA of Plasmodium berghei is difficult to manipulate in Escherichia coli by conventional restriction and ligation methods due to its high content of adenine and thymine (AT) nucleotides. This limits our ability to clone large genes and to generate complex vectors for modifying the parasite genome. We here describe a protocol for using lambda Red recombinase to modify inserts of a P. berghei genomic DNA library constructed in a linear, low-copy, phage-derived vector. The method uses primer extensions of 50 bp, which provide sufficient homology for an antibiotic resistance marker to recombine efficiently with a P. berghei genomic DNA insert in E. coli. In a subsequent in vitro Gateway reaction the bacterial marker is replaced with a cassette for selection in P. berghei. The insert is then released and used for transfection. The basic techniques we describe here can be adapted to generate highly efficient vectors for gene deletion, tagging, targeted mutagenesis, or genetic complementation with larger genomic regions. PMID:22990774

Pfander, Claudia; Anar, Burcu; Brochet, Mathieu; Rayner, Julian C; Billker, Oliver

2013-01-01

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Recombinant adenovirus-mediated gene transfer suppresses experimental arthritis  

Directory of Open Access Journals (Sweden)

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

E. Quattrocchi

2011-09-01

18

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

Directory of Open Access Journals (Sweden)

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

19

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

20

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

Science.gov (United States)

Caffeine is a widely used inhibitor of the protein kinases that play a central role in the DNA damage response. We used chemical inhibitors and genetically deficient mouse embryonic stem cell lines to study the role of DNA damage response in stable integration of the transfected DNA and found that caffeine rapidly, efficiently and reversibly inhibited homologous integration of the transfected DNA as measured by several homologous recombination-mediated gene-targeting assays. Biochemical and structural biology experiments revealed that caffeine interfered with a pivotal step in homologous recombination, homologous joint molecule formation, through increasing interactions of the RAD51 nucleoprotein filament with non-homologous DNA. Our results suggest that recombination pathways dependent on extensive homology search are caffeine-sensitive and stress the importance of considering direct checkpoint-independent mechanisms in the interpretation of the effects of caffeine on DNA repair. PMID:23666627

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

2013-01-01

 
 
 
 
21

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

2014-08-01

22

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

23

Molecular recognition of DNA structure by proteins that mediate genetic recombination.  

Science.gov (United States)

The latter half of genetic recombination is mediated by proteins that recognise the structure of the four-way DNA junction, and manipulate this structure. In solution the four-way junction adopts a stacked X-structure in the presence of metal ions. The folding is brought about by the pairwise coaxial stacking of helices in a right-handed antiparallel X-shaped structure. The four-way junction is cleaved by structure-selective resolving enzymes that have been isolated from a wide variety of sources, from eubacteria and their phages through to mammals. In addition, another class of proteins accelerate the branch migration of the junction. These proteins all appear to be divisible into a component that recognises structure and another that carries out a reaction on the junction. Thus the ability of structure-selective binding to the four-way DNA junction is a key feature of enzymes important in genetic recombination. PMID:7826676

Lilley, D M

1994-06-01

24

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

Science.gov (United States)

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

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

1997-07-01

25

Recombiner  

International Nuclear Information System (INIS)

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

26

Extended GIPQ description of the RPA excitations  

International Nuclear Information System (INIS)

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

27

RPA Systems Opportunities Final Report - ARCHIVE  

which provides critical systems supporting SPS, the largest part of RPA?s \\business is due to expire at the end of 2011 at .... The expiry of the Accenture \\contract at the end of 2011 could not come at a worse time in this context; ..... \\Desktop PCs.

28

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

International Nuclear Information System (INIS)

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

29

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

Science.gov (United States)

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

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

2014-03-01

30

DNA-binding properties of T4 UvsY recombination mediator protein: polynucleotide wrapping promotes high-affinity binding to single-stranded DNA  

Digital Repository Infrastructure Vision for European Research (DRIVER)

To carry out homologous recombination events in the cell, recombination proteins must be able to recognize and form presynaptic filaments on single-stranded DNA (ssDNA) in the presence of a vast excess of double-stranded DNA (dsDNA). Therefore recombination machineries stringently discriminate between ssDNA and dsDNA lattices. Recent single-molecule studies of bacteriophage T4 recombination proteins revealed that, surprisingly, the UvsY recombination mediator protein binds stronger to stretch...

Xu, Hang; Beernink, Hans T. H.; Morrical, Scott W.

2010-01-01

31

Toward a consistent RHA-RPA  

International Nuclear Information System (INIS)

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

32

RPA calculations with Gaussian expansion method  

Energy Technology Data Exchange (ETDEWEB)

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{sup -}, 2{sup +} and 3{sup -} 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 {sup 60}Ca are investigated in some detail.

Nakada, H. [Department of Physics, Graduate School of Science, Chiba University, Yayoi-cho 1-33, Inage, Chiba 263-8522 (Japan)], E-mail: nakada@faculty.chiba-u.jp; Mizuyama, K. [Department of Physics, University of Jyvaeskylae, PO Box 35 (YFL), FI-40014, Jyvaeskylae (Finland); Yamagami, M. [Department of Computer Science and Engineering, University of Aizu, Aizu-Wakamatsu, Fukushima 965-8580 (Japan); Matsuo, M. [Department of Physics, Faculty of Science, Niigata University, Niigata 950-2181 (Japan)

2009-09-15

33

A pink mouse reports the switch from red to green fluorescence upon Cre-mediated recombination  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Targeted genetic modification in the mouse becomes increasingly important in biomedical and basic science. This goal is most often achieved by use of the Cre/loxP system and numerous Cre-driver mouse lines are currently generated. Their initial characterization requires reporter mouse lines to study the in vivo spatiotemporal activity of Cre. Findings Here, we report a dual fluorescence reporter mouse line, which switches expression from the red fluorescent protein mCherry to eGFP after Cre-mediated recombination. Both fluorescent proteins are expressed from the ubiquitously active and strong CAGGS promoter. Among the founders, we noticed a pink mouse line, expressing high levels of the red fluorescent protein mCherry throughout the entire body. Presence of mCherry in the living animal as well as in almost all organs was clearly visible without optical equipment. Upon Cre-activity, mCherry expression was switched to eGFP, demonstrating functionality of this reporter mouse line. Conclusions The pink mouse presented here is an attractive novel reporter line for fluorescence-based monitoring of Cre-activity. The high expression of mCherry, which is visible to the naked eye, facilitates breeding and crossing, as no genotyping is required to identify mice carrying the reporter allele. The presence of two fluorescent proteins allows in vivo monitoring of recombined and non-recombined cells. Finally, the pink mouse is an eye-catching animal model to demonstrate the power of transgenic techniques in teaching courses.

Hartwich Heiner

2012-06-01

34

Recombinant AAV-mediated gene delivery to the central nervous system.  

Science.gov (United States)

Various regions of the brain have been successfully transduced by recombinant adeno-associated virus (rAAV) vectors with no detected toxicity. When using the cytomegalovirus immediate early (CMV) promoter, a gradual decline in the number of transduced cells has been described. In contrast, the use of cellular promoters such as the neuron-specific enolase promoter or hybrid promoters such as the chicken beta-actin/CMV promoter resulted in sustained transgene expression. The cellular tropism of rAAV-mediated gene transfer in the central nervous system (CNS) varies depending on the serotype used. Serotype 2 vectors preferentially transduce neurons whereas rAAV5 and rAAV1 transduce both neurons and glial cells. Recombinant AAV4-mediated gene transfer was inefficient in neurons and glial cells of the striatum (the only structure tested so far) but efficient in ependymal cells. No inflammatory response has been described following rAAV2 administration to the brain. In contrast, antibodies to AAV2 capsid and transgene product were elicited but no reduction of transgene expression was observed and readministration of vector without loss of efficiency was possible from 3 months after the first injection. Based on the success of pioneer work performed with marker genes, various strategies for therapeutic gene delivery were designed. These include enzyme replacement in lysosomal storage diseases, Canavan disease and Parkinson's disease; delivery of neuroprotective factors in Parkinson's disease, Huntington disease, Alzheimer's disease, amyotrophic lateral sclerosis, ischemia and spinal cord injury; as well as modulation of neurotransmission in epilepsy and Parkinson's disease. Several of these strategies have demonstrated promising results in relevant animal models. However, their implementation in the clinics will probably require a tight regulation and a specific targeting of therapeutic gene expression which still demands further developments of the vectors. PMID:14978764

Tenenbaum, L; Chtarto, A; Lehtonen, E; Velu, T; Brotchi, J; Levivier, M

2004-02-01

35

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)

36

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

Science.gov (United States)

Bloom syndrome (BS) is a rare genetic disorder characterized by genomic instability and a high predisposition to cancer. The gene defective in BS, BLM, encodes a member of the RecQ family of 3'-5' DNA helicases, and is proposed to function in recombinational repair during DNA replication. Here, we have utilized single-molecule fluorescence resonance energy transfer microscopy to examine the behaviour of BLM on forked DNA substrates. Strikingly, BLM unwound individual DNA molecules in a repetitive manner, unwinding a short length of duplex DNA followed by rapid reannealing and reinitiation of unwinding in several successions. Our results show that a monomeric BLM can 'measure' how many base pairs it has unwound, and once it has unwound a critical length, it reverses the unwinding reaction through strand switching and translocating on the opposing strand. Repetitive unwinding persisted even in the presence of hRPA, and interaction between wild-type BLM and hRPA was necessary for unwinding reinitiation on hRPA-coated DNA. The reported activities may facilitate BLM processing of stalled replication forks and illegitimately formed recombination intermediates. PMID:19165145

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

2009-02-18

37

Human Treg responses allow sustained recombinant adeno-associated virus-mediated transgene expression.  

Science.gov (United States)

Recombinant adeno-associated virus (rAAV) vectors have shown promise for the treatment of several diseases; however, immune-mediated elimination of transduced cells has been suggested to limit and account for a loss of efficacy. To determine whether rAAV vector expression can persist long term, we administered rAAV vectors expressing normal, M-type ?-1 antitrypsin (M-AAT) to AAT-deficient subjects at various doses by multiple i.m. injections. M-specific AAT expression was observed in all subjects in a dose-dependent manner and was sustained for more than 1 year in the absence of immune suppression. Muscle biopsies at 1 year had sustained AAT expression and a reduction of inflammatory cells compared with 3 month biopsies. Deep sequencing of the TCR V? region from muscle biopsies demonstrated a limited number of T cell clones that emerged at 3 months after vector administration and persisted for 1 year. In situ immunophenotyping revealed a substantial Treg population in muscle biopsy samples containing AAT-expressing myofibers. Approximately 10% of all T cells in muscle were natural Tregs, which were activated in response to AAV capsid. These results suggest that i.m. delivery of rAAV type 1-AAT (rAAV1-AAT) induces a T regulatory response that allows ongoing transgene expression and indicates that immunomodulatory treatments may not be necessary for rAAV-mediated gene therapy. PMID:24231351

Mueller, Christian; Chulay, Jeffrey D; Trapnell, Bruce C; Humphries, Margaret; Carey, Brenna; Sandhaus, Robert A; McElvaney, Noel G; Messina, Louis; Tang, Qiushi; Rouhani, Farshid N; Campbell-Thompson, Martha; Fu, Ann Dongtao; Yachnis, Anthony; Knop, David R; Ye, Guo-Jie; Brantly, Mark; Calcedo, Roberto; Somanathan, Suryanarayan; Richman, Lee P; Vonderheide, Robert H; Hulme, Maigan A; Brusko, Todd M; Wilson, James M; Flotte, Terence R

2013-12-01

38

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

39

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

Science.gov (United States)

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. PMID:24262008

2013-01-01

40

RPA calculations with the Skyrme interaction SIV  

International Nuclear Information System (INIS)

Most of the Skyrme interactions proposed by the Orsay group lead to imaginary eigenvalues in RPA calculations, mainly due to the too strongly attractive character of the force in the S=1 T=0 particle hole channel. The force SIV has a small S=1 part and does not lead to imaginary eigenvalues; furthermore it gives good single particle energies in light nuclei and one should expect a RPA calculation to give reasonable results for these nuclei. Some results of such a calculation in 12C, 160 and 40Ca are presented. The single particle energies and wave functions are those of the Hartree-Fock solution found with the same interaction. The continuum spectrum is discretized by diagonalizing the Hartree-Fock Hamiltonian in a large oscillator basis (7 major shells for the results presented). All the particle-hole configurations which can be constructed for a given multipolarity are taken into account; this method has been found to converge when the dimension of the basis is increased. (Auth.)

 
 
 
 
41

CtIP promotes microhomology-mediated alternative end joining during class-switch recombination.  

Science.gov (United States)

Immunoglobulin heavy chain (Igh locus) class-switch recombination (CSR) requires targeted introduction of DNA double strand breaks (DSBs) into repetitive 'switch'-region DNA elements in the Igh locus and subsequent ligation between distal DSBs. Both canonical nonhomologous end joining (C-NHEJ) that seals DNA ends with little or no homology and a poorly defined alternative end joining (A-NHEJ, also known as alt-NHEJ) process that requires microhomology ends for ligation have been implicated in CSR. Here, we show that the DNA end-processing factor CtIP is required for microhomology-directed A-NHEJ during CSR. Additionally, we demonstrate that microhomology joins that are enriched upon depletion of the C-NHEJ component Ku70 require CtIP. Finally, we show that CtIP binds to switch-region DNA in a fashion dependent on activation-induced cytidine deaminase. Our results establish CtIP as a bona fide component of microhomology-dependent A-NHEJ and unmask a hitherto unrecognized physiological role of microhomology-mediated end joining in a C-NHEJ-proficient environment. PMID:21131982

Lee-Theilen, Mieun; Matthews, Allysia J; Kelly, Dierdre; Zheng, Simin; Chaudhuri, Jayanta

2011-01-01

42

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)

43

RPA correction to the optical potential  

Directory of Open Access Journals (Sweden)

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

Bauge E.

2010-03-01

44

Self-control in DNA site-specific recombination mediated by the tyrosine recombinase TnpI.  

Science.gov (United States)

Tn4430 is a distinctive transposon of the Tn3 family that encodes a tyrosine recombinase (TnpI) to resolve replicative transposition intermediates. The internal resolution site of Tn4430 (IRS, 116 bp) contains two inverted repeats (IR1 and IR2) at the crossover core site, and two additional TnpI binding motifs (DR1 and DR2) adjacent to the core. Deletion analysis demonstrated that DR1 and DR2 are not required for recombination in vivo and in vitro. Their function is to provide resolution selectivity to the reaction by stimulating recombination between directly oriented sites on a same DNA molecule. In the absence of DR1 and/or DR2, TnpI-mediated recombination of supercoiled DNA substrates gives a mixture of topologically variable products, while deletion between two wild-type IRSs exclusively produces two-noded catenanes. This demonstrates that TnpI binding to the accessory motifs DR1 and DR2 contributes to the formation of a specific synaptic complex in which catalytically inert recombinase subunits act as architectural elements to control recombination sites pairing and strand exchange. A model for the organization of TnpI/IRS recombination complex is presented. PMID:16629665

Vanhooff, Virginie; Galloy, Christine; Agaisse, Hervé; Lereclus, Didier; Révet, Bernard; Hallet, Bernard

2006-05-01

45

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

46

Homologous recombination mediates cellular resistance and fraction size sensitivity to radiation therapy  

International Nuclear Information System (INIS)

Purpose: Cellular sensitivity to radiotherapy total dose and fraction size is strongly influenced by DNA double strand break (DSB) repair. Here, we investigate response to radiotherapy fraction size using CHO cell lines deficient in specific DNA repair pathways in response to radiation induced DNA double strand breaks (DSB). Experimental design: We irradiated CHO cell lines, AA8 (WT), irs1SF (XRCC3-), V3-3 (DNA-PKcs-) and EM9 (XRCC1-) with 16 Gy in 1 Gy daily fractions over 3 weeks or 16 Gy in 4 Gy daily fractions over 4 days, and studied clonogenic survival, DNA DSB repair kinetics (RAD51 and 53BP1 foci staining) and cell cycle profiles (flow cytometry). Results: In response to fractionated radiotherapy, wild-type and DNA repair defective cells accumulated in late S/G2 phase. In cells proficient in homologous recombination (HR), accumulation in S/G2 resulted in reduced sensitivity to fraction size and increased cellular resistance (clonogenic survival). Sensitivity to fraction size was also lost in NHEJ-defective V3-3 cells, which likely rely on functional HR. By contrast, HR-defective irs1SF cells, with functional NHEJ, remained equally sensitive to fractionation throughout the 3-week treatment. Conclusions: The high fidelity of HR, which is independent of induced DNA damage level, is postulated to explain the low fractionation sensitivity and cellular resistance of cells in S/G2 phase. In conclusion, our results suggest that HR mediates resistance to fractionated radiotherapy, an observation that may help future efforts to improve radiotherapy outcome

47

Subtraction method and stability condition in the extended RPA theories  

CERN Document Server

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

Tselyaev, V I

2013-01-01

48

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

49

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

Science.gov (United States)

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

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

2014-08-01

50

Comparison of the Structural Stability and Dynamic Properties of Recombinant Anthrax Protective Antigen and its 2-Fluorohistidine Labeled Analogue  

Science.gov (United States)

Protective antigen (PA) is the primary protein antigenic component of both the currently used anthrax vaccine and related recombinant vaccines under development. An analogue of recombinant PA (2-FHis rPA) has been recently shown to block the key steps of pore formation in the process of inducing cytotoxicity in cells, and thus can potentially be used as an antitoxin or a vaccine. This rPA analogue was produced by fermentation to incorporate the unnatural amino acid 2-fluorohistidine (2-FHis). In this study, the effects of 2-FHis labeling on rPA antigen’s conformational stability and dynamic properties were investigated by various biophysical techniques. Temperature/pH stability profiles of rPA and 2-FHis rPA were analyzed by the empirical phase diagram (EPD) approach, and physical stability differences between them were identified. Results showed that rPA and 2-FHis rPA had similar stability at pH 7–8. With decreasing solution pH, however, 2-FHis rPA was found to be more stable. Dynamic sensitive measurements of the two proteins at pH 5 found that 2-FHis rPA was more dynamic and/or differentially hydrated under acidic pH conditions. The biophysical characterization and stability data provide information useful for the potential development of 2-FHis rPA as a more stable rPA vaccine candidate. PMID:22911632

Hu, Lei; Joshi, Sangeeta B.; Andra, Kiran K.; Thakkar, Santosh V.; Volkin, David B.; Bann, James G.; Middaugh, C. Russell

2013-01-01

51

RPA equations and the instantaneous Bethe-Salpeter equation  

CERN Document Server

We give a derivation of the particle-hole RPA equations for an interacting multi-fermion system by applying the instantaneous approximation to the amputated two-fermion propagator of the system. In relativistic field theory the same approximation leads from the fermion-antifermion Bethe-Salpeter equation to the Salpeter equation. We show that RPA equations and Salpeter equation are indeed equivalent.

Resag, J

1993-01-01

52

Spin-isospin resonances with relativistic RPA approaches  

International Nuclear Information System (INIS)

The relativistic RPA approaches are applied to calculate the charge-exchange spin flip resonances. Comparing the RPA calculations based on the relativistic Hartree and relativistic Hartree-Fock theories, the different physical mechanisms in determining the Gamow-Teller resonance are investigated. Then, the theoretical descriptions of spin-dipole and spin-quadrupole resonances are presented. In particular, the energy hierarchies of different components in these resonances are focused on.

53

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

Science.gov (United States)

Armillaria root disease is one of the most damaging timber and fruit tree diseases in the world. Despite its economic importance, many basic questions about the biology of the causal fungi, Armillaria spp., are unanswered. For example, Armillaria undergoes matings between diploid and haploid mycelia, which can result in a recombinant diploid without meiosis. Evidence of such somatic recombination in natural populations suggests that this reproductive mode may affect the pathogen's ecology. Investigations of the mechanisms and adaptive consequences of somatic recombination are, however, hampered by the lack of a method to reliably synthesize somatic recombinants. Here we report the first genetic transformation system for the genus Armillaria. We transformed A. mellea with selective markers for use in diploid-haploid matings to reliably synthesize somatic recombinants. This was accomplished with Agrobacterium tumefaciens carrying pBGgHg, which carries the hygromycin phosphotransferase gene (hph). hph was integrated into transformants, as evidenced by serial transfer to selective media, PCR, reverse transcription-PCR (RT-PCR), and Southern hybridization. Nuclear and mitochondrial markers were developed to genotype synthesized mycelia. In matings between a wild-type diploid and hygromycin-resistant haploids (transgenic), we identified recombinant, hygromycin-resistant diploids and, additionally, hygromycin-resistant triploids, all with the mitochondrial haplotype of the haploid partner. Our approach created no mycelium in which the haploid nucleus was replaced by the diploid nucleus, the typical outcome of diploid-haploid matings in Armillaria. This genetic transformation system, in combination with new markers to track chromosomal and cytoplasmic inheritance in A. mellea, will advance research aimed at characterizing the significance of somatic recombination in the ecology of this important fungus. PMID:20952653

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

2010-12-01

54

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

55

Inhalation of nebulized perfluorochemical enhances recombinant adenovirus and adeno-associated virus-mediated gene expression in lung epithelium.  

Science.gov (United States)

Use of perfluorochemical liquids during intratracheal vector administration enhances recombinant adenovirus and adeno-associated virus (AAV)-mediated lung epithelial gene expression. We hypothesized that inhalation of nebulized perfluorochemical vapor would also enhance epithelial gene expression after subsequent intratracheal vector administration. Freely breathing adult C57BL/6 mice were exposed for selected times to nebulized perflubron or sterile saline in a sealed Plexiglas chamber. Recombinant adenoviral vector was administered by transtracheal puncture at selected times afterward and mice were killed 3 days after vector administration to assess transgene expression. Mice tolerated the nebulized perflubron without obvious ill effects. Vector administration 6 hr after nebulized perflubron exposure resulted in an average 540% increase in gene expression in airway and alveolar epithelium, compared with that with vector alone or saline plus vector control (pinhalation of nebulized perflubron enhanced recombinant AAV2/5 vector expression throughout the lung. Serial chest radiographs, bronchoalveolar lavages, and results of complete blood counts and serum biochemistries demonstrated no obvious adverse effects of nebulized perflubron. Further, one macaque receiving nebulized perflubron only was monitored for 1 year with no obvious adverse effects of exposure. These results demonstrate that inhalation of nebulized perflubron, a simple, clinically more feasible technique than intratracheal administration of liquid perflubron, safely enhances lung gene expression. PMID:22568624

Beckett, Travis; Bonneau, Laura; Howard, Alan; Blanchard, James; Borda, Juan; Weiner, Daniel J; Wang, Lili; Gao, Guang Ping; Kolls, Jay K; Bohm, Rudolf; Liggitt, Denny; Weiss, Daniel J

2012-04-01

56

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

Science.gov (United States)

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

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Sister Chromatid Exchanges Are Mediated by Homologous Recombination in Vertebrate Cells  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Sister chromatid exchange (SCE) frequency is a commonly used index of chromosomal stability in response to environmental or genetic mutagens. However, the mechanism generating cytologically detectable SCEs and, therefore, their prognostic value for chromosomal stability in mitotic cells remain unclear. We examined the role of the highly conserved homologous recombination (HR) pathway in SCE by measuring SCE levels in HR-defective vertebrate cells. Spontaneous and mitomycin C-induced SCE level...

Sonoda, Eiichiro; Sasaki, Masao S.; Morrison, Ciaran; Yamaguchi-iwai, Yuko; Takata, Minoru; Takeda, Shunichi

1999-01-01

58

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

59

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

60

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

Scientific Electronic Library Online (English)

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

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

 
 
 
 
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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

62

76 FR 54195 - 2010 Resources Planning Act (RPA) Assessment Draft  

Science.gov (United States)

...to the building. FOR FURTHER INFORMATION CONTACT: Dr. Linda Langner, Quantitative Sciences Staff by phone at 703-605-4886 or by email to llangner@fs.fed.us. Additional information about the RPA Assessment can be obtained on the Internet...

2011-08-31

63

Studies of the Interaction between Rad52 Protein and the Yeast Single-Stranded DNA Binding Protein RPA  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The RFA1 gene encodes the large subunit of the yeast trimeric single-stranded DNA binding protein replication protein A (RPA), which is known to play a critical role in DNA replication. A Saccharomyces cerevisiae strain carrying the rfa1-44 allele displays a number of impaired recombination and repair phenotypes, all of which are suppressible by overexpression of RAD52. We demonstrate that a rad52 mutation is epistatic to the rfa1-44 mutation, placing RFA1 and RAD52 in the same genetic pathwa...

Hays, Sharon L.; Firmenich, Antoine A.; Massey, Philip; Banerjee, Ronadip; Berg, Paul

1998-01-01

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Studies of the Interaction between Rad52 Protein and the Yeast Single-Stranded DNA Binding Protein RPA  

Science.gov (United States)

The RFA1 gene encodes the large subunit of the yeast trimeric single-stranded DNA binding protein replication protein A (RPA), which is known to play a critical role in DNA replication. A Saccharomyces cerevisiae strain carrying the rfa1-44 allele displays a number of impaired recombination and repair phenotypes, all of which are suppressible by overexpression of RAD52. We demonstrate that a rad52 mutation is epistatic to the rfa1-44 mutation, placing RFA1 and RAD52 in the same genetic pathway. Furthermore, two-hybrid analysis indicates the existence of interactions between Rad52 and all three subunits of RPA. The nature of this Rad52-RPA interaction was further explored by using two different mutant alleles of rad52. Both mutations lie in the amino terminus of Rad52, a region previously defined as being responsible for its DNA binding ability (U. H. Mortenson, C. Beudixen, I. Sunjeuaric, and R. Rothstein, Proc. Natl. Acad. Sci. USA 93:10729–10734, 1996). The yeast two-hybrid system was used to monitor the protein-protein interactions of the mutant Rad52 proteins. Both of the mutant proteins are capable of self-interaction but are unable to interact with Rad51. The mutant proteins also lack the ability to interact with the large subunit of RPA, Rfa1. Interestingly, they retain their ability to interact with the medium-sized subunit, Rfa2. Given the location of the mutations in the DNA binding domain of Rad52, a model incorporating the role of DNA in the protein-protein interactions involved in the repair of DNA double-strand breaks is presented. PMID:9632824

Hays, Sharon L.; Firmenich, Antoine A.; Massey, Philip; Banerjee, Ronadip; Berg, Paul

1998-01-01

65

Generation of TALEN-mediated GRdim knock-in rats by homologous recombination.  

Science.gov (United States)

Transcription Activator-Like Effector Nucleases (TALEN) are potential tools for precise genome engineering of laboratory animals. We report the first targeted genomic integration in the rat using TALENs (Transcription Activator-Like Effector Nucleases) by homology-derived recombination (HDR). We assembled TALENs and designed a linear donor insert targeting a pA476T mutation in the rat Glucocorticoid Receptor (Nr3c1) namely GR(dim), that prevents receptor homodimerization in the mouse. TALEN mRNA and linear double-stranded donor were microinjected into rat one-cell embryos. Overall, we observed targeted genomic modifications in 17% of the offspring, indicating high TALEN cutting efficiency in rat zygotes. PMID:24523878

Ponce de León, Verónica; Mérillat, Anne-Marie; Tesson, Laurent; Anegón, Ignacio; Hummler, Edith

2014-01-01

66

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

Science.gov (United States)

Transcription Activator-Like Effector Nucleases (TALEN) are potential tools for precise genome engineering of laboratory animals. We report the first targeted genomic integration in the rat using TALENs (Transcription Activator-Like Effector Nucleases) by homology-derived recombination (HDR). We assembled TALENs and designed a linear donor insert targeting a pA476T mutation in the rat Glucocorticoid Receptor (Nr3c1) namely GRdim, that prevents receptor homodimerization in the mouse. TALEN mRNA and linear double-stranded donor were microinjected into rat one-cell embryos. Overall, we observed targeted genomic modifications in 17% of the offspring, indicating high TALEN cutting efficiency in rat zygotes. PMID:24523878

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

2014-01-01

67

Chaperone-mediated gene therapy with recombinant AAV-PPCA in a new mouse model of type I sialidosis.  

Science.gov (United States)

The lysosomal storage disease sialidosis is caused by a primary deficiency of the sialidase N-acetyl-?-neuraminidase-1 (NEU1). Patients with type I sialidosis develop an attenuated, non-neuropathic form of the disease also named cherry red spot myoclonus syndrome, with symptoms arising during juvenile/ adult age. NEU1 requires binding to its chaperone, protective protein/cathepsin A (PPCA), for lysosomal compartmentalization, stability and catalytic activation. We have generated a new mouse model of type I sialidosis that ubiquitously expresses a NEU1 variant carrying a V54M amino acid substitution identified in an adult patient with type I sialidosis. Mutant mice developed signs of lysosomal disease after 1year of age, predominantly in the kidney, albeit low residual NEU1 activity was detected in most organs and cell types. We demonstrate that the activity of the mutant enzyme could be effectively increased in all systemic tissues by chaperone-mediated gene therapy with a liver-tropic recombinant AAV2/8 vector expressing PPCA. This resulted in clear amelioration of the disease phenotype. These results suggest that at least some of the NEU1 mutations associated with type I sialidosis may respond to PPCA-chaperone-mediated gene therapy. PMID:23770387

Bonten, Erik J; Yogalingam, Gouri; Hu, Huimin; Gomero, Elida; van de Vlekkert, Diantha; d'Azzo, Alessandra

2013-10-01

68

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

69

Recombinant peptides in thrombolysis.  

Science.gov (United States)

Recombinant thrombolytic peptides are mainly represented by recombinant forms of tissue plasminogen activator (t-PA), a proteolytic enzyme that catalyzes the conversion of plasminogen into active plasmin, which then functions to dissolve clots. The three clinically relevant recombinant thrombolytic peptides are alteplase (t-PA), reteplase (r-PA), and tenecteplase (TNK). r-PA and TNK have been structurally modified from native t-PA to increase their half-life and fibrin specificity. Thrombolytics play an important role in several diseases, including ST-segment elevation myocardial infarction (STEMI), deep vein thrombosis (DVT) and pulmonary embolism (PE), ischemic stroke, and peripheral arterial disease. Thrombolytic therapy has evolved into an alternative treatment for STEMI, reserved predominantly for patients who do not have access to timely percutaneous coronary intervention. In patients with DVT/PE or arterial related critical limb ischemia, the use of thrombolytic therapy is limited to specific patient populations. Thrombolytic therapy is the treatment of choice for ischemic stroke in patients who present

Campbell, Jennifer; Hilleman, Daniel

2010-07-01

70

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

71

Suppression of mutagenesis by Rad51D-mediated homologous recombination  

Energy Technology Data Exchange (ETDEWEB)

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

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

2005-11-15

72

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

Directory of Open Access Journals (Sweden)

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

Shin'ichi Takeda

2013-06-01

73

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

Directory of Open Access Journals (Sweden)

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

Holt Robert A

2006-04-01

74

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

Directory of Open Access Journals (Sweden)

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

Sivakumar Jeyarajan

2010-09-01

75

Architecture and ssDNA interaction of the Timeless-Tipin-RPA complex.  

Science.gov (United States)

The Timeless-Tipin (Tim-Tipin) complex, also referred to as the fork protection complex, is involved in coordination of DNA replication. Tim-Tipin is suggested to be recruited to replication forks via Replication Protein A (RPA) but details of the interaction are unknown. Here, using cryo-EM and biochemical methods, we characterized complex formation of Tim-Tipin, RPA and single-stranded DNA (ssDNA). Tim-Tipin and RPA form a 258 kDa complex with a 1:1:1 stoichiometry. The cryo-EM 3D reconstruction revealed a globular architecture of the Tim-Tipin-RPA complex with a ring-like and a U-shaped domain covered by a RPA lid. Interestingly, RPA in the complex adopts a horse shoe-like shape resembling its conformation in the presence of long ssDNA (>30 nucleotides). Furthermore, the recruitment of the Tim-Tipin-RPA complex to ssDNA is modulated by the RPA conformation and requires RPA to be in the more compact 30 nt ssDNA binding mode. The dynamic formation and disruption of the Tim-Tipin-RPA-ssDNA complex implicates the RPA-based recruitment of Tim-Tipin to the replication fork. PMID:25348395

Witosch, Justine; Wolf, Eva; Mizuno, Naoko

2014-11-10

76

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

77

Recombinase polymerase amplification (RPA) of CaMV-35S promoter and nos terminator for rapid detection of genetically modified crops.  

Science.gov (United States)

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. PMID:25310647

Xu, Chao; Li, Liang; Jin, Wujun; Wan, Yusong

2014-01-01

78

Mutant IDH1-Driven Cellular Transformation Increases RAD51-Mediated Homologous Recombination and Temozolomide Resistance.  

Science.gov (United States)

Isocitrate dehydrogenase 1 (IDH1) mutations occur in most lower grade glioma and not only drive gliomagenesis but are also associated with longer patient survival and improved response to temozolomide. To investigate the possible causative relationship between these events, we introduced wild-type (WT) or mutant IDH1 into immortalized, untransformed human astrocytes, then monitored transformation status and temozolomide response. Temozolomide-sensitive parental cells exhibited DNA damage (?-H2AX foci) and a prolonged G2 cell-cycle arrest beginning three days after temozolomide (100 ?mol/L, 3 hours) exposure and persisting for more than four days. The same cells transformed by expression of mutant IDH1 exhibited a comparable degree of DNA damage and cell-cycle arrest, but both events resolved significantly faster in association with increased, rather than decreased, clonogenic survival. The increases in DNA damage processing, cell-cycle progression, and clonogenicity were unique to cells transformed by mutant IDH1, and were not noted in cells transformed by WT IDH1 or an oncogenic form (V12H) of Ras. Similarly, these effects were not noted following introduction of mutant IDH1 into Ras-transformed cells or established glioma cells. They were, however, associated with increased homologous recombination (HR) and could be reversed by the genetic or pharmacologic suppression of the HR DNA repair protein RAD51. These results show that mutant IDH1 drives a unique set of transformative events that indirectly enhance HR and facilitate repair of temozolomide-induced DNA damage and temozolomide resistance. The results also suggest that inhibitors of HR may be a viable means to enhance temozolomide response in IDH1-mutant glioma. Cancer Res; 74(17); 4836-44. ©2014 AACR. PMID:25035396

Ohba, Shigeo; Mukherjee, Joydeep; See, Wendy L; Pieper, Russell O

2014-09-01

79

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

International Nuclear Information System (INIS)

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

80

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

Energy Technology Data Exchange (ETDEWEB)

We evaluated the feasibility of non-invasive imaging of recombinant adenovirus-mediated human sodium-iodide symporter (hNIS) gene expression by {sup 99m}TcO{sub 4}{sup -} scintigraphy in skeletal muscle of rats. Replication-defective recombinant adenovirus encoding hNIS gene [Rad-CMV-hNIS 5 x 10{sup 7}, 2 x 10{sup 8} or 1 x 10{sup 9} plaque forming units (pfu)] or {beta}-galactosidase gene (Rad-CMV-LacZ 1 x 10{sup 9} pfu) was injected into the right biceps femoris muscle of rats (n=5-6 for each group). Three days after gene transfer, scintigraphy was performed using a gamma camera 30 min after injection of {sup 99m}TcO{sub 4}{sup -} (1.85 MBq). An additional two rats injected with 1 x 10{sup 9} pfu of Rad-CMV-hNIS underwent {sup 99m}TcO{sub 4}{sup -} scintigraphy with sodium perchlorate. After the imaging studies, rats were sacrificed for assessment of the biodistribution of {sup 99m}TcO{sub 4}{sup -} and measurement of hNIS mRNA expression. In all the rats injected with 1 x 10{sup 9} pfu of Rad-CMV-hNIS, hNIS expression was successfully imaged by {sup 99m}TcO{sub 4}{sup -} scintigraphy, while rats injected with Rad-CMV-LacZ or lower doses of Rad-CMV-hNIS failed to show uptake. The biodistribution studies indicated that a significantly different amount of {sup 99m}TcO{sub 4}{sup -} was retained in the liver (p<0.001) and the right muscle (p<0.05), with the highest uptake in rats injected with 1 x 10{sup 9} pfu of Rad-CMV-hNIS. The muscular hNIS mRNA level quantified by real-time reverse transcription-polymerase chain reaction was significantly higher in rats injected with 1 x 10{sup 9} pfu of Rad-CMV-hNIS (p<0.05), with a positive correlation with the imaging counts (r=0.810, p<0.05) and the biodistribution (r=0.847, p<0.001). Hot spots in rats injected with 1 x 10{sup 9} pfu of Rad-CMV-hNIS were specifically inhibited by sodium perchlorate. This study illustrated that {sup 99m}TcO{sub 4}{sup -} scintigraphy can monitor Rad-CMV-hNIS-mediated gene expression in skeletal muscle of rats, non-invasively and quantitatively. (orig.)

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

2004-09-01

 
 
 
 
81

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

82

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

Science.gov (United States)

Background Chronic hepatitis B is a primary cause of liver-related death. Interferon alpha (IFN-?) is able to inhibit the replication of hepadnavirus, and the sustained and stable expression of IFN-? at appropriate level may be beneficial to HBV clearance. With the development of molecular cloning technology, gene therapy plays a more and more important role in clinical practice. In light of the findings, an attempt to investigate the anti-HBV effects mediated by a eukaryotic expression plasmid (pSecTagB-IFN-?) in vitro was carried out. Methods HBV positive cell line HepG2.2.15 and its parental cell HepG2 were transfected with pSecTagB-IFN-? or empty plasmid by using Lipofectamine™ 2000 reagent. The expression levels of IFN-? were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA methods. The effects of pSecTagB-IFN-? on HBV mRNA, DNA and antigens were analyzed by real-time fluorescence quantitative PCR (qRT-PCR) and ELISA assays. RT-PCR, qRT-PCR and western blot were employed to investigate the influence of pSecTagB-IFN-? on IFN-?-induced signal pathway. Furthermore, through qRT-PCR and ELISA assays, the suppressive effects of endogenously expressed IFN-? and the combination with lamivudine on HBV were also examined. Results pSecTagB-IFN-? could express efficiently in hepatoma cells, and then inhibited HBV replication, characterized by the decrease of HBV S gene (HBs) and HBV C gene (HBc) mRNA, the reduction of HBV DNA load, and the low contents of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). Mechanism research showed that the activation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signal pathway, the up-regulation of IFN-?-induced antiviral effectors and double-stranded (ds) RNA sensing receptors by delivering pSecTagB-IFN-?, could be responsible for these phenomena. Furthermore, pSecTagB-IFN-? vector revealed effectively anti-HBV effect than exogenously added IFN-?. Moreover, lamivudine combined with endogenously expressed IFN-? exhibited stronger anti-HBV effect than with exogenous IFN-?. Conclusion Our results showed that endogenously expressed IFN-? can effectively and persistently inhibit HBV replication in HBV infected cells. These observations opened a promising way to design new antiviral genetic engineering drugs based on IFN-?. PMID:23984795

2013-01-01

83

Reducing the impact of PCR-mediated recombination in molecular evolution and environmental studies using a new-generation high-fidelity DNA polymerase.  

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PCR-mediated recombination can greatly impact estimates of diversity, both in environmental studies and in analyses of gene family evolution. Here we measure chimera (PCR-mediated recombinant) formation by analyzing a mixture of eight partial actin sequences isolated from the amoeba Arcella hemisphaerica amplified under a variety of conditions that mimic standard laboratory situations. We further compare a new-generation proofreading processivity-enhanced polymerase to both a standard proofreading enzyme and previously published results. Proofreading polymerases are preferred over other polymerases in instances where evolutionary inferences must be made. Our analyses reveal that reducing the initial template concentration is as critical as reducing the number of cycles for decreasing chimera formation and improving accuracy. Furthermore, assessing the efficiency of recovery of original haplotypes demonstrates that multiple PCR reactions are required to capture the actual genetic diversity of a sample. Finally, the experiments confirm that processivity-enhanced polymerases enable a substantial decrease in PCR-mediated recombination through reducing starting template concentration, without compromising the robustness of PCR reactions. PMID:19852769

Lahr, Daniel J G; Katz, Laura A

2009-10-01

84

NMR Analysis of the Architecture and Functional Remodeling of a Modular Multi-Domain Protein, RPA.  

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Modular proteins with multiple domains tethered by flexible linkers have variable global archiectures. Using the eukaryotic ssDNA binding protein, Replication Protein A (RPA), we demonstrate that NMR spectroscopy is a powerful tool to characterize the remodeling of architecture in different functional states. The first direct evidence is obtained for the remodeling of RPA upon binding ssDNA, including an alteration in the availability of the RPA32N domain that may help explain its damage-dependent phosphorylation. PMID:19378948

Brosey, Chris A.; Chagot, Marie-Eve; Ehrhardt, Mark; Pretto, Dalyir I.; Weiner, Brian E.; Chazin, Walter J.

2009-01-01

85

Corrections to the RPA Dielectric Function Induced by Fluctuations in the Momentum Distribution  

CERN Document Server

In this article, we introduce a new dielectric function that is different even from the generalised RPA, first introduced in cond-mat/9810043, in that it pays attention to fluctuations in the momentum distribution. We argued that this is important when the momentum distribution is very nonideal. This new dielectric function reduces to the generalised RPA when the number-fluctuations are small, and the generalised RPA in turn reduces to the traditional RPA dielectric function when the momentum distribution is close to ideal.

Setlur, G S; Setlur, Girish S.; Chang, Yia-Chung

1998-01-01

86

RPA and ATR link transcriptional stress to p53.  

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The mechanisms by which DNA-damaging agents trigger the induction of the stress response protein p53 are poorly understood but may involve alterations of chromatin structure or blockage of either transcription or replication. Here we show that transcription-blocking agents can induce phosphorylation of the Ser-15 site of p53 in a replication-independent manner. Furthermore, microinjection of anti-RNA polymerase II antibodies into the nuclei of cells showed that blockage of transcription is sufficient for p53 accumulation even in the absence of DNA damage. This induction of p53 occurs by two independent mechanisms. First, accumulation of p53 is linked to diminished nuclear export of mRNA; and second, inhibition specifically of elongating RNA polymerase II complexes results in the phosphorylation of the Ser-15 site of p53 in a replication protein A (RPA)- and ATM and Rad3-related (ATR)-dependent manner. We propose that this transcription-based stress response involving RPA, ATR, and p53 has evolved as a DNA damage-sensing mechanism to safeguard cells against DNA damage-induced mutagenesis. PMID:17616578

Derheimer, Frederick A; O'Hagan, Heather M; Krueger, Heather M; Hanasoge, Sheela; Paulsen, Michelle T; Ljungman, Mats

2007-07-31

87

Quantal Brownian Motion from RPA dynamics: The master and Fokker-Planck equations  

International Nuclear Information System (INIS)

From the purely quantal RPA description of the damped harmonic oscillator and of the corresponding Brownian Motion within the full space (phonon subspace plus reservoir), a master equation (as well as a Fokker-Planck equation) for the reduced density matrix (for the reduced Wigner function, respectively) within the phonon subspace is extracted. The RPA master equation agrees with the master equation derived by the time-dependent perturbative approaches which utilize Tamm-Dancoff Hilbert spaces and invoke the rotating wave approximation. Since the RPA yields a full, as well as a contracted description, it can account for both the kinetic and the unperturbed oscillator momenta. The RPA description of the quantal Brownian Motion contrasts with the descriptions provided by the time perturbative approaches whether they invoke or not the rotating wave approximation. The RPA description also contrasts with the phenomenological phase space quantization. (orig.)

88

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

89

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

Science.gov (United States)

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

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

2013-12-01

90

p16INK4a impairs homologous recombination-mediated DNA repair in human papillomavirus-positive head and neck tumors.  

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The p16INK4a protein is a principal cyclin-dependent kinase inhibitor that decelerates the cell cycle. Abnormally high levels of p16INK4a are commonly observed in human papillomavirus (HPV)-positive head and neck squamous cell carcinomas (HNSCC). We and others found that p16INK4a overexpression is associated with improved therapy response and survival of patients with HNSCC treated with radiotherapy. However, the functional role of p16INK4a in HNSCC remains unexplored. Our results implicate p16INK4a in regulation of homologous recombination-mediated DNA damage response independently from its role in control of the cell cycle. We found that expression of p16INK4a dramatically affects radiation sensitivity of HNSCC cells. p16INK4a overexpression impairs the recruitment of RAD51 to the site of DNA damage in HPV-positive cells by downregulating of cyclin D1 protein expression. Consistent with the in vitro findings, immunostaining of HNSCC patient samples revealed that high levels p16INK4a expression significantly correlated with decreased cyclin D1 expression. In summary, these findings reveal an unexpected function of p16INK4a in homologous recombination-mediated DNA repair response and imply p16INK4a status as an independent marker to predict response of patients with HNSCC to radiotherapy. PMID:24473065

Dok, Rüveyda; Kalev, Peter; Van Limbergen, Evert Jan; Asbagh, Layka Abbasi; Vázquez, Iria; Hauben, Esther; Sablina, Anna; Nuyts, Sandra

2014-03-15

91

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

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Replication protein A (RPA) is an essential eukaryotic single-stranded DNA binding protein with a central role in DNA metabolism. RPA directly participates in DNA double-strand break repair by stimulating 5'-3' end resection by the Sgs1/BLM helicase and Dna2 endonuclease in vitro. Here we investigated the role of RPA in end resection in vivo, using a heat-inducible degron system that allows rapid conditional depletion of RPA in Saccharomyces cerevisiae. We found that RPA depletion eliminated both the Sgs1-Dna2- and Exo1-dependent extensive resection pathways and synergized with mre11? to prevent end resection. The short single-stranded DNA tails formed in the absence of RPA were unstable due to 3' strand loss and the formation of fold-back hairpin structures that required resection initiation and Pol32-dependent DNA synthesis. Thus, RPA is required to generate ssDNA, and also to protect ssDNA from degradation and inappropriate annealing that could lead to genome rearrangements. PMID:23706822

Chen, Huan; Lisby, Michael; Symington, Lorraine S

2013-05-23

92

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

Energy Technology Data Exchange (ETDEWEB)

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

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

2013-07-01

93

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

International Nuclear Information System (INIS)

0.05). 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

94

Synergistic antitumor effect of recombinant adeno-associated virus-mediated pigment epithelium-derived factor with hyperthermia on solid tumor.  

Science.gov (United States)

Adeno-associated virus (AAV) is an ideal choice for gene delivery; however, its further development has been limited owing to its low transduction efficiency. DNA-damaging agents can improve AAV-mediated transgene expression. Hyperthermia, as one of the oldest documented tumor treatment modalities, can cause DNA damage as well. However, combined treatment consisting of hyperthermia and AAV-mediated gene therapy has not been reported yet. In this work we investigated whether therapy consisting of AAV-mediated pigment epithelium-derived factor (PEDF) delivery combined with hyperthermia has synergistic antitumor effect on established solid tumors. We produced the recombinant AAV encoding PEDF (rAAV-PEDF). The therapeutic effect of rAAV-PEDF plus hyperthermia was evaluated in a subcutaneous fibrosarcoma mouse model, and the possible mechanism of antitumor effect was investigated. We found that rAAV-PEDF could infect a murine fibrosarcoma cell line (Meth-A) and express PEDF protein with bioactivity in vitro. In addition, in vivo experiments suggested that the combination of rAAV-PEDF with hyperthermia could significantly suppress tumor growth and prolong survival time of treated mice. Immunofluorescence studies indicated that the combination therapy could inhibit angiogenesis and induce apoptosis in tumor tissues. An immunohistochemistry assay of tumor tissue showed that PEDF expression in the combined treatment group was significantly higher than in the rAAV-PEDF group, which implied that hyperthermia could improve the expression of PEDF protein in vivo. No significant differences were observed in each group by hematoxylin-eosin staining of major organs, serum chemistry test, and complete blood assay. These results indicate that the combination of rAAV-PEDF with hyperthermia has synergistic therapeutic effects on established solid tumors, with no side effects. In addition, hyperthermia could improve AAV-mediated transgene expression, which suggests that hyperthermia could serve as a promising adjunctive therapy for AAV-mediated gene therapy. PMID:25003563

Wu, Qinjie; He, Shasha; Wei, Xiawei; Shao, Bin; Luo, Shuntao; Guo, Fuchun; Zhang, Hailong; Wang, Yongsheng; Gong, Changyang; Yang, Li

2014-09-01

95

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

International Nuclear Information System (INIS)

Objective: To study the combined effect of exogenous recombinant adenovirus-medicated retinoblastoma gene 94 (Ad-Rb94) combined with ?-ray on the growth of esophageal carcinoma cells. Methods: Cell culture were randomly divided into 5 groups: control group, recombinant adenovirus vector containing ?-galactosidase gene (Ad-LacZ) group, Ad-Rb94 group, ?-ray radiation group and Ad-Rb94 combined with ?-ray radiation group. EC109 cells were transfected by Ad-Rb94 and exposed to 4 Gy 137Cs ?-ray irradiation 6 hours after transfection. The inhibition rate of EC109 cells were detected by MTT assay. Results: EC109 cells transfected with Ad-Rb94 group, ?-ray radiation group and Ad-Rb94 combined with ?-ray radiation group were all inhibited. The inhibition rate of Ad-Rb94 combined with ?ray radiation group reached (40.30%±4.2%), significantly higher than Ad-Rb94 group (18.3±0.4%) and ?-ray radiation group (27.40%±2.9%) (?2=7.91, ?2=5.82, P2=5.12, P<0.05). Conclusion: The recombinant Ad-Rb94 gene transfection combined with ?-ray shows the synergism for the inhibition of the growth of EC109 cells. (authors)

96

Effective Force for Nuclear Random Phase Approximation (RPA)  

International Nuclear Information System (INIS)

The theoretical evaluation of the effective force operators for nuclear structure models is achieved within different approximate schemes. Some of the matrix elements of the force result from the study of the association energies of nucleons, which involves various assumptions of nuclear coupling. Further information on the effective force is obtained by solving equations for the reaction-matrix - a reliable operator substituting the realistic inter-nucleon force - and also from the data available on the nuclear-matter properties. When comparing this information with 'experiment' it should be remembered that each of the models is characterized by its own effective force evaluated from appropriate equations. Much attention is given to the precise definition of the effective force for a particular model - the random phase approximation (RPA) - and the relationship is established between the effective force operator and the reaction-matrix, on the one hand, and the scattering amplitude in the Fermi-liquid theory, on the other. Some estimates for the zero-range effective force are given. These are obtained from the data on the density dependence of the local part of the nuclear self-consistent potential. The resulting operator depends on the density itself. (author)

97

Conserving RPA and the response of 48Ca  

International Nuclear Information System (INIS)

The connection between the single-particle self-energy and the corresponding conserving particle-hole (ph) interaction, discussed long ago by Kadanoff and Baym, is employed to study the response of 48Ca. Second order self-energy contributions are taken into account in the construction of the energy dependent ph interaction. From this perspective it is possible to make contact with other approaches which also aim to incorporate the coupling to 2p2h excitations within the RPA framework. The method is used to study both the discrete low-energy states as well as the giant resonances in both 48Ca and 48Sc using a realistic G matrix interaction based on meson exchange. The calculated strength distribution compares favorably with experimental but the strength below 15 MeV is still somewhat too large as compared to experiment for all types of excitations. The quenching of magnetic and Gamow-Teller strength due to 2p2h admixture amounts to about 30%. (orig.)

98

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

International Nuclear Information System (INIS)

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

99

Scattering in partide-hole: simple approximations to nuclear RPA calculations in the continum  

International Nuclear Information System (INIS)

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

100

Induction of protective CD4+ T cell-mediated immunity by a Leishmania peptide delivered in recombinant influenza viruses.  

Science.gov (United States)

The available evidence suggests that protective immunity to Leishmania is achieved by priming the CD4(+) Th1 response. Therefore, we utilised a reverse genetics strategy to generate influenza A viruses to deliver an immunogenic Leishmania peptide. The single, immunodominant Leishmania-specific LACK(158-173) CD4(+) peptide was engineered into the neuraminidase stalk of H1N1 and H3N2 influenza A viruses. These recombinant viruses were used to vaccinate susceptible BALB/c mice to determine whether the resultant LACK(158-173)-specific CD4(+) T cell responses protected against live L. major infection. We show that vaccination with influenza-LACK(158-173) triggers LACK(158-173)-specific Th1-biased CD4(+) T cell responses within an appropriate cytokine milieu (IFN-?, IL-12), essential for the magnitude and quality of the Th1 response. A single intraperitoneal exposure (non-replicative route of immunisation) to recombinant influenza delivers immunogenic peptides, leading to a marked reduction (2-4 log) in parasite burden, albeit without reduction in lesion size. This correlated with increased numbers of IFN-?-producing CD4(+) T cells in vaccinated mice compared to controls. Importantly, the subsequent prime-boost approach with a serologically distinct strain of influenza (H1N1->H3N2) expressing LACK(158-173) led to a marked reduction in both lesion size and parasite burdens in vaccination trials. This protection correlated with high levels of IFN-? producing cells in the spleen, which were maintained for 6 weeks post-challenge indicating the longevity of this protective effector response. Thus, these experiments show that Leishmania-derived peptides delivered in the context of recombinant influenza viruses are immunogenic in vivo, and warrant investigation of similar vaccine strategies to generate parasite-specific immunity. PMID:22470440

Kedzierska, Katherine; Curtis, Joan M; Valkenburg, Sophie A; Hatton, Lauren A; Kiu, Hiu; Doherty, Peter C; Kedzierski, Lukasz

2012-01-01

 
 
 
 
101

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 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

102

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

Directory of Open Access Journals (Sweden)

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

Zhang Deshui

2012-11-01

103

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

Science.gov (United States)

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

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

1995-04-01

104

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

Science.gov (United States)

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

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

2014-03-01

105

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

106

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

107

Description of the 2? ?? decay within a fully-renormalized RPA approach  

International Nuclear Information System (INIS)

The RPA treatment of a many body Hamiltonian describing the states of even-even nuclei involved in a 2??? decay is revisited. One shows that re-normalizing the dipole two quasiparticle operators by accounting for new correlations in the ground state requires a similar re-normalization for the dipole density operators which results in activating new boson degrees of freedom. Possible consequences on Ikeda sum rule and Gamow-Teller (GT) transition amplitude are suggested. A numerical application for a two levels model is presented. The present formalism is hereafter referred to as the frn RPA (full re-normalised Random Phase Approximation). The equations for the new RPA amplitudes and energies were analytically derived. The solutions having the new terms amplitudes dominant define a new proton-neutron dipole mode. It is proved that the new mode appears as a result of a partial restoration of the Pauli principle. The QRPA equations and the equations defining the averages of the quasiparticle number operators are to be self consistently solved by an iterative procedure. Within the new QRPA procedure analytical formulae for the Ikeda sum rule and the GT transition amplitude were derived. The frn RPA has been applied to the case of a single level for protons and a single level for neutrons. The frn RPA equations exhibit two solutions, the lower one characterizing the new mode, i.e. that whose maximum amplitude is Z. Although the other mode has an energy higher than the energies provided by the standard and the rn RPA approaches, the frn RPA breaks down before the normal RPA and this happens due to the collapse of the new mode. Before the frn RPA breaks down the GT transition amplitude vanishes. In the new approach the Ikeda sum rule is reasonably well reproduced. Comparing the results of the present approach with those of the rn RPA it is worth enumerating the contrasting features. I) a) The rn RPA exhibits the beauty of avoiding the collapse of the mode energy. b) However the Ikeda sum rule is dramatically violated. c) The new dynamic which is added to the standard RPA is not consistently accounted for, i.e. it neglects terms which are of the same order of magnitude with those retained in addition to the standard RPA ones. d) It keeps the main feature of the standard RPA consisting in that boson is a superposition of the two quasiparticle operators A1?+ (p,n) and A1-?(p,n)(-)1-?. However the corresponding amplitudes are renormalized. II) a) The frn RPA takes into account, self consistently, all terms of the model Hamiltonian which lead to quasiparticle correlations in the ground state. In this way, the apparent advantage of the rn RPA (obtained with the price of an non-consistency) of avoiding the energy collapse is removed. b) The Ikeda sum rule is, to a large extent, restored. c) A new dipole mode in the low part of the spectrum of the intermediate odd-odd nucleus is described by the present approach. Since for the realistic value of gpp (? 1.0) the share of the ?(-) strength is reasonable large, such a state might be populated through a single ?- process. d) The specific feature of the boson consists of that it includes the scattering terms B1?+ (p,n) and B1-? (p,n)(-)1-?. The presence of these terms brings new correlations in the ground state and modifies the normalizations of operators A1?+ (p,n) and A1-?(p,n)(-)1-?. The formalism is extended to the Fermi transition. Restricting the single particle space to one state one obtains a solvable many body Hamiltonian. The energies of the RPA, rn-RPA, the second frn-RPA and the second excitation energy of H(0) are very close to each other. The first excited state provided by the diagonalization procedure is interpreted as a two frn-RPA boson state. By this, the existence of a low lying non-spurious state is once more confirmed. (authors)

108

Ab initio self-consistent total-energy calculations within the EXX/RPA formalism  

Science.gov (United States)

Calculations of exact-exchange (EXX) and random phase approximation (RPA)-correlation energies within the formally exact adiabatic connection fluctuation-dissipation theorem formalism have recently been carried out for a number of isolated and condensed systems. Unfortunately, most of the applications have been done in a non-self-consistent procedure, and for several systems it has been found that RPA correlation energies may significantly depend on the choice of input single-particle wave functions. In this work, we develop an efficient approach to compute the EXX/RPA total energy self-consistently. We derive an expression for the RPA self-consistent potential based on the density functional perturbation theory and dielectric matrix approaches and implemented it within the plane-wave pseudopotential framework. The efficiency of this approach is greatly improved by exploiting an iterative procedure to compute the inverted Kohn-Sham density-density response function. We apply our implementation to study the binding energy curves and the structural properties of rare gasses such as Ar and Kr and alkaline-earth Be dimers. In addition, the EXX and RPA-correlation potentials of these systems at different dissociation distances are analyzed.

Nguyen, Ngoc Linh; Colonna, Nicola; de Gironcoli, Stefano

2014-07-01

109

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

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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

110

Targeted Gene Addition in Human Epithelial Stem Cells by Zinc-finger Nuclease-mediated Homologous Recombination  

Science.gov (United States)

Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a “safe harbor” locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of >20% in a human keratinocyte cell line, >10% in immortalized keratinocytes, and <1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs. PMID:23760447

Coluccio, Andrea; Miselli, Francesca; Lombardo, Angelo; Marconi, Alessandra; Malagoli Tagliazucchi, Guidantonio; Goncalves, Manuel A; Pincelli, Carlo; Maruggi, Giulietta; Del Rio, Marcela; Naldini, Luigi; Larcher, Fernando; Mavilio, Fulvio; Recchia, Alessandra

2013-01-01

111

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

112

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

Science.gov (United States)

The importance of therapeutic recombinant proteins in medicine has led to a variety of tactics to increase their circulation time or to enable routes of administration other than injection. One clinically successful tactic to improve both protein circulation and delivery is to fuse the Fc domain of IgG to therapeutic proteins so that the resulting fusion proteins interact with the human neonatal Fc receptor (FcRn). As an alternative to grafting the high molecular weight Fc domain to therapeutic proteins, we have modified their N and/or C termini with a short peptide sequence that interacts with FcRn. Our strategy was motivated by results [Mezo AR, et al. (2008) Proc Natl Acad Sci USA 105:2337-2342] that identified peptides that compete with human IgG for FcRn. The small size and simple structure of the FcRn-binding peptide (FcBP) allows for expression of FcBP fusion proteins in Escherichia coli and results in their pH-dependent binding to FcRn with an affinity comparable to that of IgG. The FcBP fusion proteins are internalized, recycled, and transcytosed across cell monolayers that express FcRn. This strategy has the potential to improve protein transport across epithelial barriers, which could lead to noninvasive administration and also enable longer half-lives of therapeutic proteins. PMID:22991460

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

2012-10-01

113

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

114

Recombinant IL-21 and anti-CD4 antibodies cooperate in syngeneic neuroblastoma immunotherapy and mediate long-lasting immunity.  

Science.gov (United States)

IL-21 is an immune-enhancing cytokine, which showed promising results in cancer immunotherapy. We previously observed that the administration of anti-CD4 cell-depleting antibody strongly enhanced the anti-tumor effects of an IL-21-engineered neuroblastoma (NB) cell vaccine. Here, we studied the therapeutic effects of a combination of recombinant (r) IL-21 and anti-CD4 monoclonal antibodies (mAb) in a syngeneic model of disseminated NB. Subcutaneous rIL-21 therapy at 0.5 or 1 ?g/dose (at days 2, 6, 9, 13 and 15 after NB induction) had a limited effect on NB development. However, coadministration of rIL-21 at the two dose levels and a cell-depleting anti-CD4 mAb cured 28 and 70 % of mice, respectively. Combined immunotherapy was also effective if started 7 days after NB implant, resulting in a 30 % cure rate. Anti-CD4 antibody treatment efficiently depleted CD4(+) CD25(high) Treg cells, but alone had limited impact on NB. Combination immunotherapy by anti-CD4 mAb and rIL-21 induced a CD8(+) cytotoxic T lymphocyte response, which resulted in tumor eradication and long-lasting immunity. CD4(+) T cells, which re-populated mice after combination immunotherapy, were required for immunity to NB antigens as indicated by CD4(+) T cell depletion and re-challenge experiments. In conclusion, these data support a role for regulatory CD4(+) T cells in a syngeneic NB model and suggest that rIL-21 combined with CD4(+) T cell depletion reprograms CD4(+) T cells from immune regulatory to anti-tumor functions. These observations open new perspectives for the use of IL-21-based immunotherapy in conjunction with transient CD4(+) T cell depletion, in human metastatic NB. PMID:24647609

Rigo, Valentina; Corrias, Maria Valeria; Orengo, Anna Maria; Brizzolara, Antonella; Emionite, Laura; Fenoglio, Daniela; Filaci, Gilberto; Croce, Michela; Ferrini, Silvano

2014-05-01

115

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

Science.gov (United States)

Prior work using allogeneic bone marrow transplantation (allo-BMT) models showed that peritransplant administration of flagellin, a toll-like receptor 5 (TLR5) agonist protected murine allo-BMT recipients from CMV infection while limiting graft-vs-host disease (GvHD). However, the mechanism by which flagellin-TLR5 interaction promotes anti-CMV immunity was not defined. Here, we investigated the anti-CMV immunity of NK cells in C57BL/6 (B6) mice treated with a highly purified cGMP grade recombinant flagellin variant CBLB502 (rflagellin) followed by murine CMV (mCMV) infection. A single dose of rflagellin administered to mice between 48 to 72 hours prior to MCMV infection resulted in optimal protection from mCMV lethality. Anti-mCMV immunity in rflagellin-treated mice correlated with a significantly reduced liver viral load and increased numbers of Ly49H+ and Ly49D+ activated cytotoxic NK cells. Additionally, the increased anti-mCMV immunity of NK cells was directly correlated with increased numbers of IFN-?, granzyme B- and CD107a producing NK cells following mCMV infection. rFlagellin-induced anti-mCMV immunity was TLR5-dependent as rflagellin-treated TLR5 KO mice had ?10-fold increased liver viral load compared with rflagellin-treated WT B6 mice. However, the increased anti-mCMV immunity of NK cells in rflagellin-treated mice is regulated indirectly as mouse NK cells do not express TLR5. Collectively, these data suggest that rflagellin treatment indirectly leads to activation of NK cells, which may be an important adjunct benefit of administering rflagellin in allo-BMT recipients. PMID:24879439

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

2014-01-01

116

Immunological evaluation of patients with hematological malignancies receiving ambulatory cytokine-mediated immunotherapy with recombinant human interferon-alpha 2a and interleukin-2.  

Science.gov (United States)

Immunological parameters were evaluated in patients treated with cytokine-mediated immunotherapy (CMI) consisting of low doses of recombinant human interferon alpha 2a (rIFN alpha) and recombinant human interleukin-2 (rIL-2) administered either concomitantly or sequentially by subcutaneous self-injections in an outpatient setting. Twenty-six patients with hematological malignancies and 2 metastatic melanoma patients in a progressive stage were enrolled in this clinical trial. Of the 26 patients, 24 were at a stage of minimal residual disease, including 14 patients who had received autologous bone marrow transplantation (ABMT) 2-5 months previously, 7 chronic myelogenous leukemia (CML) and 3 acute myeloid leukemia (AML) patients. Two patients (1 CML and 1 mult. myeloma) were treated at a stage of progressive disease. Non-MHC-restricted cytotoxicity directed against natural-killer(NK)-resistant (Daudi) and NK-sensitive (K562) target cells was assessed before, during and after CMI, either in fresh peripheral blood samples (spontaneous activity) or after in vitro rIL-2 activation (induced activity). Spontaneous killing activity was low prior to treatment, but increased upon termination of treatment in 10/15 evaluated cycels. rIL-2-activated cytotoxicity in vitro was markedly elevated in 8/12 and 6/8 patients after one and two cycles, respectively, of sequential treatment, as well as in 3/8 CML and 5/6 patients after one and two cycles, respectively, of concomitant treatment. Activation of the T cell mitogenic response was demonstrated in 6/9 patients after concomitant CMI, while no such effect was observed throughout a sequential treatment in lymphoma and leukemia patients after ABMT. Although a direct correlation between immune stimulation and the in vivo antitumor response cannot yet be determined, our clinical observations support a beneficial therapeutic effect in a substantial number of patients. These results indicated that the ambulatory CMI protocol of rIL-2 and rIFN alpha could stimulate the host defense immune system and may be helpful in mediating the in vivo antitumor response in patients with minimal residual disease. PMID:1394343

Morecki, S; Revel-Vilk, S; Nabet, C; Pick, M; Ackerstein, A; Nagler, A; Naparstek, E; Ben Shahar, M; Slavin, S

1992-01-01

117

[Identification of cholinoreceptors on the soma of RPa3 and LPa3 neurons in Helix lucorum].  

Science.gov (United States)

Selective cholinomimetics and cholinolytics were employed to identify cholinoreceptors on the somatic membrane in Helix lucorum RPa3 and LPa3 neurons using recording of transmembrane ionic currents. Agonists of nicotinic and muscarinic cholinoreceptors evoked reversible activation and following nonreversible inhibition of receptors (except carbamylcholine). Selective cholinomimetics were connected with the same membrane sites that were activated by acetylcholine. Nicotinic and muscarinic cholinolytics decreased the amplitude of inward current induced by acetylcholine (scopolamine and platyphylline had no effect). It is concluded that somatic membrane of RPa3 and LPa3 Helix neurons contains nicotinic and muscarinic cholinoreceptors. These receptors have several pharmacological peculiarities as compared with those of the vertebrates. Muscarinic cholinoreceptors of RPa3 and LPa3 neurons must be included into a special subtype that differs from well-known M1 and M2 subtypes. PMID:1584310

Pivovarov, A S; Drozdova, E I

1992-01-01

118

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

119

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

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Full Text Available 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 deliver the highly homologous viral IL-10 (vIL-10, which is predominantly anti-inflammatory with minimal pro-inflammatory activities, can potentially be a more effective strategy to combat inflammatory lung diseases. In this study, we compare the use of PFC liquid versus aerosolized method to deliver adenovirus encoding the vIL-10 gene (AdvIL-10 in C57Bl6 mice. Detectable vIL-10 levels were measured from bronchoalveolar lavage fluid and lung homogenates at one, four, ten and thirty days after AdvIL-10. Furthermore, we determined if use of PFC liquid could allow for the use of a lower dose of AdvIL-10 by comparing the levels of detectable vIL-10 at different doses of AdvIL-10 delivered +/- PFC liquid. Results showed that PFC liquid enhanced detectable vIL-10 by up to ten fold and that PFC liquid allowed the use of ten-fold less vector. PFC liquid increased detectable vIL-10 in lung homogenates at all time points; however, the increase in detectable vIL-10 in BAL fluid peaked at four days and was no longer evident by thirty days after intratracheal instillation. In summary, this is the first report utilizing PFC liquid to enhance the delivery of a potentially therapeutic molecule, vIL-10. We believe this strategy can be used to perform future studies on the use of the predominantly anti-inflammatory vIL-10 to treat inflammatory lung diseases.

Zimmerman Jerry J

2007-05-01

120

Assays of dioxins and dioxin-like compounds in actually contaminated soils using transgenic tobacco plants carrying a recombinant mouse aryl hydrocarbon receptor-mediated ?-glucuronidase reporter gene expression system  

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The transgenic tobacco plant XD4V-26 carrying the recombinant mouse aryl hydrocarbon receptor XD4V-mediated ?-glucuronidase (GUS) reporter gene expression system was used for assay of dioxins and dioxin-like compounds consisting of polychlorodibenzo-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls (Co-PCBs) in actually contaminated soils. The transgenic tobacco plant XD4V-26 showed a significant dose-dependent induced GUS activity when cultured on MS medium co...

Inui, Hideyuki; Gion, Keiko; Utani, Yasushi; Wakai, Taketo; Kodama, Susumu; Eun, Heesoo; Kim, Yun-seok; Ohkawa, Hideo

2012-01-01

 
 
 
 
121

Spin waves in the 2D Hubbard model beyond the RPA  

International Nuclear Information System (INIS)

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

122

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

2014-01-01

123

Improvement in the stability and functionality of Nicotiana tabacum produced recombinant TRAIL through employment of endoplasmic reticulum expression and ascorbate buffer mediated extraction strategies  

Science.gov (United States)

Introduction: In order to employ Nicotiana tabacum cells as a profitable natural bioreactor for production of bio-functional "Soluble human TRAIL" (ShTRAIL), endoplasmic reticulum (ER) targeted expression and innovative extraction procedures were exploited. Methods: At first, the ShTRAIL encoding gene was sub-cloned into designed H2 helper vector to equip it with potent TMV omega leader sequences, ER sorting signal peptide, poly-histidine tag and ER retention signal peptide (KDEL). Then, the ER targeted ShTRAIL cassette was sequentially sub-cloned into "CaMV-35S" helper and "pGreen-0179" final expression vectors. Afterward, Agrobacterium mediated transformation method was adopted to express the ShTRAIL in the ER of N. tabacum . Next, the ShTRAIL protein was extracted through both phosphate and innovative ascorbate extraction buffers. Subsequently, oligomerization state of the ShTRAIL was evaluated through cross-linking assay and western blot analysis. Then, semi-quantitative western blot analysis was performed to estimate the ShTRAIL production. Finally, biological activity of the ShTRAIL was evaluated through MTT assay. Results: The phosphate buffer extracted ShTRAIL was produced in dimmer form, whereas the ShTRAIL extracted with ascorbate buffer generated trimer form. The ER targeted ShTRAIL strategy increased the ShTRAIL’s production level up to about 20 ?g/g of fresh weight of N. tabacum . MTT assay indicated that ascorbate buffer extracted ShTRAIL could prohibit proliferation of A549 cell line. Conclusion: Endoplasmic reticulum expression and reductive ascorbate buffer extraction procedure can be employed to enhance the stability and overall production level of bio-functional recombinant ShTRAIL from transgenic N. tabacum cells. PMID:25337465

Heidari, Hamid Reza; Bandehpour, Mojgan; Vahidi, Hossein; Barar, Jaleh; Kazemi, Bahram; Naderi-Manesh, Hossein

2014-01-01

124

TERRA and hnRNPA1 orchestrate an RPA-to-POT1 switch on telomeric single-stranded DNA.  

Science.gov (United States)

Maintenance of telomeres requires both DNA replication and telomere 'capping' by shelterin. These two processes use two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomeres 1 (POT1). Although RPA and POT1 each have a critical role at telomeres, how they function in concert is not clear. POT1 ablation leads to activation of the ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase at telomeres, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. Unexpectedly, we found that purified POT1 and its functional partner TPP1 are unable to prevent RPA binding to telomeric ssDNA efficiently. In cell extracts, we identified a novel activity that specifically displaces RPA, but not POT1, from telomeric ssDNA. Using purified protein, here we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) recapitulates the RPA displacing activity. The RPA displacing activity is inhibited by the telomeric repeat-containing RNA (TERRA) in early S phase, but is then unleashed in late S phase when TERRA levels decline at telomeres. Interestingly, TERRA also promotes POT1 binding to telomeric ssDNA by removing hnRNPA1, suggesting that the re-accumulation of TERRA after S phase helps to complete the RPA-to-POT1 switch on telomeric ssDNA. Together, our data suggest that hnRNPA1, TERRA and POT1 act in concert to displace RPA from telomeric ssDNA after DNA replication, and promote telomere capping to preserve genomic integrity. PMID:21399625

Flynn, Rachel Litman; Centore, Richard C; O'Sullivan, Roderick J; Rai, Rekha; Tse, Alice; Songyang, Zhou; Chang, Sandy; Karlseder, Jan; Zou, Lee

2011-03-24

125

Second RPA calculations of the charge-exchange quadrupole response functions in closed-shell nuclei  

International Nuclear Information System (INIS)

A microscopic model is used to evaluate the damping widts of the charge-exchange quadrupole states in five nuclei ranging from 40Ca to 208Pb. The coupling among the 1p1h states is described in the framework of the self-consistent Hartree-Fock+charge-exchange RPA theory and the coupling between the 1p1h and 2p2h configurations is obtained within the charge-exchange second RPA theory. The Skyrme III interaction is used in these calculations. We discuss the relation between the mean-field damping (Landau damping) and the one arising from the dissipative mechanism and indicative the relevance of these calculations for the analysis of the charge-exchange reactions. (orig.)

126

Second RPA calculations of the charge-exchange quadrupole response functions in closed-shell nuclei  

Energy Technology Data Exchange (ETDEWEB)

A microscopic model is used to evaluate the damping widts of the charge-exchange quadrupole states in five nuclei ranging from [sup 40]Ca to [sup 208]Pb. The coupling among the 1p1h states is described in the framework of the self-consistent Hartree-Fock+charge-exchange RPA theory and the coupling between the 1p1h and 2p2h configurations is obtained within the charge-exchange second RPA theory. The Skyrme III interaction is used in these calculations. We discuss the relation between the mean-field damping (Landau damping) and the one arising from the dissipative mechanism and indicative the relevance of these calculations for the analysis of the charge-exchange reactions. (orig.)

Ait-Tahar, S. (Theoretical Physics Department, University of Oxford (United Kingdom) Physics Department, The Open University, Milton Keynes (United Kingdom)); Brink, D.M. (Theoretical Physics Department, University of Oxford (United Kingdom))

1993-07-26

127

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

128

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

129

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)

130

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

International Nuclear Information System (INIS)

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

131

Angular-momentum projection for Hartree-Fock and RPA with realistic interactions  

International Nuclear Information System (INIS)

Hartree-Fock (HF) with a Hamiltonian constructed from similarity transformed realistic NN potentials plus 3N contact interactions provides a good starting point for the description of closed shell nuclei. In conjunction with Many-Body-Perturbation-Theory, experimental ground-state energies and radii are well reproduced. To describe collective excitations, the Random-Phase-Approximation (RPA) is the method of choice. Beyond closed shells, e.g. in the sd-shell region, ground-states might exhibit intrinsic deformation, resulting in HF states where angular-momentum ceases to be a good quantum number. Lab-frame observables, like ground-state energies or rotational bands can be recovered from the intrinsic states via angular-momentum projection. We study axially deformed even-even sd-shell nuclei, namely 20Ne, 28Si and 32S. Starting from a HF ground state obtained by exact angular-momentum projection, we use the RPA to study collective excitations. The transition strengths obtained from the RPA are projected to good angular momentum in an exact formalism, without resorting to popular approximations. We investigate the effect of deformed intrinsic states on giant resonances.

132

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

International Nuclear Information System (INIS)

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

133

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

134

The proteasome factor Bag101 binds to Rad22 and suppresses homologous recombination  

Science.gov (United States)

Although RAD52 plays a critical role in the initiation of homologous recombination (HR) by facilitating the replacement of RPA with RAD51, the mechanism controlling RAD52 remains elusive. Here, we show that Bag101, a factor implicated in proteasome functioning, regulates RAD52 protein levels and subsequent HR. LC-MS/MS analysis identified Bag101 which binds to Rad22, the fission yeast homologue of RAD52. Bag101 reduced HR frequency through its overexpression and conversely, HR frequencies were enhanced when it was deleted. Consistent with this observation, Rad22 protein levels was reduced in cells where bag101 was overexpressed even when Rad22 transcription was up-regulated, suggesting the operation of proteasome-mediated Rad22 degradation. Indeed, Rad22 protein levels were stabilized in proteasome mutants. Rad22 physically interacted with the BAG domain of Bag101, and a lack of this domain enhanced HR frequency. Similarly, radiation exposure triggered the dissociation of these proteins so that Rad22 was stabilized and able to enhance HR. PMID:23779158

Saito, Yuichiro; Takeda, Jun; Okada, Masahiro; Kobayashi, Junya; Kato, Akihiro; Hirota, Kouji; Taoka, Masato; Matsumoto, Tomohiro; Komatsu, Kenshi; Isobe, Toshiaki

2013-01-01

135

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

Science.gov (United States)

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

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

2014-05-01

136

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

DEFF Research Database (Denmark)

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

Gao, Min; Wei, Wei

2014-01-01

137

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

138

A novel checkpoint and RPA inhibitory pathway regulated by Rif1.  

Science.gov (United States)

Cells accumulate single-stranded DNA (ssDNA) when telomere capping, DNA replication, or DNA repair is impeded. This accumulation leads to cell cycle arrest through activating the DNA-damage checkpoints involved in cancer protection. Hence, ssDNA accumulation could be an anti-cancer mechanism. However, ssDNA has to accumulate above a certain threshold to activate checkpoints. What determines this checkpoint-activation threshold is an important, yet unanswered question. Here we identify Rif1 (Rap1-Interacting Factor 1) as a threshold-setter. Following telomere uncapping, we show that budding yeast Rif1 has unprecedented effects for a protein, inhibiting the recruitment of checkpoint proteins and RPA (Replication Protein A) to damaged chromosome regions, without significantly affecting the accumulation of ssDNA at those regions. Using chromatin immuno-precipitation, we provide evidence that Rif1 acts as a molecular "band-aid" for ssDNA lesions, associating with DNA damage independently of Rap1. In consequence, small or incipient lesions are protected from RPA and checkpoint proteins. When longer stretches of ssDNA are generated, they extend beyond the junction-proximal Rif1-protected regions. In consequence, the damage is detected and checkpoint signals are fired, resulting in cell cycle arrest. However, increased Rif1 expression raises the checkpoint-activation threshold to the point it simulates a checkpoint knockout and can also terminate a checkpoint arrest, despite persistent telomere deficiency. Our work has important implications for understanding the checkpoint and RPA-dependent DNA-damage responses in eukaryotic cells. PMID:22194703

Xue, Yuan; Rushton, Michael D; Maringele, Laura

2011-12-01

139

Electromagnetic transitions between giant resonances within a continuum-RPA approach  

International Nuclear Information System (INIS)

A general continuum-RPA approach is developed to describe electromagnetic transitions between giant resonances. Using a diagrammatic representation for the three-point Green's function, an expression for the transition amplitude is derived which allows one to incorporate effects of mixing of single and double giant resonances as well as to take the entire basis of particle-hole states into consideration. The radiative widths for E1 transition between the charge-exchange spin-dipole giant resonance and Gamow-Teller states are calculated for 90Nb and 208Bi. (orig.)

140

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

 
 
 
 
141

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

142

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

International Nuclear Information System (INIS)

An effective interaction is derived by fitting the matrix elements of a sum of Yukawa terms to the G-matrix elements of a sum of Yukawa terms to the G-matrix elements derived from the Hamada-Johnston potential. Central, spin-orbit and tensor components are taken into account. Numerical results are given and compared with those obtained from the Paris and Reid potentials. As an application, the excitation spectra in 16O are investigated in the framework of the TDA and RPA. (author). 12 refs, 3 tabs

143

Enhanced radiosensitivity and G2/M arrest were observed in radioresistant esophageal cancer cells by knocking down RPA expression.  

Science.gov (United States)

The aim of this study was to evaluate the changes in radiosensitivity of radioresistant esophageal cancer cells (TE-1R) after disruption of replication protein A (RPA) expression and to explore the potential mechanism. A radioresistant human esophageal cancer cell line TE-1R was established by treating TE-1 cells with the radiation. Then, siRPA1 or -2 was transfected to TE-1R cells. The untransfected group (control) and nonsense short interfering RNA (siRNA) transfected group (NC) were used as controls. To investigate the radiosensitivity changes of TE-1R cells, the dose-survival curve was established by colony-forming assay, and the cell cycle distribution was measured by flow cytometry. (1) Comparing with control and NC groups, the protein expression of RPA1 and -2 decreased significantly 48 h after siRPA1 or -2 transfection. (2) The D 0, D q, and SF2 values reduced from 2.09, 1.70, and 0.85 in NC group to 1.67, 0.71, and 0.44 and 1.82, 0.89, and 0.51 in siRNA1 and siRPA2 transfected groups, respectively. The D q sensitization enhancement ratios (SERDq) were 2.39 and 1.91 in siRNA1 and siRPA2 transfected groups, respectively. (3) The G2/M arrest was significantly caused by siRPA1 or -2 transfection as compared with that in the NC group (t value was 2.827, 2.853, p < 0.05). Post transcriptional silencing of RPA1 or -2 via RNAi can enhance the radiosensitivity of human esophageal cancer cells TE-1R, and the potential mechanism may be related to the inhibition of post-radiation sublethal damage repair and the halted cell cycle progression at G2/M phase. Therefore, RPA may become a new target for radiosensitization enhancement in esophageal cancer. PMID:24789547

Di, Zhao; Sanyuan, Sun; Hong, Lu; Dahai, Yu

2014-11-01

144

Secretion of recombinant archeal lipase mediated by SVP2 signal peptide in Escherichia coli and its optimization by response surface methodology.  

Science.gov (United States)

Towards the targeting of recombinant Thermoanaerobacter thermohydrosulfuricus lipase (TtL) for secretion into the culture medium of Escherichia coli, we have investigated a combination of the archeal lipase gene with a Salinovibrio metalloprotease (SVP2) signal peptide sequence. The SVP2 signal peptide has shown all necessary features of a leader sequence for high level secretion of a recombinant target protein in E. coli. Two sets of primers were designed for amplification of the corresponding gene fragments by PCR. Firstly, the PCR product of the TtL gene with designed restriction sites of SacI and HindIII was cloned into pQE-80L plasmid, named as pQE80L-TtL. Afterwards, the amplified fragment of SVP2 signal peptide with EcoRI and SacI restriction sites was also cloned into pQE80L-TtL and the final construct pQE-STL was obtained. A study on the extracellular expression of recombinant STL revealed that most of the enzyme activity was located in the periplasmic space. Glycine and Triton X-100 were investigated to determine whether the leakage of recombinant STL from the outer membrane was promoted, and it was revealed that glycine has a positive effect. Statistical media optimization design was then applied to optimize the effect of seven factors including glycine, Triton X-100, IPTG, yeast extract concentration, incubation time, induction time, and temperature on the extracellular expression of STL. The optimum conditions for the secretion of the lipase was obtained by incubating recombinant E. coli BL21 cells in the medium supplemented by 1.27% glycine and 24h of incubation in the presence of 0.2mM IPTG concentration. PMID:24907409

Pournejati, Roya; Karbalaei-Heidari, Hamid Reza; Budisa, Nediljko

2014-09-01

145

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(r(6)), the THC-ppRPA algorithm scales asymptotically as only O(r(4)), 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. PMID:25028011

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

2014-07-14

146

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

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

2012-01-01

147

Rescue of a Chlamydomonas inner-arm-dynein-deficient mutant by electroporation-mediated delivery of recombinant p28 light chain.  

Science.gov (United States)

We have recently shown that rabbit actin can be introduced by electroporation into the Chlamydomonas ida5 mutant lacking conventional actin and rescue its mutant phenotype [Hayashi et al., 2001: Cell Motil. Cytoskeleton 49:146-153]. In this study, we explored the possibility of using electroporation for functional assay of a recombinant protein. The p28 light chain of inner-arm dyneins was expressed in Escherichia coli, purified to homogeneity, and introduced by electroporation into a non-motile mutant ida4oda6 that lacks it. Because this protein was insoluble in the low ionic strength solution used in the previous study, electroporation was performed at physiological ionic strength in the presence of Ca(2+). Most cells shed their flagella after electroporation. Reflagellation took place within 3 h and up to 30% of the cells became motile, indicating that the introduced p28 retained its functional activity. Fluorescently-labeled p28 was equally effective; in this case fluorescence was observed along the flagella. The presence of Ca(2+) and deflagellation appeared to be important for efficient protein delivery, because a triple mutant with the fa1 mutation deficient in the flagellar shedding mechanism recovered motility only very poorly. Similar results were obtained with other combinations of recombinant proteins and mutants. This study thus demonstrates the feasibility of using electroporation for activity assays of recombinant proteins. PMID:12378537

Hayashi, Masahito; Yanagisawa, Haru-Aki; Hirono, Masafumi; Kamiya, Ritsu

2002-12-01

148

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

149

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

International Nuclear Information System (INIS)

Purpose: To determine whether or not Radiation Therapy Oncology Group (RTOG) recursive partitioning analysis (RPA) derived prognostic classes for patients with brain metastases are generally applicable and can be recommended as rational strategy for patient selection for future clinical trials. Inclusion of time to non-CNS death as additional endpoint besides death from any cause might result in further valuable information, as survival limitation due to uncontrolled extracranial disease can be explored. Methods: We performed a retrospective analysis of prognostic factors for survival and time to non-CNS death in 528 patients treated at a single institution with radiotherapy or surgery plus radiotherapy for brain metastases. For this purpose, patients were divided into groups with Karnofsky performance status (KPS) 0.05 for RPA class II versus III). However, it was 8.5 months in RPA class II patients with controlled primary tumor, which was found to be the only prognostic factor for time to non-CNS death in patients with KPS ?70%. In patients with KPS <70%, no statistically significant prognostic factors were identified for this endpoint. Conclusions: Despite some differences, this analysis essentially confirmed the value of RPA-derived prognostic classes, as published by the RTOG, when survival was chosen as endpoint. RPA class I patients seem to be most likely to profit from aggressive treatment strategies and should be included in appropriate clinical trials. However, their number appears to be very limited. Considering time to non-CNS death, our results suggest that certain patients in RPA class II also might benefit from increased local control of brain metastases

150

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

151

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

Scientific Electronic Library Online (English)

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

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

2004-06-01

152

HF-RPA theory for giant resonances and the nuclear matter incompressibility coefficient: Is there a problem?  

International Nuclear Information System (INIS)

We first review the current states of Hartree-Fock (HF) based random phase approximation (RPA) as applied to the study of the properties of isoscalar giant resonances. We then provide assessments of the consequences due to certain violations of self-consistency in implementing HF-RPA, as often encountered in the published literature. Next, we present results of microscopic calculations of excitation cross sections, ?(E), for the isoscalar giant dipole resonance (ISGDR) and provide a resolution of the long standing problem concerning the value of the nuclear matter incompressibility coefficient, ?nm, deduced from experimental data on ?(E) for the ISGDR. (author)

153

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

154

Implication of RPA32 phosphorylation in S-phase checkpoint signalling at replication forks stalled with aphidicolin in Xenopus egg extracts.  

Science.gov (United States)

Activation of the replication checkpoint relies upon uncoupling of DNA polymerases and helicase activities at replication forks, which in multicellular organism results in production of long stretches of single-stranded DNA bound by the trimeric, single stranded DNA binding protein, the RPA complex. Binding of RPA to this substrate promotes synthesis of replication intermediates that contributes to checkpoint activation by allowing binding of the 9-1-1 checkpoint clamp. The RPA32kDa subunit is also phosphorylated during this process but its role in checkpoint signalling is unclear. Here we have investigated the requirement for RPA32 phosphorylation in checkpoint activation in Xenopus egg extracts. We show that phospho-deficient mutants of RPA32 stimulate checkpoint signalling at replication forks arrested with aphidicolin at both the initiation and the elongation step of DNA replication, without affecting DNA synthesis. In contrast, we show that phospho-mimetic RPA32 mutants do not stimulate checkpoint activation at unwound forks. These results indicate that the hypophosphorylated, replication fork-associated form of RPA32 functions in S-phase-dependent checkpoint signalling at unwound forks in Xenopus egg extracts while RPA32 phosphorylation may be implicated in other pathways such as repair or restart of arrested replication forks. PMID:23047005

Recolin, Bénédicte; Maiorano, Domenico

2012-11-01

155

SETD2-dependent histone H3K36 trimethylation is required for homologous recombination repair and genome stability.  

Science.gov (United States)

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. PMID:24931610

Pfister, Sophia X; Ahrabi, Sara; Zalmas, Lykourgos-Panagiotis; Sarkar, Sovan; Aymard, François; Bachrati, Csanád Z; Helleday, Thomas; Legube, Gaëlle; La Thangue, Nicholas B; Porter, Andrew C G; Humphrey, Timothy C

2014-06-26

156

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

Science.gov (United States)

Summary 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. PMID:24931610

Pfister, Sophia X.; Ahrabi, Sara; Zalmas, Lykourgos-Panagiotis; Sarkar, Sovan; Aymard, Francois; Bachrati, Csanad Z.; Helleday, Thomas; Legube, Gaelle; La Thangue, Nicholas B.; Porter, Andrew C.G.; Humphrey, Timothy C.

2014-01-01

157

PARP-mediated Repair, Homologous Recombination, and Back-up Non-Homologous End Joining-Like Repair of Single-Strand Nicks  

Science.gov (United States)

Double-strand breaks (DSBs) in chromosomal DNA can induce both homologous recombination (HR) and non-homologous recombination (NHEJ). Recently we showed that single-strand nicks induce HR with a significant reduction in toxicity and mutagenic effects associated with NHEJ. To further investigate the differences and similarities of DSB- and nick-induced repair, we used an integrated reporter system in human cells to measure HR and NHEJ produced by the homing endonuclease I-AniI and a designed ‘nickase’ variant that nicks the same target site, focusing on the PARP and HR repair pathways. PARP inhibitors, which block single-strand break repair, increased the rate of nick-induced HR up to 1.7-fold but did not affect DSB-induced HR or mutNHEJ. Additionally, expression of the PALB2 WD40 domain in trans acted as a dominant-negative inhibitor of both DSB- and nick-induced HR, sensitized cells to PARP inhibition, and revealed an alternative mutagenic repair pathway for nicks. Thus, while both DSB- and nick-induced HR use a common pathway, their substrates are differentially processed by cellular factors. These results also suggest that the synthetic lethality of PARP and BRCA may be due to repair of nicks through an error prone, NHEJ-like mechanism that is active when both PARP and HR pathways are blocked. PMID:23684799

Metzger, Michael J.; Stoddard, Barry L.; Monnat, Raymond J.

2013-01-01

158

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

Science.gov (United States)

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

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

2013-01-01

159

Nuclear vorticity in isoscalar E1 modes: Skyrme-RPA analysis  

CERN Document Server

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

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

2013-01-01

160

RPA study of collective oscillations in the Bose-Fermi mixed gases of trapped atoms  

International Nuclear Information System (INIS)

Collective excitations of a trapped bose-fermi mixture of cold atomic gases are studied in RPA. Selfconsistent mean-field equations of coupled bosons and fermions are solved and the ground-state properties are studied for a variable boson-fermion interaction strength. Collective excitations with angular momentum 0 to 3 are calculated and the strength distributions and dynamical form factors are studied. In particular, boson-fermion out-of-phase dipole oscillation is shown to appear at low energy, which is analogous to the nuclear soft dipole oscillation. It is shown that the boson-fermion interaction gives rise to an effective fermion-fermion attraction which induces a concentration of the monopole strength. We comment also on the possible instability of the system due to the boson-fermion attractive interaction. (author)

 
 
 
 
161

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.)

162

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

Directory of Open Access Journals (Sweden)

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

Selander Martin

2007-02-01

163

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

International Nuclear Information System (INIS)

pecific 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

164

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

165

Recombinant adeno-associated virus type 2-mediated gene delivery into the Rpe65-/- knockout mouse eye results in limited rescue.  

Science.gov (United States)

BACKGROUND: Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10-15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function. METHODS: rAAV.RPE65 was injected into the subretinal space of Rpe65-/- knockout mice and control mice. Histological and immunohistological analyses were performed to evaluate any rescue of photoreceptors and to determine longevity and pattern of transgene expression. Electron microscopy was used to examine ultrastructural changes, and electroretinography was used to measure changes in visual function following rAAV.RPE65 injection. RESULTS: rAAV-mediated RPE65 expression was detected for up to 18 months post injection. The delivery of rAAV.RPE65 to Rpe65-/- mouse retinas resulted in a transient improvement in the maximum b-wave amplitude under both scotopic and photopic conditions (76% and 59% increase above uninjected controls, respectively) but no changes were observed in a-wave amplitude. However, this increase in b-wave amplitude was not accompanied by any slow down in photoreceptor degeneration or apoptotic cell death. Delivery of rAAV.RPE65 also resulted in a decrease in retinyl ester lipid droplets and an increase in short wavelength cone opsin-positive cells, suggesting that the recovery of RPE65 expression has long-term benefits for retinal health. CONCLUSION: This work demonstrated the potential benefits of using the Rpe65-/- mice to study the effects and mechanism of rAAV.RPE65-mediated gene delivery into the retina. Although the functional recovery in this model was not as robust as in the dog model, these experiments provided important clues about the long-term physiological benefits of restoration of RPE65 expression in the retina. PMID:15109394

Lai, Chooi-May; Yu, Meaghan JT; Brankov, Meliha; Barnett, Nigel L; Zhou, Xiaohuai; Redmond, T Michael; Narfstrom, Kristina; Rakoczy, P Elizabeth

2004-04-27

166

Surface reengineering of RPA70N enables cocrystallization with an inhibitor of the replication protein A interaction motif of ATR interacting protein.  

Science.gov (United States)

Replication protein A (RPA) is the primary single-stranded DNA (ssDNA) binding protein in eukaryotes. The N-terminal domain of the RPA70 subunit (RPA70N) interacts via a basic cleft with a wide range of DNA processing proteins, including several that regulate DNA damage response and repair. Small molecule inhibitors that disrupt these protein-protein interactions are therefore of interest as chemical probes of these critical DNA processing pathways and as inhibitors to counter the upregulation of DNA damage response and repair associated with treatment of cancer patients with radiation or DNA-damaging agents. Determination of three-dimensional structures of protein-ligand complexes is an important step for elaboration of small molecule inhibitors. However, although crystal structures of free RPA70N and an RPA70N-peptide fusion construct have been reported, RPA70N-inhibitor complexes have been recalcitrant to crystallization. Analysis of the P61 lattice of RPA70N crystals led us to hypothesize that the ligand-binding surface was occluded. Surface reengineering to alter key crystal lattice contacts led to the design of RPA70N E7R, E100R, and E7R/E100R mutants. These mutants crystallized in a P212121 lattice that clearly had significant solvent channels open to the critical basic cleft. Analysis of X-ray crystal structures, target peptide binding affinities, and (15)N-(1)H heteronuclear single-quantum coherence nuclear magnetic resonance spectra showed that the mutations do not result in perturbations of the RPA70N ligand-binding surface. The success of the design was demonstrated by determining the structure of RPA70N E7R soaked with a ligand discovered in a previously reported molecular fragment screen. A fluorescence anisotropy competition binding assay revealed this compound can inhibit the interaction of RPA70N with the peptide binding motif from the DNA damage response protein ATRIP. The implications of the results are discussed in the context of ongoing efforts to design RPA70N inhibitors. PMID:23962067

Feldkamp, Michael D; Frank, Andreas O; Kennedy, J Phillip; Patrone, James D; Vangamudi, Bhavatarini; Waterson, Alex G; Fesik, Stephen W; Chazin, Walter J

2013-09-17

167

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

168

DNA2 cooperates with the WRN and BLM RecQ helicases to mediate long-range DNA end resection in human cells.  

Science.gov (United States)

The 5'-3' resection of DNA ends is a prerequisite for the repair of DNA double strand breaks by homologous recombination, microhomology-mediated end joining, and single strand annealing. Recent studies in yeast have shown that, following initial DNA end processing by the Mre11-Rad50-Xrs2 complex and Sae2, the extension of resection tracts is mediated either by exonuclease 1 or by combined activities of the RecQ family DNA helicase Sgs1 and the helicase/endonuclease Dna2. Although human DNA2 has been shown to cooperate with the BLM helicase to catalyze the resection of DNA ends, it remains a matter of debate whether another human RecQ helicase, WRN, can substitute for BLM in DNA2-catalyzed resection. Here we present evidence that WRN and BLM act epistatically with DNA2 to promote the long-range resection of double strand break ends in human cells. Our biochemical experiments show that WRN and DNA2 interact physically and coordinate their enzymatic activities to mediate 5'-3' DNA end resection in a reaction dependent on RPA. In addition, we present in vitro and in vivo data suggesting that BLM promotes DNA end resection as part of the BLM-TOPOIII?-RMI1-RMI2 complex. Our study provides new mechanistic insights into the process of DNA end resection in mammalian cells. PMID:25122754

Sturzenegger, Andreas; Burdova, Kamila; Kanagaraj, Radhakrishnan; Levikova, Maryna; Pinto, Cosimo; Cejka, Petr; Janscak, Pavel

2014-09-26

169

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

International Nuclear Information System (INIS)

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

170

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

DEFF Research Database (Denmark)

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

Luo, Yonglun; Bolund, Lars

2012-01-01

171

The interacting boson model, BSC-RPA theory and the theory of pairing interactions applied to Ge nuclei  

International Nuclear Information System (INIS)

The low lying states and especialy the extraordinary behaviour of the first excited O+ state as a function of neutron number in the sequence of nuclei, 68Ge, 70Ge, 72Ge, 74Ge, 76Ge, are described (except for 76Ge) by introducing a s'-boson into the interacting boson model (IBM) which usually involves only s- and d-bosons. This introduction could be understood by considering results from BCS-RPA calculations in the neutron configuration only. BCS-RPA theory is, however, shown to be a poor approximation in the case under consideration. Results from the exact diagonalisation of the Hamiltonian for the charge independant pairing interaction in both the proton and neutron configuration are discussed. 7 figs., 2 tabs., 21 refs

172

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

173

RPA alleviates the inhibitory effect of vinylphosphonate internucleotide linkages on DNA unwinding by BLM and WRN helicases  

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Bloom (BLM) and Werner (WRN) syndrome proteins are members of the RecQ family of SF2 DNA helicases. In this paper, we show that restricting the rotational DNA backbone flexibility, by introducing vinylphosphonate internucleotide linkages in the translocating DNA strand, inhibits efficient duplex unwinding by these enzymes. The human single-stranded DNA binding protein replication protein A (RPA) fully restores the unwinding activity of BLM and WRN on vinylphosphonate-containing substrates whi...

Garcia, P. L.; Bradley, G.; Hayes, C. H.; Krintel, S. J.; Soultanas, P.; Janscak, P.

2004-01-01

174

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

Science.gov (United States)

Gimeracil (5-chloro-2, 4-dihydroxypyridine) is an inhibitor of dihydropyrimidine dehydrogenase (DPYD), which degrades pyrimidine including 5-fluorouracil in the blood. Gimeracil was originally added to an oral fluoropyrimidine derivative S-1 to yield prolonged 5-fluorouracil concentrations in serum and tumor tissues. We have already reported that gimeracil had radiosensitizing effects by partially inhibiting homologous recombination (HR) in the repair of DNA double strand breaks. We investigated the mechanisms of gimeracil radiosensitization. Comet assay and radiation-induced focus formation of various kinds of proteins involved in HR was carried out. siRNA for DPYD were transfected to HeLa cells to investigate the target protein for radiosensitization with gimeracil. SCneo assay was carried out to examine whether DPYD depletion by siRNA inhibited HR repair of DNA double strand breaks. Tail moments in neutral comet assay increased in gimeracil-treated cells. Gimeracil restrained the formation of foci of Rad51 and replication protein A (RPA), whereas it increased the number of foci of Nbs1, Mre11, Rad50, and FancD2. When HeLa cells were transfected with the DPYD siRNA before irradiation, the cells became more radiosensitive. The degree of radiosensitization by transfection of DPYD siRNA was similar to that of gimeracil. Gimeracil did not sensitize DPYD-depleted cells. Depletion of DPYD by siRNA significantly reduced the frequency of neopositive clones in SCneo assay. Gimeracil partially inhibits the early step in HR. It was found that DPYD is the target protein for radiosensitization by gimeracil. The inhibitors of DPYD, such as gimeracil, could enhance the efficacy of radiotherapy through partial suppression of HR-mediated DNA repair. PMID:21668582

Sakata, Koh-Ichi; Someya, Masanori; Matsumoto, Yoshihisa; Tauchi, Hiroshi; Kai, Masahiro; Toyota, Minoru; Takagi, Masaru; Hareyama, Masato; Fukushima, Masakazu

2011-09-01

175

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

International Nuclear Information System (INIS)

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

176

Deletion of the murine cytochrome P450 Cyp2j locus by fused BAC-mediated recombination identifies a role for Cyp2j in the pulmonary vascular response to hypoxia.  

Science.gov (United States)

Epoxyeicosatrienoic acids (EETs) confer vasoactive and cardioprotective functions. Genetic analysis of the contributions of these short-lived mediators to pathophysiology has been confounded to date by the allelic expansion in rodents of the portion of the genome syntenic to human CYP2J2, a gene encoding one of the principle cytochrome P450 epoxygenases responsible for the formation of EETs in humans. Mice have eight potentially functional genes that could direct the synthesis of epoxygenases with properties similar to those of CYP2J2. As an initial step towards understanding the role of the murine Cyp2j locus, we have created mice bearing a 626-kb deletion spanning the entire region syntenic to CYP2J2, using a combination of homologous and site-directed recombination strategies. A mouse strain in which the locus deletion was complemented by transgenic delivery of BAC sequences encoding human CYP2J2 was also created. Systemic and pulmonary hemodynamic measurements did not differ in wild-type, null, and complemented mice at baseline. However, hypoxic pulmonary vasoconstriction (HPV) during left mainstem bronchus occlusion was impaired and associated with reduced systemic oxygenation in null mice, but not in null mice bearing the human transgene. Administration of an epoxygenase inhibitor to wild-type mice also impaired HPV. These findings demonstrate that Cyp2j gene products regulate the pulmonary vascular response to hypoxia. PMID:24278032

Zhou, Guo Ling; Beloiartsev, Arkadi; Yu, Binglan; Baron, David M; Zhou, Weihua; Niedra, Rasma; Lu, Naifang; Tainsh, Laurel T; Zapol, Warren M; Seed, Brian; Bloch, Kenneth D

2013-11-01

177

Estrogen Receptors Bind to and Activate the HOXC4/HoxC4 Promoter to Potentiate HoxC4-mediated Activation-induced Cytosine Deaminase Induction, Immunoglobulin Class Switch DNA Recombination, and Somatic Hypermutation*  

Science.gov (United States)

Estrogen enhances antibody and autoantibody responses through yet to be defined mechanisms. It has been suggested that estrogen up-regulates the expression of activation-induced cytosine deaminase (AID), which is critical for antibody class switch DNA recombination (CSR) and somatic hypermutation (SHM), through direct activation of this gene. AID, as we have shown, is induced by the HoxC4 homeodomain transcription factor, which binds to a conserved HoxC4/Oct site in the AICDA/Aicda promoter. Here we show that estrogen-estrogen receptor (ER) complexes do not directly activate the AID gene promoter in B cells undergoing CSR. Rather, they bind to three evolutionarily conserved and cooperative estrogen response elements (EREs) we identified in the HOXC4/HoxC4 promoter. By binding to these EREs, ERs synergized with CD154 or LPS and IL-4 signaling to up-regulate HoxC4 expression, thereby inducing AID and CSR without affecting B cell proliferation or plasmacytoid differentiation. Estrogen administration in vivo significantly potentiated CSR and SHM in the specific antibody response to the 4-hydroxy-3-nitrophenylacetyl hapten conjugated with chicken ?-globulin. Ablation of HoxC4 (HoxC4?/?) abrogated the estrogen-mediated enhancement of AID gene expression and decreased CSR and SHM. Thus, estrogen enhances AID expression by activating the HOXC4/HoxC4 promoter and inducing the critical AID gene activator, HoxC4. PMID:20855884

Mai, Thach; Zan, Hong; Zhang, Jinsong; Hawkins, J. Seth; Xu, Zhenming; Casali, Paolo

2010-01-01

178

Recent advances in immunopathophysiology of interleukin-6: an innovative therapeutic drug, tocilizumab (recombinant humanized anti-human interleukin-6 receptor antibody), unveils the mysterious etiology of immune-mediated inflammatory diseases.  

Science.gov (United States)

Interleukin (IL)-6 cDNA was originally cloned as a terminal B cell differentiation factor into antibody-producing plasma cells. This revealed that it is a multifunctional cytokine that acts on a variety of cells. From the clinical viewpoint, it is especially important that IL-6 acts on hepatocytes to induce acute-phase reactants, including C-reactive protein, serum amyloid A protein, and fibrinogen, and to decrease serum albumin levels. Very recently, this cytokine has been found to enhance the synthesis of a peptide called hepcidin in the liver which regulates iron recycling, resulting in anemia due to hypofferemia. It has also been shown that IL-6 is responsible for various clinical symptoms, including the appearance of autoantibodies, fatigue, anemia, anorexia, fever, and increases in the erythrocyte sedimentation rate, all of which develop in patients with various chronic autoimmune inflammatory diseases. In practice, blocking the IL-6 signaling pathway with a recombinant humanized anti-IL-6 receptor antibody, tocilizumab (TCZ), has dramatically improved all the signs and symptoms of these patients. A study in mice demonstrated that IL-6 promotes the development of a new type of T-helper cells called Th17 cells that impact the pathogenesis of autoimmune diseases. This suggests that TCZ is not only an antiinflammatory agent but also might affect basic autoimmunity. In this review, recent advances in the immunobiology of interleukin-6 related to immune-mediated diseases are discussed. PMID:17978466

Ohsugi, Yoshiyuki

2007-11-01

179

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

Science.gov (United States)

Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (?4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates. PMID:24972080

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

2014-01-01

180

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

 
 
 
 
181

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

182

Increased RPA1 Gene Dosage Affects Genomic Stability Potentially Contributing to 17p13.3 Duplication Syndrome  

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A novel microduplication syndrome involving various-sized contiguous duplications in 17p13.3 has recently been described, suggesting that increased copy number of genes in 17p13.3, particularly PAFAH1B1, is associated with clinical features including facial dysmorphism, developmental delay, and autism spectrum disorder. We have previously shown that patient-derived cell lines from individuals with haploinsufficiency of RPA1, a gene within 17p13.3, exhibit an impaired ATR-dependent DNA damage ...

Outwin, Emily; Carpenter, Gillian; Bi, Weimin; Withers, Marjorie A.; Lupski, James R.; O Driscoll, Mark

2011-01-01

183

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

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

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

2009-01-01

184

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

185

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

186

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

187

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

Energy Technology Data Exchange (ETDEWEB)

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

Yannouleas, C.P.

1982-07-01

188

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

Science.gov (United States)

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.

Wildmann, N.; Mauz, M.; Bange, J.

2013-08-01

189

Thermodynamic analysis of the single-stranded DNA binding activity of the archaeal replication protein A (RPA) from Sulfolobus solfataricus.  

Science.gov (United States)

The single-stranded DNA binding protein from Sulfolobus solfataricus (Sso-RPA) binds single-stranded DNA with dissociation constants in the range of 10-30 nM at room temperature. The affinity for DNA decreases at higher temperatures. At 85 degrees C, the optimal growth temperature of the crenarchaeot S. solfataricus, the dissociation constant is only about 1 microM. We analyzed the equilibrium between Sso-RPA and a fluorescently labeled 13 nucleotide oligonucleotide by fluorescence anisotropy measurements in the presence of four different salts and in the temperature range between 10 and 60 degrees C. In the presence of potassium chloride and choline chloride, three to four ions are released upon complexation, independent of the temperature. In contrast, in the presence of potassium fluoride and potassium glutamate, we observed a significant change of the number of ions released when the temperature was varied. The binding reaction is strongly exothermic with enthalpies of about -55 to -70 kJ/mol, depending upon the salt. Van't Hoff analysis suggests that the binding enthalpy is temperature independent. PMID:16401088

Kernchen, Uwe; Lipps, Georg

2006-01-17

190

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

191

The HoxC4 homeodomain protein mediates activation of the immunoglobulin heavy chain 3' hs1,2 enhancer in human B cells. Relevance to class switch DNA recombination.  

Science.gov (United States)

The immunoglobulin heavy chain (IgH) 3' regulatory region modulates IgH locus transcription, upon induction by specific trans-acting factors, and plays a significant role in class switch DNA recombination (CSR) and, perhaps, somatic hypermutation (SHM). CSR and SHM are central to the maturation of the antibody response. In contrast to the single 5'-hs3a-hs1,2-hs3b-hs4-3 ' mouse IgH 3 ' regulatory region, the human IgH 3 ' regulatory region exists as a 5'-hs3-hs1,2-hs4-3' cluster duplicated 3 ' of Calpha1 and Calpha2. We show here that the human hs1,2 element is the strongest enhancer of transcription, as directed by a V(H)1 or the ECS-Igamma3 promoter, thereby suggesting a dominant role for hs1,2 over hs3 and hs4 in the overall activity of the 3 ' regulatory region. Within hs1,2, we identified three regions (1, 2, and 3) that are all necessary, but individually not sufficient, for enhancement of transcription. In region 2, a HoxC4 site and a HoxC4/embedded octamer (HoxC4/Oct) site are conserved across human, mouse, rat, and rabbit. These two sites recruit HoxC4 and Oct-1/Oct-2, which act synergistically with the Oca-B coactivator to effect the full hs1,2-enhancing activity. HoxC4, Oct-1/Oct-2, and Oca-B recruitment is negligible in pro-B cells, moderate in pre-B cells, and maximal in germinal center B cells and plasma cells, where HoxC4, Oct-2, and Oca-B expression correlates with hs1,2 activation and ongoing CSR. The hs1,2mediated enhancement of V(H) and C(H) promoter-driven transcription as induced by HoxC4 and Oct-1/Oct-2 suggests an important role of these homeodomain proteins in the overall regulation of the IgH locus expression. PMID:15252056

Kim, Edmund C; Edmonston, Christopher R; Wu, Xiaoping; Schaffer, András; Casali, Paolo

2004-10-01

192

PRODUCTION OF RECOMBINANT PROTEINS IN INSECT CELLS  

Directory of Open Access Journals (Sweden)

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

Christian Kollewe

2013-01-01

193

Single-stranded heteroduplex intermediates in ? Red homologous recombination  

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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

194

Improving baculovirus recombination  

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Recombinant baculoviruses have established themselves as a favoured technology for the high-level expression of recombinant proteins. The construction of recombinant viruses, however, is a time consuming step that restricts consideration of the technology for high throughput developments. Here we use a targeted gene knockout technology to inactivate an essential viral gene that lies adjacent to the locus used for recombination. Viral DNA prepared from the knockout fails to initiate an infecti...

Zhao, Yuguang; Chapman, David A. G.; Jones, Ian M.

2003-01-01

195

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

196

Recombineering Homologous Recombination Constructs in Drosophila  

Science.gov (United States)

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner. PMID:23893070

Carreira-Rosario, Arnaldo; Scoggin, Shane; Shalaby, Nevine A.; Williams, Nathan David; Hiesinger, P. Robin; Buszczak, Michael

2013-01-01

197

Experimental investigation of hole boring and light sail regimes of RPA by varying laser and target parameters  

Science.gov (United States)

Temporal evolution of plasma jets from micrometre-scale thick foils following the interaction of intense (3 × 1020 W cm-2) laser pulses is studied systematically by time resolved optical interferometry. The fluid velocity in the plasma jets is determined by comparing the data with 2D hydrodynamic simulation, which agrees with the expected hole-boring (HB) velocity due to the laser radiation pressure. The homogeneity of the plasma density across the jets has been found to be improved substantially when irradiating the laser at circular polarization compared to linear polarization. While overdense plasma jets were formed efficiently for micrometre thick targets, decreasing the target areal density and/or increasing the irradiance on the target have provided indication of transition from the ‘HB’ to the ‘light sail (LS)’ regime of RPA, characterized by the appearance of narrow-band spectral features at several MeV/nucleon in proton and carbon spectra.

Kar, S.; Kakolee, K. F.; Cerchez, M.; Doria, D.; Macchi, A.; McKenna, P.; Neely, D.; Osterholz, J.; Quinn, K.; Ramakrishna, B.; Sarri, G.; Willi, O.; Yuan, X. H.; Zepf, M.; Borghesi, M.

2013-12-01

198

Recombinant viral vectors: cancer vaccines.  

Science.gov (United States)

To date cancer vaccines have yet to show efficacy in a phase III trial. However, the clinical benefit seen with monoclonal antibody mediated therapies (e.g., Herceptin) has provided proof of principle that immune responses directed against tumour-associated antigens could have therapeutic potential. The failure of past cancer vaccine trials is likely due to several factors including the inappropriate choice of tumour antigen, use of an unoptimised antigen delivery system or vaccination schedule or selection of the wrong patient group. Any one of these variables could potentially result in the induction of an immune response of insufficient magnitude to deliver clinical benefit. Live recombinant viral vaccines have been used in the development of cancer immunotherapy approaches for the past 10 years. Though such vectors are self-adjuvanted and offer the ability to express multiple tumour-associated antigens (TAAs) along with an array of immune co-factors, arguably, they have yet to demonstrate convincing efficacy in pivotal clinical trials. However, in recent years, more coordinated studies have revealed mechanisms to optimise current vectors and have lead to the development of new advantageous vector systems. In this review, we highlight that live recombinant viral vectors provide a versatile and effective antigen delivery system and describe the optimal properties of an effective viral vector. Additionally, we discuss the advantages and disadvantages of the panel of recombinant viral systems currently available to cancer vaccinologists and how they can work in synergy in heterologous prime boost protocols and with other treatment modalities. PMID:17030074

Harrop, Richard; John, Justin; Carroll, Miles W

2006-10-01

199

Nucleotide fluctuation of radiation-resistant Halobacterium sp. NRC-1 single-stranded DNA-binding protein (RPA) genes  

Science.gov (United States)

The Single-Stranded DNA-Binding Protein (RPA) Genes in gamma ray radiation-resistant halophilic archaeon Halobacterium sp. NRC-1 were analyzed in terms of their nucleotide fluctuations. In an ATCG sequence, each base was assigned a number equal to its atomic number. The resulting numerical sequence was the basis of the statistical analysis in this study. Fractal analysis using the Higuchi method gave fractal dimensions of 2.04 and 2.06 for the gene sequences VNG2160 and VNG2162, respectively. The 16S rRNA sequence has a fractal dimension of 1.99. The di-nucleotide Shannon entropy values were found to be negatively correlated with the observed fractal dimensions (R2~ 0.992, N=3). Inclusion of Deinococcus radiodurans Rad-A in the regression analysis decreases the R2 slightly to 0.98 (N=4). A third VNG2163 RPA gene of unknown function but with upregulation activity under irradiation was found to have a fractal dimension of 2.05 and a Shannon entropy of 3.77 bits. The above results are similar to those found in bacterial Deinococcus radiodurans and suggest that their high radiation resistance property would have favored selection of CG di-nucleotide pairs. The two transcription factors TbpD (VNG7114) and TfbA (VNG 2184) were also studied. Using VNG7114, VNG2184, and VNG2163; the regression analysis of fractal dimension versus Shannon entropy shows that R2 ~ 0.997 for N =3. The VNG2163 unknown function may be related to the pathways with transcriptions closely regulated to sequences VNG7114 and VNG2184.

Holden, Todd; Tremberger, G., Jr.; Cheung, E.; Subramaniam, R.; Gadura, N.; Schneider, P.; Sullivan, R.; Flamholz, A.; Lieberman, D.; Cheung, T. D.

2009-08-01

200

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

 
 
 
 
201

Auger recombination in silicon  

International Nuclear Information System (INIS)

The Auger recombination in n-Si is studied at room temperature using the technique of probing infrared light absorption (lambda approximately 2 ?m) by nonequilibrium carriers. Experimental value of the Auger recombination coefficient is determined (? approximately 1.5x10-30 cm6/s). At room temperature the phonon mechanism of the Auger recombination is shown to be more important than the resonance one

202

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.

203

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

Science.gov (United States)

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

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

2004-01-01

204

Oxygen-hydrogen recombiner  

International Nuclear Information System (INIS)

Purpose: To improve the oxygen-hydrogen removing performance, as well as enable to maintain the high performance in a range of increasing the processing gas flow rate within a recombiner with the reduced pressure and reduced amount of charged catalyst. Constitution: A plate-like metal catalyst comprising alumina added as a binder to the surface of a sponge-like metal support made of nickel-chromium alloy and particles of platinum type novel metal such as platinum or palladium having a catalytic activity supported on alumina is contained in a cartridge and filled within an oxygen-hydrogen recombiner. The recombiner is adapted so that the exhaust gas flow rate therein is within a range from 1 nm/sec to 4 nm/sec. It is possible with such a constitution to improve the recombining performance, reduce the pressure loss, decrease the size of the recombiner and facilitate the maintenance and check thereof. (Kawakami, Y.)

205

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

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

2008-01-01

206

Topoisomerase I involvement in illegitimate recombination in Saccharomyces cerevisiae.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Chromosome aberrations may cause cancer and many heritable diseases. Topoisomerase I has been suspected of causing chromosome aberrations by mediating illegitimate recombination. The effects of deletion and of overexpression of the topoisomerase I gene on illegitimate recombination in the yeast Saccharomyces cerevisiae have been studied. Yeast transformations were carried out with DNA fragments that did not have any homology to the genomic DNA. The frequency of illegitimate integration was 6-...

Zhu, J.; Schiestl, R. H.

1996-01-01

207

Sex, not genotype, determines recombination levels in mice.  

Science.gov (United States)

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, chromosomal location, proximity to other recombination events (i.e., chiasma interference), and, intriguingly, the sex of the transmitting parent. To distinguish between simple genetic and nongenetic explanations of sex-specific recombination differences in mammals, we compared recombination in meiocytes from XY sex-reversed and XO females with that in meiocytes from XX female and XY male mice. The rate and pattern of recombination in XY and XO oocytes were virtually identical to those in normal XX females, indicating that sex, not genotype, is the primary determinant of meiotic recombination patterns in mammals. PMID:16175513

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

2005-10-01

208

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

209

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

Science.gov (United States)

Whole brain radiotherapy (WBRT) is the most widely used treatment for brain metastasis (BM), especially for patients with multiple intracranial lesions. The purpose of this study was to examine the efficacy of systemic treatments following WBRT in breast cancer patients with BM who had different clinical characteristics, based on the classification of the Radiation Therapy Oncology Group recursive partitioning analysis (RPA) and the breast cancer-specific Graded Prognostic Assessment (Breast-GPA). One hundred and one breast cancer patients with BM treated between 2006 and 2010 were analyzed. The median interval between breast cancer diagnosis and identification of BM in the triple-negative patients was shorter than in the luminal A subtype (26 vs. 36 months, respectively; P = 0.021). Univariate analysis indicated that age at BM diagnosis, Karnofsky performance status/recursive partitioning analysis (KPS/RPA) classes, number of BMs, primary tumor control, extracranial metastases and systemic treatment following WBRT were significant prognostic factors for overall survival (OS) (P interval between initial diagnosis and the development of BM than luminal A patients. Systemic treatments following WBRT improved the survival of RPA class II/III patients. PMID:23743596

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

2013-09-01

210

Activated recombinant adenovirus proteinases  

Science.gov (United States)

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

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

1999-08-10

211

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

212

Recombinant ?-phage nanobioparticles for tumor therapy in mice models  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Lambda phages have considerable potential as gene delivery vehicles due to their genetic tractability, low cost, safety and physical characteristics in comparison to other nanocarriers and gene porters. Little is known concerning lambda phage-mediated gene transfer and expression in mammalian hosts. We therefore performed experiments to evaluate lambda-ZAP bacteriophage-mediated gene transfer and expression in vitro. For this purpose, we constructed recombinant ?-phage nanobioparticles conta...

Ghaemi, Amir; Soleimanjahi, Hoorieh; Gill, Pooria; Hassan, Zuhair; Jahromi, Soodeh Razeghi M.; Roohvand, Farzin

2010-01-01

213

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

214

rpa dbffiles may08  

Protected Area Type Originating Directive Protected Area Sub Type Name Designation Date Area (m2) Length (m) Drinking Water Water Framework Directive (2000/60/EC) Lake Water Body Silent Valley Reservoir 875921.91 ...

215

RPA Review - Executive Summary  

Oct 15, 2009 ... evidence based, due diligence and make recommendations for CAP 2013 .... \\represents a good management information set with targets for the development \\and ..... The summation of these issues gave the impression of an ...

216

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

International Nuclear Information System (INIS)

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

217

Recombinant protein production by in vivo polymer inclusion display.  

Science.gov (United States)

A novel approach to produce purified recombinant proteins was established. The target protein is produced as polyhydroxyalkanoate (PHA) synthase fusion protein, which mediates intracellular formation of PHA inclusions displaying the target protein. After isolation of the PHA inclusions, the pure target protein was released by simple enterokinase digestion. PMID:21803888

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

2011-09-01

218

The dissociative recombination of ?  

Science.gov (United States)

The dissociative recombination of 0953-4075/31/8/021/img2 has been measured using a single-pass merged-beam apparatus. It is found that the cross section displays resonant structure and falls-off abruptly above 0.3 eV.

LePadellec, A.; Sheehan, C.; Mitchell, J. B. A.

1998-04-01

219

Recombinant DNA for Teachers.  

Science.gov (United States)

A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

Duvall, James G., III

1992-01-01

220

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

 
 
 
 
221

Recombinational DNA repair and human disease  

International Nuclear Information System (INIS)

We review the genes and proteins related to the homologous recombinational repair (HRR) pathway that are implicated in cancer through either genetic disorders that predispose to cancer through chromosome instability or the occurrence of somatic mutations that contribute to carcinogenesis. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and an ataxia-like disorder (ATLD), are chromosome instability disorders that are defective in the ataxia telangiectasia mutated (ATM), NBS, and Mre11 genes, respectively. These genes are critical in maintaining cellular resistance to ionizing radiation (IR), which kills largely by the production of double-strand breaks (DSBs). Bloom syndrome involves a defect in the BLM helicase, which seems to play a role in restarting DNA replication forks that are blocked at lesions, thereby promoting chromosome stability. The Werner syndrome gene (WRN) helicase, another member of the RecQ family like BLM, has very recently been found to help mediate homologous recombination. Fanconi anemia (FA) is a genetically complex chromosomal instability disorder involving seven or more genes, one of which is BRCA2. FA may be at least partially caused by the aberrant production of reactive oxidative species. The breast cancer-associated BRCA1 and BRCA2 proteins are strongly implicated in HRR; BRCA2 associates with Rad51 and appears to regulate its activity. We discuss in detail the phenotypes of the various mutant cell lines and the signaling pathways mediated by the ATM kinase. ATM's phosphorylation targets can be grouped into oxidative stress-mediated transcriptional changes, cell cycle checkpoints, and recombinational repair. We present the DNA damage response pathways by using the DSB as the prototype lesion, whose incorrect repair can initiate and augment karyotypic abnormalities

222

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

223

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

Science.gov (United States)

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

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

2008-01-01

224

Intercultural Mediation  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

Dragos Marian Radulescu; Denisa Mitrut

2012-01-01

225

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

226

Self-consistent Hartree-Fock and RPA Green's function method indicate no pygmy resonance in the monopole response of neutron-rich Ni isotopes  

Science.gov (United States)

The random phase approximation (RPA) monopole strength and the unperturbed particle-hole excitation strength are studied, in which the strength in the continuum is properly treated without discretizing unbound particle spectra. The model is the self-consistent Hartree-Fock calculation and the RPA Green's function method with Skyrme interactions. Numerical examples are the Ni isotopes, especially 2868Ni40, in which an experimental observation of a low-lying peak with an appreciable amount of monopole strength at 12.9±1.0 MeV was recently reported. In the present study it is concluded that sharp monopole peaks with the width of the order of 1 MeV can hardly be expected for 68Ni in that energy region. Instead, a broad shoulder of monopole strength consisting of neutron excitations to nonresonant one-particle states (called "threshold strength") with relatively low angular momenta (?,j) is obtained in the continuum energy region above the particle threshold, which is considerably lower than that of the isoscalar giant monopole resonance. In the case of monopole excitations of 68Ni there are no unperturbed particle-hole states below 20 MeV, in which the particle expresses a neutron (or proton) resonant state. It is emphasized that in the theoretical estimate a proper treatment of the continuum is extremely important.

Hamamoto, Ikuko; Sagawa, Hiroyuki

2014-09-01

227

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

228

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

229

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

230

Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences.  

Science.gov (United States)

V(D)J recombination is the process by which the variable region exons encoding the antigen recognition sites of receptors expressed on B and T lymphocytes are generated during early development via somatic assembly of component gene segments. In response to antigen, somatic hypermutation (SHM) and class switch recombination (CSR) induce further modifications of immunoglobulin genes in B cells. CSR changes the IgH constant region for an alternate set that confers distinct antibody effector functions. SHM introduces mutations, at a high rate, into variable region exons, ultimately allowing affinity maturation. All of these genomic alteration processes require tight regulatory control mechanisms, both to ensure development of a normal immune system and to prevent potentially oncogenic processes, such as translocations, caused by errors in the recombination/mutation processes. In this regard, transcription of substrate sequences plays a significant role in target specificity, and transcription is mechanistically coupled to CSR and SHM. However, there are many mechanistic differences in these reactions. V(D)J recombination proceeds via precise DNA cleavage initiated by the RAG proteins at short conserved signal sequences, whereas CSR and SHM are initiated over large target regions via activation-induced cytidine deaminase (AID)-mediated DNA deamination of transcribed target DNA. Yet, new evidence suggests that AID cofactors may help provide an additional layer of specificity for both SHM and CSR. Whereas repair of RAG-induced double-strand breaks (DSBs) involves the general nonhomologous end-joining DNA repair pathway, and CSR also depends on at least some of these factors, CSR requires induction of certain general DSB response factors, whereas V(D)J recombination does not. In this review, we compare and contrast V(D)J recombination and CSR, with particular emphasis on the role of the initiating enzymes and DNA repair proteins in these processes. PMID:15705419

Dudley, Darryll D; Chaudhuri, Jayanta; Bassing, Craig H; Alt, Frederick W

2005-01-01

231

Oxygen-hydrogen recombiner  

International Nuclear Information System (INIS)

Purpose: To reduce the amount of a catalyst and improve the hydrogen removing performance of a catalyst type oxygen-hydrogen recombiner in a radioactive gaseous wastes processing facility of BWR type nuclear power plants. Constitution: The catalyst (of palladium or platinum group) has a low activity at a low temperature. In view of the above, hydrogen gases injected from the outside for improving the catalyst activity even under a low temperature state such as reactor start-up and shut-down. When the hydrogen gas concentration increases, chemical combination between oxygen and hydrogen in the recombiner is increased, by which the amount of heat generation is increased to rise the temperature of the catalyst. In this way, the catalyst activity can be improved. Corresponding to the injection of the hydrogen gas, oxygen is also injected so as to attain 1 : 2 ratio. (Ikeda, J.)

232

AECL passive autocatalytic recombiners  

International Nuclear Information System (INIS)

Atomic Energy of Canada Limited's (AECL) Passive Autocatalytic Recombiner (PAR) is a passive device used for hydrogen mitigation under post-accident conditions in nuclear reactor containment. The PAR employs a proprietary AECL catalyst which promotes the exothermal reaction between hydrogen and oxygen to form water vapour. The heat of reaction combined with the PAR geometry establishes a convective flow through the recombiner, where ambient hydrogen-rich gas enters the PAR inlet and hot, humid, hydrogen-depleted gas exits the outlet. AECL's PAR has been extensively qualified for CANDU and light water reactors (LWRs), and has been supplied to France, Finland, Ukraine, South Korea and is currently being deployed in Canadian nuclear power plants. (author)

233

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

234

Site directed recombination  

Science.gov (United States)

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

Jurka, Jerzy W. (Los Altos, CA)

1997-01-01

235

Intrachromosomal recombination in plants.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Molecular evidence for intrachromosomal recombination between closely linked DNA repeats within the plant genome is presented. The non-overlapping complementary deletion derivatives of the selectable neomycin phosphotransferase gene (nptII), when intact conferring kanamycin resistance, were inserted into the genome of Nicotiana tabacum. The functional marker gene was restored with frequencies between 10(-4) and 10(-6) per proliferating cell clone. Prolonged tissue culture prior to kanamycin s...

Peterhans, A.; Schlu?pmann, H.; Basse, C.; Paszkowski, J.

1990-01-01

236

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

237

A Simplified Recombinant PSO  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

Bratton, Daniel; Blackwell, Tim M.

2008-01-01

238

Relativistic dielectronic recombination theory  

International Nuclear Information System (INIS)

Dielectronic recombination (DR) is an inverse Auger process in which a free electron is captured by a recombining ion to form a doubly excited autoionizing state. The subsequent decay of the autoionizing state to a stabilized bound state by emitting photons completes the recombination process. DR is an important recombination process for high temperature plasmas. It can affect the ionization balance and level kinetics of the hot plasmas. In addition, the dielectronic satellite lines observed in the emission spectra are frequently used as plasmas diagnostic tools. In the past decade, intense theoretical and experimental studies on the DR process have been carried out. Most of the earlier theoretical calculations on the DR rate coefficients were done either by using a term average approximation or in LS coupling without including the effects of relativity and configuration interaction. The early experimental investigations were concentrated on few times ionized low-Z ions. Recently, the development of electron beam ion trap (EBIT), electron beam ion source (EBIS) and heavy ion storage ring has become possible to produce very highly-charged heavy ions (e.g. U82+ and Xe53+)and to study the interaction between electrons and these ions. For highly-charged heavy ions, one excepts that the nonrelativistic method would be inadequate and a relativistic treatment is necessary. To meet this challenge we have developed a relativistic package based on the multiconfiguration Dirac-Fock method and have carried out systematic relativistic calculations of DR cross sections and rate coefficients and resonant transfer and excitation cross sections in ion-atom collisions. In this paper, we will briefly discuss the relativistic calculations of atomic structure and transition rates and will focus for attention on the effects of relativity and intermediate coupling on the DR cross sections and rate coefficients

239

Negative capacitance in organic semiconductor devices: bipolar injection and charge recombination mechanism  

CERN Document Server

We report negative capacitance at low frequencies in organic semiconductor based diodes and show that it appears only under bipolar injection conditions. We account quantitatively for this phenomenon by the recombination current due to electron-hole annihilation. Simple addition of the recombination current to the well established model of space charge limited current in the presence of traps, yields excellent fits to the experimentally measured admittance data. The dependence of the extracted characteristic recombination time on the bias voltage is indicative of a recombination process which is mediated by localized traps.

Ehrenfreund, E; Dennler, G; Neugebauer, H; Sariciftci, N S

2007-01-01

240

Recombinant human milk proteins.  

Science.gov (United States)

Human milk provides proteins that benefit newborn infants. They not only provide amino acids, but also facilitate the absorption of nutrients, stimulate growth and development of the intestine, modulate immune function, and aid in the digestion of other nutrients. Breastfed infants have a lower prevalence of infections than formula-fed infants. Since many women in industrialized countries choose not to breastfeed, and an increasing proportion of women in developing countries are advised not to breastfeed because of the risk of HIV transmission, incorporation of recombinant human milk proteins into infant foods is likely to be beneficial. We are expressing human milk proteins known to have anti-infective activity in rice. Since rice is a normal constituent of the diet of infants and children, limited purification of the proteins is required. Lactoferrin has antimicrobial and iron-binding activities. Lysozyme is an enzyme that is bactericidal and also acts synergistically with lactoferrin. These recombinant proteins have biological activities identical to their native counterparts. They are equally resistant to heat processing, which is necessary for food applications, and to acid and proteolytic enzymes which are needed to maintain their biological activity in the gastrointestinal tract of infants. These recombinant human milk proteins may be incorporated into infant formulas, baby foods and complementary foods, and used with the goal to reduce infectious diseases. PMID:16902336

Lönnerdal, Bo

2006-01-01

 
 
 
 
241

The Logic and Mechanism of Homologous Recombination Partner Choice  

Science.gov (United States)

SUMMARY Recombinational repair of spontaneous double-strand breaks (DSBs) exhibits sister bias. DSB-initiated meiotic recombination exhibits homolog bias. Physical analysis in yeast reveals that, in both cases, recombination intrinsically gives homolog bias. From this baseline default, cohesin intervenes to confer sister bias, likely independent of cohesion. In meiosis, cohesin’s sister-biasing effect is counteracted by RecA-homolog Rad51 and its mediators, plus meiotic RecA- homolog Dmc1, which thereby restore intrinsic homolog bias. Meiotic axis complex Red1/Mek1/Hop1 participates by cleanly switching recombination from mitotic mode to meiotic mode, concomitantly activating Dmc1. We propose that a Rad51/DNA filament at one DSB end captures the intact sister, creating an “anchor pad”. This filament extends across the DSB site on the intact partner, precluding inter-sister strand exchange, thus forcing use of the homolog. Cohesin and Dmc1 interactively modulate this extension, giving program-appropriate effects. In accord with this model, Rad51-mediated recombination in vivo requires the presence of a sister. PMID:23973374

Hong, Soogil; Sung, Youngjin; Yu, Mi; Lee, Minsu; Kleckner, Nancy; Kim, Keun P.

2014-01-01

242

Intercultural Mediation  

Directory of Open Access Journals (Sweden)

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

Dragos Marian Radulescu

2012-11-01

243

Oligonucleotide recombination in corynebacteria without the expression of exogenous recombinases.  

Science.gov (United States)

Brevibacterium lactofermentum and Corynebacterium glutamicum are important biotechnology species of the genus Corynebacterium. The single-strand DNA annealing protein (SSAP)-independent oligonucleotide-mediated recombination procedure was successfully applied to the commonly used wild-type strains B. lactofermentum AJ1511 and C. glutamicum ATCC13032. When the rpsL gene was used as a target, the optimized protocol yielded up to (1.4±0.3)×10(3) and (6.7±1.3)×10(3) streptomycin-resistant colonies per 10(8) viable cells for the corresponding strains. We tested the influence of several parameters that are known to enhance the efficiency of oligonucleotide-mediated recombination in other bacterial species. Among them, increasing the concentration of oligonucleotides and targeting the lagging strand of the chromosome have proven to have positive effects on both of the tested species. No difference in the efficiency of recombination was observed between the oligonucleotides phosphorothiorated at the 5' ends and the unmodified oligonucleotides or between the oligonucleotides with four mutated nucleotides and those with one mutated nucleotide. The described approach demonstrates that during the adaptation of the recombineering technique, testing SSAP-independent oligonucleotide-mediated recombination could be a good starting point. Such testing could decrease the probability of an incorrect interpretation of the effect of exogenous protein factors (such as SSAP and/or corresponding exonucleases) due to non-optimal experimental conditions. In addition, SSAP-independent recombination itself could be useful in combination with suitable selection/enrichment methods. PMID:25087479

Krylov, Alexander A; Kolontaevsky, Egor E; Mashko, Sergey V

2014-10-01

244

Recombinant Collagenlike Proteins  

Science.gov (United States)

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

Fertala, Andzej

2007-01-01

245

Test of Hartree-Fock-RPA neutron and proton transition densities for 206,208Pb(p,p') at 650 MeV  

International Nuclear Information System (INIS)

A microscopic description of new data for inelastic scattering of 650 MeV protons on 206,208Pb with excitation of low-lying collective states is presented. The DWIA approximation is employed to calculate the scattering amplitude with the effective ?-dependent t-matrix determined in our previous analysis of elastic proton scattering on 40Ca. In the case of 206Pb, the transition densities have been calculated in the HF+RPA theory with the Skyrme SkM' force. For 208Pb, the result of Decharge and Gogny (1980) was employed. The ground-state densities were taken from model-independent analyses of electron and proton scattering data. The theories describe the radial distributions of the scattering densities reasonably well, especially in 208Pb. However, their normalizations need readjusting. For neutron components they vary from 0.8 to 1.7 in 208Pb and they are close to unity within 20% in 206Pb. (author)

246

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

Directory of Open Access Journals (Sweden)

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

N. Wildmann

2014-04-01

247

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

248

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

249

Recombinant glucose uptake system  

Science.gov (United States)

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

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

1997-01-01

250

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

251

Dielectronic recombination lines of C+  

Science.gov (United States)

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.

Sochi, Taha; Storey, Peter J.

2013-11-01

252

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

Directory of Open Access Journals (Sweden)

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

Ellis April L

2010-09-01

253

CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri.  

Science.gov (United States)

Clustered regularly interspaced palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) nuclease protect bacteria and archeae from foreign DNA by site-specific cleavage of incoming DNA. Type-II CRISPR-Cas systems, such as the Streptococcus pyogenes CRISPR-Cas9 system, can be adapted such that Cas9 can be guided to a user-defined site in the chromosome to introduce double-stranded breaks. Here we have developed and optimized CRISPR-Cas9 function in the lactic acid bacterium Lactobacillus reuteri ATCC PTA 6475. We established proof-of-concept showing that CRISPR-Cas9 selection combined with single-stranded DNA (ssDNA) recombineering is a realistic approach to identify at high efficiencies edited cells in a lactic acid bacterium. We show for three independent targets that subtle changes in the bacterial genome can be recovered at efficiencies ranging from 90 to 100%. By combining CRISPR-Cas9 and recombineering, we successfully applied codon saturation mutagenesis in the L. reuteri chromosome. Also, CRISPR-Cas9 selection is critical to identify low-efficiency events such as oligonucleotide-mediated chromosome deletions. This also means that CRISPR-Cas9 selection will allow identification of recombinant cells in bacteria with low recombineering efficiencies, eliminating the need for ssDNA recombineering optimization procedures. We envision that CRISPR-Cas genome editing has the potential to change the landscape of genome editing in lactic acid bacteria, and other Gram-positive bacteria. PMID:25074379

Oh, Jee-Hwan; van Pijkeren, Jan-Peter

2015-01-01

254

Defects of class-switch recombination.  

Science.gov (United States)

Shaping of the secondary antibody repertoire is generated by means of class-switch recombination (CSR), which replaces IgM with other isotypes, and somatic hypermutation (SHM), which allows production of high-affinity antibodies. However, the molecular mechanisms underlying these important processes have long remained obscure. Immunodeficiency with hyper-IgM comprises a group of genetically heterogeneous defects of CSR variably associated with defects of SHM. The study of these patients has allowed us to recognize that both T-cell-B-cell interaction (resulting in CD40-mediated signaling) and intrinsic B-cell mechanisms are involved in CSR and SHM. Elucidation of the molecular defects underlying these disorders has been essential to better understand the molecular basis of Ig diversification and has offered the opportunity to define the clinical spectrum of these diseases and to prompt more accurate diagnostic and therapeutic approaches. PMID:16630945

Notarangelo, Luigi D; Lanzi, Gaetana; Peron, Sophie; Durandy, Anne

2006-04-01

255

Immunoglobulin class-switch recombination deficiencies.  

Science.gov (United States)

Immunoglobulin class-switch recombination deficiencies (Ig-CSR-Ds) are rare primary immunodeficiencies characterized by defective switched isotype (IgG/IgA/IgE) production. Depending on the molecular defect in question, the Ig-CSR-D may be combined with an impairment in somatic hypermutation (SHM). Some of the mechanisms underlying Ig-CSR and SHM have been described by studying natural mutants in humans. This approach has revealed that T cell-B cell interaction (resulting in CD40-mediated signaling), intrinsic B-cell mechanisms (activation-induced cytidine deaminase-induced DNA damage), and complex DNA repair machineries (including uracil-N-glycosylase and mismatch repair pathways) are all involved in class-switch recombination and SHM. However, several of the mechanisms required for full antibody maturation have yet to be defined. Elucidation of the molecular defects underlying the diverse set of Ig-CSR-Ds is essential for understanding Ig diversification and has prompted better definition of the clinical spectrum of diseases and the development of increasingly accurate diagnostic and therapeutic approaches. PMID:22894609

Durandy, Anne; Kracker, Sven

2012-01-01

256

Immunization of cattle with recombinant Babesia bovis merozoite surface antigen-1.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Cattle immunized with a recombinant merozoite surface antigen-1 molecule (MSA-1) produced high-titered antibody that reacted with the surface of the parasite and neutralized merozoite infectivity in vitro. However, recombinant MSA-1 immunization did not confer protection against challenge with virulent Babesia bovis. These results indicate that antibody-mediated neutralization of merozoite infectivity in vitro, at least for MSA-1-specific antibody, does not reflect in vivo protective immunity...

Hines, S. A.; Palmer, G. H.; Jasmer, D. P.; Goff, W. L.; Mcelwain, T. F.

1995-01-01

257

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

International Nuclear Information System (INIS)

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

258

Induction of homologous recombination between sequence repeats by the activation induced cytidine deaminase (AID) protein.  

Science.gov (United States)

The activation induced cytidine deaminase (AID) protein is known to initiate somatic hypermutation, gene conversion or switch recombination by cytidine deamination within the immunoglobulin loci. Using chromosomally integrated fluorescence reporter transgenes, we demonstrate a new recombinogenic activity of AID leading to intra- and intergenic deletions via homologous recombination of sequence repeats. Repeat recombination occurs at high frequencies even when the homologous sequences are hundreds of bases away from the positions of AID-mediated cytidine deamination, suggesting DNA end resection before strand invasion. Analysis of recombinants between homeologous repeats yielded evidence for heteroduplex formation and preferential migration of the Holliday junctions to the boundaries of sequence homology. These findings broaden the target and off-target mutagenic potential of AID and establish a novel system to study induced homologous recombination in vertebrate cells.DOI: http://dx.doi.org/10.7554/eLife.03110.001. PMID:25006166

Buerstedde, Jean-Marie; Lowndes, Noel; Schatz, David G

2014-01-01

259

Recombineering: genetic engineering in bacteria using homologous recombination.  

Science.gov (United States)

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

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

2014-01-01

260

Activation of recF-dependent recombination in Escherichia coli by bacteriophage lambda- and P22-encoded functions.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Escherichia coli strains bearing wild-type and mutant alleles of various recombination genes, as well as plasmids that express recombination-related genes of bacteriophages lambda and P22, were tested for their proficiency as recipients in Hfr-mediated conjugation. It was found that the homologous recombination systems of both phages could promote recombination in a recB recC mutant host. In addition, the Abc function of P22, but not the Gam function of lambda, was found to inhibit recombinat...

Poteete, A. R.; Volkert, M. R.

1988-01-01

 
 
 
 
261

Three Decades of Recombinant DNA.  

Science.gov (United States)

Discusses highlights in the development of genetic engineering, examining techniques with recombinant DNA, legal and ethical issues, GenBank (a national database of nucleic acid sequences), and other topics. (JN)

Palmer, Jackie

1985-01-01

262

Hydrogen recombiner development at AECL  

International Nuclear Information System (INIS)

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

263

Two-center dielectronic recombination  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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 den...

Mu?ller, C.; Voitkiv, A. B.; Lo?pez-urrutia, J. R. Crespo; Harman, Z.

2010-01-01

264

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

265

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.

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

266

An inverse-modelling approach for frequency response correction of capacitive humidity sensors in ABL research with small remotely piloted aircraft (RPA)  

Science.gov (United States)

The measurement of water vapour concentration in the atmosphere is an ongoing challenge in environmental research. Satisfactory solutions exist for ground-based meteorological stations and measurements of mean values. However, carrying out advanced research of thermodynamic processes aloft as well, above the surface layer and especially in the atmospheric boundary layer (ABL), requires the resolution of small-scale turbulence. Sophisticated optical instruments are used in airborne meteorology with manned aircraft to achieve the necessary fast-response measurements of the order of 10 Hz (e.g. LiCor 7500). Since these instruments are too large and heavy for the application on small remotely piloted aircraft (RPA), a method is presented in this study that enhances small capacitive humidity sensors to be able to resolve turbulent eddies of the order of 10 m. The sensor examined here is a polymer-based sensor of the type P14-Rapid, by the Swiss company Innovative Sensor Technologies (IST) AG, with a surface area of less than 10 mm2 and a negligible weight. A physical and dynamical model of this sensor is described and then inverted in order to restore original water vapour fluctuations from sensor measurements. Examples of flight measurements show how the method can be used to correct vertical profiles and resolve turbulence spectra up to about 3 Hz. At an airspeed of 25 m s-1 this corresponds to a spatial resolution of less than 10 m.

Wildmann, N.; Kaufmann, F.; Bange, J.

2014-09-01

267

Non-parallel recombination limits Cre-LoxP-based reporters as precise indicators of conditional genetic manipulation.  

Science.gov (United States)

Cre/LoxP-mediated recombination allows for conditional gene activation or inactivation. When combined with an independent lineage-tracing reporter allele, this technique traces the lineage of presumptive genetically modified Cre-expressing cells. Several studies have suggested that floxed alleles have differential sensitivities to Cre-mediated recombination, which raises concerns regarding utilization of Cre-reporters to monitor recombination of other floxed loci of interest. Here, we directly investigate the recombination correlation, at cellular resolution, between several floxed alleles induced by Cre-expressing mouse lines. The recombination correlation between different reporter alleles varied greatly in otherwise genetically identical cell types. The chromosomal location of floxed alleles, distance between LoxP sites, sequences flanking the LoxP sites, and the level of Cre activity per cell all likely contribute to observed variations in recombination correlation. These findings directly demonstrate that, due to non-parallel recombination events, commonly available Cre reporter mice cannot be reliably utilized, in all cases, to trace cells that have DNA recombination in independent-target floxed alleles, and that careful validation of recombination correlations are required for proper interpretation of studies designed to trace the lineage of genetically modified populations, especially in mosaic situations. PMID:23441020

Liu, Jing; Willet, Spencer G; Bankaitis, Eric D; Xu, Yanwen; Wright, Chris V E; Gu, Guoqiang

2013-06-01

268

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

269

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

270

Recombination and its roles in DNA repair, cellular immortalization and cancer.  

Science.gov (United States)

Genetic recombination is the creation of new gene combinations in a cell or gamete, which differ from those of progenitor cells or parental gametes. In eukaryotes, recombination may occur at mitosis or meiosis. Mitotic recombination plays an indispensable role in DNA repair, which presumably directed its early evolution; the multiplicity of recombination genes and pathways may be best understood in this context, although they have acquired important additional functions in generating diversity, both somatically (increasing the immune repertoire) and in germ line (facilitating evolution). Chromosomal homologous recombination and HsRad51 recombinase expression are increased in both immortal and preimmortal transformed cells, and may favor the occurrence of multiple oncogenic mutations. Tumorigenesis in vivo is frequently associated with karyotypic instability, locus-specific gene rearrangements, and loss of heterozygosity at tumor suppressor loci - all of which can be recombinationally mediated. Genetic defects which increase the rate of somatic mutation (several of which feature elevated recombination) are associated with early incidence and high risk for a variety of cancers. Moreover, carcinogenic agents appear to quite consistently stimulate homologous recombination. If cells with high recombination arise, either spontaneously or in response to "recombinogens," and predispose to the development of cancer, what selective advantage could favor these cells prior to the occurrence of growth-promoting mutations? We propose that the augmentation of telomere-telomere recombination may provide just such an advantage, to hyper-recombinant cells within a population of telomerase-negative cells nearing their replicative (Hayflick) limit, by extending telomeres in some progeny cells and thus allowing their continued proliferation. PMID:23604399

Shammas, M A; Shmookler Reis, R J

1999-04-01

271

Axion mediation  

Science.gov (United States)

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

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

2013-07-01

272

Recombining plasmas as laser sources  

International Nuclear Information System (INIS)

The recombination of a dense plasma into a hydrogen-like ion introduces a potential medium for the amplification of radiation over a wide spectral range from 1R to X-Rays. The recombination is a three-body process and hence favours the higher quantum levels. A time dependent calculation shows very large gains for relatively cold temperatures. The time histories of inversion densities and hence the termination of the laser action are controlled by the return of the plasma into equilibrium condition. (Auth.)

273

Calculation of Gamow-Teller #betta#-strength functions in the Rubidium region in the RPA approximation with Nilsson model wave functions  

International Nuclear Information System (INIS)

We calculate allowed Gamow-Teller and, in a few cases, Fermi #betta#strength functions in a model that is applicable to studies of nuclei throughout the periodic system. For our first study we have selected a sequence of rubidium isotopes, namely 8937Rb - 9937Rb. We develop a model that use calculated Nilsson model wave functions, spherical or deformed, as the case may be, as the starting point for determining the wave functions of the mother and daughter nuclei in the #betta# decay. Pairing is treated in the BCS approximation. To account for the retardation of low-energy GT-decay rates we add, as is customarily done, a simple residual interaction specific to GT decay, namely V sub (GT)=:#betta#-1 x #betta#1:, to the Hamiltonian. This residual interaction is treated in the RPA approximation. The strength of the interaction is adjusted to get agreement between the calculated and experimental energy of the giant Gamow-Teller resonance for 208Pb and 144Sm. Since the present model is based on calculated wave functions and single-particle levels, studies of nuclei far from stability, where little experimental information is available, are more straightforward relative to calculations where experimental levels are used. The model can treat deformed nuclei employing wave functions calculated to desired accuracy, within the framework of the model, for the deformed single-particle well. The calcualtions show that use of single-particle parameters appropriate to the region studied and taking deformation into account is important. We find good agreement between calculated and experimental spectra over the region studied, provided an appropriate choice of single-particle parameters and deformation is made. (Authors)

274

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

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

Melchert Russell B

2009-04-01

275

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

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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

276

Molecular pathways: understanding the role of Rad52 in homologous recombination for therapeutic advancement.  

Science.gov (United States)

The Rad52 protein was largely ignored in humans and other mammals when the mouse knockout revealed a largely "no-effect" phenotype. However, using synthetic lethal approaches to investigate context-dependent function, new studies have shown that Rad52 plays a key survival role in cells lacking the function of the breast cancer type 1 susceptibility protein (BRCA1)-BRCA2 pathway of homologous recombination. Biochemical studies also showed significant differences between yeast and human Rad52 (hRad52), in which yeast Rad52 can promote strand invasion of replication protein A (RPA)-coated single-stranded DNA (ssDNA) in the presence of Rad51 but hRad52 cannot. This results in the paradox of how is hRad52 providing Rad51 function: presumably there is something missing in the biochemical assays that exists in vivo, but the nature of this missing factor is currently unknown. Recent studies have suggested that Rad52 provides back-up Rad51 function for all members of the BRCA1-BRCA2 pathway, suggesting that Rad52 may be a target for therapy in BRCA pathway-deficient cancers. Screening for ways to inhibit Rad52 would potentially provide a complementary strategy for targeting BRCA-deficient cancers in addition to poly (ADP-ribose) polymerase (PARP) inhibitors. PMID:23071261

Lok, Benjamin H; Powell, Simon N

2012-12-01

277

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

278

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

Science.gov (United States)

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

Moss, Bernard

1996-10-01

279

Chemoenzymatic bio-orthogonal chemistry for site-specific double modification of recombinant thrombomodulin.  

Science.gov (United States)

Best of both worlds: A one-pot strategy for site-specific PEGylation through strain-promoted alkyne-azide cycloaddition (SPAAC) and fluorescent labeling through sortase A-mediated ligation (SML) of recombinant thrombomodulin without prior chemical modification and without diminishing the protein activity has been developed. PMID:24357004

Jiang, Rui; Wang, Lin; Weingart, Jacob; Sun, Xue-Long

2014-01-01

280

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

 
 
 
 
281

Detecting recombination in evolving nucleotide sequences  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Genetic recombination can produce heterogeneous phylogenetic histories within a set of homologous genes. These recombination events can be obscured by subsequent residue substitutions, which consequently complicate their detection. While there are many algorithms for the identification of recombination events, little is known about the effects of subsequent substitutions on the accuracy of available recombination-detection approaches. Results We assessed the effect of subsequent substitutions on the detection of simulated recombination events within sets of four nucleotide sequences under a homogeneous evolutionary model. The amount of subsequent substitutions per site, prior evolutionary history of the sequences, and reciprocality or non-reciprocality of the recombination event all affected the accuracy of the recombination-detecting programs examined. Bayesian phylogenetic-based approaches showed high accuracy in detecting evidence of recombination event and in identifying recombination breakpoints. These approaches were less sensitive to parameter settings than other methods we tested, making them easier to apply to various data sets in a consistent manner. Conclusion Post-recombination substitutions tend to diminish the predictive accuracy of recombination-detecting programs. The best method for detecting recombined regions is not necessarily the most accurate in identifying recombination breakpoints. For difficult detection problems involving highly divergent sequences or large data sets, different types of approach can be run in succession to increase efficiency, and can potentially yield better predictive accuracy than any single method used in isolation.

Ragan Mark A

2006-09-01

282

Recombination luminescence in rigid media  

International Nuclear Information System (INIS)

When separated ion-pairs result from the ?-irradiation of pure or doped matrices or from solute photoionization, some of the photoejected electrons undergo spontaneous recombination, giving rise to the so-called isothermal luminescence (ITL). Cation-anion recombination takes place when molecular diffusion is possible; that is, it appears mostly in thermoluminescence (TL), upon warming irradiated samples. Electrons are photoextracted from matrix traps or from anions, and the neutralization luminescence is designated as stimulated luminescence (SL). ITL, TL, and SL all constitute very sensitive test for the presence of charged species. ITL decay kinetics throw some light on the recombination mechanism. In TL studies, the maxima of the glow curves correlate with phase transition temperatures (crystal ? crystal or glass ? crystal), providing insight into matrix molecular dynamics. From SL spectra, positive and negative charges can be discriminated, and various characteristics of the negative ions - electrons or anions - can be attained. The global SL irrespective of its spectrum composition, SL emission spectra, and SL excitation spectra or stimulation spectra are reviewed. The SL spectra, being specific of negative charged species, complement optical absorption spectroscopy when cations and anions have undistinguishable spectra. Even though radiative charge recombination constitutes the final step of ion-pair existence, it may serve to track back the successive stages of photoelectron life: electron ejection, matrix trapping or the attachments of electrons to molecules or radicals, and the release of trapped electrons. (Yamashita, S.)

283

Inhomogeneous recombinations during cosmic reionization  

Science.gov (United States)

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

Sobacchi, Emanuele; Mesinger, Andrei

2014-05-01

284

Recombinant DNA: History of the Controversy.  

Science.gov (United States)

The hazards associated with recombinant DNA research are presented along with some social implications and the development of recombinant DNA research guidelines by the National Institutes of Health. (SA)

Vigue, Charles L.; Stanziale, William G.

1979-01-01

285

Homologous recombination contributes to the repair of zinc-finger-nuclease induced double strand breaks in pig primary cells and facilitates recombination with exogenous DNA.  

Science.gov (United States)

Site-specific nucleases have become powerful tools for genome editing by the introduction of end-joining-mediated mutations, but it is unclear to which extent induced double strand breaks will also facilitate homologous recombination with exogenous DNA. This question is, however, of particular importance for somatic cells, which have to be modified for the generation of large animal models, but, on the other hand, have also been described to be reluctant to recombination-based DNA repair. Here, we examined zinc-finger nucleases for their potential to introduce modifications in pig somatic cells via end-joining or recombination. We found that co-transfection with nuclease-encoding plasmids resulted in a dramatic boost of recombination with different targeting vectors, suggesting a much more prominent role of this repair pathway in somatic cells than was previously thought. Although recombination with any of the vectors even occurred on both alleles of the target gene, we found also evidence for distinct properties of the used vectors regarding their preference for mono-allelic or bi-allelic modification. Thus, we show that the combined usage of site-specific nucleases and targeting vectors does not only promote homologous recombination in somatic cells but might also resemble a promising tool for detailed examination of DNA repair pathways. PMID:24613297

Klymiuk, Nikolai; Fezert, Pauline; Wünsch, Annegret; Kurome, Mayuko; Kessler, Barbara; Wolf, Eckhard

2014-05-10

286

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

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

Araki Masatake

2010-03-01

287

Polymorphic Variation in Human Meiotic Recombination  

Digital Repository Infrastructure Vision for European Research (DRIVER)

In this study, our phenotype of interest is meiotic recombination. Using genotypes of ?6,000 SNP markers in members of the Centre d'Étude du Polymorphisme Humain Utah pedigrees, we found extensive individual variation in the number of female and male recombination events. The locations and frequencies of these recombination events vary along the genome. In both female and male meiosis, the regions with the most recombination events are found at the ends of the chromosomes. Our analysis als...

Cheung, Vivian G.; Burdick, Joshua T.; Hirschmann, Deborah; Morley, Michael

2007-01-01

288

Recombinational substrates designed to study recombination between unique and repetitive sequences in vivo  

International Nuclear Information System (INIS)

Three recombination events, reciprocal recombination, sister-chromatid recombination, and gene conversion, were studied using substrates designed in vitro. Each type of recombination event can be monitored at any chromosomal location. The authors have shown that sister-chromatid recombination is induced mitotically by DNA damaging agents, such as methyl methanesulfonate and ?-rays, but is decreased mitotically in strains defective in rad52. Reciprocal recombination by which circular plasmids integrate into the genome is unaffected by rad52 defective alleles and occurs by a different recombination pathway. Mechanisms are suggested by which gene conversion between sister chromatids can generate chromosome rearrangements

289

Interaction of Ddc1 and RPA with single-stranded/double-stranded DNA junctions in yeast whole cell extracts: Proteolytic degradation of the large subunit of replication protein A in ddc1? strains.  

Science.gov (United States)

To characterize proteins that interact with single-stranded/double-stranded (ss/ds) DNA junctions in whole cell free extracts of Saccharomyces cerevisiae, we used [(32)P]-labeled photoreactive partial DNA duplexes containing a 3'-ss/ds-junction (3'-junction) or a 5'-ss/ds-junction (5'-junction). Identification of labeled proteins was achieved by MALDI-TOF mass spectrometry peptide mass fingerprinting and genetic analysis. In wild-type extract, one of the components of the Ddc1-Rad17-Mec3 complex, Ddc1, was found to be preferentially photocrosslinked at a 3'-junction. On the other hand, RPAp70, the large subunit of the replication protein A (RPA), was the predominant crosslinking product at a 5'-junction. Interestingly, ddc1? extracts did not display photocrosslinking of RPAp70 at a 5'-junction. The results show that RPAp70 crosslinked to DNA with a 5'-junction is subject to limited proteolysis in ddc1? extracts, whereas it is stable in WT, rad17?, mec3? and mec1? extracts. The degradation of the RPAp70-DNA adduct in ddc1? extract is strongly reduced in the presence of the proteasome inhibitor MG 132. We also addressed the question of the stability of free RPA, using anti-RPA antibodies. The results show that RPAp70 is also subject to proteolysis without photocrosslinking to DNA upon incubation in ddc1? extract. The data point to a novel property of Ddc1, modulating the turnover of DNA binding proteins such as RPAp70 by the proteasome. PMID:25089887

Sukhanova, Maria V; D'Herin, Claudine; Boiteux, Serge; Lavrik, Olga I

2014-10-01

290

Pharmaceutical strategies utilizing recombinant human serum albumin.  

Science.gov (United States)

Gene manipulation techniques open up the possibility of making recombinant human serum albumin (rHSA) or mutants with desirable therapeutic properties and for protein fusion products. rHSA can serve as a carrier in synthetic heme protein, thus reversibly carrying oxygen. Myristoylation of insulin results in a prolonged half-life because of self aggregation and increased albumin binding. Preferential albumin uptake by tumor cells serves as the basis for albumin-anticancer drug conjugate formulation. Furthermore, drug targeting can be achieved by incorporating drugs into albumin microspheres whereas liver targeting can be achieved by conjugating drug with galactosylated or mannosylated albumin. Microspheres and nanoparticles of different sizes can, with or without drugs and/or radioisotopes, be used for drug delivery or diagnostic purposes. In vivo implantation of albumin fusion protein expressing cells encapsulated in HSA-alginate coated beads showed promising results compared to organoids in rats. Chimeric peptide strategy with cationized albumin as the transport can deliver drugs via receptor mediated transcytosis through the blood brain barrier. Gene bearing, albumin microbubbles containing ultrasound contrast agents can non-invasively deliver gene after destruction by ultrasound. Various site-directed mutants of HSA can be tailor made depending on the application required. PMID:12069157

Chuang, Victor Tuan Giam; Kragh-Hansen, Ulrich; Otagiri, Masaki

2002-05-01

291

Recombinant respiratory syncytial virus F protein expression is hindered by inefficient nuclear export and mRNA processing.  

Science.gov (United States)

Studies of the fusion activity of respiratory syncytial virus (RSV) F protein are significantly hindered by low recombinant expression levels. While infection produces F protein levels detectable by western blot, recombinant expression produces undetectable to low levels of F protein. Identifying the obstacles that hinder recombinant F protein expression may lead to improved expression and facilitate the study of F protein function. We hypothesized that nuclear localization and/or inefficient RNA polymerase II-mediated transcription contribute to poor recombinant F protein expression. This study shows a combination of stalled nuclear export, premature polyadenylation, and low mRNA abundance all contribute to low recombinant F protein expression levels. In addition, this study provides an expression optimization strategy that results in greater F protein expression levels than observed by codon-optimization of the F protein gene, which will be useful for future studies of F protein function. PMID:20111897

Huang, Kelly; Lawlor, Heather; Tang, Roderick; MacGill, Randall S; Ulbrandt, Nancy D; Wu, Herren

2010-04-01

292

Microbial factories for recombinant pharmaceuticals  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

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

2009-01-01

293

Recombinations of Busy Beaver Machines  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

Ba?tfai, Norbert

2009-01-01

294

The CMB bispectrum from recombination  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We compute the cosmic microwave background temperature bispectrum generated by nonlinearities at recombination on all scales. We use CosmoLib$2^{\\rm nd}$, a numerical Boltzmann code at second-order to compute CMB bispectra on the full sky. We consistently include all effects except gravitational lensing, which can be added to our result using standard methods. The bispectrum is peaked on squeezed triangles and agrees with the analytic approximation in the squeezed limit at t...

Huang, Zhiqi; Vernizzi, Filippo

2012-01-01

295

Homologous Recombination in Negative Sense RNA Viruses  

Directory of Open Access Journals (Sweden)

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

Michael Worobey

2011-08-01

296

Accelerated homologous recombination and subsequent genome modification in Drosophila  

Science.gov (United States)

Gene targeting by ‘ends-out’ homologous recombination enables the deletion of genomic sequences and concurrent introduction of exogenous DNA with base-pair precision without sequence constraint. In Drosophila, this powerful technique has remained laborious and hence seldom implemented. We describe a targeting vector and protocols that achieve this at high frequency and with very few false positives in Drosophila, either with a two-generation crossing scheme or by direct injection in embryos. The frequency of injection-mediated gene targeting can be further increased with CRISPR-induced double-strand breaks within the region to be deleted, thus making homologous recombination almost as easy as conventional transgenesis. Our targeting vector replaces genomic sequences with a multifunctional fragment comprising an easy-to-select genetic marker, a fluorescent reporter, as well as an attP site, which acts as a landing platform for reintegration vectors. These vectors allow the insertion of a variety of transcription reporters or cDNAs to express tagged or mutant isoforms at endogenous levels. In addition, they pave the way for difficult experiments such as tissue-specific allele switching and functional analysis in post-mitotic or polyploid cells. Therefore, our method retains the advantages of homologous recombination while capitalising on the mutagenic power of CRISPR. PMID:24154526

Baena-Lopez, Luis Alberto; Alexandre, Cyrille; Mitchell, Alice; Pasakarnis, Laurynas; Vincent, Jean-Paul

2013-01-01

297

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

Energy Technology Data Exchange (ETDEWEB)

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

Skalski, John R.; Ngouenet, Roger F.

2001-05-01

298

Recombination rate and protein evolution in yeast  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Theory and artificial selection experiments show that recombination can promote adaptation by enhancing the efficacy of natural selection, but the extent to which recombination affects levels of adaptation across the genome is still an open question. Because patterns of molecular evolution reflect long-term processes of mutation and selection in nature, interactions between recombination rate and genetic differentiation between species can be used to test the benefits of recombination. However, this approach faces a major difficulty: different evolutionary processes (i.e. negative versus positive selection produce opposing relationships between recombination rate and genetic divergence, and obscure patterns predicted by individual benefits of recombination. Results We use a combination of polymorphism and genomic data from the yeast Saccharomyces cerevisiae to infer the relative importance of nearly-neutral (i.e. slightly deleterious evolution in different gene categories. For genes with high opportunities for slightly deleterious substitution, recombination substantially reduces the rate of molecular evolution, whereas divergence in genes with little opportunity for slightly deleterious substitution is not strongly affected by recombination. Conclusion These patterns indicate that adaptation throughout the genome can be strongly influenced by each gene's recombinational environment, and suggest substantial long-term fitness benefits of enhanced purifying selection associated with sexual recombination.

Knowles L Lacey

2007-11-01

299

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

International Nuclear Information System (INIS)

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

300

[Generation and preliminary immunological efficacy of a recombinant human adenovirus-rabies virus glycoprotein].  

Science.gov (United States)

To construct a recombinant human adenovirus type 5 expressing glycoprotein (GP) of attenuated rabies virus SRV9 and testing immunological efficacy on the immunized mice. Open reading frame of rabies virus GP gene of SRV9 strain was cloned into the shuttle vector of adenovirus expression system in multiple cloning sites to construct the recombinant shuttle plasmid pacAd5 CMV-Gs9, cotransfection was performed into 293AD cells mediated by FuGENE Transfection Reagent with linearized backbone plasmid and recombinant shuttle plasmid, cell cultures were collected after CPE appearance and were identified by PCR and electronmicroscopy, virus titer was measured in 293AD cells. Kunming mice were intraperitoneally injected with 10(6) TCID50 adenovirus, blood for serum preparation was collected through caudal vein pre-immune and post-immune and tested for VNA appearance by fluorescent antibody virus neutralization test (FAVN) detection. Recombinant shuttle plasmid pacAd5 CMV-Gs9 was constructed correctly. A recombinant human adenovirus type 5 was obtained expressing GP protein of rabies virus SRV9. The virus titer reached 10(6) CFU/mL at the least. All mice developed a certain amount of the anti-rabies neutralizing antibody 14 days after intraperitoneal inoculation, while the effective protection rates were 90%. In conclusion, Recombinant adenovirus expressing the rabies virus GP was constructed successfully and a certain amount of neutralizing antibodies were induced in mice, which laid the material foundation for further development of new rabies vaccine. PMID:21998956

Wang, Ying; Zhang, Shou-Feng; Liu, Ye; Zhang, Fei; Zhang, Jin-Xia; Hu, Rong-Liang

2011-09-01

 
 
 
 
301

AFLP and single-strand conformation polymorphism studies of recombination in the entomopathogenic fungus Nomuraea rileyi.  

Science.gov (United States)

In most putative asexual fungi analysed through population genetic studies, recombination has been detected. However, the mechanism by which it is achieved is still not known. A parasexual cycle is known to occur in asexual fungi but there is no evidence, as yet, of its prevalence in natural populations. This study was undertaken to investigate the possibility of a parasexual cycle mediating recombination in the mitosporic fungus Nomuraea rileyi. The genotypic diversity in isolates sampled from an epizootic population from South India was studied through AFLP. The AFLP data were subjected to analysis of molecular variance (AMOVA) and cluster analysis. Great genetic variation was observed in the population including the isolates from a single insect. To assess the occurrence of recombination in the population, single-strand conformation polymorphism (SSCP) of partial regions of two mitochondrial (mt) genes (rRNA genes of LSU and SSU) and a nuclear gene (beta tubulin) was performed. The SSCP data were analysed using MP, the tree length permutation test, and multilocus analysis. Recombination was inferred from the SSCP analysis. The occurrence of isolates with diverse genotypes in a single insect; the fact that fungi multiply as hyphal bodies (cell wall-less) in the insect haemolymph; and the inference of recombination in mitochondrial genes (suggesting cytomixis), all indicate that recombination is accomplished by fusion of hyphal bodies of different isolates infecting the insect. PMID:17604614

Devi, Uma K; Reineke, Annette; Rao, Uma C Maheswara; Reddy, Nageswara Rao N; Khan, Akbar P Ali

2007-06-01

302

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

303

Polymorphic variation in human meiotic recombination.  

Science.gov (United States)

In this study, our phenotype of interest is meiotic recombination. Using genotypes of approximately 6,000 SNP markers in members of the Centre d'Etude du Polymorphisme Humain Utah pedigrees, we found extensive individual variation in the number of female and male recombination events. The locations and frequencies of these recombination events vary along the genome. In both female and male meiosis, the regions with the most recombination events are found at the ends of the chromosomes. Our analysis also shows that there are polymorphic differences among individuals in the activity of the recombination "jungles"; these preferred sites of meiotic recombination differ greatly among individuals. These findings have important implications for understanding genetic disorders that result from improper chromosome segregation. PMID:17273974

Cheung, Vivian G; Burdick, Joshua T; Hirschmann, Deborah; Morley, Michael

2007-03-01

304

Recombination fluorescence in ultracold neutral plasmas  

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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

305

A simplified system for generating recombinant adenoviruses  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. We report herein a strategy that simplifies the generation and production of such viruses. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, using homologous recombination in bacteria rather than in eukaryotic cells. After transfections of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid o...

He, Tong-chuan; Zhou, Shibin; Da Costa, Luis T.; Yu, Jian; Kinzler, Kenneth W.; Vogelstein, Bert

1998-01-01

306

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

International Nuclear Information System (INIS)

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

307

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

308

Microbial factories for recombinant pharmaceuticals  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Most of the hosts used to produce the 151 recombinant pharmaceuticals so far approved for human use by the Food and Drug Administration (FDA and/or by the European Medicines Agency (EMEA are microbial cells, either bacteria or yeast. This fact indicates that despite the diverse bottlenecks and obstacles that microbial systems pose to the efficient production of functional mammalian proteins, namely lack or unconventional post-translational modifications, proteolytic instability, poor solubility and activation of cell stress responses, among others, they represent convenient and powerful tools for recombinant protein production. The entering into the market of a progressively increasing number of protein drugs produced in non-microbial systems has not impaired the development of products obtained in microbial cells, proving the robustness of the microbial set of cellular systems (so far Escherichia coli and Saccharomyces cerevisae developed for protein drug production. We summarize here the nature, properties and applications of all those pharmaceuticals and the relevant features of the current and potential producing hosts, in a comparative way.

Domingo-Espín Joan

2009-03-01

309

On effective surface recombination parameters  

Science.gov (United States)

This paper examines two effective surface recombination parameters: the effective surface recombination velocity Seff and the surface saturation current density J0s. The dependence of Seff and J0s on surface charge Q, surface dopant concentration Ns, and interface parameters is derived. It is shown that for crystalline silicon at 300 K in low-injection, Seff is independent of Ns only when Q2/Ns films. Under the same conditions, J0s is independent of Ns when Q2/Ns > 1.5 × 107 cm for accumulation and Q1.85/Ns > 1.5 × 106 cm for inversion. These conditions are commonly satisfied in undiffused wafers but rarely in diffused wafers. We conclude that for undiffused silicon, J0s is superior to the conventional Seff as a metric for quantifying the surface passivation, whereas for diffused silicon, the merit in using J0s or Seff (or neither) depends on the sample. Experimental examples are given that illustrate the merits and flaws of J0s and Seff.

McIntosh, Keith R.; Black, Lachlan E.

2014-07-01

310

Experimental recombination rates for highly charged ions  

International Nuclear Information System (INIS)

Recent studies of recombination between free electrons and highly charged ions using electron coolers of heavy-ion storage rings have produced accurate rate coefficients of interest for plasma modeling and diagnostics. Some surprises were discovered which can lead to revisions of recombination models. With bare ions one finds at low energy a strong and puzzling deviation from radiative recombination theory. Dielectronic recombination with C3+, N4+) show that jj coupling gives essential contributions to the cross section also for light ions. (author)

311

How homologous recombination generates a mutable genome  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Recombination and mutation have traditionally been regarded as independent evolutionary processes: the latter generates variation, which the former reshuffles. Recent studies, however, have suggested that allelic recombination influences the underlying mutation rate, as high mutation rates are inferred in regions of high recombination. Furthermore, recombination between duplicated sequences introduces structural variation into the human genome and facilitates the formation of clustered gene families. Comparisons of wholegenome sequences reveal the expansion of gene family clusters to be an important mode of genome evolution. The negative aspect of this genomic dynamism is the contribution of these rearrangements to genetic diseases.

Hurles Matthew

2005-09-01

312

BRCA1 controls homologous recombination at Tus/Ter-stalled mammalian replication forks.  

Science.gov (United States)

Replication fork stalling can promote genomic instability, predisposing to cancer and other diseases. Stalled replication forks may be processed by sister chromatid recombination (SCR), generating error-free or error-prone homologous recombination (HR) outcomes. In mammalian cells, a long-standing hypothesis proposes that the major hereditary breast/ovarian cancer predisposition gene products, BRCA1 and BRCA2, control HR/SCR at stalled replication forks. Although BRCA1 and BRCA2 affect replication fork processing, direct evidence that BRCA gene products regulate homologous recombination at stalled chromosomal replication forks is lacking, due to a dearth of tools for studying this process. Here we report that the Escherichia coli Tus/Ter complex can be engineered to induce site-specific replication fork stalling and chromosomal HR/SCR in mouse cells. Tus/Ter-induced homologous recombination entails processing of bidirectionally arrested forks. We find that the Brca1 carboxy (C)-terminal tandem BRCT repeat and regions of Brca1 encoded by exon 11-two Brca1 elements implicated in tumour suppression-control Tus/Ter-induced homologous recombination. Inactivation of either Brca1 or Brca2 increases the absolute frequency of 'long-tract' gene conversions at Tus/Ter-stalled forks, an outcome not observed in response to a site-specific endonuclease-mediated chromosomal double-strand break. Therefore, homologous recombination at stalled forks is regulated differently from homologous recombination at double-strand breaks arising independently of a replication fork. We propose that aberrant long-tract homologous recombination at stalled replication forks contributes to genomic instability and breast/ovarian cancer predisposition in BRCA mutant cells. PMID:24776801

Willis, Nicholas A; Chandramouly, Gurushankar; Huang, Bin; Kwok, Amy; Follonier, Cindy; Deng, Chuxia; Scully, Ralph

2014-06-26

313

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

314

Role of PCNA and TLS polymerases in D-loop extension during homologous recombination in humans?  

Science.gov (United States)

Homologous recombination (HR) is essential for maintaining genomic integrity, which is challenged by a wide variety of potentially lethal DNA lesions. Regardless of the damage type, recombination is known to proceed by RAD51-mediated D-loop formation, followed by DNA repair synthesis. Nevertheless, the participating polymerases and extension mechanism are not well characterized. Here, we present a reconstitution of this step using purified human proteins. In addition to Pol ?, TLS polymerases, including Pol ? and Pol ?, also can extend D-loops. In vivo characterization reveals that Pol ? and Pol ? are involved in redundant pathways for HR. In addition, the presence of PCNA on the D-loop regulates the length of the extension tracks by recruiting various polymerases and might present a regulatory point for the various recombination outcomes. PMID:23731732

Sebesta, Marek; Burkovics, Peter; Juhasz, Szilvia; Zhang, Sufang; Szabo, Judit E.; Lee, Marietta Y.W.T.; Haracska, Lajos; Krejci, Lumir

2013-01-01

315

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

2014-01-01

316

Putative antirecombinase Srs2 DNA helicase promotes noncrossover homologous recombination avoiding loss of heterozygosity  

Science.gov (United States)

DNA damage alone or DNA replication fork arrest at damaged sites may induce DNA double-strand breaks and initiate homologous recombination. This event can result in a crossover with a homologous chromosome, causing loss of heterozygosity along the chromosome. It is known that Srs2 acts as an antirecombinase at the replication fork: it is recruited by the SUMO (a small ubiquitin-related modifier)-conjugated DNA-polymerase sliding clamp (PCNA) and interferes with Rad51/Rad52-mediated homologous recombination. Here, we report that Srs2 promotes another type of homologous recombination that produces noncrossover products only, in collaboration with PCNA and Rad51. Srs2 proteins lacking the Rad51-binding domain, PCNA-SUMO–binding motifs, or ATP hydrolysis-dependent DNA helicase activity reduce this noncrossover recombination. However, the removal of either the Rad51-binding domain or the PCNA-binding motif strongly increases crossovers. Srs2 gene mutations are epistatic to mutations in the PCNA modification-related genes encoding PCNA, Siz1 (a SUMO ligase) and Rad6 (a ubiquitin-conjugating protein). Knocking out RAD51 blocked this recombination but enhanced nonhomologous end-joining. We hypothesize that, during DNA double-strand break repair, Srs2 mediates collaboration between the Rad51 nucleofilament and PCNA-SUMO and directs the heteroduplex intermediate to DNA synthesis in a moving bubble. This Rad51/Rad52/Srs2/PCNA-mediated noncrossover pathway avoids both interchromosomal crossover and imprecise end-joining, two potential paths leading to loss of heterozygosity, and contributes to genome maintenance and human health. PMID:24043837

Miura, Tohru; Shibata, Takehiko; Kusano, Kohji

2013-01-01

317

Titania Photocatalysis beyond Recombination: A Critical Review  

Directory of Open Access Journals (Sweden)

Full Text Available This short review paper shows the significance of recombination of a photoexcited electron and a hole in conduction and valence bands, respectively, of a titania photocatalyst, since recombination has not yet been fully understood and has not been evaluated adequately during the past several decades of research on heterogeneous photocatalysis.

Bunsho Ohtani

2013-11-01

318

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.)

319

Cell biology of homologous recombination in yeast  

DEFF Research Database (Denmark)

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

Eckert-Boulet, Nadine Valerie; Rothstein, Rodney

2011-01-01

320

Recombinant DNA encoding a desulfurization biocatalyst  

Science.gov (United States)

This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous.

Rambosek, John (Seattle, WA); Piddington, Chris S. (Seattle, WA); Kovacevich, Brian R. (Seattle, WA); Young, Kevin D. (Grand Forks, ND); Denome, Sylvia A. (Thompson, ND)

1994-01-01

 
 
 
 
321

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

322

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

323

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

324

Multiple Levels of Affinity-Dependent DNA Discrimination in Cre-LoxP Recombination  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Cre recombinase residue Arg259 mediates a canonical bidentate hydrogen-bonded contact with Gua27 of its LoxP DNA substrate. Substituting Cyt8-Gua27 with the three other basepairs, to give LoxAT, LoxTA, and LoxGC, reduced Cre-mediated recombination in vitro, with the preference order of Gua27 >Ade27 ~ Thy27 ? Cyt27. While LoxAT and LoxTA exhibited 2.5-fold reduced affinity and 2.5-5-fold slower reaction rates, LoxGC was a barely functional substrate. Its maximum level of turnover was 6-fold ...

Gelato, Kathy A.; Martin, Shelley S.; Wong, Scott; Baldwin, Enoch P.

2006-01-01

325

BLM helicase facilitates RNA polymerase I-mediated ribosomal RNA transcription  

Science.gov (United States)

Bloom's syndrome (BS) is an autosomal recessive disorder that is invariably characterized by severe growth retardation and cancer predisposition. The Bloom's syndrome helicase (BLM), mutations of which lead to BS, localizes to promyelocytic leukemia protein bodies and to the nucleolus of the cell, the site of RNA polymerase I-mediated ribosomal RNA (rRNA) transcription. rRNA transcription is fundamental for ribosome biogenesis and therefore protein synthesis, cellular growth and proliferation; its inhibition limits cellular growth and proliferation as well as bodily growth. We report that nucleolar BLM facilitates RNA polymerase I-mediated rRNA transcription. Immunofluorescence studies demonstrate the dependance of BLM nucleolar localization upon ongoing RNA polymerase I-mediated rRNA transcription. In vivo protein co-immunoprecipitation demonstrates that BLM interacts with RPA194, a subunit of RNA polymerase I. 3H-uridine pulse-chase assays demonstrate that BLM expression is required for efficient rRNA transcription. In vitro helicase assays demonstrate that BLM unwinds GC-rich rDNA-like substrates that form in the nucleolus and normally inhibit progression of the RNA polymerase I transcription complex. These studies suggest that nucleolar BLM modulates rDNA structures in association with RNA polymerase I to facilitate RNA polymerase I-mediated rRNA transcription. Given the intricate relationship between rDNA metabolism and growth, our data may help in understanding the etiology of proportional dwarfism in BS. PMID:22106380

Grierson, Patrick M.; Lillard, Kate; Behbehani, Gregory K.; Combs, Kelly A.; Bhattacharyya, Saumitri; Acharya, Samir; Groden, Joanna

2012-01-01

326

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

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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

327

Erlotinib attenuates homologous recombinational repair of chromosomal breaks in human breast cancer cells  

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The epidermal growth factor receptor family (EGFR) has been implicated in a number of cancers, including breast, and its members have become the target of novel cancer therapies. In this report, we show a novel link between erlotinib, a potent EGFR inhibitor, DNA damage, and homology-directed recombinational repair (HDR) in human breast cancer cells. Erlotinib suppresses HDR. This is not secondary to erlotinib-mediated changes in cell cycle and is associated with increased ?-H2AX foci, which...

Li, Liping; Wang, Hong; Yang, Eddy S.; Arteaga, Carlos L.; Xia, Fen

2008-01-01

328

Regulation of human lung fibroblast glycosaminoglycan production by recombinant interferons, tumor necrosis factor, and lymphotoxin.  

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Mononuclear cells may be important regulators of fibroblast glycosaminoglycan (GAG) biosynthesis. However, the soluble factors mediating these effects, the importance of intercytokine interactions in this regulation and the mechanisms of these alterations remain poorly understood. We analyzed the effect of recombinant (r) tumor necrosis factor (TNF), lymphotoxin (LT), and gamma, alpha, and beta 1 interferons (INF-gamma, -alpha and -beta 1), alone and in combination, on GAG production by norma...

Elias, J. A.; Krol, R. C.; Freundlich, B.; Sampson, P. M.

1988-01-01

329

The Recombinational Anatomy of a Mouse Chromosome  

Science.gov (United States)

Among mammals, genetic recombination occurs at highly delimited sites known as recombination hotspots. They are typically 1–2 kb long and vary as much as a 1,000-fold or more in recombination activity. Although much is known about the molecular details of the recombination process itself, the factors determining the location and relative activity of hotspots are poorly understood. To further our understanding, we have collected and mapped the locations of 5,472 crossover events along mouse Chromosome 1 arising in 6,028 meioses of male and female reciprocal F1 hybrids of C57BL/6J and CAST/EiJ mice. Crossovers were mapped to a minimum resolution of 225 kb, and those in the telomere-proximal 24.7 Mb were further mapped to resolve individual hotspots. Recombination rates were evolutionarily conserved on a regional scale, but not at the local level. There was a clear negative-exponential relationship between the relative activity and abundance of hotspot activity classes, such that a small number of the most active hotspots account for the majority of recombination. Females had 1.2× higher overall recombination than males did, although the sex ratio showed considerable regional variation. Locally, entirely sex-specific hotspots were rare. The initiation of recombination at the most active hotspot was regulated independently on the two parental chromatids, and analysis of reciprocal crosses indicated that parental imprinting has subtle effects on recombination rates. It appears that the regulation of mammalian recombination is a complex, dynamic process involving multiple factors reflecting species, sex, individual variation within species, and the properties of individual hotspots. PMID:18617997

Paigen, Kenneth; Szatkiewicz, Jin P.; Sawyer, Kathryn; Leahy, Nicole; Parvanov, Emil D.; Ng, Siemon H. S.; Graber, Joel H.; Broman, Karl W.; Petkov, Petko M.

2008-01-01

330

Evidence of recombination in putative ancient asexuals.  

Science.gov (United States)

Ancient asexuals have been considered to be a contradiction of the basic tenets of evolutionary theory. Barred from rearranging genetic variation by recombination, their reduced number of gene arrangements is thought to hamper their response to changing environments. For the same reason, it should be difficult for them to avoid the build-up of deleterious mutations. Several groups of taxonomically diverse organisms are thought to be ancient asexuals, although clear evidence for or against the existence of recombination events is scarce. Several methods have recently been developed for predicting recombination events by analyzing aligned sequences of a given region of DNA that all originate from one species. The methods are based on phylogenetic, substitution, and compatibility analyses. Here we present the results of analyses of sequence data from different loci studied in several groups of evolutionarily distant species that are considered to be ancient asexuals, using seven different types of analysis. The groups of organisms were the arbuscular mycorrhizal fungi (Glomales), Darwinula stevensoni (Darwinuloidea crustacean ostracods) and the bdelloid rotifers (Bdelloidea), which are thought to have been asexual for the last 400, 25-100, and 35-40 Myr, respectively. The seven different analytical methods evaluated the evolutionary relationships among haplotypes, and these methods had previously been shown to be reliable for predicting the occurrence of recombination events. Despite the different degree of genetic variation among the different groups of organisms, at least some evidence for recombination was found in all species groups. In particular, predictions of recombination events in the arbuscular mycorrhizal fungi were frequent. Predictions of recombination were also found for sequence data that have previously been used to infer the absence of recombination in bdelloid rotifers. Although our results have to be taken with some caution because they could signal very ancient recombination events or possibly other genetic variation of nonrecombinant origin, they suggest that some cryptic recombination events may exist in these organisms. PMID:12679528

Gandolfi, Andrea; Sanders, Ian R; Rossi, Valeria; Menozzi, Paolo

2003-05-01

331

Recent Developments in Dissociative Recombination  

International Nuclear Information System (INIS)

There have been a number of recent developments in dissociative recombination research as it relates to ITER, that should be highlighted. These concern primarily experimental and modelling issues and this document will not touch upon the topics of the other scientists involved in DR studies that are present at the meeting. The topic of branching ratios in general is a topic fundamental to DR especially how it influences the formation of radical and stable neutral molecules that again might play a role in particle formation. It should be remembered that the reactions of neutral radicals to form cyclic compounds are responsible for the formation of soot in combustion, though the role played by ions in flames is at best uncertain. In the near wall plasma environment, ion processes may well be more important since neutral species are rarer. Modelling studies by Pernot and collaborators at the Universite de Paris-Sud have shown that if one compares the yields of individual neutral species in ion-chemistry models (in this particular case, the ionosphere of Titan), and if one assumes that DR reactions of hydrocarbon ions primarily decay via the ejection of a hydrogen ion (which is assumed by most Titan ionospheric models) and if one compares these predictions with those coming from a model where actual measured branching ratios are used, differences of up to 5 orders of magnitude are found. This shows very clearly the need for branching ratio studies. In early merged beam studies of DR performed in Canada in the 1970's, it was noticed that cross sections for polyatomic species typically displayed a sharp fall-off above 0.1 eV. This has since been seen in many storage ring studies and clearly this has important consequences for ITER chemistry where plasma temperatures are likely to be well above ambient. In a recent analysis, Jungen and Pratt have explained this phenomenon on the basis that the recombination is dominated by the indirect process (initial capture into a vibrationally excited, neutral Rydberg state) in which the propensity rule (+?v=1) dominates the capture. When the electron energy exceeds that between the v'=0 and v'=1 levels of the ion, where the capture must now involve a ?v=2 transition, this will be much less effective and so the cross section drops precipitously. This assumes of course that the recombining ion is primarily in the ground v=0 level. H3+ continues to be an active subject of research and a very recent experiment at the TSR ring in Heidelberg has examined the influence of rotational excitation on the rate of the recombination. This is a very beautiful study but an important outcome is that even though a cryogenically cooled storage trap was used to produce the ions, the internal rotational temperature of the ions was never found to be below 150K. This suggests that ion cooling by storage in the ring leads eventually to an equilibrium value for the internal energy of the ions as they are de-excited/re-excited by passage through the electron cooler. As observed in earlier merged beam experiments in Canada, the extraction field in the ion source plays an important role in determining the excitation state of the ions as collisions outside the source can lead to re-heating. Indeed in the TSR experiments using a conventional Penning source and a normal extraction field, the ions were found to have a rotational temperature of several thousands of degrees. This clearly has important significance for earlier measurements taken in storage rings. Finally, the world will soon have a new storage ring facility for dissociative recombination research and this will be in Langzhou in China. This machine will have a higher magnetic rigidity that previous rings used for DR and so heavier ions and higher mass resolution experiments can be performed there. Experimental operation of this new ring is expected to commence in 2012/2013. (author)

332

Recombinant I?B?-loaded curcumin nanoparticles for improved cancer therapeutics  

Science.gov (United States)

The field of recombinant protein therapeutics has been evolving rapidly, making significant impact on clinical applications for several diseases, including cancer. However, the functional aspects of proteins rely exclusively on their structural integrity, in which nanoparticle mediated delivery offers unique advantages over free proteins. In the present work, a novel strategy has been developed where the nanoparticles (NPs) used for the delivery of the recombinant protein could contribute to enhancing the therapeutic efficacy of the recombinant protein. The transcription factor, NF?B, involved in cell growth and its inhibitor, I?B?, regulates its proliferation. Another similar naturally available molecule, which inhibits the function of NF?B, is curcumin. Hence, we have developed a ‘green synthesis’ method for preparing water-soluble curcumin nanoparticles to stabilize recombinant I?B? protein. The NPs were characterized by UV-vis and fluorescence spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering before administration into human cervical carcinoma (HeLa) and glioblastoma (U87MG) cells. Experimental results demonstrated that this combined module had enhanced therapeutic efficacy, causing apoptotic cell death, which was confirmed by cytotoxicity assay and flowcytometry analyses. The expression of apoptotic genes studied by semi-quantitative reverse transcription PCR delineated the molecular pathways involved in cell death. Thus, our study revealed that the functional delivery of recombinant I?B?-loaded curcumin NPs has promise as a natural-product-based protein therapeutics against cancer cells.

Banerjee, Subhamoy; Sahoo, Amaresh Kumar; Chattopadhyay, Arun; Sankar Ghosh, Siddhartha

2014-08-01

333

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

Science.gov (United States)

Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by C31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. C31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition. PMID:24225316

Colloms, Sean D; Merrick, Christine A; Olorunniji, Femi J; Stark, W Marshall; Smith, Margaret C M; Osbourn, Anne; Keasling, Jay D; Rosser, Susan J

2014-02-01

334

Construction and characterization of recombinant Japanese encephalitis virus carrying brainspecific miRNA target sequences  

Directory of Open Access Journals (Sweden)

Full Text Available Objective?To construct the recombinant Japanese encephalitis virus ( JEV carrying brain-specific miRNA targeting sequences. Methods?The target sequences of brain-specific miR-124 and miR-125 were introduced into the infectious cDNA clone of JEV to generate recombinant plasmids based on reverse genetics technology. The recombinant plasmids were linearized with Xho ? and served as templates of transcription with SP6 RNA polymerase to generate infectious viral RNA. The RNA transcripts were then transfected into BHK-21 cells, and the supernatant was obtained after incubated at 37?, 5% CO2 for 3 days. The cytopathic changes of BHK-21 cells inoculated with the supernatant were observed after one passage. The rescued viruses carrying miRNA target sequences were validated by RT-PCR, standard plaque forming test on BHK-21 cells and growth curves analysis. Results?Two recombinant viruses carrying miR-124 or miR-125 target sequence were rescued, respectively. The insertion of miRNA target sequences was confirmed by DNA sequencing. The rescued viruses yielded similar plaque morphology and replication efficiency compared with wild type JEV. Conclusion?The recombinant JEV containing brain-specific miRNA target sequences can be obtained by reverse genetics technique, which could be used in further studies of miRNA-mediated tissue-specific attenuation mechanism of JEV. DOI: 10.11855/j.issn.0577-7402.2014.06.01

Wen-yuan CAO

2014-08-01

335

Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli  

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Full Text Available Abstract Background The overproduction of recombinant proteins in host cells often leads to their misfolding and aggregation. Previous attempts to increase the solubility of recombinant proteins by co-overproduction of individual chaperones were only partially successful. We now assessed the effects of combined overproduction of the functionally cooperating chaperone network of the E. coli cytosol on the solubility of recombinant proteins. Results A two-step procedure was found to show the strongest enhancement of solubility. In a first step, the four chaperone systems GroEL/GroES, DnaK/DnaJ/GrpE, ClpB and the small HSPs IbpA/IbpB, were coordinately co-overproduced with recombinant proteins to optimize de novo folding. In a second step, protein biosynthesis was inhibited to permit chaperone mediated refolding of misfolded and aggregated proteins in vivo. This novel strategy increased the solubility of 70% of 64 different heterologous proteins tested up to 42-fold. Conclusion The engineered E. coli strains and the two-step procedure presented here led to a remarkable increase in the solubility of a various recombinant proteins and should be applicable to a wide range of target proteins produced in biotechnology.

Mogk Axel

2007-06-01

336

A new rabies vaccine based on a recombinant ORF virus (parapoxvirus) expressing the rabies virus glycoprotein.  

Science.gov (United States)

The present study describes the generation of a new Orf virus (ORFV) recombinant, D1701-V-RabG, expressing the rabies virus (RABV) glycoprotein that is correctly presented on the surface of infected cells without the need of replication or production of infectious recombinant virus. One single immunization with recombinant ORFV can stimulate high RABV-specific virus-neutralizing antibody (VNA) titers in mice, cats, and dogs, representing all nonpermissive hosts for the ORFV vector. The protective immune response against severe lethal challenge infection was analyzed in detail in mice using different dosages, numbers, and routes for immunization with the ORFV recombinant. Long-term levels of VNA could be elicited that remained greater than 0.5 IU per ml serum, indicative for the protective status. Single applications of higher doses (10(7) PFU) can be sufficient to confer complete protection against intracranial (i.c.) challenge, whereas booster immunization was needed for protection by the application of lower dosages. Anamnestic immune responses were achieved by each of the seven tested routes of inoculation, including oral application. Finally, in vivo antibody-mediated depletion of CD4-positive and/or CD8-posititve T cell subpopulations during immunization and/or challenge infection attested the importance of CD4 T cells for the induction of protective immunity by D1701-V-RabG. This report demonstrates another example of the potential of the ORFV vector and also indicates the capability of the new recombinant for vaccination of animals. PMID:23175365

Amann, Ralf; Rohde, Jörg; Wulle, Ulrich; Conlee, Douglas; Raue, Rudiger; Martinon, Olivier; Rziha, Hanns-Joachim

2013-02-01

337

Electron Recombination in a Dense Hydrogen Plasma  

Energy Technology Data Exchange (ETDEWEB)

A high pressure hydrogen gas filled RF cavity was subjected to an intense proton beam to study the evolution of the beam induced plasma inside the cavity. Varying beam intensities, gas pressures and electric fields were tested. Beam induced ionized electrons load the cavity, thereby decreasing the accelerating gradient. The extent and duration of this degradation has been measured. A model of the recombination between ionized electrons and ions is presented, with the intent of producing a baseline for the physics inside such a cavity used in a muon accelerator. Analysis of the data taken during the summer of 2011 shows that self recombination takes place in pure hydrogen gas. The decay of the number of electrons in the cavity once the beam is turned off indicates self recombination rather than attachment to electronegative dopants or impurities. The cross section of electron recombination grows for larger clusters of hydrogen and so at the equilibrium of electron production and recombination in the cavity, processes involving H{sub 5}{sup +} or larger clusters must be taking place. The measured recombination rates during this time match or exceed the analytic predicted values. The accelerating gradient in the cavity recovers fully in time for the next beam pulse of a muon collider. Exactly what the recombination rate is and how much the gradient degrades during the 60 ns muon collider beam pulse will be extrapolated from data taken during the spring of 2012.

Jana, M.R.; Johnstone, C.; Kobilarcik, T.; Koizumi, G.M.; Moretti, A.; Popovic, M.; Tollestrup, A.V.; Yonehara, K.; /Fermilab; Leonova, M.A.; Schwarz, T.A.; /Fermilab; Chung, M.; /Unlisted /IIT, Chicago /Fermilab /MUONS Inc., Batavia /Turin Polytechnic

2012-05-01

338

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

339

Alu repeats increase local recombination rates  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Recombination rates vary widely across the human genome, but little of that variation is correlated with known DNA sequence features. The genome contains more than one million Alu mobile element insertions, and these insertions have been implicated in non-homologous recombination, modulation of DNA methylation, and transcriptional regulation. If individual Alu insertions have even modest effects on local recombination rates, they could collectively have a significant impact on the pattern of linkage disequilibrium in the human genome and on the evolution of the Alu family itself. Results We carried out sequencing, SNP identification, and SNP genotyping around 19 AluY insertion loci in 347 individuals sampled from diverse populations, then used the SNP genotypes to estimate local recombination rates around the AluY loci. The loci and SNPs were chosen so as to minimize other factors (such as SNP ascertainment bias and SNP density that could influence recombination rate estimates. We detected a significant increase in recombination rate within ~2 kb of the AluY insertions in our African population sample. To test this observation against a larger set of AluY insertions, we applied our locus- and SNP-selection design and analyses to the HapMap Phase II data. In that data set, we observed a significantly increased recombination rate near AluY insertions in both the CEU and YRI populations. Conclusion We show that the presence of a fixed AluY insertion is significantly predictive of an elevated local recombination rate within 2 kb of the insertion, independent of other known predictors. The magnitude of this effect, approximately a 6% increase, is comparable to the effects of some recombinogenic DNA sequence motifs identified via their association with recombination hot spots.

Hedges Dale J

2009-11-01

340

Recombinant IgE antibody engineering to target EGFR  

DEFF Research Database (Denmark)

Monoclonal antibodies have become a mainstay for the targeted treatment of cancer today. Some of the most successful targets of monoclonal antibodies are constituted by the epidermal growth factor receptor family spearheaded by the epidermal growth factor receptor (EGFR). Prompted by studies indicating that IgE compared to IgG may harness alternate effector functions to eradicate malignant cells, we addressed the establishment, engineering, and the potential tumoricidal effects of recombinant anti-EGFR IgE. Therefore, two different therapeutic EGFR-specific antibodies, 225 and 425, were chosen for re-cloning into different chimeric IgE and IgG formats and produced in human cells. Simultaneous antibody binding to the sEGFR demonstrated accessibility of both epitopes for recombinant IgE. Proliferation and cytotoxicity assays demonstrated signal blocking and effector mediating capability of IgE isotypes. Pronounced degranulation in the presence of sEGFR upon activation exclusively with two IgE antibodies verified the epitope proximity and provides evidence that tumor-targeting by anti-EGFR IgE is safe with regard to soluble target structures. Degranulation mediated by tumor cells expressing EGFR could be demonstrated for singular and combined IgE antibodies; however, use of two IgE specificities was not superior to use of one IgE alone. The data suggest that the surface distribution of EGFR is optimally suited to mount a robust effector cell trigger and corroborate the potential and specificity of the IgE/IgE receptor network to react to xenobiotic or pathogenic patterns for targeting malignancies.

Spillner, Edzard; Plum, Melanie

2012-01-01

 
 
 
 
341

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

342

Charges recombination in ? particle tracks in argon  

International Nuclear Information System (INIS)

The creation and evolution of (neutral) excited states and ionized states in ? particle tracks in high pressure argon are studied. The main features of recently published experimental results on the recombination luminescence can be explained and a track model is proposed. Details are given on the track radius, on the electrons thermallization, and on collisions between electrons and triplet excited states. The most important result is that at high pressure and high electron and ion densities a collective electron-ion recombination is possible, that is more efficient that the well known dissociative recombination

343

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

344

Recombination lasing in a magnetoplasmadynamic arcjet  

Science.gov (United States)

The plasmadynamic recombination laser concept is verified experimentally in a high-power quasisteady MPD arcjet operating at 4 kA and 12 g/sec of argon. Measurements of the spatial variation of electron temperature, electron density, and population densities in the arc exhaust flow confirm that inverted populations of the 4p to 4s Ar II transitions are established by collisional-radiative recombination of the Ar III ion. Using an optical cavity aligned transversely to the flow, recombination lasing of seven such transitions, 5145, 4880, 4764, 4727, 4658, 4579, and 4545 A, is observed spectrophotographically and photoelectrically over the entire 1-msec discharge.

Campbell, E. M.; Jahn, R. G.; von Jaskowsky, W. F.; Clark, K. E.

1980-01-01

345

Polynomial identities for ternary intermolecular recombination  

CERN Document Server

The operation of binary intermolecular recombination, originating in the theory of DNA computing, permits a natural generalization to n-ary operations which perform simultaneous recombination of n molecules. In the case n = 3, we use computer algebra to determine the polynomial identities of degree <= 9 satisfied by this trilinear nonassociative operation. Our approach requires computing a basis for the nullspace of a large integer matrix, and for this we compare two methods: (i) the row canonical form, and (ii) the Hermite normal form with lattice basis reduction. In the conclusion, we formulate some conjectures for the general case of n-ary intermolecular recombination.

Bremner, Murray R

2010-01-01

346

Testing for recombinant human erythropoietin  

Science.gov (United States)

ERYTHROPOIETIN (Epo) may have effects on exercise capacity and physiological regulation beyond a simple increase in red cell mass and the associated improvement in oxygen transport (4). In the context of a larger study on this topic, Lundby and colleagues (11) also asked questions about the reliability of urine testing for recombinant human Epo (rHuEpo). They studied eight healthy male subjects during a 4-wk "loading" and 2-wk "boosting" phase of Epo use followed by a 2-wk maintenance phase. In the parent study they showed that the effects of Epo on exercise performance were confined to its impact on red cell mass and not to other physiological effects of the hormone. These results were consistent with ideas about the relationship between maximal oxygen uptake and red cell mass or total body hemoglobin that emerged in the 1950s. The findings are timely and have implications for public policy relating to the control of doping practices. In this short report a number of challenges related to urine testing for Epo are highlighted.

Joris R Delanghe (Ghent University Hospital Clinical Chemistry); Michael J Joyner (Mayo Clinic Anesthesiology)

2008-08-10

347

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

348

Dissociative recombination of H3+  

Science.gov (United States)

The process of dissociative recombination (DR) of the ^H3+ ion has been studied over the past years and it was found that the coupling of vibrational and electronic degrees of freedom plays a crucial role in the mechanism: when the Janh-Teller coupling effect was incorporated into the theoretical treatmnet it yielded DR rates in much better agreement with experiments. The previous work on H3+ was performed using hyperspherical coordinates and Siegert states for the vibrational wave functions. SVD technique employed in this study provides more accurate vibrational energies than the Siegert state approach for it takes into account the non-adiabatic coupling between different adiabatic channels. Another improvement towards theory-experiment agreement was to take into account the conditions and parameters of the experiments performed. The present approach uses SVD vibrational states in the calculatiion of H3+ DR rates and accounts for experimental conditions. Incorporating averaging procedures that describe better the experimental conditions improves the agreement between theory and experiment. Results for vibrationally-excited initial states of H3+ are also presented in this work.

Santos, Samantha; Kokoouline, Viatcheslav; Greene, Chris

2007-06-01

349

Dissociative recombination of H3^+  

Science.gov (United States)

The process of dissociative recombination (DR) of the H3^+ ion has been studied over the past years and it was found that the coupling of vibrational and electronic degrees of freedom plays a crucial role in the mechanism: when the Janh-Teller coupling effect was incorporated into the theoretical treatmnet it yielded DR rates in much better agreement with experiments. The previous work on H3^+ was performed using hyperspherical coordinates and Siegert states for the vibrational wave functions. SVD technique employed in this study provides more accurate vibrational energies than the Siegert state approach for it takes into account the non-adiabatic coupling between different adiabatic channels. Another improvement towards theory-experiment agreement was to take into account the conditions and parameters of the experiments performed. The present approach uses SVD vibrational states in the calculatiion of H3^+ DR rates and accounts for experimental conditions. Incorporating averaging procedures that describe better the experimental conditions improves the agreement between theory and experiment. Results for vibrationally-excited initial states of H3^+ are also presented in this work.

Santos, Samantha; Kokoouline, Viatcheslav; Greene, Chris

2007-04-01

350

Oxidative refolding of rPA in l-ArgHCl and in ionic liquids: A correlation between hydrophobicity, salt effects, and refolding yield.  

Science.gov (United States)

The ionic liquid 1-ethyl-3-methyl imidazolium chloride (EMIM Cl) and the amino acid l-arginine hydrochloride (l-ArgHCl) have been successfully used to improve the yield of oxidative refolding for various proteins. However, the molecular mechanisms behind the actions of such solvent additives-especially of ionic liquids-are still not well understood. To analyze these mechanisms, we have determined the transfer free energies from water into ionic liquid solutions of proteinogenic amino acids and of diketopiperazine as peptide bond analogue. For EMIM Cl and 1-ethyl-3-methyl imidazolium diethyl phosphate, which had a suppressive effect on protein refolding, as well as for l-ArgHCl favorable interactions with amino acid side chains, but no favorable interactions with the peptide backbone could be observed. A quantitative analysis of other ionic liquids together with their already published effects on protein refolding showed that only solvent additives within a certain range of hydrophobicity, chaotropicity and kosmotropicity were effective for the refolding of recombinant plasminogen activator. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1129-1140, 2014. PMID:24931846

Tischer, Alexander; Lilie, Hauke; Auton, Matthew; Lange, Christian

2014-11-01

351

DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages  

Science.gov (United States)

Sequencing of the 7 kb immC region from four P1-related phages identified a novel DNA recombinase that exhibits many Cre-like characteristics, including recombination in mammalian cells, but which has a distinctly different DNA specificity. DNA sequence comparison to the P1 immC region showed that all phages had related DNA terminase, C1 repressor and DNA recombinase genes. Although these genes from phages P7, ?w39 and p15B were highly similar to those from P1, those of phage D6 showed significant divergence. Moreover, the D6 sequence showed evidence of DNA deletion and substitution in this region relative to the other phages. Characterization of the D6 site-specific DNA recombinase (Dre) showed that it was a tyrosine recombinase closely related to the P1 Cre recombinase, but that it had a distinct DNA specificity for a 32 bp DNA site (rox). Cre and Dre are heterospecific: Cre did not catalyze recombination at rox sites and Dre did not catalyze recombination at lox sites. Like Cre, Dre catalyzed both integrative and excisive recombination and required no other phage-encoded proteins for recombination. Dre-mediated recombination in mammalian cells showed that, like Cre, no host bacterial proteins are required for efficient Dre-mediated site-specific DNA recombination. PMID:15550568

Sauer, Brian; McDermott, Jeffrey

2004-01-01

352

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)

353

Stimulated radiative recombination of electrons and ions  

International Nuclear Information System (INIS)

The laser stimulated radiative recombination of electrons and protons to form hydrogen atoms in the n=11 and 12 states has been measured previously using the Merged Beams Apparatus. at the University of Western Ontario. Similar studies have been performed at the Heidelberg Storage Ring involving recombination to form R(n=2)2. A new multi-pass cavity has been installed at UWO and preliminary results indicate that the cross section has been enhanced a factor of more than ten over the previous measurements. An in-depth study of the process is underway and will be reported at the meeting. The possible importance of stimulated recombination in thermonuclear fusion plasmas and the effect of laser stimulation on molecular ion recombination will be discussed

354

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

355

Gene mapping with recombinant inbreds in maize  

International Nuclear Information System (INIS)

Recombinant inbred lines of maize have been developed for the rapid mapping of molecular probes to chromosomal location. Two recombinant inbred families have been constructed from F2 populations of T232 x CM37 and CO159 x Tx303. A genetic map based largely on isozymes and restriction fragment length polymorphisms has been produced that covers virtually the entire maize genome. In order to map a new gene, an investigator has only to determine its allelic distribution among the recombinant inbred lines and then compare it by computer with the distributions of all previously mapped loci. The availability of the recombinant inbreds and the associated data base constitute an efficient means of mapping new molecular markers in maize

356

Recombinant vaccines: experimental and applied aspects  

DEFF Research Database (Denmark)

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

Lorenzen, Niels

1999-01-01

357

The ?0 polarization and the recombination function  

International Nuclear Information System (INIS)

We study the ?O polarization in the p+p -> ?O + X reaction. In the framework of the Thomas precession model we use different recombination functions and see how their shape influences the polarization prediction. (author). 8 refs., 2 figs

358

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

359

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

360

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

 
 
 
 
361

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

362

EPR surface recombination centers in silicon  

International Nuclear Information System (INIS)

The phenomenon of spin-dependent recombination, i.e. the change of recombination rate of photoexcited carriers, and hence, their concentration in saturation of EPR-transition recombination centers, is used to investigate surface recombination centers in silicon. Silicon crystals of the n- and p-type were studied after chemical treatment in a mixture of hydrofluoric and nitric acid. New anisotropic EPR-spectrum from surface centers is obtained in magnetic field orientation along crystal axis. Silicon irradiation with 50 keV X-ray quanta results in the growth of intensity of observed EPR-sectrum of surface centers a 3-5 times for different samples. Besides, one more new EPR spectrum, which vanishes after sample etching, occurs in irradiated samples. The technique used in this paper appreciably increases the sensitivity of standard EPR-technique

363

Co-factor activated recombinant adenovirus proteinases  

Science.gov (United States)

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

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

1996-08-06

364

Coupling meiotic chromosome axis integrity to recombination  

Digital Repository Infrastructure Vision for European Research (DRIVER)

During meiosis, DNA events of recombination occur in direct physical association with underlying chromosome axes. Meiotic cohesin Rec8 and cohesin-associated Spo76/Pds5 are prominent axis components. Two observations indicate that recombination complexes can direct the local destabilization of underlying chromosome axes. First, in the absence of Rec8, Spo76/Pds5 is lost locally at sites of late-persisting Msh4 foci, with a concomitant tendency for loosening of intersister and interhomolog con...

Storlazzi, Aurora; Tesse, Sophie; Ruprich-robert, Gwenael; Gargano, Silvana; Po?ggeler, Stefanie; Kleckner, Nancy; Zickler, Denise

2008-01-01

365

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

366

Nuclear shadowing in a parton recombination model  

CERN Document Server

Deep inelastic structure functions $F_2^A(x)$ are investigated in a $Q^2$ rescaling model with parton recombination effects. We find that the model can explain experimentally measured $F_2^A(x)$ structure functions reasonably well in the wide Bjorken$-x$ range ($0.005recombination results are very sensitive to input sea-quark and gluon distributions.

Kumano, S

1993-01-01

367

Dissociative Recombination of Astrochemically Interesting Ions  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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

368

Expansion of a chromosomal repeat in Escherichia coli: roles of replication, repair, and recombination functions  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Previous studies of gene amplification in Escherichia coli have suggested that it occurs in two steps: duplication and expansion. Expansion is thought to result from homologous recombination between the repeated segments created by duplication. To explore the mechanism of expansion, a 7 kbp duplication in the chromosome containing a leaky mutant version of the lac operon was constructed, and its expansion into an amplified array was studied. Results Under selection for lac function, colonies bearing multiple copies of the mutant lac operon appeared at a constant rate of approximately 4 to 5 per million cells plated per day, on days two through seven after plating. Expansion was not seen in a recA strain; null mutations in recBCD and ruvC reduced the rate 100- and 10-fold, respectively; a ruvC recG double mutant reduced the rate 1000-fold. Expansion occurred at an increased rate in cells lacking dam, polA, rnhA, or uvrD functions. Null mutations of various other cellular recombination, repair, and stress response genes had little effect upon expansion. The red recombination genes of phage lambda could substitute for recBCD in mediating expansion. In the red-substituted cells, expansion was only partially dependent upon recA function. Conclusion These observations are consistent with the idea that the expansion step of gene amplification is closely related, mechanistically, to interchromosomal homologous recombination events. They additionally provide support for recently described models of RecA-independent Red-mediated recombination at replication forks.

Poteete Anthony R

2009-02-01

369

The role of Deinococcus radiodurans RecFOR proteins in homologous recombination.  

Science.gov (United States)

Deinococcus radiodurans exhibits extraordinary resistance to the lethal effect of DNA-damaging agents, a characteristic attributed to its highly proficient DNA repair capacity. Although the D. radiodurans genome is clearly devoid of recBC and addAB counterparts as RecA mediators, the genome possesses all genes associated with the RecFOR pathway. In an effort to gain insights into the role of D. radiodurans RecFOR proteins in homologous recombination, we generated recF, recO and recR disruptant strains and characterized the disruption effects. All the disruptant strains exhibited delayed growth relative to the wild-type, indicating that the RecF, RecO and RecR proteins play an important role in cell growth under normal growth conditions. A slight reduction in transformation efficiency was observed in the recF and recO disruptant strains compared to the wild-type strain. Interestingly, disruption of recR resulted in severe reduction of the transformation efficiency. On the other hand, the recF disruptant strain was the most sensitive phenotype to ? rays, UV irradiation and mitomycin C among the three disruptants. In the recF disruptant strain, the intracellular level of the LexA1 protein did not decrease following ? irradiation, suggesting that a large amount of the RecA protein remains inactive despite being induced. These results demonstrate that the RecF protein plays a crucial role in the homologous recombination repair process by facilitating RecA activation in D. radiodurans. Thus, the RecF and RecR proteins are involved in the RecA activation and the stability of incoming DNA, respectively, during RecA-mediated homologous recombination processes that initiated the ESDSA pathway in D. radiodurans. Possible mechanisms that involve the RecFOR complex in homologous intermolecular recombination and homologous recombination repair processes are also discussed. PMID:22321371

Satoh, Katsuya; Kikuchi, Masahiro; Ishaque, Abu M; Ohba, Hirofumi; Yamada, Mitsugu; Tejima, Kouhei; Onodera, Takefumi; Narumi, Issay

2012-04-01

370

Annexin-Mediated Calcium Signalling in Plants  

Directory of Open Access Journals (Sweden)

Full Text Available Calcium-permeable channels underpin elevations of free calcium that encode specific signals in stress adaptation, development and immunity. Identifying the genes encoding these channels remains a central goal of plant signalling research. Evidence now suggests that members of the plant annexin family function as unconventional calcium-permeable channels, with roles in development and stress signalling. Arabidopsis annexin 1 mediates a plasma membrane calcium-permeable conductance in roots that is activated by reactive oxygen species. Recombinant annexin 1 forms a very similar conductance in planar lipid bilayers, indicating that this protein could facilitate the in vivo conductance directly. The annexin 1 mutant is impaired in salinity-induced calcium signalling. Protein–protein interactions, post-translational modification and dynamic association with membranes could all influence annexin-mediated calcium signalling and are reviewed here. The prospect of annexins playing roles in calcium signalling events in symbiosis and immunity are considered.

Julia M. Davies

2014-02-01

371

Somatic recombination, gene amplification and cancer.  

Science.gov (United States)

The principle objective of this research programme, to analyse chemical induction of somatic recombination and related endpoints, i.e., mobilization of transposing elements and gene amplification, has been approached by means of several assay systems. These have included Drosophila, Saccharomyces and mammalian cell cultures. 6.1. Screening assays for mitotic recombination. A large number of chemicals have been investigated in the three Drosophila assay systems employed--the multiple wing hair/flare wing spot system developed by Graf et al., 1984, the white-ivory system developed by Green et al., 1986 and the white/white+ eye spot assay developed by Vogel (Vogel and Nivard, 1993). Particularly the screening of 181 chemicals, covering a wide array of chemical classes, by the last mentioned assay has shown that measurement of somatic recombination in Drosophila constitutes a sensitive and efficient short-term test which shows a remarkably good correlation with the agent score of 83 short-term tests analysed by ICPEMC (Mendelsohn et al., 1992; Table 2) as well as the assay performance in international collaborative programmes measuring carcinogen/non-carcinogens (de Serres and Ashby, 1981; Ashby et al., 1985, 1988). Also the wing spot assay has gained wide international recognition as a similarly sensitive test. These two assay systems in Drosophila measure both intrachromosomal events and interchromosomal recombination. The white-ivory system on the other hand is based on the loss of a tandem duplication in the white locus, the mechanism of which is less known, but probably involves intrachromosomal recombination. The difference in the mechanism between this assay and the former two was indicated by the lack of response to methotrexate in the white-ivory assay, while this compound was strongly recombinogenic in both the wing spot and white/white+ assays. The use of different strains of Drosophila with the white/white+ assay demonstrated the importance of the background genotype for the outcome of the test. Up to a 60-fold variation was found between the different genotypes in the response to procarcinogens, evidently dependent on differences in the metabolic activation of procarcinogens. In 1989 Schiestl presented results on intrachromosomal recombination in the strain RS112 of Saccharomyces, which indicated a capability to detect a range of chemical carcinogens, which gave negative results in Ames Salmonella assay. Such a test system, which could identify a larger range of potential carcinogens than conventional short-term tests evidently would be of great value and it therefore seemed of importance to follow up the observations by Schiestl. However, studies within this programme on the same strain of Saccharomyces, as well as the strains D7 (measuring intragenic recombination, intergenic recombination, and point mutation) and JD1 (measuring intragenic recombination at two loci) could not support the observations and interpretation by Schiestl (1989). The Drosophila white-ivory system, which presumably responds primarily by intrachromosomal recombination, did not give positive results with these Salmonella-negative agents either. One system to measure mitotic recombination in mammalian cell cultures was developed in the present programme. It was based on heterozygous mutations in both alleles of the adenosine deaminase gene (ADA). The system selects for proficient recombinants generated by the deficient cells. So far only pilot experiments, indicating that this experimental system operates as planned, have been performed. 6.2 Mechanisms of mitotic recombination The induction of mosaic spots in the wing spot and the white/white+ assays is predominantly dependent on interchromosomal recombination. This is evident from the fact that heterozygous inversions reduce the frequency of spots. A relationship between the length of inversions and the reduction of spots was demonstrated in the white/white+ assay--the long inversion ln(l)sc4L PMID:8692194

Ramel, C; Cederberg, H; Magnusson, J; Vogel, E; Natarajan, A T; Mullender, L H; Nivard, J M; Parry, J M; Leyson, A; Comendador, M A; Sierra, L M; Ferreiro, J A; Consuegra, S

1996-06-12

372

Evidence of recombination among early-vaccination era measles virus strains  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background The advent of live-attenuated vaccines against measles virus during the 1960'ies changed the circulation dynamics of the virus. Earlier the virus was indigenous to countries worldwide, but now it is mediated by a limited number of evolutionary lineages causing sporadic outbreaks/epidemics of measles or circulating in geographically restricted endemic areas of Africa, Asia and Europe. We expect that the evolutionary dynamics of measles virus has changed from a situation where a variety of genomic variants co-circulates in an epidemic with relatively high probabilities of co-infection of the individual to a situation where a co-infection with strains from evolutionary different lineages is unlikely. Results We performed an analysis of the partial sequences of the hemagglutinin gene of 18 measles virus strains collected in Denmark between 1965 and 1983 where vaccination was first initiated in 1987. The results were compared with those obtained with strains collected from other parts of the world after the initiation of vaccination in the given place. Intergenomic recombination among pre-/early-vaccination strains is suggested by 1 estimations of linkage disequilibrium between informative sites, 2 the decay of linkage disequilibrium with distance between informative sites and 3 a comparison of the expected number of homoplasies to the number of apparent homoplasies in the most parsimonious tree. No significant evidence of recombination could be demonstrated among strains circulating at present. Conclusion We provide evidence that recombination can occur in measles virus and that it has had a detectable impact on sequence evolution of pre-vaccination samples. We were not able to detect recombination from present-day sequence surveys. We believe that the decreased rate of visible recombination may be explained by changed dynamics, since divergent strains do not meet very often in current epidemics that are often spawned by a single sequence type. Signs of pre-vaccination recombination events in the present-day sequences are not strong enough to be detectable.

Muller Claude P

2005-10-01

373

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

374

Recombinant Skeleton Using Junction Points in Skeleton Based Images  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We perform the task of combining two skeleton images and to produce the recombinant skeleton. We propose the recombinant skeleton algorithm to produce the recombinant skeletons. The existing skeleton representation has been taken and the merge vertex detection algorithm was used before applying the recombinant skeleton algorithm. We can design and apply this recombinant skeleton in motion detection, image matching, tracking, panorama stitching, 3D modeling and object recognition. We can gener...

Mrs. Komala Lakshmi; Punithavalli, Dr M.

2011-01-01

375

Generation and Characterization of Ins1-cre-driver C57BL/6N for Exclusive Pancreatic Beta Cell-specific Cre-loxP Recombination  

Science.gov (United States)

Cre/loxP system-mediated site-specific recombination is utilized to study gene function in vivo. Successful conditional knockout of genes of interest is dependent on the availability of Cre-driver mice. We produced and characterized pancreatic ? cell-specific Cre-driver mice for use in diabetes mellitus research. The gene encoding Cre was inserted into the second exon of mouse Ins1 in a bacterial artificial chromosome (BAC). Five founder mice were produced by microinjection of linearized BAC Ins1-cre. The transgene was integrated between Mafa and the telomere on chromosome 15 in one of the founders, BAC Ins1-cre25. To investigate Cre-loxP recombination, BAC Ins1-cre25 males were crossed with two different Cre-reporters, R26R and R26GRR females. On gross observation, reporter signal after Cre-loxP recombination was detected exclusively in the adult pancreatic islets in both F1 mice. Immunohistological analysis indicated that Cre-loxP recombination-mediated reporter signal was colocalized with insulin in pancreatic islet cells of both F1 mice, but not with glucagon. Moreover, Cre-loxP recombination signal was already observed in the pancreatic islets at E13.5 in both F1 fetuses. Finally, we investigated ectopic Cre-loxP recombination for Ins1, because the ortholog Ins2 is also expressed in the brain, in addition to the pancreas. However, there was no Cre-loxP recombination-mediated reporter signal in the brain of both F1 mice. Our data suggest that BAC Ins1-cre25 mice are a useful Cre-driver C57BL/6N for pancreatic ? cell-specific Cre-loxP recombination, except for crossing with knock-in mice carrying floxed gene on chromosome 15. PMID:24770644

Hasegawa, Yoshikazu; Daitoku, Yoko; Mizuno, Seiya; Tanimoto, Yoko; Mizuno-Iijima, Saori; Matsuo, Miki; Kajiwara, Noriko; Ema, Masatsugu; Oishi, Hisashi; Miwa, Yoshihiro; Mekada, Kazuyuki; Yoshiki, Atsushi; Takahashi, Satoru; Sugiyama, Fumihiro; Yagami, Ken-ichi

2014-01-01

376

Nap1 stimulates homologous recombination by RAD51 and RAD54 in higher-ordered chromatin containing histone H1.  

Science.gov (United States)

Homologous recombination plays essential roles in mitotic DNA double strand break (DSB) repair and meiotic genetic recombination. In eukaryotes, RAD51 promotes the central homologous-pairing step during homologous recombination, but is not sufficient to overcome the reaction barrier imposed by nucleosomes. RAD54, a member of the ATP-dependent nucleosome remodeling factor family, is required to promote the RAD51-mediated homologous pairing in nucleosomal DNA. In higher eukaryotes, most nucleosomes form higher-ordered chromatin containing the linker histone H1. However, the mechanism by which RAD51/RAD54-mediated homologous pairing occurs in higher-ordered chromatin has not been elucidated. In this study, we found that a histone chaperone, Nap1, accumulates on DSB sites in human cells, and DSB repair is substantially decreased in Nap1-knockdown cells. We determined that Nap1 binds to RAD54, enhances the RAD54-mediated nucleosome remodeling by evicting histone H1, and eventually stimulates the RAD51-mediated homologous pairing in higher-ordered chromatin containing histone H1. PMID:24798879

Machida, Shinichi; Takaku, Motoki; Ikura, Masae; Sun, Jiying; Suzuki, Hidekazu; Kobayashi, Wataru; Kinomura, Aiko; Osakabe, Akihisa; Tachiwana, Hiroaki; Horikoshi, Yasunori; Fukuto, Atsuhiko; Matsuda, Ryo; Ura, Kiyoe; Tashiro, Satoshi; Ikura, Tsuyoshi; Kurumizaka, Hitoshi

2014-01-01

377

Nap1 stimulates homologous recombination by RAD51 and RAD54 in higher-ordered chromatin containing histone H1  

Science.gov (United States)

Homologous recombination plays essential roles in mitotic DNA double strand break (DSB) repair and meiotic genetic recombination. In eukaryotes, RAD51 promotes the central homologous-pairing step during homologous recombination, but is not sufficient to overcome the reaction barrier imposed by nucleosomes. RAD54, a member of the ATP-dependent nucleosome remodeling factor family, is required to promote the RAD51-mediated homologous pairing in nucleosomal DNA. In higher eukaryotes, most nucleosomes form higher-ordered chromatin containing the linker histone H1. However, the mechanism by which RAD51/RAD54-mediated homologous pairing occurs in higher-ordered chromatin has not been elucidated. In this study, we found that a histone chaperone, Nap1, accumulates on DSB sites in human cells, and DSB repair is substantially decreased in Nap1-knockdown cells. We determined that Nap1 binds to RAD54, enhances the RAD54-mediated nucleosome remodeling by evicting histone H1, and eventually stimulates the RAD51-mediated homologous pairing in higher-ordered chromatin containing histone H1. PMID:24798879

Machida, Shinichi; Takaku, Motoki; Ikura, Masae; Sun, Jiying; Suzuki, Hidekazu; Kobayashi, Wataru; Kinomura, Aiko; Osakabe, Akihisa; Tachiwana, Hiroaki; Horikoshi, Yasunori; Fukuto, Atsuhiko; Matsuda, Ryo; Ura, Kiyoe; Tashiro, Satoshi; Ikura, Tsuyoshi; Kurumizaka, Hitoshi

2014-01-01

378

Identification of key structural determinants of the IntI1 integron integrase that influence attC × attI1 recombination efficiency  

Science.gov (United States)

The integron platform codes for an integrase (IntI) from the tyrosine family of recombinases that mediates recombination between a proximal double-strand recombination site, attI and a single-strand target recombination site, attC. The attI site is only recognized by its cognate integrase, while the various tested attCs sites are recombined by several different IntI integrases. We have developed a genetic system to enrich and select mutants of IntI1 that provide a higher yield of recombination in order to identify key protein structural elements important for attC × attI1 recombination. We isolated mutants with higher activity on wild type and mutant attC sites. Interestingly, three out of four characterized IntI1 mutants selected on different substrates are mutants of the conserved aspartic acid in position 161. The IntI1 model we made based on the VchIntIA 3D structure suggests that substitution at this position, which plays a central role in multimer assembly, can increase or decrease the stability of the complex and accordingly influence the rate of attI × attC recombination versus attC × attC. These results suggest that there is a balance between the specificity of the protein and the protein/protein interactions in the recombination synapse. PMID:17884913

Demarre, Gaelle; Frumerie, Clara; Gopaul, Deshmukh N.; Mazel, Didier

2007-01-01

379

Application of tunneling recombination afterglow for EPR optical detection of recombining centres in ionic crystals  

International Nuclear Information System (INIS)

Results are reported of optically detected EPR (OD EPR) investigations of electronic and hole centres involved in the tunneling recombination process in Ag-doped NaCl, KCl, and KBr, Tl-doped KCl, and Mn-doped CaF2 crystals. Tunneling recombination afterglow spectra and OD EPR spectra are presented and discussed

380

New Arabidopsis advanced intercross recombinant inbred lines reveal female control of nonrandom mating.  

Science.gov (United States)

Female control of nonrandom mating has never been genetically established, despite being linked to inbreeding depression and sexual selection. In order to map the loci that control female-mediated nonrandom mating, we constructed a new advanced intercross recombinant inbred line (RIL) population derived from a cross between Arabidopsis (Arabidopsis thaliana) accessions Vancouver (Van-0) and Columbia (Col-0) and mapped quantitative trait loci (QTLs) responsible for nonrandom mating and seed yield traits. We genotyped a population of 490 RILs. A subset of these lines was used to construct an expanded map of 1,061.4 centimorgans with an average interval of 6.7±5.3 centimorgans between markers. QTLs were then mapped for female- and male-mediated nonrandom mating and seed yield traits. To map the genetic loci responsible for female-mediated nonrandom mating and seed yield, we performed mixed pollinations with genetically marked Col-0 pollen and Van-0 pollen on RIL pistils. To map the loci responsible for male-mediated nonrandom mating and seed yield, we performed mixed pollinations with genetically marked Col-0 and RIL pollen on Van-0 pistils. Composite interval mapping of these data identified four QTLs that control female-mediated nonrandom mating and five QTLs that control female-mediated seed yield. We also identified four QTLs that control male-mediated nonrandom mating and three QTLs that control male-mediated seed yield. Epistasis analysis indicates that several of these loci interact. To our knowledge, the results of these experiments represent the first time female-mediated nonrandom mating has been genetically defined. PMID:24623850

Fitz Gerald, Jonathan Nesbit; Carlson, Ann Louise; Smith, Evadne; Maloof, Julin N; Weigel, Detlef; Chory, Joanne; Borevitz, Justin O; Swanson, Robert John

2014-05-01

 
 
 
 
381

A microscopic model of evolution of recombination  

Science.gov (United States)

We study the evolution of recombination using a microscopic model developed within the frame of the theory of quantitative traits. Two components of fitness are considered: a static one that describes adaptation to environmental factors not related to the population itself, and a dynamic one that accounts for interactions between organisms, e.g. competition. We focus on the dynamics of colonization of an empty niche. As competition is a function of the population, selection pressure rapidly changes in time. The simulations show that both in the case of flat and steep static fitness landscapes, recombination provides a high velocity of movement in the phenotypic space thus allowing recombinants to colonize the highest fitness regions earlier than non-recombinants that are often driven to extinction. The stabilizing effects of competition and assortativity are also discussed. Finally, the analysis of phase diagrams shows that competition is the key factor for the evolution of recombination, while assortativity plays a significant role only in small populations.

Bagnoli, Franco; Guardiani, Carlo

2005-03-01

382

Mechanism and regulation of class switch recombination.  

Science.gov (United States)

Antibody class switching occurs in mature B cells in response to antigen stimulation and costimulatory si