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Sample records for human ap endonuclease

  1. Human AP Endonuclease 1: A Potential Marker for the Prediction of Environmental Carcinogenesis Risk

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    Jae Sung Park

    2014-01-01

    Full Text Available Human apurinic/apyrimidinic endonuclease 1 (APE1 functions mainly in DNA repair as an enzyme removing AP sites and in redox signaling as a coactivator of various transcription factors. Based on these multifunctions of APE1 within cells, numerous studies have reported that the alteration of APE1 could be a crucial factor in development of human diseases such as cancer and neurodegeneration. In fact, the study on the combination of an individual’s genetic make-up with environmental factors (gene-environment interaction is of great importance to understand the development of diseases, especially lethal diseases including cancer. Recent reports have suggested that the human carcinogenic risk following exposure to environmental toxicants is affected by APE1 alterations in terms of gene-environment interactions. In this review, we initially outline the critical APE1 functions in the various intracellular mechanisms including DNA repair and redox regulation and its roles in human diseases. Several findings demonstrate that the change in expression and activity as well as genetic variability of APE1 caused by environmental chemical (e.g., heavy metals and cigarette smoke and physical carcinogens (ultraviolet and ionizing radiation is likely associated with various cancers. These enable us to ultimately suggest APE1 as a vital marker for the prediction of environmental carcinogenesis risk.

  2. Identification of APN2, the Saccharomyces cerevisiae homolog of the major human AP endonuclease HAP1, and its role in the repair of abasic sites

    OpenAIRE

    Johnson, Robert E.; Torres-Ramos, Carlos A.; Izumi, Tadahide; Mitra, Sankar; Prakash, Satya; Prakash, Louise

    1998-01-01

    Abasic (AP) sites arise in DNA through spontaneous base loss and enzymatic removal of damaged bases. APN1 encodes the major AP-endonuclease of Saccharomyces cerevisiae. Human HAP1 (REF1) encodes the major AP endonuclease which, in addition to its role in DNA repair, functions as a redox regulatory protein. We identify APN2, the yeast homolog of HAP1 and provide evidence that Apn1 and Apn2 represent alternate pathways for repairing AP sites. The apn1Δ apn2Δ strain displays a highly elevated le...

  3. Identification of APN2, the Saccharomyces cerevisiae homolog of the major human AP endonuclease HAP1, and its role in the repair of abasic sites.

    Science.gov (United States)

    Johnson, R E; Torres-Ramos, C A; Izumi, T; Mitra, S; Prakash, S; Prakash, L

    1998-10-01

    Abasic (AP) sites arise in DNA through spontaneous base loss and enzymatic removal of damaged bases. APN1 encodes the major AP-endonuclease of Saccharomyces cerevisiae. Human HAP1 (REF1) encodes the major AP endonuclease which, in addition to its role in DNA repair, functions as a redox regulatory protein. We identify APN2, the yeast homolog of HAP1 and provide evidence that Apn1 and Apn2 represent alternate pathways for repairing AP sites. The apn1Delta apn2Delta strain displays a highly elevated level of MMS-induced mutagenesis, which is dependent on the REV3, REV7, and REV1 genes. Our findings indicate that AP sites are highly cytotoxic and mutagenic in eukaryotes, and that the REV3, REV7-encoded DNA polymerase zeta mediates the mutagenic bypass of AP sites.

  4. The major role of human AP-endonuclease homolog Apn2 in repair of abasic sites in Schizosaccharomyces pombe

    OpenAIRE

    Ribar, Balazs; Izumi, Tadahide; Mitra, Sankar

    2004-01-01

    The abasic (AP) sites, the major mutagenic and cytotoxic genomic lesions, induced directly by oxidative stress and indirectly after excision of damaged bases by DNA glycosylases, are repaired by AP-endonucleases (APEs). Among two APEs in Saccharomyces cerevisiae, Apn1 provides the major APE activity, and Apn2, the ortholog of the mammalian APE, provides back-up activity. We have cloned apn1 and apn2 genes of Schizosaccharomyces pombe, and have shown that inactivation of Apn2 and not Apn1 sens...

  5. Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines

    DEFF Research Database (Denmark)

    Mohammed, M Z; Vyjayanti, V N; Laughton, C A

    2011-01-01

    Modulation of DNA base excision repair (BER) has the potential to enhance response to chemotherapy and improve outcomes in tumours such as melanoma and glioma. APE1, a critical protein in BER that processes potentially cytotoxic abasic sites (AP sites), is a promising new target in cancer. In the....... In the current study, we aimed to develop small molecule inhibitors of APE1 for cancer therapy....

  6. The major role of human AP-endonuclease homolog Apn2 in repair of abasic sites in Schizosaccharomyces pombe.

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    Ribar, Balazs; Izumi, Tadahide; Mitra, Sankar

    2004-01-01

    The abasic (AP) sites, the major mutagenic and cytotoxic genomic lesions, induced directly by oxidative stress and indirectly after excision of damaged bases by DNA glycosylases, are repaired by AP-endonucleases (APEs). Among two APEs in Saccharomyces cerevisiae, Apn1 provides the major APE activity, and Apn2, the ortholog of the mammalian APE, provides back-up activity. We have cloned apn1 and apn2 genes of Schizosaccharomyces pombe, and have shown that inactivation of Apn2 and not Apn1 sensitizes this fission yeast to alkylation and oxidative damage-inducing agents, which is further enhanced by Apn1 inactivation. We also show that Uve1, present in S.pombe but not in S.cerevisiae, provides the back-up APE activity together with Apn1. We confirmed the presence of APE activity in recombinant Apn2 and in crude cell extracts. Thus S.pombe is distinct from S.cerevisiae, and is similar to mammalian cells in having Apn2 as the major APE.

  7. Schizosaccharomyces pombe encodes a mutated AP endonuclease 1.

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    Laerdahl, Jon K; Korvald, Hanne; Nilsen, Line; Dahl-Michelsen, Kristin; Rognes, Torbjørn; Bjørås, Magnar; Alseth, Ingrun

    2011-03-07

    Mutagenic and cytotoxic apurinic/apyrimidinic (AP) sites are among the most frequent lesions in DNA. Repair of AP sites is initiated by AP endonucleases and most organisms possess two or more of these enzymes. Saccharomyces cerevisiae has AP endonuclease 1 (Apn1) as the major enzymatic activity with AP endonuclease 2 (Apn2) being an important backup. Schizosaccharomyces pombe also encodes two potential AP endonucleases, and Apn2 has been found to be the main repair activity, while Apn1 has no, or only a limited role in AP site repair. Here we have identified a new 5' exon (exon 1) in the apn1 gene and show that the inactivity of S. pombe Apn1 is due to a nonsense mutation in the fifth codon of this new exon. Reversion of this mutation restored the AP endonuclease activity of S. pombe Apn1. Interestingly, the apn1 nonsense mutation was only found in laboratory strains derived from L972 h(-) and not in unrelated isolates of S. pombe. Since all S. pombe laboratory strains originate from L972 h(-), it appears that all experiments involving S. pombe have been conducted in an apn1(-) mutant strain with a corresponding DNA repair deficiency. These observations have implications both for future research in S. pombe and for the interpretation of previously conducted epistatis analysis. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. AP endonuclease independent repair of abasic sites in Schizosaccharomyces pombe

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    Nilsen, Line; Forstrøm, Rune J.; Bjørås, Magnar; Alseth, Ingrun

    2012-01-01

    Abasic (AP) sites are formed spontaneously and are inevitably intermediates during base excision repair of DNA base damages. AP sites are both mutagenic and cytotoxic and key enzymes for their removal are AP endonucleases. However, AP endonuclease independent repair initiated by DNA glycosylases performing β,δ-elimination cleavage of the AP sites has been described in mammalian cells. Here, we describe another AP endonuclease independent repair pathway for removal of AP sites in Schizosaccharomyces pombe that is initiated by a bifunctional DNA glycosylase, Nth1 and followed by cleavage of the baseless sugar residue by tyrosyl phosphodiesterase Tdp1. We propose that repair is completed by the action of a polynucleotide kinase, a DNA polymerase and finally a DNA ligase to seal the gap. A fission yeast double mutant of the major AP endonuclease Apn2 and Tdp1 shows synergistic increase in MMS sensitivity, substantiating that Apn2 and Tdp1 process the same substrate. These results add new knowledge to the complex cellular response to AP sites, which could be exploited in chemotherapy where synthetic lethality is a key strategy of treatment. PMID:22084197

  9. AP endonuclease independent repair of abasic sites in Schizosaccharomyces pombe.

    Science.gov (United States)

    Nilsen, Line; Forstrøm, Rune J; Bjørås, Magnar; Alseth, Ingrun

    2012-03-01

    Abasic (AP) sites are formed spontaneously and are inevitably intermediates during base excision repair of DNA base damages. AP sites are both mutagenic and cytotoxic and key enzymes for their removal are AP endonucleases. However, AP endonuclease independent repair initiated by DNA glycosylases performing β,δ-elimination cleavage of the AP sites has been described in mammalian cells. Here, we describe another AP endonuclease independent repair pathway for removal of AP sites in Schizosaccharomyces pombe that is initiated by a bifunctional DNA glycosylase, Nth1 and followed by cleavage of the baseless sugar residue by tyrosyl phosphodiesterase Tdp1. We propose that repair is completed by the action of a polynucleotide kinase, a DNA polymerase and finally a DNA ligase to seal the gap. A fission yeast double mutant of the major AP endonuclease Apn2 and Tdp1 shows synergistic increase in MMS sensitivity, substantiating that Apn2 and Tdp1 process the same substrate. These results add new knowledge to the complex cellular response to AP sites, which could be exploited in chemotherapy where synthetic lethality is a key strategy of treatment.

  10. Lys98 substitution in human AP endonuclease 1 affects the kinetic mechanism of enzyme action in base excision and nucleotide incision repair pathways.

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    Nadezhda A Timofeyeva

    Full Text Available Human apurinic/apyrimidinic endonuclease 1 (APE1 is a key enzyme in the base excision repair (BER and nucleotide incision repair (NIR pathways. We recently analyzed the conformational dynamics and kinetic mechanism of wild-type (wt protein, in a stopped-flow fluorescence study. In this study, we investigated the mutant enzyme APE1K98A using the same approach. Lys98 was known to hydrogen bond to the carboxyl group of Asp70, a residue implicated in binding the divalent metal ion. Our data suggested that the conformational selection and induced fit occur during the enzyme action. We expanded upon the evidence that APE1 can pre-exist in two conformations. The isomerization of an enzyme-product complex in the BER process and the additional isomerization stage of enzyme-substrate complex in the NIR process were established for APE1K98A. These stages had not been registered for the wtAPE1. We found that the K98A substitution resulted in a 12-fold reduction of catalytic constant of 5'-phosphodiester bond hydrolysis in (3-hydroxytetrahydrofuran-2-ylmethyl phosphate (F, tetrahydrofuran containing substrate, and in 200-fold reduction in 5,6-dihydrouridine (DHU containing substrate. Thus, the K98A substitution influenced NIR more than BER. We demonstrated that the K98A mutation influenced the formation of primary unspecific enzyme-substrate complex in a complicated manner, depending on the Mg(2+ concentration and pH. This mutation obstructed the induced fit of enzyme in the complex with undamaged DNA and F-containing DNA and appreciably decreased the stability of primary complex upon interaction of enzyme with DNA, containing the natural apurinic/apyrimidinic (AP site. Furthermore, it significantly delayed the activation of the less active form of enzyme during NIR and slowed down the conformational conversion of the complex of enzyme with the cleavage product of DHU-substrate. Our data revealed that APE1 uses the same active site to catalyze the cleavage

  11. Second human protein with homology to the Escherichia coli abasic endonuclease exonuclease III.

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    Hadi, M Z; Wilson, D M

    2000-01-01

    There are two major apurinic/apyrimidinic (AP) endonuclease/3'-diesterase families designated after the Escherichia coli proteins exonuclease III (ExoIII) and endonuclease IV (EndoIV). These repair proteins function to excise mutagenic and cytotoxic AP sites or 3'-phosphate/phosphoglycolate groups from DNA. In mammals, the predominant repair endonuclease is Ape1, a homolog of ExoIII, whereas a mammalian homolog to EndoIV has not been identified to date. We have identified a human protein termed Ape2 that represents a subclass of the ExoIII family (exhibiting highest similarity to the Saccharomyces cerevisiae ETH1/APN2 gene product) and maintains many of the essential functional residues of the ExoIII-like proteins. The human protein is 518 amino acids with a predicted molecular mass of 57.3 kDa and a pI of 8.65. Unlike Ape1, this protein exhibited only weak ability to complement the repair defects of AP endonuclease/3'-repair-defective bacteria and yeast. Similarly, a weak, but specific, DNA-binding and incision activity for abasic site-containing substrates was observed with partially purified Ape2 protein. APE2 is located on the X chromosome at position p11.21 and consists of six exons. The transcript for APE2 is ubiquitously expressed, suggesting an important function for the encoded protein. An Ape2 green fluorescent fusion protein localized predominantly to the nucleus of HeLa cells, indicating a nuclear function; this localization was dependent on the C-terminal domain. We discuss our results in the context of the evolutionary conservation of the AP endonuclease families and their divergent activities and biological contributions.

  12. An AP endonuclease 1-DNA polymerase beta complex: theoretical prediction of interacting surfaces.

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    Abyzov, Alexej; Uzun, Alper; Strauss, Phyllis R; Ilyin, Valentin A

    2008-04-25

    Abasic (AP) sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1) cleaves the phosphodiester backbone 5' to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-beta). While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-beta, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-beta based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-beta located downstream of APEX1 (3' to the damaged site) and three with pol-beta located upstream of APEX1 (5' to the damaged site). Molecular dynamics (MD) simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (approximately -10.0 kcal/mol) to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-beta at the 3'-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-beta in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-beta in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3' side. The method described here can be used for analysis in any DNA

  13. An AP endonuclease 1-DNA polymerase beta complex: theoretical prediction of interacting surfaces.

    Directory of Open Access Journals (Sweden)

    Alexej Abyzov

    2008-04-01

    Full Text Available Abasic (AP sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1 cleaves the phosphodiester backbone 5' to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-beta. While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-beta, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-beta based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-beta located downstream of APEX1 (3' to the damaged site and three with pol-beta located upstream of APEX1 (5' to the damaged site. Molecular dynamics (MD simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (approximately -10.0 kcal/mol to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-beta at the 3'-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-beta in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-beta in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3' side. The method described here can be used for analysis in

  14. Deletion of the MAG1 DNA glycosylase gene suppresses alkylation-induced killing and mutagenesis in yeast cells lacking AP endonucleases.

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    Xiao, W; Chow, B L; Hanna, M; Doetsch, P W

    2001-12-19

    DNA base excision repair (BER) is initiated by DNA glycosylases that recognize and remove damaged bases. The phosphate backbone adjacent to the resulting apurinic/apyrimidinic (AP) site is then cleaved by an AP endonuclease or glycosylase-associated AP lyase to invoke subsequent BER steps. We have used a genetic approach in Saccharomyces cerevisiae to address whether AP sites are blocks to DNA replication and the biological consequences if AP sites persist in the genome. We found that yeast cells deficient in the two AP endonucleases (apn1 apn2 double mutant) are extremely sensitive to killing by methyl methanesulfonate (MMS), a model DNA alkylating agent. Interestingly, this sensitivity can be reduced up to 2500-fold by deleting the MAG1 3-methyladenine DNA glycosylase gene, suggesting that Mag1 not only removes lethal base lesions, but also benign lesions and possibly normal bases, and that the resulting AP sites are highly toxic to the cells. This rescuing effect appears to be specific for DNA alkylation damage, since the mag1 mutation reduces killing effects of two other DNA alkylating agents, but does not alter the sensitivity of apn cells to killing by UV, gamma-ray or H(2)O(2). Our mutagenesis assays indicate that nearly half of spontaneous and almost all MMS-induced mutations in the AP endonuclease-deficient cells are due to Mag1 DNA glycosylase activity. Although the DNA replication apparatus appears to be incapable of replicating past AP sites, Polzeta-mediated translesion synthesis is able to bypass AP sites, and accounts for all spontaneous and MMS-induced mutagenesis in the AP endonuclease-deficient cells. These results allow us to delineate base lesion flow within the BER pathway and link AP sites to other DNA damage repair and tolerance pathways.

  15. AP Human Geography and Success on the AP Test

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    Roncone, John; Newhalfen, Nate

    2013-01-01

    Classroom projects that explore culture and globalization enhance the curriculum and help students see how geography directly connects to their lives. These authors contend that a project-based approach can supplement the teaching of an AP Human Geography course, and visualize this course as an essential tool for students to truly understand how…

  16. An AP endonuclease functions in active DNA demethylation and gene imprinting in Arabidopsis [corrected].

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    Yan Li

    2015-01-01

    Full Text Available Active DNA demethylation in plants occurs through base excision repair, beginning with removal of methylated cytosine by the ROS1/DME subfamily of 5-methylcytosine DNA glycosylases. Active DNA demethylation in animals requires the DNA glycosylase TDG or MBD4, which functions after oxidation or deamination of 5-methylcytosine, respectively. However, little is known about the steps following DNA glycosylase action in the active DNA demethylation pathways in plants and animals. We show here that the Arabidopsis APE1L protein has apurinic/apyrimidinic endonuclease activities and functions downstream of ROS1 and DME. APE1L and ROS1 interact in vitro and co-localize in vivo. Whole genome bisulfite sequencing of ape1l mutant plants revealed widespread alterations in DNA methylation. We show that the ape1l/zdp double mutant displays embryonic lethality. Notably, the ape1l+/-zdp-/- mutant shows a maternal-effect lethality phenotype. APE1L and the DNA phosphatase ZDP are required for FWA and MEA gene imprinting in the endosperm and are important for seed development. Thus, APE1L is a new component of the active DNA demethylation pathway and, together with ZDP, regulates gene imprinting in Arabidopsis.

  17. Antibody to a human DNA repair protein allows for cloning of a Drosophila cDNA that encodes an apurinic endonuclease

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    Kelley, M.R. (Dept. of Biochemistry and Biophysics, Loyola Univ. Medical School, Maywood, IL (US)); Venugopal, S.; Harless, J.; Deutsch, W.A. (Louisiana State Univ., Baton Rouge, LA (USA). Dept. of Biochemistry)

    1989-03-01

    The cDNA of a Drosophila DNA repair gene, AP3, was cloned by screening an embryonic lambda gt11 expression library with an antibody that was originally prepared against a purified human apurinicapyrimidine (AP) endonuclease. The 1.2-kilobase (kb) AP3 cDNA mapped to a region on the third chromosome where a number of mutagen-sensitive alleles were located. The cDNA clone yielded an in vitro translation product of 35,000 daltons, in agreement with the predicted size of the translation product of the only open reading frame of AP3, and identical to the molecular size of an AP endonuclease activity recovered following sodium dodecyl sulfate-polyacrymalide gel electrophoresis of Drosophilia extracts. The C-terminal portion of the predicted protein contained regions of presumptive DNA-binding domains, while the DNA sequence at the amino end of AP3 showed similarity to the Escherichia coli recA gene. AP3 is expressed as an abundant 1.3-kb mRNA that is detected throughout the life cycle of Drosophila melanogaster. Another 3.5-klb mRNA also hybridized to the AP3 cDNA, but species was restricted to the early stages of development.

  18. Gapminder: An AP Human Geography Lab Assignment

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    Keller, Kenneth H.

    2012-01-01

    This lesson is designed as a lab assignment for Advanced Placement (AP) Human Geography students wherein they use the popular Gapminder web site to compare levels of development in countries from different world regions. For this lesson, it is important for the teacher to practice with Gapminder before giving the assignment to students. (Contains…

  19. Insights into the DNA cleavage mechanism of human LINE-1 retrotransposon endonuclease

    NARCIS (Netherlands)

    Repanas, K.; Fuentes, G.; Cohen, S.; Bonvin, A.M.J.J.; Perrakis, A.

    2008-01-01

    The human LINE-1 endonuclease (L1-EN) contributes in defining the genomic integration sites of the abundant human L1 and Alu retrotransposons. LINEs have been considered as possible vehicles for gene delivery and understanding the mechanism of L1-EN could help engineering them as genetic tools. We

  20. Identification and characterization of inhibitors of human apurinic/apyrimidinic endonuclease APE1.

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    Anton Simeonov

    2009-06-01

    Full Text Available APE1 is the major nuclease for excising abasic (AP sites and particular 3'-obstructive termini from DNA, and is an integral participant in the base excision repair (BER pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5-fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC(1280, a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays -- a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen -- and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals.

  1. Alkylation base damage is converted into repairable double-strand breaks and complex intermediates in G2 cells lacking AP endonuclease.

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    Wenjian Ma

    2011-04-01

    Full Text Available DNA double-strand breaks (DSBs are potent sources of genome instability. While there is considerable genetic and molecular information about the disposition of direct DSBs and breaks that arise during replication, relatively little is known about DSBs derived during processing of single-strand lesions, especially for the case of single-strand breaks (SSBs with 3'-blocked termini generated in vivo. Using our recently developed assay for detecting end-processing at random DSBs in budding yeast, we show that single-strand lesions produced by the alkylating agent methyl methanesulfonate (MMS can generate DSBs in G2-arrested cells, i.e., S-phase independent. These derived DSBs were observed in apn1/2 endonuclease mutants and resulted from aborted base excision repair leading to 3' blocked single-strand breaks following the creation of abasic (AP sites. DSB formation was reduced by additional mutations that affect processing of AP sites including ntg1, ntg2, and, unexpectedly, ogg1, or by a lack of AP sites due to deletion of the MAG1 glycosylase gene. Similar to direct DSBs, the derived DSBs were subject to MRX (Mre11, Rad50, Xrs2-determined resection and relied upon the recombinational repair genes RAD51, RAD52, as well as on the MCD1 cohesin gene, for repair. In addition, we identified a novel DNA intermediate, detected as slow-moving chromosomal DNA (SMD in pulsed field electrophoresis gels shortly after MMS exposure in apn1/2 cells. The SMD requires nicked AP sites, but is independent of resection/recombination processes, suggesting that it is a novel structure generated during processing of 3'-blocked SSBs. Collectively, this study provides new insights into the potential consequences of alkylation base damage in vivo, including creation of novel structures as well as generation and repair of DSBs in nonreplicating cells.

  2. Defective mitochondrial respiration, altered dNTP pools and reduced AP endonuclease 1 activity in peripheral blood mononuclear cells of Alzheimer's disease patients.

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    Maynard, Scott; Hejl, Anne-Mette; Dinh, Thuan-Son T; Keijzers, Guido; Hansen, Åse M; Desler, Claus; Moreno-Villanueva, Maria; Bürkle, Alexander; Rasmussen, Lene J; Waldemar, Gunhild; Bohr, Vilhelm A

    2015-10-01

    Accurate biomarkers for early diagnosis of Alzheimer's disease (AD) are badly needed. Recent reports suggest that dysfunctional mitochondria and DNA damage are associated with AD development. In this report, we measured various cellular parameters, related to mitochondrial bioenergetics and DNA damage, in peripheral blood mononuclear cells (PBMCs) of AD and control participants, for biomarker discovery. PBMCs were isolated from 53 patients with AD of mild to moderate degree and 30 age-matched healthy controls. Tests were performed on the PBMCs from as many of these participants as possible. We measured glycolysis and mitochondrial respiration fluxes using the Seahorse Bioscience flux analyzer, mitochondrial ROS production using flow cytometry, dNTP levels by way of a DNA polymerization assay, DNA strand breaks using the Fluorometric detection of Alkaline DNA Unwinding (FADU) assay, and APE1 incision activity (in cell lysates) on a DNA substrate containing an AP site (to estimate DNA repair efficiency). In the PBMCs of AD patients, we found reduced basal mitochondrial oxygen consumption, reduced proton leak, higher dATP level, and lower AP endonuclease 1 activity, depending on adjustments for gender and/or age. This study reveals impaired mitochondrial respiration, altered dNTP pools and reduced DNA repair activity in PBMCs of AD patients, thus suggesting that these biochemical activities may be useful as biomarkers for AD.

  3. Simple and cost-effective restriction endonuclease analysis of human adenoviruses.

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    Adhikary, Arun Kumar; Hanaoka, Nozomu; Fujimoto, Tsuguto

    2014-01-01

    Restriction endonuclease analyses (REAs) constitute the only inexpensive molecular approach capable of typing and characterizing human adenovirus (HAdV) strains based on the entire genome. However, the application of this method is limited by the need for time-consuming and labor-intensive procedures. We herein developed a simple and cost-effective REA for assessing HAdV. The method consists of (1) simple and cost-effective DNA extraction, (2) fast restriction endonuclease (RE) digestion, and (3) speedy mini agarose gel electrophoresis. In this study, DNA was isolated according to the kit-based method and 21.0 to 28.0  μg of viral DNA was extracted from prototypes (HAdV-1, HAdV-3, HAdV-4, and HAdV-37) in each flask. The amount of DNA ranged from 11.4 to 57.0  μg among the HAdV-3 (n=73) isolates. The obtained viral DNA was found to be applicable to more than 10 types of REAs. Fast-cut restriction endonucleases (REs) were able to digest the DNA within 15 minutes, and restriction fragments were easily separated via horizontal mini agarose gel electrophoresis. The whole procedure for 10 samples can be completed within approximately six hours (the conventional method requires at least two days). These results show that our REA is potentially applicable in many laboratories in which HAdVs are isolated.

  4. Human RECQL5beta stimulates flap endonuclease 1

    DEFF Research Database (Denmark)

    Speina, Elzbieta; Dawut, Lale; Hedayati, Mohammad

    2010-01-01

    Human RECQL5 is a member of the RecQ helicase family which is implicated in genome maintenance. Five human members of the family have been identified; three of them, BLM, WRN and RECQL4 are associated with elevated cancer risk. RECQL1 and RECQL5 have not been linked to any human disorder yet; cells...... dramatically stimulates the rate of FEN1 cleavage of flap DNA substrates. Moreover, we show that RECQL5beta and FEN1 interact physically and co-localize in the nucleus in response to DNA damage. Our findings, together with the previous literature on WRN, BLM and RECQL4's stimulation of FEN1, suggests...

  5. OpenAPS Data Commons on Open Humans

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    Lewis, Dana M.; Ball, Madeleine

    2017-01-01

    Poster describing OpenAPS, Open Humans, and joint work creating a data commons for OpenAPS data in the Open Humans platform. Presented at the 2017 Sage Assembly Bionetworks Assembly and recipient of a Young Innovator/Investigator award.

  6. A physical map of human Alu repeats cleavage by restriction endonucleases

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    Chernukhin Valery A

    2008-06-01

    Full Text Available Abstract Background Alu repetitive elements are the abundant sequences in human genome. Diversity of DNA sequences of these elements makes difficult the construction of theoretical patterns of Alu repeats cleavage by restriction endonucleases. We have proposed a method of restriction analysis of Alu repeats sequences in silico. Results Simple software to analyze Alu repeats database has been suggested and Alu repeats digestion patterns for several restriction enzymes' recognition sites have been constructed. Restriction maps of Alu repeats cleavage for corresponding restriction enzymes have been calculated and plotted. Theoretical data have been compared with experimental results on DNA hydrolysis with restriction enzymes, which we obtained earlier. Conclusion Alu repeats digestions provide the main contribution to the patterns of human chromosomal DNA cleavage. This corresponds to the experimental data on total human DNA hydrolysis with restriction enzymes.

  7. A domain in human EXOG converts apoptotic endonuclease to DNA-repair exonuclease

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    Szymanski, Michal R.; Yu, Wangsheng; Gmyrek, Aleksandra M.; White, Mark A.; Molineux, Ian J.; Lee, J. Ching; Yin, Y. Whitney

    2017-05-03

    Human EXOG (hEXOG) is a 5'-exonuclease that is crucial for mitochondrial DNA repair; the enzyme belongs to a nonspecific nuclease family that includes the apoptotic endonuclease EndoG. Here we report biochemical and structural studies of hEXOG, including structures in its apo form and in a complex with DNA at 1.81 and 1.85 Å resolution, respectively. A Wing domain, absent in other ββα-Me members, suppresses endonuclease activity, but confers on hEXOG a strong 5'-dsDNA exonuclease activity that precisely excises a dinucleotide using an intrinsic ‘tape-measure’. The symmetrical apo hEXOG homodimer becomes asymmetrical upon binding to DNA, providing a structural basis for how substrate DNA bound to one active site allosterically regulates the activity of the other. These properties of hEXOG suggest a pathway for mitochondrial BER that provides an optimal substrate for subsequent gap-filling synthesis by DNA polymerase γ.

  8. The basic N-terminal domain of TRF2 limits recombination endonuclease action at human telomeres.

    Science.gov (United States)

    Saint-Léger, Adélaïde; Koelblen, Melanie; Civitelli, Livia; Bah, Amadou; Djerbi, Nadir; Giraud-Panis, Marie-Josèphe; Londoño-Vallejo, Arturo; Ascenzioni, Fiorentina; Gilson, Eric

    2014-01-01

    The stability of mammalian telomeres depends upon TRF2, which prevents inappropriate repair and checkpoint activation. By using a plasmid integration assay in yeasts carrying humanized telomeres, we demonstrated that TRF2 possesses the intrinsic property to both stimulate initial homologous recombination events and to prevent their resolution via its basic N-terminal domain. In human cells, we further showed that this TRF2 domain prevents telomere shortening mediated by the resolvase-associated protein SLX4 as well as GEN1 and MUS81, 2 different types of endonucleases with resolvase activities. We propose that various types of resolvase activities are kept in check by the basic N-terminal domain of TRF2 in order to favor an accurate repair of the stalled forks that occur during telomere replication.

  9. Alternative nucleophilic substrates for the endonuclease activities of human immunodeficiency virus type 1 integrase

    Energy Technology Data Exchange (ETDEWEB)

    Ealy, Julie B. [Department of Medicine, Penn State College of Medicine, Milton S. Hershey Medical Center, 500 University Drive, PO Box 850, Mail Services H036, Hershey, PA 17033 (United States); Department of Chemistry, Penn State Lehigh Valley, 2809 E. Saucon Valley Road, Center Valley, PA 18034 (United States); Sudol, Malgorzata [Department of Medicine, Penn State College of Medicine, Milton S. Hershey Medical Center, 500 University Drive, PO Box 850, Mail Services H036, Hershey, PA 17033 (United States); Krzeminski, Jacek; Amin, Shantu [Department of Pharmacology, Penn State College of Medicine, Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033 (United States); Katzman, Michael, E-mail: mkatzman@psu.edu [Department of Medicine, Penn State College of Medicine, Milton S. Hershey Medical Center, 500 University Drive, PO Box 850, Mail Services H036, Hershey, PA 17033 (United States); Department of Microbiology and Immunology, Penn State College of Medicine, Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033 (United States)

    2012-11-10

    Retroviral integrase can use water or some small alcohols as the attacking nucleophile to nick DNA. To characterize the range of compounds that human immunodeficiency virus type 1 integrase can accommodate for its endonuclease activities, we tested 45 potential electron donors (having varied size and number or spacing of nucleophilic groups) as substrates during site-specific nicking at viral DNA ends and during nonspecific nicking reactions. We found that integrase used 22 of the 45 compounds to nick DNA, but not all active compounds were used for both activities. In particular, 13 compounds were used for site-specific and nonspecific nicking, 5 only for site-specific nicking, and 4 only for nonspecific nicking; 23 other compounds were not used for either activity. Thus, integrase can accommodate a large number of nucleophilic substrates but has selective requirements for its different activities, underscoring its dynamic properties and providing new information for modeling and understanding integrase.

  10. Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1

    KAUST Repository

    Sobhy, M.

    2013-06-06

    Human flap endonuclease 1 (FEN1), one of the structure-specific 5\\' nucleases, is integral in replication, repair, and recombination of cellular DNA. The 5\\' nucleases share significant unifying features yet cleave diverse substrates at similar positions relative to 5\\' end junctions. Using single-molecule Förster resonance energy transfer, we find a multistep mechanism that verifies all substrate features before inducing the intermediary-DNA bending step that is believed to unify 5\\' nuclease mechanisms. This is achieved by coordinating threading of the 5\\' flap of a nick junction into the conserved capped-helical gateway, overseeing the active site, and bending by binding at the base of the junction. We propose that this sequential and multistep substrate recognition process allows different 5\\' nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. Such mechanisms would also ensure the protection ofDNA junctions from nonspecific bending and cleavage. 2013 The Authors.

  11. Endonuclease G, a candidate human enzyme for the initiation of genomic inversion in herpes simplex type 1 virus.

    Science.gov (United States)

    Huang, Ke-Jung; Zemelman, Boris V; Lehman, I Robert

    2002-06-07

    The herpes simplex virus type 1 (HSV-1) a sequence is present as a direct repeat at the two termini of the 152-kilobase viral genome and as an inverted repeat at the junction of the two unique components L and S. During replication, the HSV-1 genome undergoes inversion of L and S, producing an equimolar mixture of the four possible isomers. Isomerization is believed to result from recombination triggered by breakage at the a sequence, a recombinational hot spot. We have identified an enzyme in HeLa cell extracts that preferentially cleaves the a sequence and have purified it to near homogeneity. Microsequencing showed it to be human endonuclease G, an enzyme with a strong preference for G+C-rich sequences. Endonuclease G appears to be the only cellular enzyme that can specifically cleave the a sequence. Endonuclease G also showed the predicted recombination properties in an in vitro recombination assay. Based on these findings, we propose that endonuclease G initiates the a sequence-mediated inversion of the L and S components during HSV-1 DNA replication.

  12. Cloning and characterisation of hAps1 and hAps2, human diadenosine polyphosphate-metabolising Nudix hydrolases

    Directory of Open Access Journals (Sweden)

    Safrany Stephen T

    2002-07-01

    Full Text Available Abstract Background The human genome contains at least 18 genes for Nudix hydrolase enzymes. Many have similar functions to one another. In order to understand their roles in cell physiology, these proteins must be characterised. Results We have characterised two novel human gene products, hAps1, encoded by the NUDT11 gene, and hAps2, encoded by the NUDT10 gene. These cytoplasmic proteins are members of the DIPP subfamily of Nudix hydrolases, and differ from each other by a single amino acid. Both metabolise diadenosine-polyphosphates and, weakly, diphosphoinositol polyphosphates. An apparent polymorphism of hAps1 has also been identified, which leads to the point mutation S39N. This has also been characterised. The favoured nucleotides were diadenosine 5',5"'-pentaphosphate (kcat/Km = 11, 8 and 16 × 103M-1s-1 respectively for hAps1, hAps1-39N and hAps2 and diadenosine 5',5"'-hexaphosphate (kcat/Km = 13, 14 and 11 × 103M-1s-1 respectively for hAps1, hAps1-39N and hAps2. Both hAps1 and hAps2 had pH optima of 8.5 and an absolute requirement for divalent cations, with manganese (II being favoured. Magnesium was not able to activate the enzymes. Therefore, these enzymes could be acutely regulated by manganese fluxes within the cell. Conclusions Recent gene duplication has generated the two Nudix genes, NUDT11 and NUDT10. We have characterised their gene products as the closely related Nudix hydrolases, hAps1 and hAps2. These two gene products complement the activity of previously described members of the DIPP family, and reinforce the concept that Ap5A and Ap6A act as intracellular messengers.

  13. Genomic Disruption of VEGF-A Expression in Human Retinal Pigment Epithelial Cells Using CRISPR-Cas9 Endonuclease.

    Science.gov (United States)

    Yiu, Glenn; Tieu, Eric; Nguyen, Anthony T; Wong, Brittany; Smit-McBride, Zeljka

    2016-10-01

    To employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells. CRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcuspyogenes Cas9 endonuclease (SpCas9) gene. The lentiviral vectors were used to infect ARPE-19 cells, a human RPE cell line. Frequency of insertion or deletion (indel) mutations was assessed by T7 endonuclease 1 mismatch detection assay; mRNA levels were assessed with quantitative real-time PCR; and VEGF-A protein levels were determined by ELISA. In vitro angiogenesis was measured using an endothelial cell tube formation assay. Five gRNAs targeting VEGF-A were selected based on the highest predicted on-target probabilities, lowest off-target probabilities, or combined average of both scores. Lentiviral delivery of the top-scoring gRNAs with SpCas9 resulted in indel formation in the VEGF-A gene at frequencies up to 37.0% ± 4.0% with corresponding decreases in secreted VEGF-A protein up to 41.2% ± 7.4% (P CRISPR-Cas9 endonuclease system may reduce VEGF-A secretion from human RPE cells and suppress angiogenesis, supporting the possibility of employing gene editing for antiangiogenesis therapy in ocular diseases.

  14. The human homolog of Escherichia coli endonuclease V is a nucleolar protein with affinity for branched DNA structures.

    Directory of Open Access Journals (Sweden)

    Cathrine Fladeby

    Full Text Available Loss of amino groups from adenines in DNA results in the formation of hypoxanthine (Hx bases with miscoding properties. The primary enzyme in Escherichia coli for DNA repair initiation at deaminated adenine is endonuclease V (endoV, encoded by the nfi gene, which cleaves the second phosphodiester bond 3' of an Hx lesion. Endonuclease V orthologs are widespread in nature and belong to a family of highly conserved proteins. Whereas prokaryotic endoV enzymes are well characterized, the function of the eukaryotic homologs remains obscure. Here we describe the human endoV ortholog and show with bioinformatics and experimental analysis that a large number of transcript variants exist for the human endonuclease V gene (ENDOV, many of which are unlikely to be translated into functional protein. Full-length ENDOV is encoded by 8 evolutionary conserved exons covering the core region of the enzyme, in addition to one or more 3'-exons encoding an unstructured and poorly conserved C-terminus. In contrast to the E. coli enzyme, we find recombinant ENDOV neither to incise nor bind Hx-containing DNA. While both enzymes have strong affinity for several branched DNA substrates, cleavage is observed only with E. coli endoV. We find that ENDOV is localized in the cytoplasm and nucleoli of human cells. As nucleoli harbor the rRNA genes, this may suggest a role for the protein in rRNA gene transactions such as DNA replication or RNA transcription.

  15. Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1

    Directory of Open Access Journals (Sweden)

    Mohamed A. Sobhy

    2013-06-01

    Full Text Available Human flap endonuclease 1 (FEN1, one of the structure-specific 5′ nucleases, is integral in replication, repair, and recombination of cellular DNA. The 5′ nucleases share significant unifying features yet cleave diverse substrates at similar positions relative to 5′ end junctions. Using single-molecule Förster resonance energy transfer, we find a multistep mechanism that verifies all substrate features before inducing the intermediary-DNA bending step that is believed to unify 5′ nuclease mechanisms. This is achieved by coordinating threading of the 5′ flap of a nick junction into the conserved capped-helical gateway, overseeing the active site, and bending by binding at the base of the junction. We propose that this sequential and multistep substrate recognition process allows different 5′ nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. Such mechanisms would also ensure the protection of DNA junctions from nonspecific bending and cleavage.

  16. Altered endoribonuclease activity of apurinic/apyrimidinic endonuclease 1 variants identified in the human population.

    Directory of Open Access Journals (Sweden)

    Wan Cheol Kim

    Full Text Available Apurinic/apyrimidinic endonuclease 1 (APE1 is the major mammalian enzyme in the DNA base excision repair pathway and cleaves the DNA phosphodiester backbone immediately 5' to abasic sites. APE1 also has 3'-5' DNA exonuclease and 3' DNA phosphodiesterase activities, and regulates transcription factor DNA binding through its redox regulatory function. The human APE1 has recently been shown to endonucleolytically cleave single-stranded regions of RNA. Towards understanding the biological significance of the endoribonuclease activity of APE1, we examined eight different amino acid substitution variants of APE1 previously identified in the human population. Our study shows that six APE1 variants, D148E, Q51H, I64V, G241R, R237A, and G306A, exhibit a 76-85% reduction in endoribonuclease activity against a specific coding region of the c-myc RNA, yet fully retain the ability to cleave apurinic/apyrimidinic DNA. We found that two APE1 variants, L104R and E126D, exhibit a unique RNase inhibitor-resistant endoribonuclease activity, where the proteins cleave c-myc RNA 3' of specific single-stranded guanosine residues. Expression of L104R and E126D APE1 variants in bacterial Origami cells leads to a 60-80% reduction in colony formation and a 1.5-fold increase in cell doubling time, whereas the other variants, which exhibit diminished endoribonuclease activity, had no effect. These data indicate that two human APE1 variants exhibit a unique endoribonuclease activity, which correlates with their ability to induce cytotoxicity or slow down growth in bacterial cells and supports the notion of their biological functionality.

  17. Molecular Recognition of DNA Damage Sites by Apurinic/Apyrimidinic Endonucleases

    Energy Technology Data Exchange (ETDEWEB)

    Braun, W. A.

    2005-07-28

    The DNA repair/redox factor AP endonuclease 1 (APE1) is a multifunctional protein which is known to to be essential for DNA repair activity in human cells. Structural/functional analyses of the APE activity is thus been an important research field to assess cellular defense mechanisms against ionizing radiation.

  18. DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction*

    Science.gov (United States)

    Algasaier, Sana I.; Exell, Jack C.; Bennet, Ian A.; Thompson, Mark J.; Gotham, Victoria J. B.; Shaw, Steven J.; Craggs, Timothy D.; Finger, L. David; Grasby, Jane A.

    2016-01-01

    Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5′-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5′-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5′-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5′-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Tyr40, Asp181, and Arg100 and a reacting duplex 5′-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5′-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage. PMID:26884332

  19. Opposite effects of cell differentiation and apoptosis on Ap3A/Ap4A ratio in human cell cultures.

    Science.gov (United States)

    Vartanian, A; Prudovsky, I; Suzuki, H; Dal Pra, I; Kisselev, L

    1997-09-29

    The biological role of diadenosine oligophosphates (DAOP) remains obscure in spite of numerous attempts to solve this enigma. It is known that Ap3A contrary to Ap4A accumulates in human cultured cells treated with interferons (IFNs) alpha or gamma. Since IFNs are considered as antiproliferative regulators, we assumed that different cell status may be associated with varying intracellular levels of DAOP. Promyelocytic human cell line HL60 induced by phorbol ester (TPA) to differentiate to macrophage-like cells in culture exhibits a profound loss of proliferative potential. Here we have shown a 4-5-fold increase in Ap3A concentration in HL60 cells induced by TPA, similar to the effect of IFN, while the Ap4A concentration remained unchanged. On the contrary, in cells undergoing apoptosis induced by VP16, a topoisomerase II inhibitor, the Ap3A concentration considerably decreased, while the Ap4A concentration increased. These findings combined with earlier results suggest an involvement of the Ap3A/Ap4A ratio in signal transduction pathways controlling the cell status.

  20. Apurinic endonuclease activity of yeast Apn2 protein.

    Science.gov (United States)

    Unk, I; Haracska, L; Johnson, R E; Prakash, S; Prakash, L

    2000-07-21

    Abasic (apurinic/apyrimidinic; AP) sites are generated in vivo through spontaneous base loss and by enzymatic removal of bases damaged by alkylating agents and reactive oxygen species. In Saccharomyces cerevisiae, the APN1 and APN2 genes function in alternate pathways of AP site removal. Apn2-like proteins have been identified in other eukaryotes including humans, and these proteins form a distinct subfamily within the exonuclease III (ExoIII)/Ape1/Apn2 family of proteins. Apn2 and other members of this subfamily contain a carboxyl-terminal extension not present in the ExoIII/Ape1-like proteins. Here, we purify the Apn2 protein from yeast and show that it is a class II AP endonuclease. Deletion of the carboxyl terminus does not affect the AP endonuclease activity of the protein, but this protein is defective in the removal of AP sites in vivo. The carboxyl terminus may enable Apn2 to complex with other proteins, and such a multiprotein assembly may be necessary for the efficient recognition and cleavage of AP sites in vivo.

  1. Biochemical Characterization of AP Lyase and m6A Demethylase Activities of Human AlkB Homologue 1 (ALKBH1).

    Science.gov (United States)

    Müller, Tina A; Tobar, Michael A; Perian, Madison N; Hausinger, Robert P

    2017-04-04

    Alkbh1 is one of nine mammalian homologues of Escherichia coli AlkB, a 2-oxoglutarate-dependent dioxygenase that catalyzes direct DNA repair by removing alkyl lesions from DNA. Six distinct enzymatic activities have been reported for Alkbh1, including hydroxylation of variously methylated DNA, mRNA, tRNA, or histone substrates along with the cleavage of DNA at apurinic/apyrimidinic (AP) sites followed by covalent attachment to the 5'-product. The studies described here extend the biochemical characterization of two of these enzymatic activities using human ALKBH1: the AP lyase and 6-methyl adenine DNA demethylase activities. The steady-state and single-turnover kinetic parameters for ALKBH1 cleavage of AP sites in DNA were determined and shown to be comparable to those of other AP lyases. The α,β-unsaturated aldehyde of the 5'-product arising from DNA cleavage reacts predominantly with C129 of ALKBH1, but secondary sites also generate covalent adducts. The 6-methyl adenine demethylase activity was examined with a newly developed assay using a methylation-sensitive restriction endonuclease, and the enzymatic rate was found to be very low. Indeed, the demethylase activity was less than half that of the AP lyase activity when ALKBH1 samples were assayed using identical buffer conditions. The two enzymatic activities were examined using a series of site-directed variant proteins, revealing the presence of distinct but partially overlapping active sites for the two reactions. We postulate that the very low 6-methyl adenine oxygenase activity associated with ALKBH1 is unlikely to represent the major function of the enzyme in the cell, while the cellular role of the lyase activity (including its subsequent covalent attachment to DNA) remains uncertain.

  2. Analysis of human reliability in the APS of fire. Application of NUREG-1921; Analisis de Fiabilidad Humana en el APS de Incendios. Aplicacion del NUREG-1921

    Energy Technology Data Exchange (ETDEWEB)

    Perez Torres, J. L.; Celaya Meler, M. A.

    2014-07-01

    An analysis of human reliability in a probabilistic safety analysis (APS) of fire aims to identify, describe, analyze and quantify, in a manner traceable, human actions that can affect the mitigation of an initiating event produced by a fire. (Author)

  3. The Freshman Nine: Helping High School Freshmen Be Successful in AP Human Geography

    Science.gov (United States)

    Garner, Jennifer

    2012-01-01

    Teaching AP Human Geography to freshmen seems like a daunting task and while there are many arguments both for and against offering the course to freshmen, for many teachers it is reality. In this article, the author offers nine tips to help high school freshmen be successful in the course and on the AP exam.

  4. Knockdown of CDK2AP1 in primary human fibroblasts induces p53 dependent senescence.

    Directory of Open Access Journals (Sweden)

    Khaled N Alsayegh

    Full Text Available Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1 is known to be a tumor suppressor that plays a role in cell cycle regulation by sequestering monomeric CDK2, and targeting it for proteolysis. A reduction of CDK2AP1 expression is considered to be a negative prognostic indicator in patients with oral squamous cell carcinoma and also associated with increased invasion in human gastric cancer tissue. CDK2AP1 overexpression was shown to inhibit growth, reduce invasion and increase apoptosis in prostate cancer cell lines. In this study, we investigated the effect of CDK2AP1 downregulation in primary human dermal fibroblasts. Using a short-hairpin RNA to reduce its expression, we found that knockdown of CDK2AP1 in primary human fibroblasts resulted in reduced proliferation and in the induction of senescence associated beta-galactosidase activity. CDK2AP1 knockdown also resulted in a significant reduction in the percentage of cells in the S phase and an accumulation of cells in the G1 phase of the cell cycle. Immunocytochemical analysis also revealed that the CDK2AP1 knockdown significantly increased the percentage of cells that exhibited γ-H2AX foci, which could indicate presence of DNA damage. CDK2AP1 knockdown also resulted in increased mRNA levels of p53, p21, BAX and PUMA and p53 protein levels. In primary human fibroblasts in which p53 and CDK2AP1 were simultaneously downregulated, there was: (a no increase in senescence associated beta-galactosidase activity, (b decrease in the number of cells in the G1-phase and increase in number of cells in the S-phase of the cell cycle, and (c decrease in the mRNA levels of p21, BAX and PUMA when compared with CDK2AP1 knockdown only fibroblasts. Taken together, this suggests that the observed phenotype is p53 dependent. We also observed a prominent increase in the levels of ARF protein in the CDK2AP1 knockdown cells, which suggests a possible role of ARF in p53 stabilization following CDK2AP1

  5. Conserved structural chemistry for incision activity in structurally non-homologous apurinic/apyrimidinic endonuclease APE1 and endonuclease IV DNA repair enzymes.

    Energy Technology Data Exchange (ETDEWEB)

    Tsutakawa, Susan E.; Shin, David S.; Mol, Clifford D.; Izum, Tadahide; Arvai, Andrew S.; Mantha, Anil K.; Szczesny, Bartosz; Ivanov, Ivaylo N.; Hosfield, David J.; Maiti, Buddhadev; Pique, Mike E.; Frankel, Kenneth A.; Hitomi, Kenichi; Cunningham, Richard P.; Mitra, Sankar; Tainer, John A.

    2013-03-22

    Non-coding apurinic/apyrimidinic (AP) sites in DNA form spontaneously and as DNA base excision repair intermediates are the most common toxic and mutagenic in vivo DNA lesion. For repair, AP sites must be processed by 5' AP endonucleases in initial stages of base repair. Human APE1 and bacterial Nfo represent the two conserved 5' AP endonuclease families in the biosphere; they both recognize AP sites and incise the phosphodiester backbone 5' to the lesion, yet they lack similar structures and metal ion requirements. Here, we determined and analyzed crystal structures of a 2.4 ? resolution APE1-DNA product complex with Mg(2+) and a 0.92 Nfo with three metal ions. Structural and biochemical comparisons of these two evolutionarily distinct enzymes characterize key APE1 catalytic residues that are potentially functionally similar to Nfo active site components, as further tested and supported by computational analyses. We observe a magnesium-water cluster in the APE1 active site, with only Glu-96 forming the direct protein coordination to the Mg(2+). Despite differences in structure and metal requirements of APE1 and Nfo, comparison of their active site structures surprisingly reveals strong geometric conservation of the catalytic reaction, with APE1 catalytic side chains positioned analogously to Nfo metal positions, suggesting surprising functional equivalence between Nfo metal ions and APE1 residues. The finding that APE1 residues are positioned to substitute for Nfo metal ions is supported by the impact of mutations on activity. Collectively, the results illuminate the activities of residues, metal ions, and active site features for abasic site endonucleases.

  6. Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases

    Directory of Open Access Journals (Sweden)

    Carmen F Bjurström

    2016-01-01

    Full Text Available We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs, transcriptional activator-like effector nucleases (TALENs, and clustered regularly interspaced short palindromic repeat (CRISPR/Cas9 systems designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34+ progenitor cells. ZFNs and TALENs were delivered as in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA (pU6.g1 or in vitro transcribed gRNA (gR.1. Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34+ cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34+ cells had the capacity to engraft in NOD-PrkdcSCID-IL2Rγnull mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34+ cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs, highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs.

  7. AP-2γ Induces p21 Expression, Arrests Cell Cycle, Inhibits the Tumor Growth of Human Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Hualei Li

    2006-07-01

    Full Text Available Activating enhancer-binding protein 2γ (AP-2γ is a member of the developmentally regulated AP-2 transcription factor family that regulates the expression of many downstream genes. Whereas the effects of AP-2α overexpression on cell growth are fairly well established, the cellular effects of AP-2γ overexpression are less well studied. Our new findings show that AP-2γ significantly upregulates p21 mRNA and proteins, inhibits cell growth, decreases clonogenic survival. Cell cycle analysis revealed that forced AP-2γ expression induced G1-phase arrest, decreased DNA synthesis, decreased the fraction of cells in S phase. AP-2γ expression also led to cyclin D1 repression, decreased Rb phosphorylation, decreased E2F activity in breast carcinoma cells. AP-2γ binding to the p21 promoter was observed in vivo, the absence of growth inhibition in response to AP-2γ expression in p21 (-/- cells demonstrated that p21 caused, at least in part, AP-2-induced cell cycle arrest. Finally, the tumor growth of human breast carcinoma cells in vivo was inhibited by the expression of AP-2γ relative to empty vector-infected cells, suggesting that AP-2γ acts as a tumor suppressor. In summary, expression of either AP-2γ or AP-2α inhibited breast carcinoma cell growth; thus, these genes may be therapeutic targets for breast cancer.

  8. Interaction of apurinic/apyrimidinic endonuclease 2 (Apn2) with Myh1 DNA glycosylase in fission yeast.

    Science.gov (United States)

    Jin, Jin; Hwang, Bor-Jang; Chang, Po-Wen; Toth, Eric A; Lu, A-Lien

    2014-03-01

    Oxidative DNA damage is repaired primarily by the base excision repair (BER) pathway in a process initiated by removal of base lesions or mismatched bases by DNA glycosylases. MutY homolog (MYH, MUTYH, or Myh1) is a DNA glycosylase which excises adenine paired with the oxidative lesion 8-oxo-7,8-dihydroguanine (8-oxoG, or G°), thus reducing G:C to T:A mutations. The resulting apurinic/apyrimidinic (AP) site is processed by an AP-endonuclease or a bifunctional glycosylase/lyase. We show here that the major Schizosaccharomyces pombe AP endonuclease, Apn2, binds to the inter-domain connector located between the N- and C-terminal domains of Myh1. This Myh1 inter-domain connector also interacts with the Hus1 subunit of the Rad9-Rad1-Hus1 checkpoint clamp. Mutagenesis studies indicate that Apn2 and Hus1 bind overlapping but different sequence motifs on Myh1. Mutation on I(261) of Myh1 reduces its interaction with Hus1, but only slightly attenuates its interaction with Apn2. However, E(262) of Myh1 is a key determinant for both Apn2 and Hus1 interactions. Like human APE1, Apn2 has 3'-phosphodiesterase activity. However, unlike hAPE1, Apn2 has a weak AP endonuclease activity which cleaves the AP sites generated by Myh1 glycosylase. Functionally, Apn2 stimulates Myh1 glycosylase activity and Apn2 phosphodiesterase activity is stimulated by Myh1. The cross stimulation of Myh1 and Apn2 enzymatic activities is dependent on their physical interaction. Thus, Myh1 and Apn2 constitute an initial BER complex. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Lundep, a sand fly salivary endonuclease increases Leishmania parasite survival in neutrophils and inhibits XIIa contact activation in human plasma.

    Directory of Open Access Journals (Sweden)

    Andrezza C Chagas

    2014-02-01

    Full Text Available Neutrophils are the host's first line of defense against infections, and their extracellular traps (NET were recently shown to kill Leishmania parasites. Here we report a NET-destroying molecule (Lundep from the salivary glands of Lutzomyia longipalpis. Previous analysis of the sialotranscriptome of Lu. longipalpis showed the potential presence of an endonuclease. Indeed, not only was the cloned cDNA (Lundep shown to encode a highly active ss- and dsDNAse, but also the same activity was demonstrated to be secreted by salivary glands of female Lu. longipalpis. Lundep hydrolyzes both ss- and dsDNA with little sequence specificity with a calculated DNase activity of 300000 Kunitz units per mg of protein. Disruption of PMA (phorbol 12 myristate 13 acetate- or parasite-induced NETs by treatment with recombinant Lundep or salivary gland homogenates increases parasite survival in neutrophils. Furthermore, co-injection of recombinant Lundep with metacyclic promastigotes significantly exacerbates Leishmania infection in mice when compared with PBS alone or inactive (mutagenized Lundep. We hypothesize that Lundep helps the parasite to establish an infection by allowing it to escape from the leishmanicidal activity of NETs early after inoculation. Lundep may also assist blood meal intake by lowering the local viscosity caused by the release of host DNA and as an anticoagulant by inhibiting the intrinsic pathway of coagulation.

  10. Constitutive activation of transcription factor AP-2 is associated with decreased MnSOD expression in transformed human lung fibroblasts.

    Science.gov (United States)

    Zhu, C; Huang, Y; Weydert, C J; Oberley, L W; Domann, F E

    2001-06-01

    Activator protein-2 (AP-2) is a transcription factor with transactivating and transrepressing potential in different promoter contexts. AP-2 contains seven cysteines, and its in vitro DNA binding activity is redox-sensitive. Superoxide dismutase-2 (SOD2), which encodes the antioxidant enzyme manganese superoxide dismutase (MnSOD), is a putative tumor suppressor gene whose loss of expression is associated with the malignant phenotype. SOD2 promoter mutations that generate new AP-2 sites are associated with loss of MnSOD expression in cancer cells. In the current study, we have identified an inverse expression pattern between AP-2 and MnSOD in normal versus transformed human cells. MRC5 cells are a normal human lung fibroblast cell strain that is mortal and senesces after a certain number of passages in vitro. MRC5-VA is a simian virus transformed variant of MRC5. We determined the levels of expression of MnSOD and AP-2 in these two cell types at the levels of mRNA, protein, and activity. Our results indicated that MnSOD expression was significantly decreased in MRC5-VA cells compared with MRC5 cells at each level of investigation, whereas AP-2 showed an opposing pattern of expression and DNA binding activity. These results suggest that AP-2 may participate in the mechanism(s) underlying decreased expression of SOD2 in transformed cells.

  11. AP Music Theory Applied

    Science.gov (United States)

    Spieker, Matthew H.

    2016-01-01

    Some American high schools include Advanced Placement (AP) Music Theory within their course offerings. Students who pass the AP exam can receive college credit either as a music or humanities credit. An AP class, however, offers music students more than future college credit; it ultimately improves musicianship skills and promotes deeper…

  12. Involvement of two endonuclease III homologs in the base excision repair pathway for the processing of DNA alkylation damage in Saccharomyces cerevisiae.

    Science.gov (United States)

    Hanna, Michelle; Chow, Barbara L; Morey, Natalie J; Jinks-Robertson, Sue; Doetsch, Paul W; Xiao, Wei

    2004-01-05

    DNA base excision repair (BER) is initiated by DNA glycosylases that recognize and remove damaged bases. The phosphate backbone adjacent to the resulting apurinic/apyrimidinic (AP) site is then cleaved by an AP endonuclease or glycosylase-associated AP lyase to invoke subsequent BER steps. We have used a genetic approach in Saccharomyces cerevisiae to determine whether or not AP sites are blocks to DNA replication and the biological consequences if AP sites persist in the genome. We previously reported that yeast cells deficient in the two AP endonucleases (apn1 apn2 double mutant) are extremely sensitive to killing by a model DNA alkylating agent methyl methanesulfonate (MMS) and that this sensitivity can be reduced by deleting the MAG1 3-methyladenine DNA glycosylase gene. Here we report that in the absence of the AP endonucleases, deletion of two Escherichia coli endonuclease III homologs, NTG1 and NTG2, partially suppresses MMS-induced killing, which indicates that the AP lyase products are deleterious unless they are further processed by an AP endonuclease. The severe MMS sensitivity seen in AP endonuclease deficient strains can also be rescued by treatment of cells with the AP lyase inhibitor methoxyamine, which suggests that the product of AP lyase action on an AP site is indeed an extremely toxic lesion. In addition to the AP endonuclease interactions, deletion of NTG1 and NTG2 enhances the mag1 mutant sensitivity to MMS, whereas overexpression of MAG1 in either the ntg1 or ntg2 mutant severely affects cell growth. These results help to delineate alkylation base lesion flow within the BER pathway.

  13. Antioxidant-induced changes of the AP-1 transcription complex are paralleled by a selective suppression of human papillomavirus transcription.

    Science.gov (United States)

    Rösl, F; Das, B C; Lengert, M; Geletneky, K; zur Hausen, H

    1997-01-01

    Considering the involvement of a redox-regulatory pathway in the expression of human papillomaviruses (HPVs), HPV type 16 (HPV-16)-immortalized human keratinocytes were treated with the antioxidant pyrrolidine-dithiocarbamate (PDTC). PDTC induces elevated binding of the transcription factor AP-1 to its cognate recognition site within the viral regulatory region. Despite of increased AP-1 binding, normally indispensable for efficient HPV-16 transcription, viral gene expression was selectively suppressed at the level of initiation of transcription. Electrophoretic mobility supershift assays showed that the composition of the AP-1 complex, predominantly consisting of Jun homodimers in untreated cells, was altered. Irrespective of enhanced c-fos expression, c-jun was phosphorylated and became primarily heterodimerized with fra-1, which was also induced after PDTC incubation. Additionally, there was also an increased complex formation between c-jun and junB. Because both fra-1 and junB overexpression negatively interferes with c-jun/c-fos trans-activation of AP-1-responsive genes, our results suggest that the observed block in viral transcription is mainly the consequence of an antioxidant-induced reconstitution of the AP-1 transcription complex. Since expression of the c-jun/c-fos gene family is tightly regulated during cellular differentiation, defined reorganization of a central viral transcription factor may represent a novel mechanism controlling the transcription of pathogenic HPVs during keratinocyte differentiation and in the progression to cervical cancer.

  14. Ginkgolide B revamps neuroprotective role of apurinic/apyrimidinic endonuclease 1 and mitochondrial oxidative phosphorylation against Aβ25-35 -induced neurotoxicity in human neuroblastoma cells.

    Science.gov (United States)

    Kaur, Navrattan; Dhiman, Monisha; Perez-Polo, J Regino; Mantha, Anil K

    2015-06-01

    Accumulating evidence points to roles for oxidative stress, amyloid beta (Aβ), and mitochondrial dysfunction in the pathogenesis of Alzheimer's disease (AD). In neurons, the base excision repair pathway is the predominant DNA repair (BER) pathway for repairing oxidized base lesions. Apurinic/apyrimidinic endonuclease 1 (APE1), a multifunctional enzyme with DNA repair and reduction-oxidation activities, has been shown to enhance neuronal survival after oxidative stress. This study seeks to determine 1) the effect of Aβ25-35 on reactive oxygen species (ROS)/reactive nitrogen species (RNS) levels, 2) the activities of respiratory complexes (I, III, and IV), 3) the role of APE1 by ectopic expression, and 4) the neuromodulatory role of ginkgolide B (GB; from the leaves of Ginkgo biloba). The pro-oxidant Aβ25-35 peptide treatment increased the levels of ROS/RNS in human neuroblastoma IMR-32 and SH-SY5Y cells, which were decreased after pretreatment with GB. Furthermore, the mitochondrial APE1 level was found to be decreased after treatment with Aβ25-35 up to 48 hr, and the level was increased significantly in cells pretreated with GB. The oxidative phosphorylation (OXPHOS; activities of complexes I, III, and IV) indicated that Aβ25-35 treatment decreased activities of complexes I and IV, and pretreatment with GB and ectopic APE1 expression enhanced these activities significantly compared with Aβ25-35 treatment. Our results indicate that ectopic expression of APE1 potentiates neuronal cells to overcome the oxidative damage caused by Aβ25-35 . In addition, GB has been shown to modulate the mitochondrial OXPHOS against Aβ25-35 -induced oxidative stress and also to regulate the levels of ROS/RNS in the presence of ectopic APE1. This study presents findings from a new point of view to improve therapeutic potential for AD via the synergistic neuroprotective role played by APE1 in combination with the phytochemical GB. © 2015 Wiley Periodicals, Inc.

  15. Restriction glycosylases: involvement of endonuclease activities in the restriction process.

    Science.gov (United States)

    Zhang, Yingbiao; Matsuzaka, Tomoyuki; Yano, Hirokazu; Furuta, Yoshikazu; Nakano, Toshiaki; Ishikawa, Ken; Fukuyo, Masaki; Takahashi, Noriko; Suzuki, Yutaka; Sugano, Sumio; Ide, Hiroshi; Kobayashi, Ichizo

    2017-02-17

    All restriction enzymes examined are phosphodiesterases generating 3΄-OH and 5΄-P ends, but one restriction enzyme (restriction glycosylase) excises unmethylated bases from its recognition sequence. Whether its restriction activity involves endonucleolytic cleavage remains unclear. One report on this enzyme, R.PabI from a hyperthermophile, ascribed the breakage to high temperature while another showed its weak AP lyase activity generates atypical ends. Here, we addressed this issue in mesophiles. We purified R.PabI homologs from Campylobacter coli (R.CcoLI) and Helicobacter pylori (R.HpyAXII) and demonstrated their DNA cleavage, DNA glycosylase and AP lyase activities in vitro at 37°C. The AP lyase activity is more coupled with glycosylase activity in R.CcoLI than in R.PabI. R.CcoLI/R.PabI expression caused restriction of incoming bacteriophage/plasmid DNA and endogenous chromosomal DNA within Escherichia coli at 37°C. The R.PabI-mediated restriction was promoted by AP endonuclease action in vivo or in vitro. These results reveal the role of endonucleolytic DNA cleavage in restriction and yet point to diversity among the endonucleases. The cleaved ends are difficult to repair in vivo, which may indicate their biological significance. These results support generalization of the concept of restriction–modification system to the concept of self-recognizing epigenetic system, which combines any epigenetic labeling and any DNA damaging.

  16. Selfish DNA: Homing Endonucleases Find a Home

    National Research Council Canada - National Science Library

    Edgell, David R

    2009-01-01

    ...] . Intriguingly, many self-splicing introns (and inteins) are also mobile genetic elements at the DNA level because they encode mobility-promoting proteins termed homing endonucleases that have the interesting property of being site-specific but sequence-tolerant DNA endonucleases [4–7] . Intron-encoded homing endonucleases recognize a site, the homing ...

  17. Human Papillomavirus 16 (HPV-16), HPV-18, and HPV-31 E6 Override the Normal Phosphoregulation of E6AP Enzymatic Activity.

    Science.gov (United States)

    Thatte, Jayashree; Banks, Lawrence

    2017-11-15

    The human papillomavirus (HPV) E6 oncoproteins recruit the cellular ubiquitin ligase E6AP/UBE3A to target cellular substrates for proteasome-mediated degradation, and one consequence of this activity is the E6 stimulation of E6AP autoubiquitination and degradation. Recent studies identified an autism-linked mutation within E6AP at T485, which was identified as a protein kinase A phosphoacceptor site and which could directly regulate E6AP ubiquitin ligase activity. In this study, we have analyzed how T485-mediated regulation of E6AP might affect E6 targeting of some of its known substrates. We show that modulation of T485 has no effect on the ability of E6 to direct either p53 or Dlg for degradation. Furthermore, T485 regulation has no effect on HPV-16 or HPV-31 E6-induced autodegradation of E6AP but does affect HPV-18 E6-induced autodegradation of E6AP. In cells derived from cervical cancers, we find low levels of both phosphorylated and nonphosphorylated E6AP in the nucleus. However, ablation of E6 results in a dramatic accumulation of phospho-E6AP in the cytoplasm, whereas nonphosphorylated E6AP accumulates primarily in the nucleus. Interestingly, E6AP phosphorylation at T485 confers association with 14-3-3 proteins, and this interaction seems to be important, in part, for the ability of E6 to recruit phospho-E6AP into the nucleus. These results demonstrate that HPV E6 overrides the normal phosphoregulation of E6AP, both in terms of its enzymatic activity and its subcellular distribution.IMPORTANCE Recent reports demonstrate the importance of phosphoregulation of E6AP for its normal enzymatic activity. Here, we show that HPV E6 is capable of overriding this regulation and can promote degradation of p53 and Dlg regardless of the phosphorylation status of E6AP. Furthermore, E6 interaction with E6AP also significantly alters how E6AP is subject to autodegradation and suggests that this is not a simple stimulation of an already-existing activity but rather a

  18. Determination of L-AP4-bound human mGlu8 receptor amino terminal domain structure and the molecular basis for L-AP4's group III mGlu receptor functional potency and selectivity.

    Science.gov (United States)

    Schkeryantz, Jeffery M; Chen, Qi; Ho, Joseph D; Atwell, Shane; Zhang, Aiping; Vargas, Michelle C; Wang, Jing; Monn, James A; Hao, Junliang

    2018-02-15

    L-2-Amino-4-phosphonobutyric acid (L-AP4) is a known potent and selective agonist for the Group III mGlu receptors. However, it does not show any selectivity among the individual group III mGlu subtypes. In order to understand the molecular basis for this group selectivity, we solved the first human mGlu8 amino terminal domain (ATD) crystal structures in complex with L-glu and L-AP4. In comparison with other published L-glu-bound mGlu ATD structures, we have observed L-glu binds in a significantly different manner in mGlu1. Furthermore, these new structures provided evidence that both the electronic and steric nature of the distal phosphate of L-AP4 contribute to its exquisite Group III functional agonist potency and selectivity. Copyright © 2018 Elsevier Ltd. All rights reserved.

  19. Clustered DNA lesions containing 5-formyluracil and AP site: repair via the BER system.

    Science.gov (United States)

    Belousova, Ekaterina A; Vasil'eva, Inna A; Moor, Nina A; Zatsepin, Timofey S; Oretskaya, Tatiana S; Lavrik, Olga I

    2013-01-01

    Lesions in the DNA arise under ionizing irradiation conditions or various chemical oxidants as a single damage or as part of a multiply damaged site within 1-2 helical turns (clustered lesion). Here, we explored the repair opportunity of the apurinic/apyrimidinic site (AP site) composed of the clustered lesion with 5-formyluracil (5-foU) by the base excision repair (BER) proteins. We found, that if the AP site is shifted relative to the 5-foU of the opposite strand, it could be repaired primarily via the short-patch BER pathway. In this case, the cleavage efficiency of the AP site-containing DNA strand catalyzed by human apurinic/apyrimidinic endonuclease 1 (hAPE1) decreased under AP site excursion to the 3'-side relative to the lesion in the other DNA strand. DNA synthesis catalyzed by DNA polymerase lambda was more accurate in comparison to the one catalyzed by DNA polymerase beta. If the AP site was located exactly opposite 5-foU it was expected to switch the repair to the long-patch BER pathway. In this situation, human processivity factor hPCNA stimulates the process.

  20. Problem-Solving Test: Restriction Endonuclease Mapping

    Science.gov (United States)

    Szeberenyi, Jozsef

    2011-01-01

    The term "restriction endonuclease mapping" covers a number of related techniques used to identify specific restriction enzyme recognition sites on small DNA molecules. A method for restriction endonuclease mapping of a 1,000-basepair (bp)-long DNA molecule is described in the fictitious experiment of this test. The most important fact needed to…

  1. Isothermal detection of RNA with restriction endonucleases.

    Science.gov (United States)

    Yan, Lei; Nakayama, Shizuka; Yitbarek, Saron; Greenfield, Isabel; Sintim, Herman O

    2011-01-07

    Herein, we demonstrate how to detect nucleic acids that do not contain restriction endonuclease recognition sites with restriction endonucleases. We show that the topology of DNA probes used in this detection strategy remarkably affects the efficiency of RNA/DNA detection.

  2. Selection of restriction endonucleases using artificial cells.

    Science.gov (United States)

    Zheng, Yu; Roberts, Richard J

    2007-01-01

    We describe in this article an in vitro system for the selection of restriction endonucleases using artificial cells. The artificial cells are generated in the form of a water-in-oil emulsion by in vitro compartmentalization. Each aqueous compartment contains a reconstituted transcription/translation mix along with the dispersed DNA templates. In the compartments containing endonuclease genes, an endonuclease expressed in vitro cleaves its own DNA template adjacent to the gene, leaving a sticky end. The pooled DNA templates are then ligated to an adaptor with a compatible end. The endonuclease genes are then enriched by adaptor-specific PCR on the ligation mix. We demonstrate that the system can achieve at least 100-fold enrichment in a single round of selection. It is sensitive enough to enrich an active endonuclease gene from a 1:10(5) model library in 2-3 rounds of selection. Finally, we describe experiments where we selected endonuclease genes directly from a bacterial genomic DNA source in three rounds of selections: the known PstI gene from Providencia stuartii and the new TspMI gene from Thermus sp. manalii. This method provides a unique tool for cloning restriction endonuclease genes and has many other potential applications.

  3. Massively parallel characterization of restriction endonucleases.

    Science.gov (United States)

    Kamps-Hughes, Nick; Quimby, Aine; Zhu, Zhenyu; Johnson, Eric A

    2013-06-01

    Restriction endonucleases are highly specific in recognizing the particular DNA sequence they act on. However, their activity is affected by sequence context, enzyme concentration and buffer composition. Changes in these factors may lead to either ineffective cleavage at the cognate restriction site or relaxed specificity allowing cleavage of degenerate 'star' sites. Additionally, uncharacterized restriction endonucleases and engineered variants present novel activities. Traditionally, restriction endonuclease activity is assayed on simple substrates such as plasmids and synthesized oligonucleotides. We present and use high-throughput Illumina sequencing-based strategies to assay the sequence specificity and flanking sequence preference of restriction endonucleases. The techniques use fragmented DNA from sequenced genomes to quantify restriction endonuclease cleavage on a complex genomic DNA substrate in a single reaction. By mapping millions of restriction site-flanking reads back to the Escherichia coli and Drosophila melanogaster genomes we were able to quantitatively characterize the cognate and star site activity of EcoRI and MfeI and demonstrate genome-wide decreases in star activity with engineered high-fidelity variants EcoRI-HF and MfeI-HF, as well as quantify the influence on MfeI cleavage conferred by flanking nucleotides. The methods presented are readily applicable to all type II restriction endonucleases that cleave both strands of double-stranded DNA.

  4. Saccharomyces cerevisiae MutLα IS A MISMATCH REPAIR ENDONUCLEASE*

    Science.gov (United States)

    Kadyrov, Farid A.; Holmes, Shannon F.; Arana, Mercedes E.; Lukianova, Olga A.; O’Donnell, Mike; Kunkel, Thomas A.; Modrich, Paul

    2008-01-01

    MutL homologs are crucial for mismatch repair and genetic stability, but their function is not well understood. Human MutLα (MLH1-PMS2 heterodimer) harbors a latent endonuclease that is dependent on integrity of a PMS2 DQHA(X)2E(X)4E motif (Kadyrov et al. (2006) Cell 126, 297-308). This sequence element is conserved in many MutL homologs, including the PMS1 subunit of Saccharomyces cerevisiae MutLα, but is absent in MutL proteins from bacteria like Escherichia coli that rely on d(GATC) methylation for strand directionality. We show that yeast MutLα is a strand-directed endonuclease that incises DNA in a reaction that depends on a mismatch, yMutSα, yRFC, yPCNA, ATP, and a pre-existing strand break, whereas E. coli MutL is not. Amino acid substitution within the PMS1 DQHA(X)2E(X)4E motif abolishes yMutLα endonuclease activity in vitro and confers strong genetic instability in vivo, but does not affect yMutLα ATPase activity or the ability of the protein to support assembly of the yMutLα•yMutSα•heteroduplex ternary complex. The loaded form of yPCNA may play an important effector role in directing yMutLα incision to the discontinuous strand of a nicked heteroduplex. PMID:17951253

  5. Ultraviolet A-induced cathepsin K expression is mediated via MAPK/AP-1 pathway in human dermal fibroblasts.

    Directory of Open Access Journals (Sweden)

    Qingfang Xu

    Full Text Available Cathepsin K (CatK, a cysteine protease with the potent elastolytic activity, plays a predominant role in intracellular elastin degradation in human dermal fibroblasts (HDFs, and contributes to solar elastosis. In previous studies, CatK expression was downregulated in photoaged skin and fibroblasts, but upregulated in acute UVA-irradiated skin and fibroblasts. The underlying mechanisms regulating UVA-induced CatK expression remain elusive.This study investigates mechanisms involved in the regulation of UVA-induced CatK expression in HDFs.Primary HDFs were exposed to UVA. Cell proliferation was analyzed using a colorimetric assay of relative cell number. Quantitative real-time RT-PCR and Western blot were performed to detect CatK expression in HDFs on three consecutive days after 10 J/cm2 UVA irradiation, or cells treated with increasing UVA doses. UVA-activated MAPK/AP-1 pathway was examined by Western blot. Effects of inhibition of MAPK pathway and knockdown of Jun and Fos on UVA-induced CatK expression were also measured by real-time RT-PCR and Western blot.UVA significantly increased CatK mRNA and protein expression in a dose-dependent manner. UVA-induced CatK expression occurred along with UVA-activated phosphorylation of JNK, p38 and Jun, UVA-increased expression of Fos. Inactivation of JNK and p38MAPK pathways both remarkably decreased UVA-induced CatK expression, which was suppressed more by inhibition of JNK pathway. Furthermore, knockdown of Jun and Fos significantly attenuated basal and UVA-induced CatK expression.UVA is capable of increasing CatK expression in HDFs, most likely by activation of MAPK pathway and of AP-1, which has been shown to be the case for matrix metalloproteinases. As current strategies for selecting anti-photoaging agents focus on their ability to decrease MMPs' expression through inhibiting UV- activated MAPK pathway, future strategies should also consider their effect on CatK expression.

  6. Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 {mu}1A (AP-1 mu1A)

    Energy Technology Data Exchange (ETDEWEB)

    Sawasdee, Nunghathai; Junking, Mutita [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Ngaojanlar, Piengpaga [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Department of Immunology and Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Sukomon, Nattakan; Ungsupravate, Duangporn [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Limjindaporn, Thawornchai [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Akkarapatumwong, Varaporn [Institute of Molecular Biosciences, Mahidol University at Salaya Campus, Nakorn Pathom 73170 (Thailand); Noisakran, Sansanee [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Yenchitsomanus, Pa-thai, E-mail: grpye@mahidol.ac.th [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand)

    2010-10-08

    Research highlights: {yields} Trafficking defect of kAE1 is a cause of dRTA but trafficking pathway of kAE1 has not been clearly described. {yields} Adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) was firstly reported to interact with kAE1. {yields} The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. {yields} AP-1 mu1A knockdown showed a marked reduction of kAE1 on the cell membrane and its accumulation in endoplasmic reticulum. {yields} AP-1 mu1A has a critical role in kAE1 trafficking to the plasma membrane. -- Abstract: Kidney anion exchanger 1 (kAE1) mediates chloride (Cl{sup -}) and bicarbonate (HCO{sub 3}{sup -}) exchange at the basolateral membrane of kidney {alpha}-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl{sup -}/HCO{sub 3}{sup -} exchange at the basolateral membrane and failure of proton (H{sup +}) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1

  7. SLX4 Assembles a Telomere Maintenance Toolkit by Bridging Multiple Endonucleases with Telomeres

    Directory of Open Access Journals (Sweden)

    Bingbing Wan

    2013-09-01

    Full Text Available SLX4 interacts with several endonucleases to resolve structural barriers in DNA metabolism. SLX4 also interacts with telomeric protein TRF2 in human cells. The molecular mechanism of these interactions at telomeres remains unknown. Here, we report the crystal structure of the TRF2-binding motif of SLX4 (SLX4TBM in complex with the TRFH domain of TRF2 (TRF2TRFH and map the interactions of SLX4 with endonucleases SLX1, XPF, and MUS81. TRF2 recognizes a unique HxLxP motif on SLX4 via the peptide-binding site in its TRFH domain. Telomeric localization of SLX4 and associated nucleases depend on the SLX4-endonuclease and SLX4-TRF2 interactions and the protein levels of SLX4 and TRF2. SLX4 assembles an endonuclease toolkit that negatively regulates telomere length via SLX1-catalyzed nucleolytic resolution of telomere DNA structures. We propose that the SLX4-TRF2 complex serves as a double-layer scaffold bridging multiple endonucleases with telomeres for recombination-based telomere maintenance.

  8. The AP-2 family of transcription factors

    OpenAIRE

    Eckert, Dawid; Buhl, Sandra; Weber, Susanne; Jäger, Richard; Schorle, Hubert

    2005-01-01

    The AP-2 family of transcription factors consists of five different proteins in humans and mice: AP-2α, AP-2β, AP-2γ, AP-2δ and AP-2ε. Frogs and fish have known orthologs of some but not all of these proteins, and homologs of the family are also found in protochordates, insects and nematodes. The proteins have a characteristic helix-span-helix motif at the carboxyl terminus, which, together with a central basic region, mediates dimerization and DNA binding. The amino terminus contains the tra...

  9. Crystallization of the xeroderma pigmentosum group F endonuclease from Aeropyrum pernix.

    Science.gov (United States)

    Lally, John; Newman, Matthew; Murray-Rust, Judith; Fadden, Andrew; Kawarabayasi, Yutaka; McDonald, Neil

    2004-09-01

    The xeroderma pigmentosa group F protein (XPF) is a founding member of a family of 3'-flap endonucleases that play an essential role in nucleotide-excision repair, DNA replication and recombination. The XPF gene has been cloned from Aeropyrum pernix, encoding a 254-residue protein (apXPF). Recombinant protein was produced in Escherichia coli and purified by three chromatographic steps. Three different crystal forms of apXPF were grown in trigonal, monoclinic and triclinic systems. The trigonal crystals diffracted to 2.8 A and were grown in the presence of double-stranded DNA. Monoclinic crystals were grown without DNA and diffracted to 3.2 A. Triclinic crystals were grown from a truncated apXPF protein lacking the tandem helix-hairpin-helix motifs and diffracted to 2.1 A.

  10. Heparin (GAG-hed inhibits LCR activity of Human Papillomavirus type 18 by decreasing AP1 binding

    Directory of Open Access Journals (Sweden)

    López-Bayghen Esther

    2006-08-01

    Full Text Available Abstract Background High risk HPVs are causative agents of anogenital cancers. Viral E6 and E7 genes are continuously expressed and are largely responsible for the oncogenic activity of these viruses. Transcription of the E6 and E7 genes is controlled by the viral Long Control Region (LCR, plus several cellular transcription factors including AP1 and the viral protein E2. Within the LCR, the binding and activity of the transcription factor AP1 represents a key regulatory event in maintaining E6/E7 gene expression and uncontrolled cell proliferation. Glycosaminoglycans (GAGs, such as heparin, can inhibit tumour growth; they have also shown antiviral effects and inhibition of AP1 transcriptional activity. The purpose of this study was to test the heparinoid GAG-hed, as a possible antiviral and antitumoral agent in an HPV18 positive HeLa cell line. Methods Using in vivo and in vitro approaches we tested GAG-hed effects on HeLa tumour cell growth, cell proliferation and on the expression of HPV18 E6/E7 oncogenes. GAG-hed effects on AP1 binding to HPV18-LCR-DNA were tested by EMSA. Results We were able to record the antitumoral effect of GAG-hed in vivo by using as a model tumours induced by injection of HeLa cells into athymic female mice. The antiviral effect of GAG-hed resulted in the inhibition of LCR activity and, consequently, the inhibition of E6 and E7 transcription. A specific diminishing of cell proliferation rates was observed in HeLa but not in HPV-free colorectal adenocarcinoma cells. Treated HeLa cells did not undergo apoptosis but the percentage of cells in G2/M phase of the cell cycle was increased. We also detected that GAG-hed prevents the binding of the transcription factor AP1 to the LCR. Conclusion Direct interaction of GAG-hed with the components of the AP1 complex and subsequent interference with its ability to correctly bind specific sites within the viral LCR may contribute to the inhibition of E6/E7 transcription and cell

  11. Loss of p12CDK2-AP1 Expression in Human Oral Squamous Cell Carcinoma with Disrupted Transforming Growth Factor-β-Smad Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Hui Peng

    2006-12-01

    Full Text Available We examined correlations between TGF-β1, TβR-I and TβR-II, p12CDK2-AP1 p21WAF1 p27KIP1 Smad2, and p-Smad2 in 125 cases of human oral squamous cell carcinoma (OSCC to test the hypothesis that resistance to TGF-β1-induced growth suppression is due to the disruption of its signaling pathway as a consequence of reduced or lost p12CDK2-AP1. Immunoreactivity for TβR-II decreased in OSCC with increasing disease aggressiveness; however, no differences were observed for TβR-I and TGF-β1. The expression of TβR-II significantly correlated with p12CDK2-AP1 and p27KIP1 (P<.001 and P<.01, respectively. Furthermore, there was a significant relationship between TβR-II expression and p-Smad2 (P < .001. The in vivo correlation of the levels of TβR-II, p12CDK2-AP1 and p27 KIP1 was confirmed in normal and OSCC cell lines. Additionally, in vitro analysis of TGF-β-treated cells showed that TGF-β1 treatment of normal keratinocytes suppressed cell growth with upregulation of p-Smad2, p12CDK2-API and p21WAF1 expression, whereas there was no effect on OSCC cell lines. These results provide evidence of a link between a disrupted TGF-β-Smad signaling pathway and loss of induction of cell cycle-inhibitory proteins, especially p12CDK2-AP1 in OSCC, which may lead to the resistance of TGF-β1 growth-inhibitory effect on OSCC.

  12. Alpha-melanocyte-stimulating hormone modulates activation of NF-kappa B and AP-1 and secretion of interleukin-8 in human dermal fibroblasts.

    Science.gov (United States)

    Böhm, M; Schulte, U; Kalden, H; Luger, T A

    1999-10-20

    Alpha-melanocyte-stimulating hormone (alpha-MSH) has evolved as a mediator of diverse biological activities in an ever-growing number of non-melanocytic cell types. One mechanism by which alpha-MSH exerts its effects is modulation of AP-1 and NF-kappa B. These two transcription factors also play an important role in fibroblasts, in extracellular matrix composition, and in cytokine expression. By use of electric mobility shift assays, we demonstrate that alpha-MSH (10(-6) to 10(-14) M) activates AP-1 in human dermal fibroblasts, whereas coincubation with interleukin-1 beta (IL-1 beta) results in suppression of its activation. alpha-MSH also induces activation of NF-kappa B but does not modulate DNA binding on costimulation with IL-1 beta. Since AP-1 and NF-kappa B are key elements in controlling interleukin-8 (IL-8) transcription, human fibroblasts were treated with alpha-MSH and IL-1 beta for 24 hours, and cytokine levels in the supernatants were measured by ELISA. alpha-MSH alone had little effect, whereas coincubation with IL-1 beta led to marked downregulation of IL-8 secretion (at most 288 +/- 152 ng/mL) when compared to treatment with IL-1 beta alone (919 +/- 157 ng/mL). Our results indicate that alpha-MSH exerts modulatory effects on the activation of NF-kappa B and AP-1, and that it can regulate chemokine secretion in human dermal fibroblasts. These effects of alpha-MSH may have important regulatory functions in extracellular matrix composition, wound healing, or angiogenesis.

  13. Increased expression of AP2 and Sp1 transcription factors in human thyroid tumors: a role in NIS expression regulation?

    Directory of Open Access Journals (Sweden)

    Schlumberger Martin

    2002-12-01

    Full Text Available Abstract Background Sodium/iodide symporter (NIS is a key protein in iodide transport by thyroid cells and this activity is a prerequisite for effective radioiodide treatment of thyroid cancer. In the majority of thyroid cancers, however, iodide uptake is reduced, probably as a result of decreased NIS protein expression. Methods To identify the mechanisms that negatively affect NIS expression in thyroid tumors, we performed electrophoresis mobility shift assays and immunoblot analysis of nuclear protein extracts from normal and tumoral thyroid tissues from 14 unrelated patients. Results Two proteins closely related to the transcription factors AP2 and Sp1 were identified in the nuclear extracts. Expression of both AP2 and Sp1 in nuclear extracts from thyroid tumors was significantly higher than that observed in corresponding normal tissues. Conclusion These observations raise the possibility that NIS expression, and subsequently iodide transport, are reduced in thyroid tumors at least in part owing to alterations in the binding activity of AP2 and Sp1 transcription factors to NIS promoter.

  14. Resveratrol inhibits BK-induced COX-2 transcription by suppressing acetylation of AP-1 and NF-κB in human rheumatoid arthritis synovial fibroblasts.

    Science.gov (United States)

    Yang, Chuen-Mao; Chen, Yu-Wen; Chi, Pei-Ling; Lin, Chih-Chung; Hsiao, Li-Der

    2017-05-15

    Bradykinin (BK) induces inflammation in rheumatoid arthritis (RA). Resveratrol is a potent activator of Sirt1 which could modulate inflammation through deacetylating histones of transcription factors. Here, we investigated the mechanisms underlying BK-induced COX-2 expression which is modulated by resveratrol/Sirt1 in human rheumatoid arthritis synovial fibroblasts (RASFs). We found that BK-induced COX-2 protein and mRNA expression associated with PGE2 synthesis, and promoter activity was mediated through B2R receptors, which were attenuated by selective B2R antagonist Hoe140 or transfection with B2R siRNA. BK-induced responses were mediated through PKCμ, MAPKs, AP-1 and NF-κB which were inhibited by their respective inhibitors or siRNAs. Up-regulation of Sirt1 by resveratrol suppressed the BK-induced COX-2/PGE2 production through inhibiting the interaction of AP-1 and NF-κB with COX-2 promoter in RASFs. BK-induced COX-2/PGE2 expression is mediated through a B2R-PKCμ-dependent MAPKs, AP-1, and NF-κB cascade. Resveratrol inhibited the phosphorylation and acetylation of p65, c-Jun, and Fos and reduced the binding to the COX-2 promoter, thereby attenuated the COX-2 expression. Therefore, resveratrol may be a promising therapeutic intervention for treatment of inflammatory arthritis. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Interaction of apurinic/apyrimidinic endonuclease 2 (Apn2) with Myh1 DNA glycosylase in fission yeast

    OpenAIRE

    Jin, Jin; Hwang, Bor-Jang; Chang, Po-Wen; Toth, Eric A.; Lu, A-Lien

    2014-01-01

    Oxidative DNA damage is repaired primarily by the base excision repair (BER) pathway in a process initiated by removal of base lesions or mismatched bases by DNA glycosylases. MutY homolog (MYH, MUTYH, or Myh1) is a DNA glycosylase which excises adenine paired with the oxidative lesion 8-oxo-7,8-dihydroguanine (8-oxoG, or Go), thus reducing G:C to T:A mutations. The resulting apurinic/apyrimidinic (AP) site is processed by an AP-endonuclease or a bifunctional glycosylase/lya...

  16. Overexpression of transcription factor AP-2 stimulates the PA promoter of the human uracil-DNA glycosylase (UNG) gene through a mechanism involving derepression

    DEFF Research Database (Denmark)

    Aas, Per Arne; Pena Diaz, Javier; Liabakk, Nina Beate

    2009-01-01

    RNA. Chromatin immunoprecipitation with AP-2 antibody demonstrated that endogenous AP-2 binds to the PA promoter in vivo. Overexpression of AP-2alpha, -beta or -gamma all stimulated expression from a PA-luciferase reporter gene construct approximately 3- to 4-fold. Interestingly, an N-terminally truncated AP-2...... within the region of DNA marked by PA. Footprinting analysis and electrophoretic mobility shift assays of PA and putative AP-2 binding regions with HeLa cell nuclear extract and recombinant AP-2alpha protein indicate that AP-2 transcription factors are central in the regulated expression of UNG2 m......alpha, lacking the activation domain but retaining the DNA binding and dimerization domains, stimulated PA to a level approaching that of full-length AP-2, suggesting that AP-2 overexpression stimulates PA activity by a mechanism involving derepression rather than activation, possibly by neutralizing...

  17. [Flap endonuclease-1 and its role in the processes of DNA metabolism in eucaryotic cells].

    Science.gov (United States)

    Nazarkina, Zh K; Lavrik, O I; Khodyreva, S N

    2008-01-01

    Flap endonuclease-1 (FEN1) is a structure specific endonuclease. The natural substrates of FEN1 are 5'-flap structures formed by three DNA chains one of them has unannealed flapped 5'-end (flap). Flap structures are the intermediates of different processes of DNA metabolism, such as DNA recombination, Okazaki fragment maturation during replication of lagging strand, as well as strand displacement DNA synthesis in base excision repair. FEN1 also possesses 5'-exonuclease activity and newly discovered gap endonuclease activity. FEN1 is known to interact physically and functionally with a number of DNA replication and repair proteins such as the proliferating cell nuclear antigen, helicase/nuclease Dna2, WRN and BLM proteins, replication protein A, apurinic/apyrimidinic endonuclease 1, DNA polymerase beta, poly(ADP-riboso) polymerase 1, high mobility group protein 1, integrase of human immunodeficiency virus, transcription coactivator p300, chromatin proteins, cyclin-dependent kinases (Cdk1, Cdk2, Cyclin A). FEN1 activity is significant for maintaining the integrity of repeat sequences in genome. Recent data suppose the correlation between the abnormality of hFEN1 activity and arising/progression of neurodegenerative and cancer diseases. FEN1 has the dramatic effect on cell growth and development thereby attracting the interest to this enzyme.

  18. Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

    Science.gov (United States)

    Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.; Lashkov, A. A.

    2012-05-01

    Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized by interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.

  19. Structural aspects of catalytic mechanisms of endonucleases and their binding to nucleic acids

    Energy Technology Data Exchange (ETDEWEB)

    Zhukhlistova, N. E.; Balaev, V. V.; Lyashenko, A. V.; Lashkov, A. A., E-mail: alashkov83@gmail.com [Russian Academy of Sciences, Shubnikov Institute of Crystallography (Russian Federation)

    2012-05-15

    Endonucleases (EC 3.1) are enzymes of the hydrolase class that catalyze the hydrolytic cleavage of deoxyribonucleic and ribonucleic acids at any region of the polynucleotide chain. Endonucleases are widely used both in biotechnological processes and in veterinary medicine as antiviral agents. Medical applications of endonucleases in human cancer therapy hold promise. The results of X-ray diffraction studies of the spatial organization of endonucleases and their complexes and the mechanism of their action are analyzed and generalized. An analysis of the structural studies of this class of enzymes showed that the specific binding of enzymes to nucleic acids is characterized by interactions with nitrogen bases and the nucleotide backbone, whereas the nonspecific binding of enzymes is generally characterized by interactions only with the nucleic-acid backbone. It should be taken into account that the specificity can be modulated by metal ions and certain low-molecular-weight organic compounds. To test the hypotheses about specific and nonspecific nucleic-acid-binding proteins, it is necessary to perform additional studies of atomic-resolution three-dimensional structures of enzyme-nucleic-acid complexes by methods of structural biology.

  20. AP1000 status overview

    Energy Technology Data Exchange (ETDEWEB)

    Cummins, W.E.; Wright, R.F.; Schulz, T.L. [Westinghouse Electric Company, Pittsburgh, PA (United States)

    2001-07-01

    The AP1000 is a two-loop, 1000 MWe pressurized water reactor (PWR) with passive safety features and extensive plant simplifications to enhance the construction, operation, and maintenance. The AP1000 design is derived directly from the AP600, a two-loop, 600 MWe PWR. The AP600 uses proven technology, which builds on the over 30 years of operating PWR experience. The AP600 design received Final Design Approval from the U.S. NRC in September 1998 and Design Certification in December 1999. The AP600 meets all of the U.S. electric utility requirements including their cost goals. Although the AP600 is the most cost-effective plant ready for deployment, it is still more expensive than the $1000/kw needed to compete in the United States today In order to develop a cost competitive nuclear power plant Westinghouse has completed design studies which demonstrate that it is feasible to increase the power output of the AP600 to at least 1000 MWe, maintaining its current design configuration, use of proven components and licensing basis. In order to achieve these objectives the AP1000 has been designed within the space constraints of the AP600, while retaining the credibility of proven components and substantial safety margins. This paper describes the changes made to up-rate the AP600 and gives an overview of the plant design. It also summarizes the basis for the AP600 testing program and computer codes being sufficient for the AP1000. (authors)

  1. Restriction endonuclease EcaI from Enterobacter cloacae

    OpenAIRE

    Hobom, G.; Schwarz, E.; Melzer, M.; Mayer, H.

    1981-01-01

    Restriction endonuclease EcaI obtained from Enterobacter cloacae DSM30056 recognizes the group of heptanucleotide palindromes 5′-G[unk]G-T-N-A-C-C-3′, and on cleavage (arrow) produces fragments with 5′-terminal pentanucleotide extensions. It is identical in specificity with restriction endonuclease BstEII from Bacillus stearothermophilus ET.

  2. Effects of gemcitabine on APE/ref-1 endonuclease activity in pancreatic cancer cells, and the therapeutic potential of antisense oligonucleotides

    OpenAIRE

    Lau, J P; Weatherdon, K L; Skalski, V; Hedley, D. W.

    2004-01-01

    Apurinic/apyrimidinic endonuclease (APE) is a key enzyme involved in DNA base excision repair (BER) that is often expressed at elevated levels in human cancers. Pancreatic cancer cells treated with the nucleoside analogue gemcitabine (2?, 2?-difluoro-2?deoxycytidine) showed increases in APE/redox effector factor (ref-1) protein levels (approximately two-fold for Panc-1 and six-fold for MiaPaCa-2), with corresponding increases in endonuclease activity. These results suggested that the activati...

  3. Migration of the model HCR to the SAIC/TRC in the analysis of human reliability of the APS de CN Vandellos II; Migracion del modelo HCR al SAIC/TRC en el analisis de fiabilidad humana del APS de CN Vandellos II

    Energy Technology Data Exchange (ETDEWEB)

    Nos, V.; Rosa, J. C. de la; Hernandez, H.

    2011-07-01

    This paper describes the systematics of adaptation and conversion of models, to migrate the detailed analysis of human actions from the APS (in operation at power) of NPP Vandellos, which uses the HCR to SAIC-CRT model. Are offered the results achieved after the migration and the main conclusions.

  4. Catalytic domain of restriction endonuclease BmrI as a cleavage module for engineering endonucleases with novel substrate specificities.

    Science.gov (United States)

    Chan, Siu-hong; Bao, Yongming; Ciszak, Ewa; Laget, Sophie; Xu, Shuang-yong

    2007-01-01

    Creating endonucleases with novel sequence specificities provides more possibilities to manipulate DNA. We have created a chimeric endonuclease (CH-endonuclease) consisting of the DNA cleavage domain of BmrI restriction endonuclease and C.BclI, a controller protein of the BclI restriction-modification system. The purified chimeric endonuclease, BmrI198-C.BclI, cleaves DNA at specific sites in the vicinity of the recognition sequence of C.BclI. Double-strand (ds) breaks were observed at two sites: 8 bp upstream and 18 bp within the C-box sequence. Using DNA substrates with deletions of C-box sequence, we show that the chimeric endonuclease requires the 5' half of the C box only for specific cleavage. A schematic model is proposed for the mode of protein-DNA binding and DNA cleavage. The present study demonstrates that the BmrI cleavage domain can be used to create combinatorial endonucleases that cleave DNA at specific sequences dictated by the DNA-binding partner. The resulting endonucleases will be useful in vitro and in vivo to create ds breaks at specific sites and generate deletions.

  5. Different effects of five depigmentary compounds, rhododendrol, raspberry ketone, monobenzone, rucinol and AP736 on melanogenesis and viability of human epidermal melanocytes.

    Science.gov (United States)

    Lee, Chang Seok; Joo, Yung Hyup; Baek, Heung Soo; Park, Miyoung; Kim, Jeong-Hwan; Shin, Hong-Ju; Park, Nok-Hyun; Lee, John Hwan; Park, Young-Ho; Shin, Song Seok; Lee, Hae-Kwang

    2016-01-01

    Numerous medications are used to treat hyperpigmentation. However, several reports have indicated that repeated application of some agents, such as rhododendrol (RD), raspberry ketone (RK) and monobenzone (MB), can be toxic to melanocytes. Although these agents had severe side effects in human trials, no current in vitro methods can predict the safety of such drugs. This study assessed the in vitro effects of five depigmentary compounds including leukoderma-inducing agents. In particular, we determined the effects of different concentrations and exposure times of different depigmentary agents on cell viability and melanogenesis in the presence and absence of ultraviolet B (UVB) radiation. Concentrations of RD, RK and MB that inhibit melanogenesis are similar to concentrations that are cytotoxic; however, concentrations of rucinol (RC) and AP736 that inhibit melanogenesis are much lower than concentrations that are cytotoxic. Furthermore, the concentrations that cause toxic effects depend on exposure duration, and prolonged exposure to RD, RK and MB had more cytotoxic effects than prolonged exposure to RC and AP736. The cytotoxic effects of RD and RK appear to be mediated by apoptosis due to increased expression of caspase-3 and caspase-8; UVB radiation increased the cytotoxicity of these agents and also increased caspase activity. Our results indicate that different leukoderma-inducing compounds have different effects on the viability of normal epidermal melanocytes and suggest that the in vitro assay used here can be used to predict whether an investigational compound that induces leukoderma may lead to adverse effects in human trials. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Ultrasensitive electrochemical detection of microRNA with star trigon structure and endonuclease mediated signal amplification.

    Science.gov (United States)

    Miao, Peng; Wang, Bidou; Yu, Zhiqiang; Zhao, Jing; Tang, Yuguo

    2015-01-15

    MicroRNAs play important roles in gene regulation. They can be used as effective biomarkers for diagnosis and prognosis of diseases like cancers. Due to their intrinsic properties of short length, low abundance and sequence homology among family members, it is difficult to realize sensitive and selective detection with economical use of time and cost. Herein, we report an ultrasensitive electrochemical method for microRNA analysis employing two oligonucleotides and one endonuclease. Generally, a glassy carbon electrode is first covered with gold nanoparticles (AuNPs) mediated by poly(diallyldimethylammonium chloride) (PDDA). Then, thiolated capture probe (CP) with methylene blue (MB) labeled at 5' end is modified on the pretreated electrode. Hybridization occurs among target microRNA, CP and auxiliary probe (AP), forming a star trigon structure on the electrode surface. Subsequently, endonuclease recognizes and cleaves CP on CP/AP duplex, releasing microRNA and AP back to the solution. The two regenerated elements can then form another star trigon with other CP molecules, initiating cycles of CP cleavage and MB departure. Significant decrease of electrochemical signals is thus observed, which can be used to reflect the concentration of microRNA. This proposed method has a linear response to microRNA in a wide range from 100 aM to 1 nM and the sensitivity of attomolar level can be achieved. Moreover, it has high selectivity against single-base mismatch sequences and can be used directly in serum samples. Therefore, this method shows great feasibility for the detection of microRNA and may have potential applications in cancer diagnosis and prognosis. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Selective microbial genomic DNA isolation using restriction endonucleases.

    Science.gov (United States)

    Barnes, Helen E; Liu, Guohong; Weston, Christopher Q; King, Paula; Pham, Long K; Waltz, Shannon; Helzer, Kimberly T; Day, Laura; Sphar, Dan; Yamamoto, Robert T; Forsyth, R Allyn

    2014-01-01

    To improve the metagenomic analysis of complex microbiomes, we have repurposed restriction endonucleases as methyl specific DNA binding proteins. As an example, we use DpnI immobilized on magnetic beads. The ten minute extraction technique allows specific binding of genomes containing the DpnI Gm6ATC motif common in the genomic DNA of many bacteria including γ-proteobacteria. Using synthetic genome mixtures, we demonstrate 80% recovery of Escherichia coli genomic DNA even when only femtogram quantities are spiked into 10 µg of human DNA background. Binding is very specific with less than 0.5% of human DNA bound. Next Generation Sequencing of input and enriched synthetic mixtures results in over 100-fold enrichment of target genomes relative to human and plant DNA. We also show comparable enrichment when sequencing complex microbiomes such as those from creek water and human saliva. The technique can be broadened to other restriction enzymes allowing for the selective enrichment of trace and unculturable organisms from complex microbiomes and the stratification of organisms according to restriction enzyme enrichment.

  8. Advanced Photon Source (APS)

    Data.gov (United States)

    Federal Laboratory Consortium — The Advanced Photon Source (APS) at the U.S. Department of Energy's Argonne National Laboratoryprovides this nation's (in fact, this hemisphere's) brightest storage...

  9. PCR-based bioprospecting for homing endonucleases in fungal mitochondrial rRNA genes.

    Science.gov (United States)

    Hafez, Mohamed; Guha, Tuhin Kumar; Shen, Chen; Sethuraman, Jyothi; Hausner, Georg

    2014-01-01

    Fungal mitochondrial genomes act as "reservoirs" for homing endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing and gene therapy applications. Bioprospecting and characterization of naturally occurring homing endonucleases offers an alternative to synthesizing artificial endonucleases. Here, we describe methods for PCR-based screening of fungal mitochondrial rRNA genes for homing endonuclease encoding sequences, and we also provide protocols for the purification and biochemical characterization of putative native homing endonucleases.

  10. Characterization of the restriction enzyme-like endonuclease encoded by the Entamoeba histolytica non-long terminal repeat retrotransposon EhLINE1.

    Science.gov (United States)

    Yadav, Vijay Pal; Mandal, Prabhat Kumar; Rao, Desirazu N; Bhattacharya, Sudha

    2009-12-01

    The genome of the human pathogen Entamoeba histolytica, a primitive protist, contains non-long terminal repeat retrotransposable elements called EhLINEs. These encode reverse transcriptase and endonuclease required for retrotransposition. The endonuclease shows sequence similarity with bacterial restriction endonucleases. Here we report the salient enzymatic features of one such endonuclease. The kinetics of an EhLINE1-encoded endonuclease catalyzed reaction, determined under steady-state and single-turnover conditions, revealed a significant burst phase followed by a slower steady-state phase, indicating that release of product could be the slower step in this reaction. For circular supercoiled DNA the K(m) was 2.6 x 10(-8) M and the k(cat) was 1.6 x 10(-2) sec(-1). For linear E. histolytica DNA substrate the K(m) and k(cat) values were 1.3 x 10(-8) M and 2.2 x 10(-4) sec(-1) respectively. Single-turnover reaction kinetics suggested a noncooperative mode of hydrolysis. The enzyme behaved as a monomer. While Mg(2+) was required for activity, 60% activity was seen with Mn(2+) and none with other divalent metal ions. Substitution of PDX(12-14)D (a metal-binding motif) with PAX(12-14)D caused local conformational change in the protein tertiary structure, which could contribute to reduced enzyme activity in the mutated protein. The protein underwent conformational change upon the addition of DNA, which is consistent with the known behavior of restriction endonucleases. The similarities with bacterial restriction endonucleases suggest that the EhLINE1-encoded endonuclease was possibly acquired from bacteria through horizontal gene transfer. The loss of strict sequence specificity for nicking may have been subsequently selected to facilitate spread of the retrotransposon to intergenic regions of the E. histolytica genome.

  11. Age and sex alone are insufficient to predict human rib structural response to dynamic A-P loading.

    Science.gov (United States)

    Schafman, Michelle A; Kang, Yun-Seok; Moorhouse, Kevin; White, Susan E; Bolte, John H; Agnew, Amanda M

    2016-10-03

    Thoracic injuries from motor vehicle crashes (MVCs) are common in children and the elderly and are associated with a high rate of mortality for both groups. Rib fractures, in particular, are linked to high mortality rates which increase with the number of fractures sustained. Anthropomorphic test devices (ATDs) and computational models have been developed to improve vehicle safety, however these tools are constructed based on limited physical datasets. To-date, no study has explored variation of rib structural properties across the entire age spectrum with data obtained using the same experimental methodology to allow for comparison. One-hundred eighty-four ribs from 93 post mortem human subjects (PMHS) (70 male, 23 female; ages 4-99) were subjected to dynamic bending tests simulating a frontal impact to the thorax. Structural mechanical properties were calculated and a multi-level statistical model quantified the sample variance as explained by age and sex. Displacement (δ X ), peak force (F peak ), linear structural stiffness (K), energy absorption to fracture (U tot ), and plastic properties including post-yield energy absorption (U Pl ), plastic displacement (δ Pl ), and the ratio of elastic to secant stiffness (K-ratio) all showed negative relationships with age, while only F peak , K, and U tot were dependent on sex. Despite these relationships being statistically significant, only 7-39% of variance is explained by age and only 3-17% of variance is explained by sex. This demonstrates that variability in bone properties is more complex than simply chronological age- and sex-dependence and should be explored in the context of biological mechanisms instead. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. In silico analysis of evolutionary patterns in restriction endonucleases.

    Science.gov (United States)

    Singh, Tiratha Raj; Pardasani, Kamal Raj

    2009-01-01

    Restriction endonucleases represent one of the best studied examples of DNA binding proteins. Type II restriction endonucleases recognize short sequences of foreign DNA and cleave the target on both strands with remarkable sequence specificity. Type II restriction endonucleases are part of restriction modification systems. Restriction modification systems occur ubiquitously among bacteria and archaea. Restriction endonucleases are indispensable tools in molecular biology and biotechnology. They are important model system for specific protein-nucleic acid interactions and also serve as good example for investigating structural, functional and evolutionary relationships among various biomolecules. The interaction between restriction endonucleases and their recognition sequences plays a crucial role in biochemical activities like catalytic site/metal binding, DNA repair and recombination etc. We study various patterns in restriction endonucleases type II and analyzed their structural, functional and evolutionary role. Our studies support X-ray crystallographic studies, arguing for divergence and molecular evolution. Conservation patterns of the nuclease superfamily have also been analyzed by estimating site-specific evolutionary rates for the analyzed structures related to respective chains in this study.

  13. Structure of the endonuclease domain of MutL: unlicensed to cut

    Science.gov (United States)

    Pillon, Monica C.; Lorenowicz, Jessica J.; Uckelmann, Michael; Klocko, Andrew D.; Mitchell, Ryan R.; Chung, Yu Seon; Modrich, Paul; Walker, Graham C.; Simmons, Lyle A.; Friedhoff, Peter; Guarné, Alba

    2010-01-01

    Summary DNA mismatch repair corrects errors that have escaped polymerase proofreading, increasing replication fidelity 100- to 1000-fold in organisms ranging from bacteria to humans. The MutL protein plays a central role in mismatch repair by coordinating multiple protein-protein interactions that signal strand removal upon mismatch recognition by MutS. Here we report the crystal structure of the endonuclease domain of Bacillus subtilis MutL. The structure is organized in dimerization and regulatory subdomains connected by a helical lever spanning the conserved endonuclease motif. Additional conserved motifs cluster around the lever and define a Zn2+-binding site that is critical for MutL function in vivo. The structure unveils a powerful inhibitory mechanism to prevent undesired DNA nicking and allows us to propose a model describing how the interaction with MutS and the processivity clamp could license the endonuclease activity of MutL. The structure also provides a molecular framework to propose and test additional roles of MutL in mismatch repair. PMID:20603082

  14. APS Science 2006.

    Energy Technology Data Exchange (ETDEWEB)

    Gibson, J. M.; Fenner, R. B.; Long, G.; Borland, M.; Decker, G.

    2007-05-24

    In my five years as the Director of the Advanced Photon Source (APS), I have been fortunate to see major growth in the scientific impact from the APS. This year I am particularly enthusiastic about prospects for our longer-term future. Every scientific instrument must remain at the cutting edge to flourish. Our plans for the next generation of APS--an APS upgrade--got seriously in gear this year with strong encouragement from our users and sponsors. The most promising avenue that has emerged is the energy-recovery linac (ERL) (see article on page xx), for which we are beginning serious R&D. The ERL{at}APS would offer revolutionary performance, especially for x-ray imaging and ultrafast science, while not seriously disrupting the existing user base. I am very proud of our accelerator physics and engineering staff, who not only keep the current APS at the forefront, but were able to greatly impress our international Machine Advisory Committee with the quality of their work on the possible upgrade option (see page xx). As we prepare for long-term major upgrades, our plans to develop and optimize all the sectors at APS in the near future are advancing. Several new beamlines saw first light this year, including a dedicated powder diffraction beamline (11-BM), two instruments for inelastic x-ray scattering at sector 30, and the Center for Nanoscale Materials (CNM) Nanoprobe beamline at sector 26. Our partnership in the first x-ray free-electron laser (LCLS) to be built at Stanford contributes to revolutionary growth in ultrafast science (see page xx), and we are developing a pulse chirping scheme to get ps pulses at sector 7 of the APS within a year or so. In this report, you will find selected highlights of scientific research at the APS from calendar year 2006. The highlighted work covers diverse disciplines, from fundamental to applied science. In the article on page xx you can see the direct impact of APS research on technology. Several new products have emerged from

  15. AP statistics crash course

    CERN Document Server

    D'Alessio, Michael

    2012-01-01

    AP Statistics Crash Course - Gets You a Higher Advanced Placement Score in Less Time Crash Course is perfect for the time-crunched student, the last-minute studier, or anyone who wants a refresher on the subject. AP Statistics Crash Course gives you: Targeted, Focused Review - Study Only What You Need to Know Crash Course is based on an in-depth analysis of the AP Statistics course description outline and actual Advanced Placement test questions. It covers only the information tested on the exam, so you can make the most of your valuable study time. Our easy-to-read format covers: exploring da

  16. The cellular protein MCM3AP is required for inhibition of cellular DNA synthesis by the IE86 protein of human cytomegalovirus.

    Directory of Open Access Journals (Sweden)

    Emma Poole

    Full Text Available Like all DNA viruses, human cytomegalovirus (HCMV infection is known to result in profound effects on host cell cycle. Infection of fibroblasts with HCMV is known to induce an advance in cell cycle through the G(0-G(1 phase and then a subsequent arrest of cell cycle in early S-phase, presumably resulting in a cellular environment optimum for high levels of viral DNA replication whilst precluding replication of cellular DNA. Although the exact mechanisms used to arrest cell cycle by HCMV are unclear, they likely involve a number of viral gene products and evidence points to the ability of the virus to prevent licensing of cellular DNA synthesis. One viral protein known to profoundly alter cell cycle is the viral immediate early 86 (IE86 protein--an established function of which is to initially drive cells into early S phase but then inhibit cellular DNA synthesis. Here we show that, although IE86 interacts with the cellular licensing factor Cdt1, it does not inhibit licensing of cellular origins. Instead, IE86-mediated inhibition of cellular DNA synthesis requires mini-chromosome-maintenance 3 (MCM3 associated protein (MCM3AP, which can cause subsequent inhibition of initiation of cellular DNA synthesis in a licensing-independent manner.

  17. Sequential treatment with AT-101 enhances cisplatin chemosensitivity in human non-small cell lung cancer cells through inhibition of apurinic/apyrimidinic endonuclease 1-activated IL-6/STAT3 signaling pathway

    Directory of Open Access Journals (Sweden)

    Ren T

    2014-12-01

    Full Text Available Tao Ren,1,2,* Jinlu Shan,1,* Yi Qing,1 Chengyuan Qian,1 Qing Li,1 Guoshou Lu,1 Mengxia Li,1 Chongyi Li,1 Yu Peng,1 Hao Luo,1 Shiheng Zhang,1 Weiwei Zhang,1 Dong Wang,1 Shu-Feng Zhou3 1Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing, People’s Republic of China; 2Oncology Department, The Affiliated Hospital, North Sichuan Medical College, Nanchong, People’s Republic of China; 3Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA *These authors contributed equally to this work Abstract: AT-101, known as R-(–-gossypol, is a potent anticancer agent, but its chemosensitizing effects remain elusive. The present study aimed to examine whether AT-101 could increase the sensitivity of non-small cell lung cancer A549 cells to cisplatin (CDDP and the underlying mechanisms. We evaluated the efficacy of the sequential treatment with AT-101 and CDDP using both in vitro and in vivo models. Our results showed that as compared to AT-101 or CDDP monotherapy, or AT-101 plus CDDP concurrent treatment, the sequential treatment significantly inhibited cell proliferation and migration and induced tumor cell death. Moreover, the efficacy of the sequential treatment was also confirmed in a mouse A549 xenograft model. Our study revealed that AT-101 inhibited the reduced status of apurinic/apyrimidinic endonuclease 1 (APE1 and attenuated APE1-mediated IL-6/STAT3 signaling activation by decreasing IL-6 protein expression; suppressing the STAT3–DNA binding; and reducing the expression of the downstream antiapoptotic proteins Bcl-2 and Bcl-xL. In conclusion, AT-101 enhances the sensitivity of A549 cells to CDDP in vitro and in vivo through the inhibition of APE1-mediated IL-6/STAT3 signaling activation, providing a rationale for the combined use of AT-101 and CDDP in non-small cell lung cancer chemotherapy. Keywords: AT101, NSCLC, cisplatin

  18. RNA aptamer inhibitors of a restriction endonuclease.

    Science.gov (United States)

    Mondragón, Estefanía; Maher, L James

    2015-09-03

    Restriction endonucleases (REases) recognize and cleave short palindromic DNA sequences, protecting bacterial cells against bacteriophage infection by attacking foreign DNA. We are interested in the potential of folded RNA to mimic DNA, a concept that might be applied to inhibition of DNA-binding proteins. As a model system, we sought RNA aptamers against the REases BamHI, PacI and KpnI using systematic evolution of ligands by exponential enrichment (SELEX). After 20 rounds of selection under different stringent conditions, we identified the 10 most enriched RNA aptamers for each REase. Aptamers were screened for binding and specificity, and assayed for REase inhibition. We obtained eight high-affinity (Kd ∼12-30 nM) selective competitive inhibitors (IC50 ∼20-150 nM) for KpnI. Predicted RNA secondary structures were confirmed by in-line attack assay and a 38-nt derivative of the best anti-KpnI aptamer was sufficient for inhibition. These competitive inhibitors presumably act as KpnI binding site analogs, but lack the primary consensus KpnI cleavage sequence and are not cleaved by KpnI, making their potential mode of DNA mimicry fascinating. Anti-REase RNA aptamers could have value in studies of REase mechanism and may give clues to a code for designing RNAs that competitively inhibit DNA binding proteins including transcription factors. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Solitary restriction endonucleases in prokaryotic genomes.

    Science.gov (United States)

    Ershova, Anna S; Karyagina, Anna S; Vasiliev, Mikhail O; Lyashchuk, Alexander M; Lunin, Vladimir G; Spirin, Sergey A; Alexeevski, Andrei V

    2012-11-01

    Prokaryotic restriction-modification (R-M) systems defend the host cell from the invasion of a foreign DNA. They comprise two enzymatic activities: specific DNA cleavage activity and DNA methylation activity preventing cleavage. Typically, these activities are provided by two separate enzymes: a DNA methyltransferase (MTase) and a restriction endonuclease (RE). In the absence of a corresponding MTase, an RE of Type II R-M system is highly toxic for the cell. Genes of the R-M system are linked in the genome in the vast majority of annotated cases. There are only a few reported cases in which the genes of MTase and RE from one R-M system are not linked. Nevertheless, a few hundreds solitary RE genes are present in the Restriction Enzyme Database (http://rebase.neb.com) annotations. Using the comparative genomic approach, we analysed 272 solitary RE genes. For 57 solitary RE genes we predicted corresponding MTase genes located distantly in a genome. Of the 272 solitary RE genes, 99 are likely to be fragments of RE genes. Various explanations for the existence of the remaining 116 solitary RE genes are also discussed.

  20. APS Science 2009.

    Energy Technology Data Exchange (ETDEWEB)

    Gibson, J. M; Mills, D. M.; Gerig, R.

    2010-05-01

    It is my pleasure to introduce the 2009 annual report of the Advanced Photon Source. This was a very good year for us. We operated with high reliability and availability, despite growing problems with obsolete systems, and our users produced a record output of publications. The number of user experiments increased by 14% from 2008 to more than 3600. We congratulate the recipients of the 2009 Nobel Prize in Chemistry-Venkatraman Ramakrishnan (Cambridge Institute for Medical Research), Thomas Steitz (Yale University), and Ada Yonath (Weizmann Institute) - who did a substantial amount of this work at APS beamlines. Thanks to the efforts of our users and staff, and the ongoing counsel of the APS Scientific Advisory Committee, we made major progress in advancing our planning for the upgrade of the APS (APS-U), producing a proposal that was positively reviewed. We hope to get formal approval in 2010 to begin the upgrade. With advocacy from our users and the support of our sponsor, the Office of Basic Energy Sciences in the Department of Energy (DOE) Office of Science, our operating budgets have grown to the level needed to more adequately staff our beamlines. We were also extremely fortunate to have received $7.9 M in American Recovery and Reinvestment Act ('stimulus') funding to acquire new detectors and improve several of our beamlines. The success of the new Linac Coherent Light Source at Stanford, the world's first x-ray free-electron laser, made us particularly proud since the undulators were designed and built by the APS. Among other highlights, we note that more than one-quarter of the 46 Energy Frontier Research Centers, funded competitively across the U.S. in 2009 by the DOE, included the Advanced Photon Source in their proposed work, which shows that synchrotron radiation, and the APS in particular, are central to energy research. While APS research covers everything from fundamental to applied science (reflected by the highlights in this report

  1. Screening for restriction endonucleases in methane-oxidizing bacteria.

    Science.gov (United States)

    Romanovskaya, V A; Alexeyev, M F; Gun'kovskaya, N V; Stolyar, S M; Shatohina, E S; Malashenko Yu, R

    1992-01-01

    51 methane-oxidizing bacteria strains such as Methylomonas methanica, M. rubra, Methylococcus capsulatus, M. thermophilus, M. luteus, M. ucrainicus, M. whittenburyi, Methylosinus trichosporium, M. sporium, Methylocystis parvus isolated from various ecological niches and geographical regions of the Ukraine and also the strains received from R. Whittenbury and Y. Heyer were screened for restriction endonucleases. Type II restriction endonucleases were detected in IMV B-3112 (= 12 b), IMV B-3027 (= 26), IMV B-3019 (= 9 c), IMV B-3017 (= 17 c), IMV B-3226 (= 26 v), IMV B-3033 (= Y), IMV B-3100 (= 100) and IMV B-3494 (= 1E494). The results obtained were indicative of relatively high frequency of restriction enzymes occurrence in methane-oxidizing bacteria. There were Kpn I (Asp 7181) restriction endonuclease isoschizomers in crude extracts of IMV B-3112, B-3017, B-3019, B-3027 isolated from fresh-water silt as well as in IMV B-3226 strain isolated from waste-water silt. Although these isolates had bee previously considered as untypical strains of M. ucrainicus, more detailed study of their properties allowed placing them with Methylovarius luteus (= Methylococcus luteus). IMV B-3494 strain was identified as Methylococcus capsulatus. Strain IMV B-3033 had earlier been allocated to Methylovarius whittenburyi (= Methylococcus whittenburyi). Specificity of restriction endonucleases of this strain was not tested. Therefore, for the first time restriction endonucleases were detected in methane-oxidizing bacteria. 8 strains (3 species) among 51 strains (13 species) were found to produce restriction endonucleases displaying three different types of specificity in the least. Producers of restriction endonucleases having Kpn I (Asp 7181) specificity were isolated from different water and silt samples of the Dnieper flood-land more than 20 years ago.

  2. Berberine Targets AP-2/hTERT, NF-κB/COX-2, HIF-1α/VEGF and Cytochrome-c/Caspase Signaling to Suppress Human Cancer Cell Growth.

    Directory of Open Access Journals (Sweden)

    Lingyi Fu

    Full Text Available Berberine (BBR, an isoquinoline derivative alkaloid isolated from Chinese herbs, has a long history of uses for the treatment of multiple diseases, including cancers. However, the precise mechanisms of actions of BBR in human lung cancer cells remain unclear. In this study, we investigated the molecular mechanisms by which BBR inhibits cell growth in human non-small-cell lung cancer (NSCLC cells. Treatment with BBR promoted cell morphology change, inhibited cell migration, proliferation and colony formation, and induced cell apoptosis. Further molecular mechanism study showed that BBR simultaneously targeted multiple cell signaling pathways to inhibit NSCLC cell growth. Treatment with BBR inhibited AP-2α and AP-2β expression and abrogated their binding on hTERT promoters, thereby inhibiting hTERT expression. Knockdown of AP-2α and AP-2β by siRNA considerably augmented the BBR-mediated inhibition of cell growth. BBR also suppressed the nuclear translocation of p50/p65 NF-κB proteins and their binding to COX-2 promoter, causing inhibition of COX-2. BBR also downregulated HIF-1α and VEGF expression and inhibited Akt and ERK phosphorylation. Knockdown of HIF-1α by siRNA considerably augmented the BBR-mediated inhibition of cell growth. Moreover, BBR treatment triggered cytochrome-c release from mitochondrial inter-membrane space into cytosol, promoted cleavage of caspase and PARP, and affected expression of BAX and Bcl-2, thereby activating apoptotic pathway. Taken together, these results demonstrated that BBR inhibited NSCLC cell growth by simultaneously targeting AP-2/hTERT, NF-κB/COX-2, HIF-1α/VEGF, PI3K/AKT, Raf/MEK/ERK and cytochrome-c/caspase signaling pathways. Our findings provide new insights into understanding the anticancer mechanisms of BBR in human lung cancer therapy.

  3. APS SCIENCE 2016

    Energy Technology Data Exchange (ETDEWEB)

    Fenner, Richard B. [Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

    2017-05-01

    The Advanced Photon Source (APS) occupies an 80-acre site on the Argonne national laboratory campus, about 25 miles from downtown chicago, illinois. it shares the site with the center for nanoscale materials and the Advanced Protein characterization facility. for directions to Argonne, see http://www.anl.gov/directions-and-visitor-information. The APS, a national synchrotron radiation research facility operated by Argonne for the u.S. department of energy (doe) office of Science, provides this nation’s brightest high-energy x-ray beams for science. research by APS users extends from the center of the earth to outer space, from new information on combustion engines and microcircuits to new drugs and nanotechnologies whose scale is measured in billionths of a meter. The APS helps researchers illuminate answers to the challenges of our high-tech world, from developing new forms of energy, to sustaining our nation’s technological and economic competitiveness, to pushing back against the ravages of disease. research at the APS promises to have far-reaching

  4. Restriction endonucleases: natural and directed evolution.

    Science.gov (United States)

    Gupta, Richa; Capalash, Neena; Sharma, Prince

    2012-05-01

    Type II restriction endonucleases (REs) are highly sequence-specific compared with other classes of nucleases. PD-(D/E)XK nucleases, initially represented by only type II REs, now comprise a large and extremely diverse superfamily of proteins and, although sharing a structurally conserved core, typically display little or no detectable sequence similarity except for the active site motifs. Sequence similarity can only be observed in methylases and few isoschizomers. As a consequence, REs are classified according to combinations of functional properties rather than on the basis of genetic relatedness. New alignment matrices and classification systems based on structural core connectivity and cleavage mechanisms have been developed to characterize new REs and related proteins. REs recognizing more than 300 distinct specificities have been identified in RE database (REBASE: http://rebase.neb.com/cgi-bin/statlist ) but still the need for newer specificities is increasing due to the advancement in molecular biology and applications. The enzymes have undergone constant evolution through structural changes in protein scaffolds which include random mutations, homologous recombinations, insertions, and deletions of coding DNA sequences but rational mutagenesis or directed evolution delivers protein variants with new functions in accordance with defined biochemical or environmental pressures. Redesigning through random mutation, addition or deletion of amino acids, methylation-based selection, synthetic molecules, combining recognition and cleavage domains from different enzymes, or combination with domains of additional functions change the cleavage specificity or substrate preference and stability. There is a growing number of patents awarded for the creation of engineered REs with new and enhanced properties.

  5. Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein.

    Directory of Open Access Journals (Sweden)

    Yaiza Fernández-García

    2016-06-01

    Full Text Available Andes virus (ANDV is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1-200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure-function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening.

  6. Creation of a novel telomere-cutting endonuclease based on the EN domain of telomere-specific non-long terminal repeat retrotransposon, TRAS1

    Directory of Open Access Journals (Sweden)

    Yoshitake Kazutoshi

    2010-04-01

    Full Text Available Abstract Background The ends of chromosomes, termed telomeres consist of repetitive DNA. The telomeric sequences shorten with cell division and, when telomeres are critically abbreviated, cells stop proliferating. However, in cancer cells, by the expression of telomerase which elongates telomeres, the cells can continue proliferating. Many approaches for telomere shortening have been pursued in the past, but to our knowledge, cutting telomeres in vivo has not so far been demonstrated. In addition, there is lack of information on the cellular effects of telomere shortening in human cells. Results Here, we created novel chimeric endonucleases to cut telomeres by fusing the endonuclease domain (TRAS1EN of the silkworm's telomere specific non-long terminal repeat retrotransposon TRAS1 to the human telomere-binding protein, TRF1. An in vitro assay demonstrated that the TRAS1EN-TRF1 chimeric endonucleases (T-EN and EN-T cut the human (TTAGGGn repeats specifically. The concentration of TRAS1EN-TRF1 chimeric endonucleases necessary for the cleavage of (TTAGGGn repeats was about 40-fold lower than that of TRAS1EN alone. When TRAS1EN-TRF1 endonucleases were introduced into human U2OS cancer cells using adenovirus vectors, the enzymes localized at telomeres of nuclei, cleaved and shortened the telomeric DNA by double-strand breaks. When human U2OS and HFL-1 fibroblast cells were infected with EN-T recombinant adenovirus, their cellular proliferation was suppressed for about 2 weeks after infection. In contrast, the TRAS1EN mutant (H258A chimeric endonuclease fused with TRF1 (ENmut-T did not show the suppression effect. The EN-T recombinant adenovirus induced telomere shortening in U2OS cells, activated the p53-dependent pathway and caused the senescence associated cellular responses, while the ENmut-T construct did not show such effects. Conclusions A novel TRAS1EN-TRF1 chimeric endonuclease (EN-T cuts the human telomeric repeats (TTAGGGn specifically in

  7. Aaptamines from the Marine Sponge Aaptos sp. Display Anticancer Activities in Human Cancer Cell Lines and Modulate AP-1-, NF-κB-, and p53-Dependent Transcriptional Activity in Mouse JB6 Cl41 Cells

    Directory of Open Access Journals (Sweden)

    Sergey A. Dyshlovoy

    2014-01-01

    Full Text Available Aaptamine (8,9-dimethoxy-1H-benzo[de][1,6]naphthyridine is a marine natural compound possessing antioxidative, antimicrobial, antifungal, and antiretroviral activity. Earlier, we have found that aaptamine and its derivatives demonstrate equal anticancer effects against the human germ cell cancer cell lines NT2 and NT2-R and cause some changes in the proteome of these cells. In order to explore further the mechanism of action of aaptamine and its derivatives, we studied the effects of aaptamine (1, demethyl(oxyaaptamine (2, and isoaaptamine (3 on human cancer cell lines and on AP-1-, NF-κB-, and p53-dependent transcriptional activity in murine JB6 Cl41 cells. We showed that compounds 1–3 demonstrate anticancer activity in THP-1, HeLa, SNU-C4, SK-MEL-28, and MDA-MB-231 human cancer cell lines. Additionally, all compounds were found to prevent EGF-induced neoplastic transformation of murine JB6 Cl41 cells. Nuclear factors AP-1, NF-κB, and p53 are involved in the cellular response to high and nontoxic concentrations of aaptamine alkaloids 1–3. Furthermore, inhibition of EGF-induced JB6 cell transformation, which is exerted by the compounds 1–3 at low nontoxic concentrations of 0.7–2.1 μM, cannot be explained by activation of AP-1 and NF-κB.

  8. DNA Modification Methylase Activity of Escherichia coli Restriction Endonucleases K and P

    Science.gov (United States)

    Haberman, Allan; Heywood, Janet; Meselson, Matthew

    1972-01-01

    The highly purified restriction endonucleases of E. coli K and coliphage P1 transfer methyl groups from S-adenosylmethionine to adenine residues of unmodified DNA. Incubation of unmodified DNA with endonucleases K or P and S-adenosylmethionine renders the DNA resistant to restriction. The enzymes, therefore, have both restriction endonuclease and modification methylase activities. PMID:4564204

  9. Catalytic and non-catalytic roles of the CtIP endonuclease in double-strand break end resection

    Science.gov (United States)

    Makharashvili, Nodar; Tubbs, Anthony T.; Yang, Soo-Hyun; Wang, Hailong; Barton, Olivia; Zhou, Yi; Deshpande, Rajashree A.; Lee, Ji-Hoon; Lobrich, Markus; Sleckman, Barry P.; Wu, Xiaohua; Paull, Tanya T.

    2014-01-01

    Summary The CtIP protein is known to function in 5′ strand resection during homologous recombination similar to the budding yeast Sae2 protein, although its role in this process is unclear. Here we characterize recombinant human CtIP and find that it exhibits 5′ flap endonuclease activity on branched DNA structures, independent of the MRN complex. Phosphorylation of CtIP at known ATM-dependent sites and other sites is essential for its catalytic activity, although the S327 and T847 phosphorylation sites are dispensable. A catalytic mutant of CtIP that is deficient in endonuclease activity exhibits wild-type levels of homologous recombination at restriction enzyme-generated breaks but is deficient in processing topoisomerase adducts and radiation-induced breaks in human cells, suggesting that the nuclease activity of CtIP is specifically required for the removal of DNA adducts at sites of DNA breaks. PMID:24837676

  10. A CRE/AP-1-like motif is essential for induced syncytin-2 expression and fusion in human trophoblast-like model.

    Directory of Open Access Journals (Sweden)

    Chirine Toufaily

    Full Text Available Syncytin-2 is encoded by the envelope gene of Endogenous Retrovirus-FRD (ERVFRD-1 and plays a critical role in fusion of placental trophoblasts leading to the formation of the multinucleated syncytiotrophoblast. Its expression is consequently regulated in a strict manner. In the present study, we have identified a forskolin-responsive region located between positions -300 to -150 in the Syncytin-2 promoter region. This 150 bp region in the context of a minimal promoter mediated an 80-fold induction of promoter activity following forskolin stimulation. EMSA analyses with competition experiments with nuclear extracts from forskolin-stimulated BeWo cells demonstrated that the -211 to -177 region specifically bound two forskolin-induced complexes, one of them containing a CRE/AP-1-like motif. Site-directed mutagenesis of the CRE/AP-1 binding site in the context of the Syncytin-2 promoter or a heterologous promoter showed that this motif was mostly essential for forskolin-induced promoter activity. Transfection experiments with dominant negative mutants and constitutively activated CREB expression vectors in addition to Chromatin Immunoprecipitation suggested that a CREB family member, CREB2 was binding and acting through the CRE/AP-1 motif. We further demonstrated the binding of JunD to this same motif. Similar to forskolin and soluble cAMP, CREB2 and JunD overexpression induced Syncytin-2 promoter activity in a CRE/AP-1-dependent manner and Syncytin-2 expression. In addition, BeWo cell fusion was induced by both CREB2 and JunD overexpression, while being repressed following silencing of either gene. These results thereby demonstrate that induced expression of Syncytin-2 is highly dependent on the interaction of bZIP-containing transcription factors to a CRE/AP-1 motif and that this element is important for the regulation of Syncytin-2 expression, which results in the formation of the peripheral syncytiotrophoblast layer.

  11. Blimp1 activation by AP-1 in human lung cancer cells promotes a migratory phenotype and is inhibited by the lysyl oxidase propeptide.

    Directory of Open Access Journals (Sweden)

    Ziyang Yu

    Full Text Available B lymphocyte-induced maturation protein 1 (Blimp1 is a master regulator of B cell differentiation, and controls migration of primordial germ cells. Recently we observed aberrant Blimp1 expression in breast cancer cells resulting from an NF-κB RelB to Ras signaling pathway. In order to address the question of whether the unexpected expression of Blimp1 is seen in other epithelial-derived tumors, we selected lung cancers as they are frequently driven by Ras signaling. Blimp1 was detected in all five lung cancer cell lines examined and shown to promote lung cancer cell migration and invasion. Interrogation of microarray datasets demonstrated elevated BLIMP1 RNA expression in lung adenocarcinoma, pancreatic ductal carcinomas, head and neck tumors as well as in glioblastomas. Involvement of Ras and its downstream kinase c-Raf was confirmed using mutant and siRNA strategies. We next addressed the issue of mechanism of Blimp1 activation in lung cancer. Using knockdown and ectopic expression, the role of the Activator Protein (AP-1 family of transcription factors was demonstrated. Further, chromatin immunoprecipitation assays confirmed binding to identified AP-1 elements in the BLIMP1 promoter of ectopically expressed c-Jun and of endogenous AP-1 subunits following serum stimulation. The propeptide domain of lysyl oxidase (LOX-PP was identified as a tumor suppressor, with ability to reduce Ras signaling in lung cancer cells. LOX-PP reduced expression of Blimp1 by binding to c-Raf and inhibiting activation of AP-1, thereby attenuating the migratory phenotype of lung cancer cells. Thus, Blimp1 is a mediator of Ras/Raf/AP-1 signaling that promotes cell migration, and is repressed by LOX-PP in lung cancer.

  12. The mRNA decay factor tristetraprolin (TTP) induces senescence in human papillomavirus-transformed cervical cancer cells by targeting E6-AP ubiquitin ligase.

    Science.gov (United States)

    Sanduja, Sandhya; Kaza, Vimala; Dixon, Dan A

    2009-09-10

    The RNA-binding protein tristetraprolin (TTP) regulates expression of many cancer-associated and proinflammatory factors through binding AU-rich elements (ARE) in the 3'-untranslated region (3'UTR) and facilitating rapid mRNA decay. Here we report on the ability of TTP to act in an anti-proliferative capacity in HPV18-positive HeLa cells by inducing senescence. HeLa cells maintain a dormant p53 pathway and elevated telomerase activity resulting from HPV-mediated transformation, whereas TTP expression counteracted this effect by stabilizing p53 protein and inhibiting hTERT expression. Presence of TTP did not alter E6 and E7 viral mRNA levels indicating that these are not TTP targets. It was found that TTP promoted rapid mRNA decay of the cellular ubiquitin ligase E6-associated protein (E6-AP). RNA-binding studies demonstrated TTP and E6-AP mRNA interaction and deletion of the E6-AP mRNA ARE-containing 3'UTR imparts resistance to TTP-mediated downregulation. Similar results were obtained with high-risk HPV16-positive cells that employ the E6-AP pathway to control p53 and hTERT levels. Furthermore, loss of TTP expression was consistently observed in cervical cancer tissue compared to normal tissue. These findings demonstrate the ability of TTP to act as a tumor suppressor by inhibiting the E6-AP pathway and indicate TTP loss to be a critical event during HPV-mediated carcinogenesis.

  13. Type I restriction endonucleases are true catalytic enzymes.

    Science.gov (United States)

    Bianco, Piero R; Xu, Cuiling; Chi, Min

    2009-06-01

    Type I restriction endonucleases are intriguing, multifunctional complexes that restrict DNA randomly, at sites distant from the target sequence. Restriction at distant sites is facilitated by ATP hydrolysis-dependent, translocation of double-stranded DNA towards the stationary enzyme bound at the recognition sequence. Following restriction, the enzymes are thought to remain associated with the DNA at the target site, hydrolyzing copious amounts of ATP. As a result, for the past 35 years type I restriction endonucleases could only be loosely classified as enzymes since they functioned stoichiometrically relative to DNA. To further understand enzyme mechanism, a detailed analysis of DNA cleavage by the EcoR124I holoenzyme was done. We demonstrate for the first time that type I restriction endonucleases are not stoichiometric but are instead catalytic with respect to DNA. Further, the mechanism involves formation of a dimer of holoenzymes, with each monomer bound to a target sequence and, following cleavage, each dissociates in an intact form to bind and restrict subsequent DNA molecules. Therefore, type I restriction endonucleases, like their type II counterparts, are true enzymes. The conclusion that type I restriction enzymes are catalytic relative to DNA has important implications for the in vivo function of these previously enigmatic enzymes.

  14. The Restriction Endonuclease Cleavage Map of Rat Liver Mitochondrial DNA

    NARCIS (Netherlands)

    Bakker, H.; Holtrop, M.; Terpstra, P.

    1977-01-01

    Mitochondrial DNA from rat liver contains six sites for cleavage by the restriction endonucleases Hind III and EcoRI. A large stretch of DNA, comprising about 40% of the mitochondrial genome is not cleaved by either of the enzymes; eight cleavage sites are located on a DNA stretch of 35% of the

  15. Identification of Campylobacter pyloridis isolates by restriction endonuclease DNA analysis

    NARCIS (Netherlands)

    Langenberg, W.; Rauws, E. A.; Widjojokusumo, A.; Tytgat, G. N.; Zanen, H. C.

    1986-01-01

    Campylobacter pyloridis isolates recovered from gastric biopsy specimens of 16 patients were examined by restriction endonuclease DNA analysis with HindIII. For 8 of these 16 patients two different isolates were compared to study the persistence of the colonizing strains and the stability of their

  16. APS Science 2007.

    Energy Technology Data Exchange (ETDEWEB)

    2008-05-30

    This report provides research highlights from the Advanced Photon Source (APS). Although these highlights represent less than 10% of the published work from the APS in 2007, they give a flavor of the diversity and impact of user research at the facility. In the strategic planning the aim is to foster the growth of existing user communities and foresee new areas of research. This coming year finds the APS engaged in putting together, along with the users, a blueprint for the next five years, and making the case for a set of prioritized investments in beamlines, the accelerator, and infrastructure, each of which will be transformational in terms of scientific impact. As this is written plans are being formulated for an important user workshop on October 20-21, 2008, to prioritize strategic plans. The fruit from past investments can be seen in this report. Examples include the creation of a dedicated beamline for x-ray photon correlation spectroscopy at Sector 8, the evolution of dedicated high-energy x-ray scattering beamlines at sectors 1 and 11, a dedicated imaging beamline at Sector 32, and new beamlines for inelastic scattering and powder diffraction. A single-pulse facility has been built in collaboration with Sector 14 (BioCARS) and Phil Anfinrud at the National Institutes of Health, which will offer exceptionally high flux for single-pulse diffraction. The nanoprobe at Sector 26, built and operated jointly by the Argonne Center for Nanoscale Materials and the X-ray Operations and Research (XOR) section of the APS X-ray Science Division, has come on line to define the state of the art in nanoscience.

  17. A Complete Cleavage Map of Neurospora crassa mtDNA Obtained with Endonucleases Eco RI and Bam HI

    NARCIS (Netherlands)

    Terpstra, P.; Holtrop, M.

    1977-01-01

    A physical map of Neurospora crassa mitochondrial DNA has been constructed using specific fragments obtained with restriction endonucleases. The DNA has 5 cleavage sites for endonuclease Bam HI, 12 for endonuclease Eco RI and more than 30 for endonuclease Hind III. The sequence of the Eco RI and Bam

  18. Crystal structure and DNA-binding property of the ATPase domain of bacterial mismatch repair endonuclease MutL from Aquifex aeolicus.

    Science.gov (United States)

    Fukui, Kenji; Iino, Hitoshi; Baba, Seiki; Kumasaka, Takashi; Kuramitsu, Seiki; Yano, Takato

    2017-09-01

    DNA mismatch repair (MMR) system corrects mismatched bases that are generated mainly by DNA replication errors. The repair system excises the error-containing single-stranded region and enables the re-synthesis of the strand. In the early reactions of MMR, MutL endonuclease incises the newly-synthesized/error-containing strand of the duplex to initiate the downstream excision reaction. MutL endonuclease consists of the N-terminal ATPase and C-terminal endonuclease domains. In this study, we report the crystal structure of the ATPase domain of MutL endonuclease from Aquifex aeolicus. The overall structure of the domain was similar to those of human MutL homologs and Escherichia coli MutL, although E. coli MutL has no endonuclease activity. The ATPase domain was comprised of two subdomains: the N-terminal ATP-binding subdomain and the C-terminal α-β sandwich subdomain. Site-directed mutagenesis experiment identified DNA-interacting eight basic amino acid residues, which were distributed across both the two subdomains and formed a DNA-binding cleft. Docking simulation between the structures of the ATPase and endonuclease domains generated a reliable model structure for the full-length A. aeolicus MutL, which satisfies our previous result of small-angle X-ray scattering analysis. On the basis of the model structure and further experimental results, we concluded that the two separate DNA-binding sites in the full-length A. aeolicus MutL simultaneously bind a dsDNA molecule. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Synergistic Antiviral Activity of S-033188/S-033447, a Novel Inhibitor of Influenza Virus Cap-Dependent Endonuclease, in Combination with Neuraminidase Inhibitors In Vitro

    OpenAIRE

    Kitano, Mitsutaka; Yamamoto, Atsuko; Noshi, Takeshi; Kawai, Makoto; Yoshida, Ryu; Sato, Akihiko; Shishido, Takao; Naito, Akira

    2017-01-01

    Abstract Background S-033447, an active form of orally available prodrug S-033188, is a novel small molecule inhibitor of cap-dependent endonuclease that is essential for influenza virus transcription and replication. In this study, we evaluated the inhibitory effect of S-033188 in combination with neuraminidase inhibitors on the replication of influenza A/H1N1 virus in cultured cells. Methods The inhibitory effects of S-033447 in combination with NA inhibitors on the cytopathic effect of A/P...

  20. Advanced glycation end products upregulate lysyl oxidase and endothelin-1 in human aortic endothelial cells via parallel activation of ERK1/2-NF-κB and JNK-AP-1 signaling pathways.

    Science.gov (United States)

    Adamopoulos, Christos; Piperi, Christina; Gargalionis, Antonios N; Dalagiorgou, Georgia; Spilioti, Eliana; Korkolopoulou, Penelope; Diamanti-Kandarakis, Evanthia; Papavassiliou, Athanasios G

    2016-04-01

    Endothelial dysfunction involves deregulation of the key extracellular matrix (ECM) enzyme lysyl oxidase (LOX) and the vasoconstrictor protein, endothelin-1 (ET-1), whose gene expression can be modulated by the transcriptional activators nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1). Advanced glycation end products (AGEs) present an aggravating factor of endothelial dysfunction which upon engagement to their receptor RAGE induce upregulation of mitogen-activated protein kinases (MAPKs), leading to NF-κB and AP-1 potentiation. We hypothesized that AGEs could induce NF-κΒ- and AP-1-dependent regulation of LOX and ET-1 expression via the AGE/RAGE/MAPK signaling axis. Western blot, real-time qRT-PCR, FACS analysis and electrophoretic mobility-shift assays were employed in human aortic endothelial cells (HAECs) following treatment with AGE-bovine serum albumin (AGE-BSA) to investigate the signaling pathway towards this hypothesis. Furthermore, immunohistochemical analysis of AGEs, RAGE, LOX and ET-1 expression was conducted in aortic endothelium of a rat experimental model exposed to high- or low-AGE content diet. HAECs exposed to AGE-BSA for various time points exhibited upregulation of LOX and ET-1 mRNA levels in a dose- and time-dependent manner. Exposure of HAECs to AGE-BSA also showed specific elevation of phospho(p)-ERK1/2 and p-JNK levels in a dose- and time-dependent fashion. AGE administration significantly increased NF-κΒ- and AP-1-binding activity to both LOX and ET-1 cognate promoter regions. Moreover, LOX and ET-1 overexpression in rat aortic endothelium upon high-AGE content diet confirmed the functional interrelation of these molecules. Our findings demonstrate that AGEs trigger NF-κΒ- and AP-1-mediated upregulation of LOX and ET-1 via the AGE/RAGE/MAPK signaling cascade in human endothelial cells, thus contributing to distorted endothelial homeostasis by impairing endothelial barrier function, altering ECM biomechanical properties

  1. Endonuclease IV cleaves apurinic/apyrimidinic sites in single-stranded DNA and its application for biosensing.

    Science.gov (United States)

    Kong, Xiang-Juan; Wu, Shuang; Cen, Yao; Chen, Ting-Ting; Yu, Ru-Qin; Chu, Xia

    2016-07-21

    Endonuclease IV (Endo IV), as a DNA repairing enzyme, plays a crucial role in repairing damaged DNA comprising abasic sites to maintain genomic integrity. The cleaving capability of Endo IV to apurinic/apyrimidinic sites (AP) in single-stranded DNA (ssDNA) was demonstrated. It was found that Endo IV has considerably high cleaving activity to AP sites in ssDNA compared with that in double-stranded DNA (dsDNA). The unique feature of Endo IV in cleaving AP sites in ssDNA was further applied to construct a novel dual signal amplified sensing system for highly sensitive enzyme and protein detection by a combination of exonuclease III (Exo III)-aided cyclic amplification reaction and a rolling circle replication (RCR) technique, which showed a good sensing performance with a detection limit of 0.008 U mL(-1) for Endo IV and 2.5 pM for streptavidin. In addition, the developed method had considerably high specificity for Endo IV and streptavidin over other potential interferences. The developed strategy indeed provides a novel platform for protein and enzyme assays and may find a broad spectrum of applications in bioanalysis, disease diagnosis, and drug development.

  2. YB-1 represses AP1-dependent gene transactivation and interacts with an AP-1 DNA sequence.

    Science.gov (United States)

    Samuel, Shaija; Twizere, Jean-Claude; Bernstein, Lori R

    2005-06-15

    Involvement of the AP-1 (activator protein-1) transcription factor has been demonstrated previously in the regulation of cell proliferation and cell-cycle progression, in the control of cell migration, invasion and metastasis, and in signal transduction, stress responsiveness, DNA replication and DNA repair. YB-1 (Y-box-binding protein-1) has also been implicated in many of these processes. However, the mechanism by which YB-1 mediates these processes is poorly understood. In the present study, we report that overexpression of a transfected gene encoding YB-1 in human HeLa cervical carcinoma cells significantly represses the transactivation of a minimal AP-1 reporter construct in response to the tumour promoter PMA. YB-1 also represses mRNA expression and PMA-induced promoter transactivation of the endogenous AP-1 target gene encoding matrix metalloproteinase-12 (metalloelastase). YB-1 transrepression of both the minimal and matrix metalloproteinase-12 promoter reporter constructs is dependent on the AP-1 sequence. To identify new nuclear proteins that bind specifically to the AP-1 DNA-binding site, we devised a DNA-affinity-chromatography-based assay termed NAPSTER (nucleotide-affinity preincubation specificity test of recognition) and discovered a 49 kDa protein from human cancer cells that binds in a sequence-specific manner to the AP-1 DNA sequence. By tandem MS fragmentation sequencing analyses we determined that p49 is a YB-1. Immunoblotting of the NAPSTER-purified p49 protein using anti-YB-1 antibodies confirmed YB-1 binding to the AP-1 DNA sequence, as did gel mobility-supershift assays using YB-1 antibodies. This is the first report of YB-1 transrepression and interaction at the AP-1 DNA-binding site.

  3. Genotyping with CRISPR-Cas-derived RNA-guided endonucleases.

    Science.gov (United States)

    Kim, Jong Min; Kim, Daesik; Kim, Seokjoong; Kim, Jin-Soo

    2014-01-01

    Restriction fragment length polymorphism (RFLP) analysis is one of the oldest, most convenient and least expensive methods of genotyping, but is limited by the availability of restriction endonuclease sites. Here we present a novel method of employing CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) in RFLP analysis. We prepare RGENs by complexing recombinant Cas9 protein derived from Streptococcus pyogenes with in vitro transcribed guide RNAs that are complementary to the DNA sequences of interest. Then, we genotype recurrent mutations found in cancer and small insertions or deletions (indels) induced in cultured cells and animals by RGENs and other engineered nucleases such as transcription activator-like effector nucleases (TALENs). Unlike T7 endonuclease I or Surveyor assays that are widely used for genotyping engineered nuclease-induced mutations, RGEN-mediated RFLP analysis can detect homozygous mutant clones that contain identical biallelic indel sequences and is not limited by sequence polymorphisms near the nuclease target sites.

  4. Identification of leptospiral isolates by bacterial restriction endonuclease analysis (Brenda

    Directory of Open Access Journals (Sweden)

    Venkatesha M

    2001-01-01

    Full Text Available DNA samples from 19 reference serovars belonging to 19 different serogroups of Leptospira interrogans and two serovars belonging to Leptospira biflexa were examined by bacterial restriction endonuclease analysis using EcoR I and Hae III enzymes. All the serovars gave unique restriction patterns that differed from each other. DNA from 10 local isolates digested with these enzymes produced patterns which on comparison with the standard patterns produced by reference strains could be identified to serovar level.

  5. A Central Role for JNK/AP-1 Pathway in the Pro-Oxidant Effect of Pyrrolidine Dithiocarbamate through Superoxide Dismutase 1 Gene Repression and Reactive Oxygen Species Generation in Hematopoietic Human Cancer Cell Line U937.

    Directory of Open Access Journals (Sweden)

    Humberto Riera

    Full Text Available Pyrrolidine dithiocarbamate (PDTC known as antioxidant and specific inhibitor of NF-κB was also described as pro-oxidant by inducing cell death and reactive oxygen species (ROS accumulation in cancer. However, the mechanism by which PDTC indices its pro-oxidant effect is unknown. Therefore, we aimed to evaluate the effect of PDTC on the human Cu/Zn superoxide dismutase 1 (SOD1 gene transcription in hematopoietic human cancer cell line U937. We herein show for the first time that PDTC decreases SOD1 transcripts, protein and promoter activity. Furthermore, SOD1 repression by PDTC was associated with an increase in oxidative stress as evidenced by ROS production. Electrophoretic mobility-shift assays (EMSA show that PDTC increased binding of activating protein-1 (AP-1 in dose dependent-manner suggesting that the MAPkinase up-stream of AP-1 is involved. Ectopic NF-κB p65 subunit overexpression had no effect on SOD1 transcription. In contrast, in the presence of JNK inhibitor (SP600125, p65 induced a marked increase of SOD1 promoter, suggesting that JNK pathway is up-stream of NF-κB signaling and controls negatively its activity. Indeed, using JNK deficient cells, PDTC effect was not observed nether on SOD1 transcription or enzymatic activity, nor on ROS production. Finally, PDTC represses SOD1 in U937 cells through JNK/c-Jun phosphorylation. Taken together, these results suggest that PDTC acts as pro-oxidant compound in JNK/AP-1 dependent-manner by repressing the superoxide dismutase 1 gene leading to intracellular ROS accumulation.

  6. CGM ApS Årsberetning til DANAK

    DEFF Research Database (Denmark)

    De Chiffre, Leonardo

    Denne årsberetning omfatter CGM ApS' akkrediterede virksomhed i kalenderåret 2003. Årsberetningen er udarbejdet til DANAK (Dansk Akkreditering, ErhvervsfremmeStyrelsen), som led i opfyldelsen af laboratoriets informationspligt i henhold til gældende regler.......Denne årsberetning omfatter CGM ApS' akkrediterede virksomhed i kalenderåret 2003. Årsberetningen er udarbejdet til DANAK (Dansk Akkreditering, ErhvervsfremmeStyrelsen), som led i opfyldelsen af laboratoriets informationspligt i henhold til gældende regler....

  7. An alternatively spliced mRNA from the AP-2 gene encodes a negative regulator of transcriptional activation by AP-2.

    OpenAIRE

    Buettner, R; Kannan, P.; Imhof, A.; Bauer, R.; Yim, S O; Glockshuber, R; Van Dyke, M W; Tainsky, M A

    1993-01-01

    AP-2 is a retinoic acid-inducible and developmentally regulated activator of transcription. We have cloned an alternative AP-2 transcript (AP-2B) from the human teratocarcinoma cell line PA-1, which encodes a protein differing in the C terminus from the previously isolated AP-2 protein (AP-2A). This protein contains the activation domain of AP-2 and part of the DNA binding domain but lacks the dimerization domain which is necessary for DNA binding. Analysis of overlapping genomic clones spann...

  8. Tethering of the conserved piggyBac transposase fusion protein CSB-PGBD3 to chromosomal AP-1 proteins regulates expression of nearby genes in humans.

    Directory of Open Access Journals (Sweden)

    Lucas T Gray

    2012-09-01

    Full Text Available The CSB-PGBD3 fusion protein arose more than 43 million years ago when a 2.5-kb piggyBac 3 (PGBD3 transposon inserted into intron 5 of the Cockayne syndrome Group B (CSB gene in the common ancestor of all higher primates. As a result, full-length CSB is now coexpressed with an abundant CSB-PGBD3 fusion protein by alternative splicing of CSB exons 1-5 to the PGBD3 transposase. An internal deletion of the piggyBac transposase ORF also gave rise to 889 dispersed, 140-bp MER85 elements that were mobilized in trans by PGBD3 transposase. The CSB-PGBD3 fusion protein binds MER85s in vitro and induces a strong interferon-like innate antiviral immune response when expressed in CSB-null UVSS1KO cells. To explore the connection between DNA binding and gene expression changes induced by CSB-PGBD3, we investigated the genome-wide DNA binding profile of the fusion protein. CSB-PGBD3 binds to 363 MER85 elements in vivo, but these sites do not correlate with gene expression changes induced by the fusion protein. Instead, CSB-PGBD3 is enriched at AP-1, TEAD1, and CTCF motifs, presumably through protein-protein interactions with the cognate transcription factors; moreover, recruitment of CSB-PGBD3 to AP-1 and TEAD1 motifs correlates with nearby genes regulated by CSB-PGBD3 expression in UVSS1KO cells and downregulated by CSB rescue of mutant CS1AN cells. Consistent with these data, the N-terminal CSB domain of the CSB-PGBD3 fusion protein interacts with the AP-1 transcription factor c-Jun and with RNA polymerase II, and a chimeric CSB-LacI construct containing only the N-terminus of CSB upregulates many of the genes induced by CSB-PGBD3. We conclude that the CSB-PGBD3 fusion protein substantially reshapes the transcriptome in CS patient CS1AN and that continued expression of the CSB-PGBD3 fusion protein in the absence of functional CSB may affect the clinical presentation of CS patients by directly altering the transcriptional program.

  9. Tethering of the Conserved piggyBac Transposase Fusion Protein CSB-PGBD3 to Chromosomal AP-1 Proteins Regulates Expression of Nearby Genes in Humans

    Science.gov (United States)

    Gray, Lucas T.; Fong, Kimberly K.; Pavelitz, Thomas; Weiner, Alan M.

    2012-01-01

    The CSB-PGBD3 fusion protein arose more than 43 million years ago when a 2.5-kb piggyBac 3 (PGBD3) transposon inserted into intron 5 of the Cockayne syndrome Group B (CSB) gene in the common ancestor of all higher primates. As a result, full-length CSB is now coexpressed with an abundant CSB-PGBD3 fusion protein by alternative splicing of CSB exons 1–5 to the PGBD3 transposase. An internal deletion of the piggyBac transposase ORF also gave rise to 889 dispersed, 140-bp MER85 elements that were mobilized in trans by PGBD3 transposase. The CSB-PGBD3 fusion protein binds MER85s in vitro and induces a strong interferon-like innate antiviral immune response when expressed in CSB-null UVSS1KO cells. To explore the connection between DNA binding and gene expression changes induced by CSB-PGBD3, we investigated the genome-wide DNA binding profile of the fusion protein. CSB-PGBD3 binds to 363 MER85 elements in vivo, but these sites do not correlate with gene expression changes induced by the fusion protein. Instead, CSB-PGBD3 is enriched at AP-1, TEAD1, and CTCF motifs, presumably through protein–protein interactions with the cognate transcription factors; moreover, recruitment of CSB-PGBD3 to AP-1 and TEAD1 motifs correlates with nearby genes regulated by CSB-PGBD3 expression in UVSS1KO cells and downregulated by CSB rescue of mutant CS1AN cells. Consistent with these data, the N-terminal CSB domain of the CSB-PGBD3 fusion protein interacts with the AP-1 transcription factor c-Jun and with RNA polymerase II, and a chimeric CSB-LacI construct containing only the N-terminus of CSB upregulates many of the genes induced by CSB-PGBD3. We conclude that the CSB-PGBD3 fusion protein substantially reshapes the transcriptome in CS patient CS1AN and that continued expression of the CSB-PGBD3 fusion protein in the absence of functional CSB may affect the clinical presentation of CS patients by directly altering the transcriptional program. PMID:23028371

  10. The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases.

    Science.gov (United States)

    Wei, Hua; Therrien, Caitlin; Blanchard, Aine; Guan, Shengxi; Zhu, Zhenyu

    2008-05-01

    Restriction endonucleases are the basic tools of molecular biology. Many restriction endonucleases show relaxed sequence recognition, called star activity, as an inherent property under various digestion conditions including the optimal ones. To quantify this property we propose the concept of the Fidelity Index (FI), which is defined as the ratio of the maximum enzyme amount showing no star activity to the minimum amount needed for complete digestion at the cognate recognition site for any particular restriction endonuclease. Fidelity indices for a large number of restriction endonucleases are reported here. The effects of reaction vessel, reaction volume, incubation mode, substrate differences, reaction time, reaction temperature and additional glycerol, DMSO, ethanol and Mn(2+) on the FI are also investigated. The FI provides a practical guideline for the use of restriction endonucleases and defines a fundamental property by which restriction endonucleases can be characterized.

  11. Histopathology of human American cutaneous leishmaniasis before and after treatment Histopatologia de leishmaniose tegumentar americana humana, antes e após o tratamento

    Directory of Open Access Journals (Sweden)

    A.C.C. Botelho

    1998-02-01

    cicatriz poderá ser de grande valia. Este trabalho propõe caracterizar o padrão histopatológico de casos humanos de leishmaniose tegumentar americana, em 32 pacientes do município de Caratinga-MG, antes e após o tratamento com os seguintes métodos terapêuticos: 1 leishvacin + glucantime; 2 leishvacin + BCG associado ao glucantime; 3 glucantime; 4 leishvacin + BCG. Foram colhidos fragmentos das lesões de todos os pacientes, através de biópsias, antes e após o tratamento, com diagnóstico de cura. Após análise das lâminas, as preparações foram descritas, do ponto de vista histopatológico, e agrupadas levando em conta a prevalência e a significância do elemento característico. Tal processo resultou na classificação: 1. reação exsudativa; 2. reação exsudativa giganto-celular; 3. reação exsudativa produtiva; 4. reação exsudativa produtiva giganto-celular; 5. reação exsudativa produtiva necrótica; 6. reação necrótica exsudativa; 7. reação produtiva exsudativa; 8. reação produtiva giganto-celular; 9. reação produtiva exsudativa giganto-celular; 10. reação produtiva exsudativa giganto-celular granulomatosa; 11. reação produtiva e 12. reação produtiva cicatricial (cura histopatológica. Observamos após tal análise, que nem sempre a cura clínica coincide com a cura histopatológica.

  12. Center for Geometrisk Metrologi, CGM ApS

    DEFF Research Database (Denmark)

    De Chiffre, Leonardo

    Denne årsberetning omfatter CGM ApS' akkrediterede virksomhed i kalenderåret 2002. Årsberetningen er udarbejdet til DANAK (Dansk Akkreditering, Erhvervsfremme Styrelsen), som led i opfyldelsen af laboratoriets informationspligt i henhold til gældende regler (Teknisk Forskrift Nr. TF4 af 2000...

  13. Eupafolin inhibits PGE2 production and COX2 expression in LPS-stimulated human dermal fibroblasts by blocking JNK/AP-1 and Nox2/p47{sup phox} pathway

    Energy Technology Data Exchange (ETDEWEB)

    Tsai, Ming-Horng [Department of Pediatrics, Division of Neonatology and Pediatric Hematology/Oncology, Chang Gung Memorial Hospital, Yunlin, Taiwan (China); Lin, Zih-Chan [Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Liang, Chan-Jung [Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Yen, Feng-Lin [Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Institute of Biomedical Sciences, Sun Yat-Sen University, 70 Lienhai Rd., Kaohsiung, Taiwan (China); Chiang, Yao-Chang [Center for Drug Abuse and Addiction, China Medical University Hospital, Taichung, Taiwan (China); China Medical University, Taichung, Taiwan (China); Lee, Chiang-Wen, E-mail: cwlee@gw.cgust.edu.tw [Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Chia-Yi, Taiwan (China); Chronic Diseases and Health Promotion Research Center, Chang Gung University of Science and Technology, Chia-Yi, Taiwan (China); Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Taoyuan, Taiwan (China)

    2014-09-01

    Eupafolin, a major active component found in the methanol extracts of Phyla nodiflora, has been used to treat inflammation of skin. We examined its effects on cyclooxygenase-2 (COX-2) expression in LPS-treated human dermal fibroblasts. Lipopolysaccharide (LPS) significantly increased prostaglandin-E2 (PGE2) production associated with increased COX-2 expression in Hs68 cells. This effect was blocked by eupafolin, TLR-4 antibody, antioxidants (APO and NAC), as well as inhibitors, including U0126 (ERK1/2), SB202190 (p38), SP600125 (JNK1/2), and Tanshinone IIA (AP-1). In gene regulation level, qPCR and promoter assays revealed that COX-2 expression was attenuated by eupafolin. In addition, eupafolin also ameliorated LPS-induced p47 phox activation and decreased reactive oxygen species (ROS) generation and NADPH oxidase (Nox) activity. Moreover, pretreatment with eupafolin and APO led to reduced LPS-induced phosphorylation of ERK1/2, JNK, and p38. Further, eupafolin attenuated LPS-induced increase in AP-1 transcription factor binding activity as well as the increase in the phosphorylation of c-Jun and c-Fos. In vivo studies have shown that in dermal fibroblasts of LPS treated mice, eupafolin exerted anti-inflammation effects by decreasing COX-2 protein levels. Our results reveal a novel mechanism for anti-inflammatory and anti-oxidative effects of eupafolin that involved inhibition of LPS-induced ROS generation, suppression of MAPK phosphorylation, diminished DNA binding activity of AP-1 and attenuated COX-2 expression leading to reduced production of prostaglandin E2 (PGE2). Our results demonstrate that eupafolin may be used to treat inflammatory responses associated with dermatologic diseases. - Highlights: • LPS activates the Nox2/p47{sup phox}/JNK/AP-1 and induces COX2 expression in Hs68 cells. • Eupafolin inhibits LPS-induced COX-2 expression via Nox2/p47{sup phox} inhibition. • Eupafolin may be used in the treatment of skin diseases involving inflammation.

  14. Bifunctional TaqII restriction endonuclease: redefining the prototype DNA recognition site and establishing the Fidelity Index for partial cleaving

    National Research Council Canada - National Science Library

    Zylicz-Stachula, Agnieszka; Zołnierkiewicz, Olga; Sliwińska, Katarzyna; Jeżewska-Frąckowiak, Joanna; Skowron, Piotr M

    2011-01-01

    The TaqII enzyme is a member of the Thermus sp. enzyme family that we propounded previously within Type IIS restriction endonucleases, containing related thermophilic bifunctional endonucleases-methyltransferases from various Thermus sp...

  15. Visualizing phosphodiester-bond hydrolysis by an endonuclease

    DEFF Research Database (Denmark)

    Molina, Rafael; Stella, Stefano; Redondo, Pilar

    2015-01-01

    The enzymatic hydrolysis of DNA phosphodiester bonds has been widely studied, but the chemical reaction has not yet been observed. Here we follow the generation of a DNA double-strand break (DSB) by the Desulfurococcus mobilis homing endonuclease I-DmoI, trapping sequential stages of a two....... This third metal ion has a crucial role, triggering the consecutive hydrolysis of the targeted phosphodiester bonds in the DNA strands and leaving its position once the DSB is generated. The multiple structures show the orchestrated conformational changes in the protein residues, nucleotides and metals...

  16. Substrate generation for endonucleases of CRISPR/cas systems.

    Science.gov (United States)

    Zoephel, Judith; Dwarakanath, Srivatsa; Richter, Hagen; Plagens, André; Randau, Lennart

    2012-09-08

    The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) (1-3). Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems (4). The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster (5). The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs (6-8). These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence (9). A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity(10). Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA (11,12) . These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases (4). Here, we present methods to generate crRNAs and precursor-cRNAs for

  17. Nucleosomes Inhibit Cas9 Endonuclease Activity in Vitro.

    Science.gov (United States)

    Hinz, John M; Laughery, Marian F; Wyrick, John J

    2015-12-08

    During Cas9 genome editing in eukaryotic cells, the bacterial Cas9 enzyme cleaves DNA targets within chromatin. To understand how chromatin affects Cas9 targeting, we characterized Cas9 activity on nucleosome substrates in vitro. We find that Cas9 endonuclease activity is strongly inhibited when its target site is located within the nucleosome core. In contrast, the nucleosome structure does not affect Cas9 activity at a target site within the adjacent linker DNA. Analysis of target sites that partially overlap with the nucleosome edge indicates that the accessibility of the protospacer-adjacent motif (PAM) is the critical determinant of Cas9 activity on a nucleosome.

  18. Engineering a Nickase on the Homing Endonuclease I-DmoI Scaffold

    DEFF Research Database (Denmark)

    Molina, Rafael; Marcaida, María José; Redondo, Pilar

    2015-01-01

    Homing endonucleases are useful tools for genome modification because of their capability to recognize and cleave specifically large DNA targets. These endonucleases generate a DNA double strand break that can be repaired by the DNA damage response machinery. The break can be repaired by homologo...

  19. Next-generation sequencing of multiple individuals per barcoded library by deconvolution of sequenced amplicons using endonuclease fragment analysis

    DEFF Research Database (Denmark)

    Andersen, Jeppe D; Pereira, Vania; Pietroni, Carlotta

    2014-01-01

    The simultaneous sequencing of samples from multiple individuals increases the efficiency of next-generation sequencing (NGS) while also reducing costs. Here we describe a novel and simple approach for sequencing DNA from multiple individuals per barcode. Our strategy relies on the endonuclease...... digestion of PCR amplicons prior to library preparation, creating a specific fragment pattern for each individual that can be resolved after sequencing. By using both barcodes and restriction fragment patterns, we demonstrate the ability to sequence the human melanocortin 1 receptor (MC1R) genes from 72...

  20. Next-generation sequencing of multiple individuals per barcoded library by deconvolution of sequenced amplicons using endonuclease fragment analysis.

    Science.gov (United States)

    Andersen, Jeppe D; Pereira, Vania; Pietroni, Carlotta; Mikkelsen, Martin; Johansen, Peter; Børsting, Claus; Morling, Niels

    2014-08-01

    The simultaneous sequencing of samples from multiple individuals increases the efficiency of next-generation sequencing (NGS) while also reducing costs. Here we describe a novel and simple approach for sequencing DNA from multiple individuals per barcode. Our strategy relies on the endonuclease digestion of PCR amplicons prior to library preparation, creating a specific fragment pattern for each individual that can be resolved after sequencing. By using both barcodes and restriction fragment patterns, we demonstrate the ability to sequence the human melanocortin 1 receptor (MC1R) genes from 72 individuals using only 24 barcoded libraries.

  1. Inteins, introns, and homing endonucleases: recent revelations about the life cycle of parasitic genetic elements

    Directory of Open Access Journals (Sweden)

    Hilario Elena

    2006-11-01

    Full Text Available Abstract Self splicing introns and inteins that rely on a homing endonuclease for propagation are parasitic genetic elements. Their life-cycle and evolutionary fate has been described through the homing cycle. According to this model the homing endonuclease is selected for function only during the spreading phase of the parasite. This phase ends when the parasitic element is fixed in the population. Upon fixation the homing endonuclease is no longer under selection, and its activity is lost through random processes. Recent analyses of these parasitic elements with functional homing endonucleases suggest that this model in its most simple form is not always applicable. Apparently, functioning homing endonuclease can persist over long evolutionary times in populations and species that are thought to be asexual or nearly asexual. Here we review these recent findings and discuss their implications. Reasons for the long-term persistence of a functional homing endonuclease include: More recombination (sexual and as a result of gene transfer than previously assumed for these organisms; complex population structures that prevent the element from being fixed; a balance between active spreading of the homing endonuclease and a decrease in fitness caused by the parasite in the host organism; or a function of the homing endonuclease that increases the fitness of the host organism and results in purifying selection for the homing endonuclease activity, even after fixation in a local population. In the future, more detailed studies of the population dynamics of the activity and regulation of homing endonucleases are needed to decide between these possibilities, and to determine their relative contributions to the long term survival of parasitic genes within a population. Two outstanding publications on the amoeba Naegleria group I intron (Wikmark et al. BMC Evol Biol 2006, 6:39 and the PRP8 inteins in ascomycetes (Butler et al.BMC Evol Biol 2006, 6:42 provide

  2. Peculiar Traits of Coarse AP

    Science.gov (United States)

    2014-01-01

    explosive event. Part of the problem with end of slow cookoff rocket motor trials is the motor case containment factor. With a fixed nozzle ...C. E. Merrill Air Force Research Laboratory Rocket Site, Edwards AFB, CA PA Clearance Number 14285   DISTRIBUTION A...started by electrically heated Nichrome wires . With AP crystals mounted in relatively massive copper jaws AP crystal combustion quenched due to thermal

  3. PolyADP-ribose polymerase is a coactivator for AP-2-mediated transcriptional activation.

    OpenAIRE

    Kannan, P.; Yu, Y.; Wankhade, S; Tainsky, M A

    1999-01-01

    Overexpression of transcription factor AP-2 has been implicated in the tumorigenicity of the human teratocarcinoma cell lines PA-1 that contain an activated ras oncogene. Here we show evidence that overexpression of AP-2 sequesters transcriptional coactivators which results in self-inhibition. We identified AP-2-interacting proteins and determined whether these proteins were coactivators for AP-2-mediated transcription. One such interacting protein is polyADP-ribose polymerase (PARP). PARP su...

  4. Psychosocial intervention, as a contribution to local human development within Medellin’s public scope. Case study: project aps - “buenvivir” (good living into a family. sponsor: City hall of Medellin, Colombia

    Directory of Open Access Journals (Sweden)

    J. J. García

    2013-10-01

    Full Text Available This article provides information about a research held on the psychosocial intervention offered from social projects such as educational formative spaces. A critical analysis is made by means of a government project conducted by the local administration of the city of Medellin known as “APS Buenvivir en Familia, 2011”, compiled by the Social Welfare Town Council Office, in association with the Municipality’s Health Department. Its purpose was to examine the logic and approach of psychosocial intervention as an input to human development. It was carried out through a qualitative focusing, taking in consideration the following categories: The psychosocial intervention has a definite impact on local human development, psychosocial praxis from the point of view of a public action strategy, community involvement taken as a true political act. The results showed that the psychosocial as a public exercise makes a contribution to local human development starting from the social involvement of communities and, in its everyday doing, presents structural and formal contradictions between the way they are designed and how the projects are planned and implemented among the people. 

  5. AP2 suppresses osteoblast differentiation and mineralization through down-regulation of Frizzled-1.

    Science.gov (United States)

    Yu, Shibing; Yerges-Armstrong, Laura M; Chu, Yanxia; Zmuda, Joseph M; Zhang, Yingze

    2015-02-01

    Transcription factor activating protein 2 (AP2) plays an important role in cellular differentiation. Although profound craniofacial and long bone developmental abnormalities have been observed in AP2-knockout mice, the molecular effects of AP2 on osteoblasts are poorly defined. We demonstrated that AP2 regulates the expression of human Frizzled 1 (FZD1), a co-receptor for the Wnt signalling pathway, in human osteoblast cell lines and primary bone marrow stromal cells (BMSCs). We also identified a putative AP2-binding site in the FZD1 proximal promoter in silico and characterized this binding element further in Saos2 in vitro by ChIP, electrophoretic mobility shift and promoter reporter assays. The transcriptional repression of the FZD1 promoter by AP2 was confirmed in normal human fetal osteoblasts (hFOB). Furthermore, overexpression of AP2 resulted in a significant reduction in both differentiation and mineralization of Saos2 cells. Knockdown of FZD1 expression before AP2 up-regulation diminished the AP2-dependent inhibition of Saos2 cell differentiation and mineralization. Similarly, overexpressing FZD1 before AP2 treatment in both Saos2 and BMSCs diminished the inhibitory effect of AP2 on osteoblast differentiation and mineralization. Taken together, these results demonstrate that AP2 is a negative regulator of osteoblast differentiation and mineralization, and its inhibitory effect may be mediated in part through down-regulation of FZD1 expression.

  6. Creation of a type IIS restriction endonuclease with a long recognition sequence.

    Science.gov (United States)

    Lippow, Shaun M; Aha, Patti M; Parker, Matthew H; Blake, William J; Baynes, Brian M; Lipovsek, Dasa

    2009-05-01

    Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6 nt from the target site to generate homogeneous, 5', four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases.

  7. Catalytic activity control of restriction endonuclease--triplex forming oligonucleotide conjugates.

    Science.gov (United States)

    Silanskas, Arunas; Zaremba, Mindaugas; Sasnauskas, Giedrius; Siksnys, Virginijus

    2012-02-15

    Targeting of individual genes in complex genomes requires endonucleases of extremely high specificity. To direct cleavage at the unique site(s) in the genome, both naturally occurring and artificial enzymes have been developed. These include homing endonucleases, zinc-finger nucleases, transcription activator-like effector nucleases, and restriction or chemical nucleases coupled to a triple-helix forming oligonucleotide (TFO). The desired cleavage has been demonstrated both in vivo and in vitro for several model systems. However, to limit cleavage strictly to unique sites and avoid undesired reactions, endonucleases with controlled activity are highly desirable. In this study we present a proof-of-concept demonstration of two strategies to generate restriction endonuclease-TFO conjugates with controllable activity. First, we combined the restriction endonuclease caging and TFO coupling procedures to produce a caged MunI-TFO conjugate, which can be activated by UV-light upon formation of a triple helix. Second, we coupled TFO to a subunit interface mutant of restriction endonuclease Bse634I which shows no activity due to impaired dimerization but is assembled into an active dimer when two Bse634I monomers are brought into close proximity by triple helix formation at the targeted site. Our results push the restriction endonuclease-TFO conjugate technology one step closer to potential in vivo applications.

  8. Extrahelical (CAG)/(CTG) triplet repeat elements support proliferating cell nuclear antigen loading and MutLα endonuclease activation.

    Science.gov (United States)

    Pluciennik, Anna; Burdett, Vickers; Baitinger, Celia; Iyer, Ravi R; Shi, Kevin; Modrich, Paul

    2013-07-23

    MutLα endonuclease can be activated on covalently continuous DNA that contains a MutSα- or MutSβ-recognizable lesion and a helix perturbation that supports proliferating cell nuclear antigen (PCNA) loading by replication factor C, providing a potential mechanism for triggering mismatch repair on nonreplicating DNA. Because mouse models for somatic expansion of disease-associated (CAG)n/(CTG)n triplet repeat sequences have implicated both MutSβ and MutLα and have suggested that expansions can occur in the absence of replication, we have asked whether an extrahelical (CAG)n or (CTG)n element is sufficient to trigger MutLα activation. (CAG)n and (CTG)n extrusions in relaxed closed circular DNA do in fact support MutSβ-, replication factor C-, and PCNA-dependent activation of MutLα endonuclease, which can incise either DNA strand. Extrahelical elements of two or three repeat units are the preferred substrates for MutLα activation, and extrusions of this size also serve as moderately effective sites for loading the PCNA clamp. Relaxed heteroduplex DNA containing a two or three-repeat unit extrusion also triggers MutSβ- and MutLα-endonuclease-dependent mismatch repair in nuclear extracts of human cells. This reaction occurs without obvious strand bias at about 10% the rate of that observed with otherwise identical nicked heteroduplex DNA. These findings provide a mechanism for initiation of triplet repeat processing in nonreplicating DNA that is consistent with several features of the model of Gomes-Pereira et al. [Gomes-Pereira M, Fortune MT, Ingram L, McAbney JP, Monckton DG (2004) Hum Mol Genet 13(16):1815-1825]. They may also have implications for triplet repeat processing at a replication fork.

  9. Extrahelical (CAG)/(CTG) triplet repeat elements support proliferating cell nuclear antigen loading and MutLα endonuclease activation

    Science.gov (United States)

    Pluciennik, Anna; Burdett, Vickers; Baitinger, Celia; Iyer, Ravi R.; Shi, Kevin; Modrich, Paul

    2013-01-01

    MutLα endonuclease can be activated on covalently continuous DNA that contains a MutSα- or MutSβ-recognizable lesion and a helix perturbation that supports proliferating cell nuclear antigen (PCNA) loading by replication factor C, providing a potential mechanism for triggering mismatch repair on nonreplicating DNA. Because mouse models for somatic expansion of disease-associated (CAG)n/(CTG)n triplet repeat sequences have implicated both MutSβ and MutLα and have suggested that expansions can occur in the absence of replication, we have asked whether an extrahelical (CAG)n or (CTG)n element is sufficient to trigger MutLα activation. (CAG)n and (CTG)n extrusions in relaxed closed circular DNA do in fact support MutSβ-, replication factor C-, and PCNA-dependent activation of MutLα endonuclease, which can incise either DNA strand. Extrahelical elements of two or three repeat units are the preferred substrates for MutLα activation, and extrusions of this size also serve as moderately effective sites for loading the PCNA clamp. Relaxed heteroduplex DNA containing a two or three-repeat unit extrusion also triggers MutSβ- and MutLα-endonuclease-dependent mismatch repair in nuclear extracts of human cells. This reaction occurs without obvious strand bias at about 10% the rate of that observed with otherwise identical nicked heteroduplex DNA. These findings provide a mechanism for initiation of triplet repeat processing in nonreplicating DNA that is consistent with several features of the model of Gomes-Pereira et al. [Gomes-Pereira M, Fortune MT, Ingram L, McAbney JP, Monckton DG (2004) Hum Mol Genet 13(16):1815–1825]. They may also have implications for triplet repeat processing at a replication fork. PMID:23840062

  10. Engraftment of PBMC from SLE and APS Donors into BALB-Rag2−/−IL2Rgc−/− Mice: a Promising Model for Studying Human Disease

    Science.gov (United States)

    Andrade, Danieli; Redecha, Patricia B.; Vukelic, Milena; Qing, Xiaoping; Perino, Giorgio; Salmon, Jane E.; Koo, Gloria C.

    2011-01-01

    Purpose To construct a humanized SLE mouse that resembles the human disease to define pathophysiology and targeted for treatments. Methods We infused peripheral blood mononuclear cells (PBMC) from SLE patients into BALB-Rag2−/−IL2Rgc−/−mice (DKO), which lack T, B and NK cells. PBMC from 5 SLE patients and 4 normal donors (ND) at 3–5×106/mouse were infused IV/IP to non-irradiated 4–5 weeks old mice. We evaluated the engraftment of human CD45+cells and monitored the plasma human IgG, anti-dsDNA, anti-cardiolipin (aCL) antibodies, proteinuria, and kidney histology. Results We found 100% successful engraftment of 40 DKO mice infused with human PBMC. In both SLE-DKO and ND-DKO mice, 50–80% human CD45+ cells were observed in PBMC fraction 4–6 weeks post engraftment, with 70–90% CD3+ cells. There were fewer CD3+4+cells (5.5±2.1%) and more CD3+8+cells (79.4±3.6%) in the SLE-DKO mice, as in the SLE patients. CD19+B cells and CD11c+Monocytic cells were found in the spleen, lung, liver and bone marrow. There was no significant difference in plasma human IgG levels and anti-dsDNA antibodies between SLE-DKO and ND-DKO mice. Levels of aCL antibody were significantly higher in all SLE-DKO mice infused with PBMC from a SLE patient with high titers of aCL antibodies. SLE-DKO mice had proteinuria, human IgG deposits in the kidneys and shorter life span. In SLE- DKO mice engrafted from the aCL-positive patient, we found micro-thrombi and infiltration of CD3+, CD8+ and CD19+ cells in the glomeruli, recapitulating APS in these mice. Conclusion A novel humanized SLE-DKO mouse is established, exhibiting many characteristics of immunologic and clinical features of SLE. PMID:21560114

  11. The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit.

    Science.gov (United States)

    Dias, Alexandre; Bouvier, Denis; Crépin, Thibaut; McCarthy, Andrew A; Hart, Darren J; Baudin, Florence; Cusack, Stephen; Ruigrok, Rob W H

    2009-04-16

    The influenza virus polymerase, a heterotrimer composed of three subunits, PA, PB1 and PB2, is responsible for replication and transcription of the eight separate segments of the viral RNA genome in the nuclei of infected cells. The polymerase synthesizes viral messenger RNAs using short capped primers derived from cellular transcripts by a unique 'cap-snatching' mechanism. The PB2 subunit binds the 5' cap of host pre-mRNAs, which are subsequently cleaved after 10-13 nucleotides by the viral endonuclease, hitherto thought to reside in the PB2 (ref. 5) or PB1 (ref. 2) subunits. Here we describe biochemical and structural studies showing that the amino-terminal 209 residues of the PA subunit contain the endonuclease active site. We show that this domain has intrinsic RNA and DNA endonuclease activity that is strongly activated by manganese ions, matching observations reported for the endonuclease activity of the intact trimeric polymerase. Furthermore, this activity is inhibited by 2,4-dioxo-4-phenylbutanoic acid, a known inhibitor of the influenza endonuclease. The crystal structure of the domain reveals a structural core closely resembling resolvases and type II restriction endonucleases. The active site comprises a histidine and a cluster of three acidic residues, conserved in all influenza viruses, which bind two manganese ions in a configuration similar to other two-metal-dependent endonucleases. Two active site residues have previously been shown to specifically eliminate the polymerase endonuclease activity when mutated. These results will facilitate the optimisation of endonuclease inhibitors as potential new anti-influenza drugs.

  12. Catalytic and noncatalytic roles of the CtIP endonuclease in double-strand break end resection.

    Science.gov (United States)

    Makharashvili, Nodar; Tubbs, Anthony T; Yang, Soo-Hyun; Wang, Hailong; Barton, Olivia; Zhou, Yi; Deshpande, Rajashree A; Lee, Ji-Hoon; Lobrich, Markus; Sleckman, Barry P; Wu, Xiaohua; Paull, Tanya T

    2014-06-19

    The carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) is known to function in 5' strand resection during homologous recombination, similar to the budding yeast Sae2 protein, but its role in this process is unclear. Here, we characterize recombinant human CtIP and find that it exhibits 5' flap endonuclease activity on branched DNA structures, independent of the MRN complex. Phosphorylation of CtIP at known damage-dependent sites and other sites is essential for its catalytic activity, although the S327 and T847 phosphorylation sites are dispensable. A catalytic mutant of CtIP that is deficient in endonuclease activity exhibits wild-type levels of homologous recombination at restriction enzyme-generated breaks but is deficient in processing topoisomerase adducts and radiation-induced breaks in human cells, suggesting that the nuclease activity of CtIP is specifically required for the removal of DNA adducts at sites of DNA breaks. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. APS Education and Diversity Efforts

    Science.gov (United States)

    Prestridge, Katherine; Hodapp, Theodore

    2015-11-01

    American Physical Society (APS) has a wide range of education and diversity programs and activities, including programs that improve physics education, increase diversity, provide outreach to the public, and impact public policy. We present the latest programs spearheaded by the Committee on the Status of Women in Physics (CSWP), with highlights from other diversity and education efforts. The CSWP is working to increase the fraction of women in physics, understand and implement solutions for gender-specific issues, enhance professional development opportunities for women in physics, and remedy issues that impact gender inequality in physics. The Conferences for Undergraduate Women in Physics, Professional Skills Development Workshops, and our new Professional Skills program for students and postdocs are all working towards meeting these goals. The CSWP also has site visit and conversation visit programs, where department chairs request that the APS assess the climate for women in their departments or facilitate climate discussions. APS also has two significant programs to increase participation by underrepresented minorities (URM). The newest program, the APS National Mentoring Community, is working to provide mentoring to URM undergraduates, and the APS Bridge Program is an established effort that is dramatically increasing the number of URM PhDs in physics.

  14. Pterocarpus santalinus L. Regulated Ultraviolet B Irradiation-induced Procollagen Reduction and Matrix Metalloproteinases Expression Through Activation of TGF-β/Smad and Inhibition of the MAPK/AP-1 Pathway in Normal Human Dermal Fibroblasts.

    Science.gov (United States)

    Gao, Wei; Lin, Pei; Hwang, Eunson; Wang, Yushuai; Yan, Zhengfei; Ngo, Hien T T; Yi, Tae-Hoo

    2018-01-01

    Ultraviolet light-induced reactive oxygen species (ROS) damage human skin and prematurely cause aging. A growing body of research is focusing on considering plants and plant-derived compounds as antiphotoaging therapeutic material. Pterocarpus santalinus L., as an Indian traditional medicine, possesses antidiabetic, anti-inflammatory and antioxidative effects. Here, we studied the antiphotoaging effects of ethanolic extract of P. santalinus L. heartwood (EPS) on ultraviolet radiation B (UVB)-irradiated normal human dermal fibroblasts (NHDFs). Results showed that EPS significantly inhibited the upregulation of matrix metalloproteinases and IL-6 caused by UVB irradiation, and suppressed UVB-induced phosphorylation of extracellular signal-regulated kinase, Jun N-terminal kinase and p38, as well as the activation of AP-1 transcription factors. Further study indicated that UVB-induced production of MMP-1 and IL-6 could be inhibited by PD 98059 (an ERK inhibitor) and SP600125 (A JNK inhibitor), implied that EPS inhibited UVB-induced MMP-1 and IL-6 secretion by inactivating MAPK signaling pathway. In addition, EPS possessed an excellent antioxidant activity, which could increase cytoprotective antioxidants such as HO-1, NQ-O1 expression by facilitating the nuclear accumulation of Nrf2. Treatment of NHDFs with EPS also recovered UVB-induced procollagen type I reduction by activating TGF-β/Smad pathway. These findings demonstrated that EPS had a potential effect against UVB-induced skin photoaging. © 2017 The American Society of Photobiology.

  15. Restoration of ultraviolet-induced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendai virus).

    Science.gov (United States)

    Tanaka, K; Sekiguchi, M; Okada, Y

    1975-01-01

    Ultraviolet (UV)-induced unscheduled DNA synthesis of xeroderma pigmentosum cells, belonging to complementation groups A, B, C, D, and E, was restored to the normal level by concomitant treatment of the cells with T4 endonuclease V and UV-inactivated HVJ (Sendai virus). The present results suggest that (1) T4 endonuclease molecules were inserted effectively into the cells by the interaction of HVJ with the cell membranes, (2) the enzyme was functional on human chromosomal DNA which had been damaged by UV irradiation in the viable cells, (3) all the studied groups of xeroderma pigmentosum ("variant" was not tested) were defective in the first step (incision) of excision repair. Images PMID:172893

  16. Cofactor requirement of HpyAV restriction endonuclease.

    Directory of Open Access Journals (Sweden)

    Siu-Hong Chan

    Full Text Available BACKGROUND: Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M systems in microorganisms. PRINCIPAL FINDINGS: We have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg(++. The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity. CONCLUSIONS/SIGNIFICANCE: Some HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms.

  17. Cofactor requirement of HpyAV restriction endonuclease.

    Science.gov (United States)

    Chan, Siu-Hong; Opitz, Lars; Higgins, Lauren; O'loane, Diana; Xu, Shuang-Yong

    2010-02-05

    Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M) systems in microorganisms. We have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg(++). The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity. Some HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms.

  18. Saturated free fatty acids and apoptosis in microvascular mesangial cells: palmitate activates pro-apoptotic signaling involving caspase 9 and mitochondrial release of endonuclease G

    Directory of Open Access Journals (Sweden)

    Simonson Michael S

    2005-01-01

    Full Text Available Abstract Background In type 2 diabetes, free fatty acids (FFA accumulate in microvascular cells, but the phenotypic consequences of FFA accumulation in the microvasculature are incompletely understood. Here we investigated whether saturated FFA induce apoptosis in human microvascular mesangial cells and analyzed the signaling pathways involved. Methods Saturated and unsaturated FFA-albumin complexes were added to cultured human mesangial cells, after which the number of apoptotic cells were quantified and the signal transduction pathways involved were delineated. Results The saturated FFA palmitate and stearate were apoptotic unlike equivalent concentrations of the unsaturated FFA oleate and linoleate. Palmitate-induced apoptosis was potentiated by etomoxir, an inhibitor of mitochondrial β-oxidation, but was prevented by an activator of AMP-kinase, which increases fatty acid β-oxidation. Palmitate stimulated an intrinsic pathway of pro-apoptotic signaling as evidenced by increased mitochondrial release of cytochrome-c and activation of caspase 9. A caspase 9-selective inhibitor blocked caspase 3 activation but incompletely blocked apoptosis in response to palmitate, suggesting an additional caspase 9-independent pathway. Palmitate stimulated mitochondrial release of endonuclease G by a caspase 9-independent mechanism, thereby implicating endonuclease G in caspase 9-indpendent regulation of apoptosis by saturated FFA. We also observed that the unsaturated FFA oleate and linoleate prevented palmitate-induced mitochondrial release of both cytochrome-c and endonuclease G, which resulted in complete protection from palmitate-induced apoptosis. Conclusions Taken together, these results demonstrate that palmitate stimulates apoptosis by evoking an intrinsic pathway of proapoptotic signaling and identify mitochondrial release of endonuclease G as a key step in proapoptotic signaling by saturated FFA and in the anti-apoptotic actions of unsaturated FFA.

  19. Nucleolin binds specifically to an AP-1 DNA sequence and represses AP1-dependent transactivation of the matrix metalloproteinase-13 gene.

    Science.gov (United States)

    Samuel, Shaija; Twizere, Jean-Claude; Beifuss, Katherine K; Bernstein, Lori R

    2008-01-01

    Transcriptional regulation via activator protein-1 (AP-1) protein binding to AP-1 binding sites within gene promoter regions of AP-1 target genes plays a key role in controlling cellular invasion, proliferation, and oncogenesis, and is important to pathogenesis of arthritis and cardiovascular disease. To identify new proteins that interact with the AP-1 DNA binding site, we performed the DNA affinity chromatography-based Nucleotide Affinity Preincubation Specificity TEst of Recognition (NAPSTER) assay, and discovered a 97 kDa protein that binds in vitro to a minimal AP-1 DNA sequence element. Mass spectrometric fragmentation sequencing determined that p97 is nucleolin. Immunoblotting of DNA affinity-purified material with anti-nucleolin antibodies confirmed this identification. Nucleolin also binds the AP-1 site in gel shift assays. Nucleolin interacts in NAPSTER with the AP-1 site within the promoter sequence of the metalloproteinase-13 gene (MMP-13), and binds in vivo in chromatin immunoprecipitation assays in the vicinity of the AP-1 site in the MMP-13 promoter. Overexpression of nucleolin in human HeLa cervical carcinoma cells significantly represses AP-1 dependent gene transactivation of a minimal AP-1 reporter construct and of an MMP-13 promoter reporter sequence. This is the first report of nucleolin binding and transregulation at the AP-1 site. (c) 2007 Wiley-Liss, Inc.

  20. AP1000 - outlook for new orders

    Energy Technology Data Exchange (ETDEWEB)

    Cummins, E. [Westinghouse Electric Company, Monroeville, Pennsylvania (United States)

    2006-07-01

    This paper gives an overview of the resurgence in nuclear power recently. The major driving factor is the price of natural gas which has more than doubled, the volatility of natural gas prices and high long-term projection for natural gas prices. Additional consideration are energy security, uncertainty in the future emission legislation, availability of advanced nuclear plant designs, relative stability of regulatory environment and public policy support. The challenges ahead are spent fuel disposal, resource availability (human and supply chain) and project management. This paper also describes the Westinghouse AP1000 nuclear power plant.

  1. Gene expression of immediate early genes of AP-1 transcription factor in human peripheral blood mononuclear cells in response to ionizing radiation.

    Science.gov (United States)

    Nishad, S; Ghosh, Anu

    2016-11-01

    Ionizing radiation (IR) is considered ubiquitous in nature. The immediate early genes are considered the earliest nuclear targets of IR and are induced in the absence of de novo protein synthesis. Many of these genes encode transcription factors that constitute the first step in signal transduction to couple cytoplasmic effects with long-term cellular response. In this paper, coordinated transcript response of fos and jun family members which constitute activator protein 1 transcription factor was studied in response to IR in human peripheral blood lymphocytes at the G0 stage. Gene expression was monitored 5 min, 1 h and 4 h post-irradiation with Co(60) γ-rays (dose rate of 0.417 Gy/min) and compared with sham-irradiated controls. When gene expression was analyzed at the early time point of 5 min post-irradiation with 0.3 Gy, the studied samples showed two distinct trends. Six out of ten individuals (called 'Group I responders') showed transient, but significant up-regulation for fosB, fosL1, fosL2 and c-jun with an average fold change (FC) ≥1.5 as compared to sham-irradiated controls. The Students's t test p value for all four genes was ≤0.001, indicating strong up-regulation. The remaining four individuals (called Group II responders) showed down-regulation for these same four genes. The average FC with 0.3 Gy in Group II individuals was 0.53 ± 0.22 (p = 0.006) for fosB, 0.60 ± 0.14 (p = 0.001) for fosL1, 0.52 ± 0.16 (p = 0.001) for fosL2 and 0.59 ± 0.28 (p = 0.03) for c-jun. The two groups could be clearly distinguished at this dose/time point using principal component analysis. Both Group I and Group II responders did not show any change in expression for three genes (c-fos, junB and junD) as compared to sham-irradiated controls. Though a similar trend was seen 5 min post-irradiation with a relatively high dose of 1 Gy, the average FC was lower and change in gene expression was not statistically significant (at p regulation at

  2. Three new active members of the I-OnuI family of homing endonucleases.

    Science.gov (United States)

    Bilto, Iman M; Guha, Tuhin K; Wai, Alvan; Hausner, Georg

    2017-08-01

    In vitro characterization of 3 LAGLIDADG-type homing endonucleases (HEs) (I-CcaI, I-CcaII, and I-AstI) that belong to the I-OnuI family showed that they are functional HEs that cleave their respective cognate target sites. These endonucleases are encoded within group ID introns and appear to be orthologues that have inserted into 3 different mitochondrial genes: rns, rnl, and cox3. The endonuclease activity of I-CcaI was tested using various substrates, and its minimum DNA recognition sequence was estimated to be 26 nt. This set of HEs may provide some insight into how these types of mobile elements can migrate into new locations. This study provides additional endonucleases that can be added to the catalog of currently available HEs that may have various biotechnology applications.

  3. AP Geography, Environmental Science Thrive

    Science.gov (United States)

    Robelen, Erik W.

    2012-01-01

    Geography may not be particularly known as a hot topic among today's students--even some advocates suggest it suffers from an image problem--but by at least one measure, the subject is starting to come into its own. Across more than 30 topics covered in the Advanced Placement (AP) program, participation in geography is rising faster than any…

  4. Restriction endonucleases from invasive Neisseria gonorrhoeae cause double-strand breaks and distort mitosis in epithelial cells during infection.

    Science.gov (United States)

    Weyler, Linda; Engelbrecht, Mattias; Mata Forsberg, Manuel; Brehwens, Karl; Vare, Daniel; Vielfort, Katarina; Wojcik, Andrzej; Aro, Helena

    2014-01-01

    The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.

  5. Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases

    DEFF Research Database (Denmark)

    Crouzier, Lucile; Dubois, Camille; Wengel, Jesper

    2012-01-01

    Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving...... LNA-modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI....

  6. Apn1 and Apn2 endonucleases prevent accumulation of repair-associated DNA breaks in budding yeast as revealed by direct chromosomal analysis.

    Science.gov (United States)

    Ma, Wenjian; Resnick, Michael A; Gordenin, Dmitry A

    2008-04-01

    Base excision repair (BER) provides relief from many DNA lesions. While BER enzymes have been characterized biochemically, BER functions within cells are much less understood, in part because replication bypass and double-strand break (DSB) repair can also impact resistance to base damage. To investigate BER in vivo, we examined the repair of methyl methanesulfonate (MMS) induced DNA damage in haploid G1 yeast cells, so that replication bypass and recombinational DSB repair cannot occur. Based on the heat-lability of MMS-induced base damage, an assay was developed that monitors secondary breaks in full-length yeast chromosomes where closely spaced breaks yield DSBs that are observed by pulsed-field gel electrophoresis. The assay detects damaged bases and abasic (AP) sites as heat-dependent breaks as well as intermediate heat-independent breaks that arise during BER. Using a circular chromosome, lesion frequency and repair kinetics could be easily determined. Monitoring BER in single and multiple glycosylase and AP-endonuclease mutants confirmed that Mag1 is the major enzyme that removes MMS-damaged bases. This approach provided direct physical evidence that Apn1 and Apn2 not only repair cellular base damage but also prevent break accumulation that can result from AP sites being channeled into other BER pathway(s).

  7. A novel berbamine derivative inhibits cell viability and induces apoptosis in cancer stem-like cells of human glioblastoma, via up-regulation of miRNA-4284 and JNK/AP-1 signaling.

    Directory of Open Access Journals (Sweden)

    Fan Yang

    Full Text Available Glioblastoma (GBM is the most common primary brain tumor, accounting for approximately 40% of all central nervous system malignancies. Despite standard treatment consisting of surgical resection, radiotherapy and/or chemotherapy, the prognosis for GBM is poor; with a median survival of 14.6 months. The cancer stem cell or cancer-initiating cell model has provided a new paradigm for understanding development and recurrence of GBM following treatment. Berbamine (BBM is a natural compound derived from the Berberis amurensis plant, and along with its derivatives, has been shown to exhibit antitumor activity in several cancers. Here, we reported that a novel synthetic Berbamine derivative, BBMD3, inhibits cell viability and induces apoptosis of cancer stem-like cells (CSCs in a time- and dose-dependent manner when the CSCs from four GBM patients (PBT003, PBT008, PBT022, and PBT030 were cultured. These CSCs grew in neurospheres and expressed CD133 and nestin as markers. Treatment with BBMD3 destroyed the neurosphere morphology, and led to the induction of apoptosis in the CSCs. Induction of apoptosis in these CSCs is dependent upon activation of caspase-3 and cleavage of poly (ADP-ribose polymerase (PARP. MicroRNA-4284 (miR-4284 was shown to be over-expressed about 4-fold in the CSCs following BBMD3 treatment. Furthermore, transfection of synthetic anti-sense oligonucleotide against human miR-4284 partially blocked the anticancer effects of BBMD3 on the GBM derived CSCs. BBMD3 also increased phosphorylation of the c-Jun N-terminal kinase (JNK/stress-activated protein kinase (SAPK, resulting in an increase expression of phosphorylated c-Jun and total c-Fos; the major components of transcriptional factor AP-1. The JNK-c-Jun/AP-1 signaling pathway plays an important role in the induction of apoptosis in response to UV irradiation and some drug treatments. Targeting glioblastoma stem-like cells with BBMD3 is therefore novel, and may have promise as an

  8. Human white blood cells contain cyclobutyl pyrimidine dimer photolyase

    Energy Technology Data Exchange (ETDEWEB)

    Sutherland, B.M.; Bennett, P.V. [Brookhaven National Lab., Upton, NY (United States)

    1995-10-10

    Although enzymatic photoreactivation of cyclobutyl pyrimidine dimers in DNA is present in almost all organisms, its presence in placental mammals is controversial. We tested human white blood cells for photolyase by using three defined DNAs (suprecoiled pET-2, nonsupercoiled bacteriphage {lambda}, and a defined-sequence 287-bp oligonucleotide), two dimer-specific endonucleases (T4 endonuclease V and UV endonuclease from Micrococcus luteus), and three assay methods. We show that human white blood cells contain photolyase that can photorepair pyrimidine dimers in defined supercoiled and linear DNAs and in a 287-bp oligonucleotide and that human photolyase is active on genomic DNA in intact human cells. 44 refs., 3 figs.

  9. [Endonuclease modified comet assay for oxidative DNA damage induced by detection of genetic toxicants].

    Science.gov (United States)

    Zhao, Jian; Li, Hongli; Zhai, Qingfeng; Qiu, Yugang; Niu, Yong; Dai, Yufei; Zheng, Yuxin; Duan, Huawei

    2014-03-01

    The aim of this study was to investigate the use of the lesion-specific endonucleases-modified comet assay for analysis of DNA oxidation in cell lines. DNA breaks and oxidative damage were evaluated by normal alkaline and formamidopyrimidine-DNA-glycosylase (FPG) modified comet assays. Cytotoxicity were assessed by MTT method. The human bronchial epithelial cell (16HBE) were treated with benzo (a) pyrene (B(a)P), methyl methanesulfonate (MMS), colchicine (COL) and vincristine (VCR) respectively, and the dose is 20 µmol/L, 25 mg/ml, 5 mg/L and 0.5 mg/L for 24 h, respectively. Oxidative damage was also detected by levels of reactive oxygen species in treated cells. Four genotoxicants give higher cytotoxicity and no significant changes on parameters of comet assay treated by enzyme buffer. Cell survival rate were (59.69 ± 2.60) %, (54.33 ± 2.81) %, (53.11 ± 4.00) %, (51.43 ± 3.92) % in four groups, respectively. There was the direct DNA damage induced by test genotoxicants presented by tail length, Olive tail moment (TM) and tail DNA (%) in the comet assay. The presence of FPG in the assays increased DNA migration in treated groups when compared to those without it, and the difference was statistically significant which indicated that the clastogen and aneugen could induce oxidative damage in DNA strand. In the three parameters, the Olive TM was changed most obviously after genotoxicants treatment. In the contrast group, the Olive TM of B(a) P,MMS, COL,VCR in the contrast groups were 22.99 ± 17.33, 31.65 ± 18.86, 19.86 ± 9.56 and 17.02 ± 9.39, respectively, after dealing with the FPG, the Olive TM were 34.50 ± 17.29, 43.80 ± 10.06, 33.10 ± 12.38, 28.60 ± 10.53, increased by 58.94%, 38.48%, 66.86% and 68.21%, respectively (t value was 3.91, 3.89, 6.66 and 3.87, respectively, and all P comet assay appears more specific for detecting oxidative DNA damage induced by genotoxicants exposure, and the application of comet assay will be expanded. The endonuclease

  10. Type II restriction endonucleases--a historical perspective and more.

    Science.gov (United States)

    Pingoud, Alfred; Wilson, Geoffrey G; Wende, Wolfgang

    2014-07-01

    This article continues the series of Surveys and Summaries on restriction endonucleases (REases) begun this year in Nucleic Acids Research. Here we discuss 'Type II' REases, the kind used for DNA analysis and cloning. We focus on their biochemistry: what they are, what they do, and how they do it. Type II REases are produced by prokaryotes to combat bacteriophages. With extreme accuracy, each recognizes a particular sequence in double-stranded DNA and cleaves at a fixed position within or nearby. The discoveries of these enzymes in the 1970s, and of the uses to which they could be put, have since impacted every corner of the life sciences. They became the enabling tools of molecular biology, genetics and biotechnology, and made analysis at the most fundamental levels routine. Hundreds of different REases have been discovered and are available commercially. Their genes have been cloned, sequenced and overexpressed. Most have been characterized to some extent, but few have been studied in depth. Here, we describe the original discoveries in this field, and the properties of the first Type II REases investigated. We discuss the mechanisms of sequence recognition and catalysis, and the varied oligomeric modes in which Type II REases act. We describe the surprising heterogeneity revealed by comparisons of their sequences and structures. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. The wonders of flap endonucleases: structure, function, mechanism and regulation.

    Science.gov (United States)

    Finger, L David; Atack, John M; Tsutakawa, Susan; Classen, Scott; Tainer, John; Grasby, Jane; Shen, Binghui

    2012-01-01

    Processing of Okazaki fragments to complete lagging strand DNA synthesis requires coordination among several proteins. RNA primers and DNA synthesised by DNA polymerase α are displaced by DNA polymerase δ to create bifurcated nucleic acid structures known as 5'-flaps. These 5'-flaps are removed by Flap Endonuclease 1 (FEN), a structure-specific nuclease whose divalent metal ion-dependent phosphodiesterase activity cleaves 5'-flaps with exquisite specificity. FENs are paradigms for the 5' nuclease superfamily, whose members perform a wide variety of roles in nucleic acid metabolism using a similar nuclease core domain that displays common biochemical properties and structural features. A detailed review of FEN structure is undertaken to show how DNA substrate recognition occurs and how FEN achieves cleavage at a single phosphate diester. A proposed double nucleotide unpairing trap (DoNUT) is discussed with regards to FEN and has relevance to the wider 5' nuclease superfamily. The homotrimeric proliferating cell nuclear antigen protein (PCNA) coordinates the actions of DNA polymerase, FEN and DNA ligase by facilitating the hand-off intermediates between each protein during Okazaki fragment maturation to maximise through-put and minimise consequences of intermediates being released into the wider cellular environment. FEN has numerous partner proteins that modulate and control its action during DNA replication and is also controlled by several post-translational modification events, all acting in concert to maintain precise and appropriate cleavage of Okazaki fragment intermediates during DNA replication.

  12. Peculiarities of Crystallization of the Restriction Endonuclease EcoRII

    Science.gov (United States)

    Karpove, Elizaveta; Pusey, M.arc L.

    1998-01-01

    Nucleases interfere with most standard molecular biology procedures. We have purified and crystallized the restriction endonuclease EcoRII, which belongs to the type II of restriction- modification enzyme, to study the protein crystallization process using a "non standard" macromolecule. A procedure for the purification of EcoRII was developed and 99% pure protein as determined by SDS PAGE electrophoresis obtained. Light scattering experiments were performed to assist in screening protein suitable crystallization conditions. The second virial coefficient was determined as a function of precipitating salt concentration, using sodium chloride, ammonium sulfate, and sodium sulfate. Small (maximum size approximately 0.2 mm) well shaped crystals have been obtained. Larger poorly formed crystals (ca 0.5 mm) have also been obtained, but we have been unable to mount them for diff-raction analysis due to their extreme fragility. Crystallization experiments with PEG have shown that using this precipitant, the best crystals are obtained from slightly over-saturated solutions. Use of higher precipitant concentration leads to dendritic crystal formation. EcoRII is difficult to solubilize and meticulous attention must be paid to the presence of reducing agents.

  13. Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

    KAUST Repository

    Rashid, Fahad

    2017-02-23

    Human flap endonuclease 1 (FEN1) and related structure-specific 5\\'nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5\\'nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually \\'locks\\' protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.

  14. Low Earth orbit assessment of proton anisotropy using AP8 and AP9 trapped proton models.

    Science.gov (United States)

    Badavi, Francis F; Walker, Steven A; Santos Koos, Lindsey M

    2015-04-01

    The completion of the International Space Station (ISS) in 2011 has provided the space research community with an ideal evaluation and testing facility for future long duration human activities in space. Ionized and secondary neutral particles radiation measurements inside ISS form the ideal tool for validation of radiation environmental models, nuclear reaction cross sections and transport codes. Studies using thermo-luminescent detectors (TLD), tissue equivalent proportional counter (TPEC), and computer aided design (CAD) models of early ISS configurations confirmed that, as input, computational dosimetry at low Earth orbit (LEO) requires an environmental model with directional (anisotropic) capability to properly describe the exposure of trapped protons within ISS. At LEO, ISS encounters exposure from trapped electrons, protons and geomagnetically attenuated galactic cosmic rays (GCR). For short duration studies at LEO, one can ignore trapped electrons and ever present GCR exposure contributions during quiet times. However, within the trapped proton field, a challenge arises from properly estimating the amount of proton exposure acquired. There exist a number of models to define the intensity of trapped particles. Among the established trapped models are the historic AE8/AP8, dating back to the 1980s and the recently released AE9/AP9/SPM. Since at LEO electrons have minimal exposure contribution to ISS, this work ignores the AE8 and AE9 components of the models and couples a measurement derived anisotropic trapped proton formalism to omnidirectional output from the AP8 and AP9 models, allowing the assessment of the differences between the two proton models. The assessment is done at a target point within the ISS-11A configuration (circa 2003) crew quarter (CQ) of Russian Zvezda service module (SM), during its ascending and descending nodes passes through the south Atlantic anomaly (SAA). The anisotropic formalism incorporates the contributions of proton narrow

  15. Preparing Students for the AP Psychology Exam

    Science.gov (United States)

    Whitlock, Kristin

    2013-01-01

    The Advanced Placement Psychology exam is one of the fastest growing exams offered by the College Board. The average percent of change in the number of students taking this exam over the past five years is 12.4%. With 238,962 students taking the exam in 2013, the AP Psychology exam is the sixth largest exam, surpassing AP Biology and AP World…

  16. An AP Calculus Classroom Amusement Park

    Science.gov (United States)

    Ferguson, Sarah

    2016-01-01

    Throughout the school year, AP Calculus teachers strive to teach course content comprehensively and swiftly in an effort to finish all required material before the AP Calculus exam. As early May approaches and the AP Calculus test looms, students and teachers nervously complete lessons, assignments, and assessments to ensure student preparation.…

  17. Molecular evolution of the AP2 subfamily.

    Science.gov (United States)

    Shigyo, Mikao; Hasebe, Mitsuyasu; Ito, Motomi

    2006-02-01

    The AP2 (APETALA2)/EREBP (Ethylene Responsive Element Binding Protein) multigene family includes developmentally and physiologically important transcription factors. AP2/EREBP genes are divided into two subfamilies: AP2 genes with two AP2 domains and EREBP genes with a single AP2/ERF (Ethylene Responsive Element Binding Factor) domain. Based on previous phylogenetic analyses, AP2 genes can be divided into two clades, AP2 and ANT groups. To clarify the molecular evolution of the AP2 subfamily, we isolated and sequenced genes with two AP2 domains from three gymnosperms, Cycas revoluta, Ginkgo biloba, and Gnetum parvifolium,as well as from the moss Physcomitrella patens. Expressions of AP2-like genes, including AP2, in Arabidopsis thaliana are regulated by the microRNA miR172. We found that the target site of miR172 is significantly conserved in gymnosperm AP2 homologs, suggesting that regulatory mechanisms of gene expression using microRNA have been conserved over the three hundred million years since the divergence of gymnosperm and flowering plant lineages. We inferred a phylogenetic relationship of these genes with the green alga Chlamydomonas reinhardtii and seed-plant genes available in public DNA databases. The phylogenetic tree showed that the AP2 subfamily diverged into the AP2 and ANT groups before the last common ancestor of land plants and after C. reinhardtii diverged from the land-plant lineage. The tree also indicated that each AP2 and ANT group further diverged into several clades through gene duplications prior to the divergence of gymnosperms and angiosperms.

  18. Investigation of mutations induced by radiation and restriction endonucleases

    Science.gov (United States)

    Haworth, Kim E.

    The effects of gamma radiation and restriction endonuclease (RE) induced DNA double strand breaks (dsb) upon the mutation frequency and the surviving fraction of three Chinese hamster cell lines V79-4, CHO-K1 and an X-ray sensitive dsb repair deficient cell line xrs-5 were studied. The X-ray sensitive xrs-5 cell line was shown to be more sensitive to both the lethal and the mutagenic effects of gamma radiation having a substantially lower surviving fraction and a higher thymidine kinase (tk) mutation frequency per unit dose than the parental CHO-K1 cells. The frequency of induced hprt- mutations in the V79-4 cell line was comparable to the induced frequency of tk mutations in the CHO-K1 cells. The effect of blunt- and cohesive- ended dsb upon the surviving fraction and the induced mutation frequency was studied by porating different Chinese hamster cell lines (CHO-K1, V79-4 and xrs-5) with RE using Streptolysin O (SLO). The surviving fraction of the different cell lines was reduced with increasing concentrations of Pvu II. Increases in the concentration of Pvu II produced increases in the frequency of hypoxyanthine guanine phosphoribosyl transferase (hprt) mutations in the V79-4 cells and tk mutations in the CHO-K1 and xrs-5 cells. However, the xrs-5 cells were shown to be hypomutable to Pvu II compared with the parental CHO-K1 cells. EcoR1 was ineffective at inducing tk mutations in the CHO-Kl cells but was as effective as Pvu II at inducing hprt mutations in the V79-4 cells. None of the spontaneously induced V79-4 hprt- mutant cells were shown to have observable molecular deletions when analysed by PCR deletion screening. One third of the radiation induced hprt - mutants were shown to be deletions. However, too few mutant cells were analysed for any non-random distribution of deletions to be observed. Half of the hprt- mutants induced by SLO poration alone were shown to be due to deletions of oi\\e or more exons. The distribution of the DNA deletions in SLO hprt

  19. Fellow's Apéro

    CERN Multimedia

    Staff Association

    2017-01-01

    Let's get together, meet each other, exchange experiences and ideas, and share useful information on CERN and the Staff Association. Join us for Fellow's Apéro, organised by the Staff Association on Tuesday 21 February at 16.30 in Restaurant 1. There will be drinks and snacks for everybody! We look forward to seeing you there! Please confirm your participation on Doodle http://doodle.com/poll/skvm7ucm2z78i6bt or alternatively on Facebook https://www.facebook.com/events/1862757017340069/. Your delegates in the Staff Association, Barbora & Jiri

  20. Testing of Solar Heated Domestic Hot Water System for Solahart Scandinavia ApS

    DEFF Research Database (Denmark)

    Andersen, Elsa

    1997-01-01

    The solar heating system marketed by Solahart Scandinavia ApS was tested in the Institutes test facility for SDHWsystems. The test results are described in the report.......The solar heating system marketed by Solahart Scandinavia ApS was tested in the Institutes test facility for SDHWsystems. The test results are described in the report....

  1. APS high heat load monochromator

    Energy Technology Data Exchange (ETDEWEB)

    Lee, W.K.; Mills, D.

    1993-02-01

    This document contains the design specifications of the APS high heat load (HHL) monochromator and associated accessories as of February 1993. It should be noted that work is continuing on many parts of the monochromator including the mechanical design, crystal cooling designs, etc. Where appropriate, we have tried to add supporting documentation, references to published papers, and calculations from which we based our decisions. The underlying philosophy behind performance specifications of this monochromator was to fabricate a device that would be useful to as many APS users as possible, that is, the design should be as generic as possible. In other words, we believe that this design will be capable of operating on both bending magnet and ID beamlines (with the appropriate changes to the cooling and crystals) with both flat and inclined crystal geometries and with a variety of coolants. It was strongly felt that this monochromator should have good energy scanning capabilities over the classical energy range of about 4 to 20 keywith Si (111) crystals. For this reason, a design incorporating one rotation stage to drive both the first and second crystals was considered most promising. Separate rotary stages for the first and second crystals can sometimes provide more flexibility in their capacities to carry heavy loads (for heavily cooled first crystals or sagittal benders of second crystals), but their tuning capabilities were considered inferior to the single axis approach.

  2. Simple and sensitive fluorescence assay of restriction endonuclease on graphene oxide

    Energy Technology Data Exchange (ETDEWEB)

    Gang, Jong Back [Dept. of Nano Chemistry, Gachon University, Incheon (Korea, Republic of)

    2015-09-15

    Restriction endonucleases hydrolyze internal phosphodiester bonds at specific sites in a DNA sequence. These enzymes are essential in a variety of fields, such as biotechnology and clinical diagnostics. It is of great importance and necessity for the scientific and biomedical use of enzymes to measure endonuclease activity. In this study, graphene oxide (GO) has been used as a platform to measure enzyme activity with high sensitivity. To increase the detection sensitivity of Hinf I, the endonuclease-digested reaction was treated with exonuclease III (Exo III) and a fluorescence assay was conducted to measure the emission. Results showed that Exo III treatment enhanced 2.7-fold signal-to-background ratio for the detection of Hinf I compared with that done without Exo III in the presence of GO.

  3. PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance

    Science.gov (United States)

    van Oers, Johanna M. M.; Roa, Sergio; Werling, Uwe; Liu, Yiyong; Genschel, Jochen; Sellers, Rani S.; Modrich, Paul; Scharff, Matthew D.; Edelmann, Winfried

    2010-01-01

    The DNA mismatch repair protein PMS2 was recently found to encode a novel endonuclease activity. To determine the biological functions of this activity in mammals, we generated endonuclease-deficient Pms2E702K knock-in mice. Pms2EK/EK mice displayed increased genomic mutation rates and a strong cancer predisposition. In addition, class switch recombination, but not somatic hypermutation, was impaired in Pms2EK/EK B cells, indicating a specific role in Ig diversity. In contrast to Pms2−/− mice, Pms2EK/EK male mice were fertile, indicating that this activity is dispensable in spermatogenesis. Therefore, the PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance and tumor suppression. PMID:20624957

  4. A site-specific endonuclease encoded by a typical archaeal intron

    DEFF Research Database (Denmark)

    Dalgaard, Jacob; Garrett, Roger Antony; Belfort, Malene

    1993-01-01

    . Additionally, although the archaeal and group I introns have entirely different structural properties and splicing pathways, I-Dmo I shares sequence similarity, in the form of the LAGLI-DADG motif, with group I intron endonucleases of eukaryotes. These observations support the independent evolutionary origin......The protein encoded by the archaeal intron in the 23S rRNA gene of the hyperthermophile Desulfurococcus mobilis is a double-strand DNase that, like group I intron homing endonucleases, is capable of cleaving an intronless allele of the gene. This enzyme, I-Dmo I, is unusual among the intron...... endonucleases in that it is thermostable and is expressed only from linear and cyclized intron species and not from the precursor RNA. However, in analogy to its eukaryotic counterparts, but unlike the bacteriophage enzymes, I-Dmo I makes a staggered double-strand cut that generates 4-nt 3' extensions...

  5. Protein NCRII-18: the role of gene fusion in the molecular evolution of restriction endonucleases.

    Science.gov (United States)

    Ibryashkina, Elena M; Solonin, Alexander S; Zakharova, Marina V

    2017-06-01

    This work first constructed the fusion protein NCRII-18 by fusing the restriction endonuclease Ecl18kI gene and part of the gene coding for the N-terminal domain of the endonuclease EcoRII. The fusion of the EcoRII N-terminal domain leads to a change in the properties of the recombinant protein. Unlike Ecl18kI, which made the basis of NCRII-18, the fusion protein predominantly recognizes the CCWGG sites, having lost the capability of interacting with the CCSGG sites. Experimental data support the hypothesis of a close evolutionary relationship between type IIE and IIP restriction endonucleases via a recombination between domains with active site structure and elements for recognition with domains responsible for recognition of DNA sequences. © 2017 Federation of European Biochemical Societies.

  6. The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage

    Science.gov (United States)

    Fu, Haiqing; Martin, Melvenia M.; Regairaz, Marie; Huang, Liang; You, Yang; Lin, Chi-Mei; Ryan, Michael; Kim, RyangGuk; Shimura, Tsutomu; Pommier, Yves; Aladjem, Mirit I.

    2015-01-01

    The Mus81 endonuclease resolves recombination intermediates and mediates cellular responses to exogenous replicative stress. Here, we show that Mus81 also regulates the rate of DNA replication during normal growth by promoting replication fork progression while reducing the frequency of replication initiation events. In the absence of Mus81 endonuclease activity, DNA synthesis is slowed and replication initiation events are more frequent. In addition, Mus81 deficient cells fail to recover from exposure to low doses of replication inhibitors and cell viability is dependent on the XPF endonuclease. Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells. Therefore, decelerated DNA replication in Mus81 deficient cells does not initiate from cryptic or latent origins not used during normal growth. These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins. PMID:25879486

  7. Cell-autonomous progeroid changes in conditional mouse models for repair endonuclease XPG deficiency.

    Directory of Open Access Journals (Sweden)

    Sander Barnhoorn

    2014-10-01

    Full Text Available As part of the Nucleotide Excision Repair (NER process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS, or the infantile lethal cerebro-oculo-facio-skeletal (COFS syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional Xpg-/- mouse model which -in a C57BL6/FVB F1 hybrid genetic background- displays many progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4-5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the Xpg-/- mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging.

  8. DNA-hosted Hoechst dyes: application for label-free fluorescent monitoring of endonuclease activity and inhibition.

    Science.gov (United States)

    Jiang, Xiao-Qin; Guo, Su-Miao; Zhang, Min; Zhou, Ming; Ye, Bang-Ce

    2014-11-21

    A simple and facile approach was developed for monitoring EcoRI endonuclease activity and inhibition, in which a hairpin-like DNA containing restriction cutting site for EcoRI endonuclease acts as the sensing element and Hoechst dyes as the signal indicator in a label-free format.

  9. Polymorphic restriction endonuclease fragment segregates and correlates with the gene for HLA-B8.

    OpenAIRE

    Cann, H M; Ascanio, L; Paul, P; Marcadet, A; Dausset, J; Cohen, D

    1983-01-01

    Cellular DNA from HLA-typed individuals was digested with the restriction endonuclease EcoRV. After electrophoresis and transfer to a hybridization membrane, the restriction endonuclease fragments were probed with cDNA carrying the nucleotide sequence encoding a class 1 HLA gene. Polymorphism for presence or absence of various EcoRV fragments was noted in a panel of unrelated HLA-typed individuals. A polymorphic 8.6-kilobase pair EcoRV fragment was found which correlated in the panel with the...

  10. Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases.

    Science.gov (United States)

    Crouzier, Lucile; Dubois, Camille; Wengel, Jesper; Veedu, Rakesh N

    2012-07-15

    Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving LNA-modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Heterologous expression in Tritrichomonas foetus of functional Trichomonas vaginalis AP65 adhesin

    Directory of Open Access Journals (Sweden)

    Alderete JF

    2005-03-01

    Full Text Available Abstract Background Trichomonosis, caused by Trichomonas vaginalis, is the number one, nonviral sexually transmitted infection that has adverse consequences for the health of women and children. The interaction of T. vaginalis with vaginal epithelial cells (VECs, a step preparatory to infection, is mediated in part by the prominent surface protein AP65. The bovine trichomonad, Tritrichomonas foetus, adheres poorly to human VECs. Thus, we established a transfection system for heterologous expression of the T. vaginalis AP65 in T. foetus, as an alternative approach to confirm adhesin function for this virulence factor. Results In this study, we show stable transfection and expression of the T. vaginalis ap65 gene in T. foetus from an episomal pBS-ap65-neo plasmid. Expression of the gene and protein was confirmed by RT-PCR and immunoblots, respectively. AP65 in transformed T. foetus bound to host cells. Specific mAbs revealed episomally-expressed AP65 targeted to the parasite surface and hydrogenosome organelles. Importantly, surface-expression of AP65 in T. foetus paralleled increased levels of adherence of transfected bovine trichomonads to human VECs. Conclusion The T. vaginalis AP65 adhesin was stably expressed in T. foetus, and the data obtained using this heterologous system strongly supports the role of AP65 as a prominent adhesin for T. vaginalis. In addition, the heterologous expression in T. foetus of a T. vaginalis gene offers an important, new approach for confirming and characterizing virulence factors.

  12. Apéndice C

    OpenAIRE

    2015-01-01

    Fuente: AAL, Divorcios, Leg. 1-63. Este cuadro se basa en una muestra de 20% de todos los juicios de divorcio entre 1569 y 1713. Los totales de 20% por periodo son: 1569-1650 (No = 81); 1651-1700 (No = 225); 1701-1713 (No = 28). Al calcular datos específicos sobre el lugar de depósito, algunos casos no los tenían. Entre 1569 y 1650, 24 de 81 (29%) no tenían datos; entre 1651 y 1713, 71 de 253 (28%) no los tenían. Para los totales numéricos, véase en el apéndice F una discusión de los métodos ...

  13. APS: Lighting up the future

    Energy Technology Data Exchange (ETDEWEB)

    Potent, V.J.

    1993-09-01

    Work on the Advanced Photon Source (APS) at Argonne National Laboratory (ANL) involves the construction and supporting research and development for a national user facility for synchrotron radiation research in the x-ray region. The facility, when operational in 1997, will provide super-intense x-ray beams for many areas of basic research and will serve the entire US x-ray research community of several thousand users. This paper describes the pertinent features of the design, construction and planned operation of the facility; and the impact quality has had in these areas. In addition, the introduction of several quality management techniques such as total quality management, reliability/availability planning, and user interface are discussed concerning their status and success.

  14. Positioning the 5'-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage

    KAUST Repository

    Song, Bo

    2018-02-09

    Flap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair and recombination, and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca2+ have suggested that base unpairing at the 5\\'-flap:template junction is an important step in the reaction, but the new structural data suggest otherwise. To clarify the role of DNA changes in the kinetic mechanism, we measured a series of transient steps - from substrate binding to product release - during the hFEN1-catalyzed reaction in the presence of Mg2+. We found that while hFEN1 binds and bends DNA at a fast, diffusion-limited rate, much slower Mg2+-dependent conformational changes in DNA around the active site are subsequently necessary and rate-limiting for 5\\'-flap cleavage. These changes are reported overall by fluorescence of 2-aminopurine at the 5\\'-flap:template junction, indicating that local DNA distortion (e.g., disruption of base stacking observed in structures), associated with positioning the 5\\'-flap scissile phosphodiester bond in the hFEN1 active site, controls catalysis. hFEN1 residues with distinct roles in the catalytic mechanism, including those binding metal ions (Asp-34, Asp-181), steering the 5\\'-flap through the active site and binding the scissile phosphate (Lys-93, Arg-100), and stacking against the base 5\\' to the scissile phosphate (Tyr-40), all contribute to these rate-limiting conformational changes, ensuring efficient and specific cleavage of 5\\'-flaps.

  15. AP calculus AB & BC crash course

    CERN Document Server

    Rosebush, J

    2012-01-01

    AP Calculus AB & BC Crash Course - Gets You a Higher Advanced Placement Score in Less Time Crash Course is perfect for the time-crunched student, the last-minute studier, or anyone who wants a refresher on the subject. AP Calculus AB & BC Crash Course gives you: Targeted, Focused Review - Study Only What You Need to Know Crash Course is based on an in-depth analysis of the AP Calculus AB & BC course description outline and actual AP test questions. It covers only the information tested on the exams, so you can make the most of your valuable study time. Written by experienced math teachers, our

  16. Consumo de antimicrobianos en APS

    Directory of Open Access Journals (Sweden)

    María Cristina Lara Bastanzuri

    2003-08-01

    Full Text Available Con el propósito de describir el patrón de uso de los medicamentos para el tratamiento con antimicrobianos en APS en Cuba en el período de 1989 al 2000, se diseñó un estudio descriptivo, observacional y retrospectivo clasificado dentro de los estudios de utilización de medicamentos como de consumo. Para ello se calcularon las dosis diarias definidas cada día de determinado fármaco de los grupos clasificados según ATC como tetraciclinas, anfenicoles, penicilinas de amplio espectro, cefalosporinas, sulfonamidas y trimetoprim, macrólidos, estreptomicinas y quinolonas. Además, se tomaron las cifras de pacientes inscriptos a penicilina benzatínica y se comparó con la población expuesta obtenida a partir de las DHD. Las penicilinas son las de mayor consumo con tendencia al aumento, igual que los aminoglucósidos, mientras que la tetraciclina presenta cifras mayores de DHD. La tendencia del cloranfenicol es a disminuir. La población expuesta está muy por debajo de los pacientes inscriptos en penicilina benzatínica y las líneas de tendencia no son similares. Excepto la docixiclina, el resto de los antimicrobianos recomendados en la Guía Terapéutica para APS se encuentran en el Listado Básico de Medicamentos del país para el nivel primario de atención médica.With the objective of describing the pattern of drug use in antimicrobial-based treatment in the primary health care in Cuba from 1989 to 2000, we designed a retrospective descriptive and observational study, classified into the study of drug use as consumption. To this end, the daily doses of certain drugs from the ATC-classified groups were calculated, which included tetracycline, amphenicols, broad spectrum penicilins, cephalosporins, sulfonamides, trimethoprim, macrolides, streptomycin and quinolones. Also, the number of patients registered as benzathine peniciline consumers was taken and compared to the exposed population data obtained from the DHD. Penicillins are the

  17. Antibiotic resistance and restriction endonucleases in fecal enterococci of chamois (Rupicapra rupicapra Linnaeus, 1758).

    Science.gov (United States)

    Vandžurová, A; Hrašková, I; Júdová, J; Javorský, P; Pristaš, P

    2012-07-01

    Two hundred eighty-four isolates of enterococci from feces of wild living chamois from alpine environments were tested for sensitivity to three antibiotics. Low frequency of resistance was observed in studied enterococcal populations (about 5 % for tetracycline and erythromycin and 0 % for ampicillin). In six animals, the population of enterococci lacked any detectable resistance. Our data indicated that enterococcal population in feces of the majority of studied animals did not encounter mobile genetic elements encoding antibiotic resistance probably due to spatial separation and/or due to low exposure to the antibiotics. Based on resistance profiles observed, three populations were analyzed for the presence of restriction endonucleases. The restriction enzymes from two isolates-31K and 1K-were further purified and characterized. Restriction endonuclease Efa1KI recognizes CCWGG sequence and is an isoschizomer of BstNI. Endonuclease Efc31KI, a BsmAI isoschizomer, recognizes the sequence GTCTC and it is a first restriction endonuclease identified in Enterococcus faecium. Our data indicate that restriction-modification (R-M) systems do not represent an efficient barrier for antibiotic resistance spreading; enterococcal populations colonized by antibiotics resistance genes were also colonized by the R-M systems.

  18. Structural studies on metal-containing enzymes: T4 endonuclease VII and D. gigas formate dehydrogenase

    NARCIS (Netherlands)

    Raaijmakers, H.C.A.

    2001-01-01

    Many biological processes require metal ions, and many of these metal-ion functions involve metalloproteins. The metal ions in metalloproteins are often critical to the protein's function, structure, or stability. This thesis focuses on two of these proteins, bacteriophage T4 endonuclease

  19. Altered UV resistance and UV mutational spectrum in repair-proficient murine fibroblasts expressing endonuclease V.

    Science.gov (United States)

    Kusewitt, D F; Dyble, J; Sherburn, T E; Ryan, S L; Ji, J Y

    1998-03-01

    In previously reported studies, we transfected repair-proficient murine fibroblasts with the denV gene of bacteriophage T4 and showed that expression of encoded endonuclease V markedly enhanced cyclobutane pyrimidine dimer (CPD) repair and reduced the frequency of ultraviolet radiation (UV)-induced mutations. In the present studies, we compared the spectra of UV-induced mutations at the hprt locus in denV-transfected and control cells. A significant difference in mutation types was observed. While multiple base deletions and single base insertions were found in denV-transfected but not control cells, multiple tandem and non-tandem point mutations identified in control cells were absent in denV-transfected cells. When we compared colony survival following UV exposure in the two cell lines, it appeared that endonuclease V expression did not enhance UV resistance, instead denV-transfected cells had increased susceptibility to low fluences of UV. The effects of endonuclease V expression on UV resistance and on UV mutational spectrum are likely to be due both to the removal of CPDs and to the novel enzymatic activity of endonuclease V.

  20. Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

    Science.gov (United States)

    Aouida, Mustapha; Eid, Ayman; Ali, Zahir; Cradick, Thomas; Lee, Ciaran; Deshmukh, Harshavardhan; Atef, Ahmed; AbuSamra, Dina; Gadhoum, Samah Zeineb; Merzaban, Jasmeen; Bao, Gang; Mahfouz, Magdy

    2015-01-01

    The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells. PMID:26225561

  1. PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair

    Science.gov (United States)

    Pluciennik, Anna; Dzantiev, Leonid; Iyer, Ravi R.; Constantin, Nicoleta; Kadyrov, Farid A.; Modrich, Paul

    2010-01-01

    MutLα (MLH1–PMS2) is a latent endonuclease that is activated in a mismatch-, MutSα-, proliferating cell nuclear antigen (PCNA)-, replication factor C (RFC)-, and ATP-dependent manner, with nuclease action directed to the heteroduplex strand that contains a preexisting break. RFC depletion experiments and use of linear DNAs indicate that RFC function in endonuclease activation is limited to PCNA loading. Whereas nicked circular heteroduplex DNA is a good substrate for PCNA loading and for endonuclease activation on the incised strand, covalently closed, relaxed circular DNA is a poor substrate for both reactions. However, covalently closed supercoiled or bubble-containing relaxed heteroduplexes, which do support PCNA loading, also support MutLα activation, but in this case cleavage strand bias is largely abolished. Based on these findings we suggest that PCNA has two roles in MutLα function: The clamp is required for endonuclease activation, an effect that apparently involves interaction of the two proteins, and by virtue of its loading orientation, PCNA determines the strand direction of MutLα incision. These results also provide a potential mechanism for activation of mismatch repair on nonreplicating DNA, an effect that may have implications for the somatic phase of triplet repeat expansion. PMID:20713735

  2. Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

    KAUST Repository

    Aouida, Mustapha

    2015-07-30

    The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

  3. Assaying multiple restriction endonucleases functionalities and inhibitions on DNA microarray with multifunctional gold nanoparticle probes.

    Science.gov (United States)

    Ma, Lan; Zhu, Zhijun; Li, Tao; Wang, Zhenxin

    2014-02-15

    Herein, a double-stranded (ds) DNA microarray-based resonance light scattering (RLS) assay with multifunctional gold nanoparticle (GNP) probes has been developed for studying restriction endonuclease functionality and inhibition. Because of decreasing significantly melting temperature, the enzyme-cleaved dsDNAs easily unwind to form single-stranded (ss) DNAs. The ssDNAs are hybridized with multiplex complementary ssDNAs functionalized GNP probes followed by silver enhancement and RLS detection. Three restriction endonucleases (EcoRI, BamHI and EcoRV) and three potential inhibitors (doxorubicin hydrochloride (DOX), ethidium bromide (EB) and an EcoRI-derived helical peptide (α4)) were selected to demonstrate capability of the assay. Enzyme activities of restriction endonucleases are detected simultaneously with high specificity down to the limits of 2.0 × 10(-2)U/mL for EcoRI, 1.1 × 10(-2)U/mL for BamHI and 1.6 × 10(-2)U/mL for EcoRV, respectively. More importantly, the inhibitory potencies of three inhibitors are showed quantitatively, indicating that our approach has great promise for high-throughput screening of restriction endonuclease inhibitors. © 2013 Elsevier B.V. All rights reserved.

  4. Arthrobacter luteus restriction endonuclease cleavage map of X174 RF DNA

    NARCIS (Netherlands)

    Vereijken, J.M.; Mansfeld, A.D.M. van; Baas, P.D.; Jansz, H.S.

    1975-01-01

    Cleavage of X174 RF DNA with the restriction endonuclease from Arthrobacter luteus (Alu I) produces 23 fragments of approximately 24–1100 base pairs in length. The order of most of these fragments has been established by digestion of Haemophilus influenzae Rd (Hind II) and Haemophilus aegyptius (Hae

  5. Sequence-dependent cleavage of mismatched DNA by Ban I restriction endonuclease.

    Science.gov (United States)

    Gao, Weimin; Zhu, Dan; Keohavong, Phouthone

    2017-10-01

    Restriction enzymes have previously shown the ability to cleave DNA substrates with mismatched base(s) in recognition sequences; in this study, Ban I endonuclease demonstrated this same ability. Single base substitutions were introduced, and fragments containing various types of unpaired base(s) (heteroduplex fragments) within the Ban I endonuclease recognition sequence, 5'-G|GPyPuCC-3', were generated. Each of the heteroduplex fragments was treated with Ban I endonuclease and analyzed by denaturing gradient gel electrophoresis. Our results showed that heteroduplex fragments containing mismatched bases at either the first or third position of the Ban I recognition sequence or, because of the symmetrical structure of the sequence, the sixth or fourth position on the opposite strand were cleaved by the enzyme. Furthermore, these cleaved fragments contained at least one strand corresponding to the original Ban I recognition sequence. Fragments with mismatches formed by an A (noncanonical, nc) opposite a purine (canonical, ca) or a T (nc) opposite a pyrimidine (ca) were cleaved more efficiently than other types of mismatched bases. These results may help elucidate the mechanisms by which DNA and protein interact during the process of DNA cleavage by Ban I endonuclease. Copyright © 2017 John Wiley & Sons, Ltd.

  6. Problem-solving test: digestion of a plasmid with restriction endonucleases.

    Science.gov (United States)

    Szeberényi, József

    2013-01-01

    Terms to be familiar with before you start to solve the test: plasmid, restriction endonuclease, agarose gel electrophoresis, ethidium bromide staining, autoradiography, Coomassie staining, Southern blotting, linear and circular DNA, superhelical DNA, exonuclease, modification methylase, palindrome, sticky and blunt ends, nicked circular DNA. Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  7. BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism.

    Science.gov (United States)

    Raskó, Tamás; Dér, András; Klement, Eva; Slaska-Kiss, Krystyna; Pósfai, Eszter; Medzihradszky, Katalin F; Marshak, Daniel R; Roberts, Richard J; Kiss, Antal

    2010-11-01

    The GGCC-specific restriction endonuclease BspRI is one of the few Type IIP restriction endonucleases, which were suggested to be a monomer. Amino acid sequence information obtained by Edman sequencing and mass spectrometry analysis was used to clone the gene encoding BspRI. The bspRIR gene is located adjacently to the gene of the cognate modification methyltransferase and encodes a 304 aa protein. Expression of the bspRIR gene in Escherichia coli was dependent on the replacement of the native TTG initiation codon with an ATG codon, explaining previous failures in cloning the gene using functional selection. A plasmid containing a single BspRI recognition site was used to analyze kinetically nicking and second-strand cleavage under steady-state conditions. Cleavage of the supercoiled plasmid went through a relaxed intermediate indicating sequential hydrolysis of the two strands. Results of the kinetic analysis of the first- and second-strand cleavage are consistent with cutting the double-stranded substrate site in two independent binding events. A database search identified eight putative restriction-modification systems in which the predicted endonucleases as well as the methyltransferases share high sequence similarity with the corresponding protein of the BspRI system. BspRI and the related putative restriction endonucleases belong to the PD-(D/E)XK nuclease superfamily.

  8. Direct endonuclease digestion and multi-analysis of restriction fragment length polymorphisms by microchip electrophoresis.

    Science.gov (United States)

    Akamine, Rie; Yatsushiro, Shouki; Yamamura, Shouhei; Kido, Jun-ichi; Shinohara, Yasuo; Baba, Yoshinobu; Kataoka, Masatoshi

    2009-12-05

    A high-performance multi-analysis system for genotypic mutation by means of restriction fragment length polymorphisms (RFLP) involving endonuclease treatment of PCR-amplified DNA on a microchip and subsequent analysis by microchip electrophoresis for DNA sizing was developed. A Hitachi SV1210 system, with which 12 samples can be analyzed on a plastic chip with good accuracy as to DNA sizing between 25 and 300 bp, was employed for RFLP analysis. We performed RFLP analysis of the ABO genotypes of blood donors for whom the ABO type was known. Six blood samples were analyzed by PCR to amplify two different regions of the genomic DNA, each of the amplified DNAs containing a different nucleotide polymorphism. To analyze the genes at polymorphic sites 261 and 526, restriction endonucleases Kpn I and Ban I were employed, respectively. When an amplified DNA was digested with each endonuclease on a microchip for 20 min, sequential analysis revealed the presence or absence of the respective restriction site. This analysis was performed within 7 min using a 1/10 volume of a DNA sample in comparison with the conventional method, and the estimated DNA size differed from the predicted size by less than 10 bp. The results indicate the potential of microchip electrophoresis for RFLP with on-chip direct endonuclease digestion and sequential analysis, offering high resolution in a short time.

  9. Accurate scanning of the BssHII endonuclease in search for its DNA cleavage site

    NARCIS (Netherlands)

    Berkhout, B.; van Wamel, J.

    1996-01-01

    A facilitated diffusion mechanism has been proposed to account for the kinetic efficiency with which restriction endonucleases are able to locate DNA recognition sites. Such a mechanism involves the initial formation of a nonspecific complex upon collision of the protein with the DNA, with the

  10. Biological and molecular characterization of Streptococcus crista strains isolated from human dental biofilm by means of arbitrary primers - PCR (AP-PCR Caracterização biológica e molecular de linhagens de Streptococcus crista isoladas do biofilme dental de seres humanos através de iniciadores arbitrários - PCR (AP-PCR

    Directory of Open Access Journals (Sweden)

    Michelle Angelini

    2006-06-01

    Full Text Available Streptococcus ssp are important components of the dental biofilm and Streptococcus crista is considered to be an interesting model of bacterial interactions taking place in this biofilm. In the present work, S. crista strains were isolated from the dental biofilm of Brazilian individuals and studied with respect to their biological characteristics and their molecular profile by means of AP-PCR techniques, using the RR2, 434, OPR2, OPR8, and OPR13 primers. Results allowed us to build a similarity dendrogram. Analysis of the similarity dendrogram allowed the separation of the studied strains into similarity groups. All isolates presented fibril tufts by Transmission Electron Microscopy (TEM. These isolates were able to bind to salivary amylase and to adhere to mouth epithelial cells. Some strains displaying fibril tufts and positive adherence were not able to co-aggregate with Fusobacterium nucleatum, suggesting that different adhesin groups are present in these strains.Streptococcus spp são importantes componentes do biofilme dental sendo Streptococus crista considerado um interessante modelo de interações bacterianas que nele ocorrem. No presente trabalho linhagens de S. crista, foram isoladas do biofilme dental de indivíduos brasileiros, e estudadas em relação a suas características biológicas e ao seu perfil molecular através da técnica do AP-PCR, usando-se os iniciadores RR2, 434, OPR2, OPR8 e OPR13. Os resultados nos permitiram construir um dendrograma de similaridade. A análise do dendrograma de similaridade permitiu a separação das linhagens estudadas em grupos de similaridade. Todos os isolados apresentaram tufo de fibrilas, quando estudados por Microscopia Eletrônica de Transmissão (MET. Estes isolados foram capazes de se ligar à amilase salivar e de se aderir a células epiteliais bucais. Algumas linhagens, que apresentam tufo de fibrilas e aderência positiva, não foram capazes de coagregar com a Fusobacterium

  11. Adipocyte lipid chaperone AP2 is a secreted adipokine regulating hepatic glucose production.

    Science.gov (United States)

    Cao, Haiming; Sekiya, Motohiro; Ertunc, Meric Erikci; Burak, M Furkan; Mayers, Jared R; White, Ariel; Inouye, Karen; Rickey, Lisa M; Ercal, Baris C; Furuhashi, Masato; Tuncman, Gürol; Hotamisligil, Gökhan S

    2013-05-07

    Proper control of hepatic glucose production is central to whole-body glucose homeostasis, and its disruption plays a major role in diabetes. Here, we demonstrate that although established as an intracellular lipid chaperone, aP2 is in fact actively secreted from adipocytes to control liver glucose metabolism. Secretion of aP2 from adipocytes is regulated by fasting- and lipolysis-related signals, and circulating aP2 levels are markedly elevated in mouse and human obesity. Recombinant aP2 stimulates glucose production and gluconeogenic activity in primary hepatocytes in vitro and in lean mice in vivo. In contrast, neutralization of secreted aP2 reduces glucose production and corrects the diabetic phenotype of obese mice. Hyperinsulinemic-euglycemic and pancreatic clamp studies upon aP2 administration or neutralization demonstrated actions of aP2 in liver. We conclude that aP2 is an adipokine linking adipocytes to hepatic glucose production and that neutralizing secreted aP2 may represent an effective therapeutic strategy against diabetes. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Apoptotic DNA Degradation into Oligonucleosomal Fragments, but Not Apoptotic Nuclear Morphology, Relies on a Cytosolic Pool of DFF40/CAD Endonuclease*

    Science.gov (United States)

    Iglesias-Guimarais, Victoria; Gil-Guiñon, Estel; Gabernet, Gisela; García-Belinchón, Mercè; Sánchez-Osuna, María; Casanelles, Elisenda; Comella, Joan X.; Yuste, Victor J.

    2012-01-01

    Apoptotic cell death is characterized by nuclear fragmentation and oligonucleosomal DNA degradation, mediated by the caspase-dependent specific activation of DFF40/CAD endonuclease. Here, we describe how, upon apoptotic stimuli, SK-N-AS human neuroblastoma-derived cells show apoptotic nuclear morphology without displaying concomitant internucleosomal DNA fragmentation. Cytotoxicity afforded after staurosporine treatment is comparable with that obtained in SH-SY5Y cells, which exhibit a complete apoptotic phenotype. SK-N-AS cell death is a caspase-dependent process that can be impaired by the pan-caspase inhibitor q-VD-OPh. The endogenous inhibitor of DFF40/CAD, ICAD, is correctly processed, and dff40/cad cDNA sequence does not reveal mutations altering its amino acid composition. Biochemical approaches show that both SH-SY5Y and SK-N-AS resting cells express comparable levels of DFF40/CAD. However, the endonuclease is poorly expressed in the cytosolic fraction of healthy SK-N-AS cells. Despite this differential subcellular distribution of DFF40/CAD, we find no differences in the subcellular localization of both pro-caspase-3 and ICAD between the analyzed cell lines. After staurosporine treatment, the preferential processing of ICAD in the cytosolic fraction allows the translocation of DFF40/CAD from this fraction to a chromatin-enriched one. Therefore, the low levels of cytosolic DFF40/CAD detected in SK-N-AS cells determine the absence of DNA laddering after staurosporine treatment. In these cells DFF40/CAD cytosolic levels can be restored by the overexpression of their own endonuclease, which is sufficient to make them proficient at degrading their chromatin into oligonucleosome-size fragments after staurosporine treatment. Altogether, the cytosolic levels of DFF40/CAD are determinants in achieving a complete apoptotic phenotype, including oligonucleosomal DNA degradation. PMID:22253444

  13. Engineering strand-specific DNA nicking enzymes from the type IIS restriction endonucleases BsaI, BsmBI, and BsmAI.

    Science.gov (United States)

    Zhu, Zhenyu; Samuelson, James C; Zhou, Jing; Dore, Andrew; Xu, Shuang-Yong

    2004-03-26

    More than 80 type IIA/IIS restriction endonucleases with different recognition specificities are now known. In contrast, only a limited number of strand-specific nicking endonucleases are currently available. To overcome this limitation, a novel genetic screening method was devised to convert type IIS restriction endonucleases into strand-specific nicking endonucleases. The genetic screen consisted of four steps: (1) random mutagenesis to create a plasmid library, each bearing an inactivated endonuclease gene; (2) restriction digestion of plasmids containing the wild-type and the mutagenized endonuclease gene; (3) back-crosses with the wild-type gene by ligation to the wild-type N-terminal or C-terminal fragment; (4) transformation of the ligated DNA into a pre-modified host and screening for nicking endonuclease activity in total cell culture or cell extracts of the transformants. Nt.BsaI and Nb.BsaI nicking endonucleases were isolated from BsaI using this genetic screen. In addition, site-directed mutagenesis was carried out to isolate BsaI nicking variants with minimal double-stranded DNA cleavage activity. The equivalent amino acid substitutions were introduced into BsmBI and BsmAI restriction endonucleases with similar recognition sequence and significant amino acid sequence identity and their nicking variants were successfully isolated. This work provides strong evidence that some type IIS restriction endonucleases carry two separate active sites. When one of the active sites is inactivated, the type IIS restriction endonuclease may nick only one strand.

  14. Coaching Strategies for AP: Building a Successful AP European History Program

    Science.gov (United States)

    Fornaciari, Jim

    2014-01-01

    The October 2013 special issue of "Social Education" dealt with almost all AP social studies subjects, but omitted AP European History. This is one of the most fascinating AP subjects for students and teachers alike. In this article, the author shares his experiences since hewas given the responsibility of building his school's Advanced…

  15. The NF1 gene contains hotspots for L1 endonuclease-dependent de novo insertion.

    Directory of Open Access Journals (Sweden)

    Katharina Wimmer

    2011-11-01

    Full Text Available Long interspersed (L1 and Alu elements are actively amplified in the human genome through retrotransposition of their RNA intermediates by the -100 still retrotranspositionally fully competent L1 elements. Retrotransposition can cause inherited disease if such an element is inserted near or within a functional gene. Using direct cDNA sequencing as the primary assay for comprehensive NF1 mutation analysis, we uncovered in 18 unrelated index patients splicing alterations not readily explained at the genomic level by an underlying point-mutation or deletion. Improved PCR protocols avoiding allelic drop-out of the mutant alleles uncovered insertions of fourteen Alu elements, three L1 elements, and one poly(T stretch to cause these splicing defects. Taken together, the 18 pathogenic L1 endonuclease-mediated de novo insertions represent the largest number of this type of mutations characterized in a single human gene. Our findings show that retrotransposon insertions account for as many as -0.4% of all NF1 mutations. Since altered splicing was the main effect of the inserted elements, the current finding was facilitated by the use of RNA-based mutation analysis protocols, resulting in improved detection compared to gDNA-based approaches. Six different insertions clustered in a relatively small 1.5-kb region (NF1 exons 21(16-23(18 within the 280-kb NF1 gene. Furthermore, three different specific integration sites, one of them located in this cluster region, were each used twice, i.e. NM_000267.3(NF1:c.1642-1_1642 in intron 14(10c, NM_000267.3(NF1:c.2835_2836 in exon 21(16, and NM_000267.3(NF1:c.4319_4320 in exon 33(25. Identification of three loci that each served twice as integration site for independent retrotransposition events as well as 1.5-kb cluster region harboring six independent insertions supports the notion of non-random insertion of retrotransposons in the human genome. Currently, little is known about which features make sites

  16. RAD51AP2, a novel vertebrate- and meiotic-specific protein, sharesa conserved RAD51-interacting C-terminal domain with RAD51AP1/PIR51

    Energy Technology Data Exchange (ETDEWEB)

    Kovalenko, Oleg V.; Wiese, Claudia; Schild, David

    2006-07-25

    Many interacting proteins regulate and/or assist the activities of RAD51, a recombinase which plays a critical role in both DNA repair and meiotic recombination. Yeast two-hybrid screening of a human testis cDNA library revealed a new protein, RAD51AP2 (RAD51 Associated Protein 2), that interacts strongly with RAD51. A full-length cDNA clone predicts a novel vertebrate specific protein of 1159 residues, and the RAD51AP2 transcript was observed only in meiotic tissue (i.e. adult testis and fetal ovary), suggesting a meiotic-specific function for RAD51AP2. In HEK293 cells the interaction of RAD51 with an ectopically-expressed recombinant large fragment of RAD51AP2 requires the C-terminal 57 residues of RAD51AP2. This RAD51-binding region shows 81% homology to the C-terminus of RAD51AP1/PIR51, an otherwise totally unrelated RAD51-binding partner that is ubiquitously expressed. Analyses using truncations and point mutations in both RAD51AP1 and RAD51AP2 demonstrate that these proteins use the same structural motif for RAD51 binding. RAD54 shares some homology with this RAD51-binding motif, but this homologous region plays only an accessory role to the adjacent main RAD51-interacting region, which has been narrowed here to 40 amino acids. A novel protein, RAD51AP2, has been discovered that interacts with RAD51 through a C-terminal motif also present in RAD51AP1.

  17. Two peptides, TsAP-1 and TsAP-2, from the venom of the Brazilian yellow scorpion, Tityus serrulatus: evaluation of their antimicrobial and anticancer activities.

    Science.gov (United States)

    Guo, Xiaoxiao; Ma, Chengbang; Du, Qiang; Wei, Ran; Wang, Lei; Zhou, Mei; Chen, Tianbao; Shaw, Chris

    2013-09-01

    Here we report two novel 17-mer amidated linear peptides (TsAP-1 and TsAP-2) whose structures were deduced from cDNAs cloned from a venom-derived cDNA library of the Brazilian yellow scorpion, Tityus serrulatus. Both mature peptides were structurally-characterised following their location in chromatographic fractions of venom and synthetic replicates of each were subjected to a range of biological assays. The peptides were each active against model test micro-organisms but with different potencies. TsAP-1 was of low potency against all three test organisms (MICs 120-160 μM), whereas TsAP-2 was of high potency against the Gram-positive bacterium, Staphylococcus aureus (MIC 5 μM) and the yeast, Candida albicans (10 μM). Haemolytic activity of TsAP-1 was low (4% at 160 μM) and in contrast, that of TsAP-2 was considerably higher (18% at 20 μM). Substitution of four neutral amino acid residues with Lys residues in each peptide had dramatic effects on their antimicrobial potencies and haemolytic activities, particularly those of TsAP-1. The MICs of the enhanced cationic analogue (TsAP-S1) were 2.5 μM for S. aureus/C. albicans and 5 μM for E. coli but with an associated large increase in haemolytic activity (30% at 5 μM). The same Lys residue substitutions in TsAP-2 produced a dramatic effect on its MIC for E. coli lowering this from >320 μM to 5 μM. TsAP-1 was ineffective against three of the five human cancer cell lines tested while TsAP-2 inhibited the growth of all five. Lys residue substitution of both peptides enhanced their potency against all five cell lines with TsAp-S2 being the most potent with IC50 values ranging between 0.83 and 2.0 μM. TsAP-1 and TsAP-2 are novel scorpion venom peptides with broad spectrum antimicrobial and anticancer cell activities the potencies of which can be significantly enhanced by increasing their cationicity. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  18. ELECTROCHEMICAL CORROSION TESTING OF TANKS 241-AN-102 & 241-AP-107 & 241-AP-108 IN SUPPORT OF ULTRASONIC TESTING

    Energy Technology Data Exchange (ETDEWEB)

    WYRWAS RB; DUNCAN JB

    2008-11-20

    This report presents the results of the corrosion rates that were measured using electrochemical methods for tanks 241-AN-102 (AN-102), 241-AP-107 (AP 107), and 241-AP-108 (AP-108) performed under test plant RPP-PLAN-38215. The steel used as materials of construction for AN and AP tank farms was A537 Class 1. Test coupons of A537 Class 1 carbon steel were used for corrosion testing in the AN-107, AP-107, and AP-108 tank waste. Supernate will be tested from AN-102, AP-107, and Ap-108. Saltcake testing was performed on AP-108 only.

  19. Dynamics of Intracellular Clathrin/AP1- and Clathrin/AP3-Containing Carriers

    Directory of Open Access Journals (Sweden)

    Comert Kural

    2012-11-01

    Full Text Available Clathrin/AP1- and clathrin/AP3-coated vesicular carriers originate from endosomes and the trans-Golgi network. Here, we report the real-time visualization of these structures in living cells reliably tracked by rapid, three-dimensional imaging with the use of a spinning-disk confocal microscope. We imaged relatively sparse, diffraction-limited, fluorescent objects containing chimeric fluorescent protein (clathrin light chain, σ adaptor subunits, or dynamin2 with a spatial precision of up to ∼30 nm and a temporal resolution of ∼1 s. The dynamic characteristics of the intracellular clathrin/AP1 and clathrin/AP3 carriers are similar to those of endocytic clathrin/AP2 pits and vesicles; the clathrin/AP1 coats are, on average, slightly shorter-lived than their AP2 and AP3 counterparts. We confirmed that although dynamin2 is recruited as a burst to clathrin/AP2 pits immediately before their budding from the plasma membrane, we found no evidence supporting a similar association of dynamin2 with clathrin/AP1 or clathrin/AP3 carriers at any stage during their lifetime. We found no effects of chemical inhibitors of dynamin function or the K44A dominant-negative mutant of dynamin on AP1 and AP3 dynamics. This observation suggests that an alternative budding mechanism, yet to be discovered, is responsible for the scission step of clathrin/AP1 and clathrin/AP3 carriers.

  20. New Protein Vector ApE1 for Targeted Delivery of Anticancer Drugs

    Directory of Open Access Journals (Sweden)

    N. V. Pozdniakova

    2012-01-01

    Full Text Available A new chimeric gene ApE1 encoding the receptor-binding domain of the human alpha-fetoprotein fused to a sequence of 22 glutamic acid residues was constructed. A new bacterial producer strain E. coli SHExT7 ApE1 was selected for ApE1 production in a soluble state. A simplified method was developed to purify ApE1 from bacterial biomass. It was shown that the new vector protein selectively interacts with AFP receptors on the tumor cell surface and can be efficiently accumulated in tumor cells. In addition, ApE1 was shown to be stable in storage and during its chemical modification. An increased number of carboxyl groups in the molecule allows the production of cytotoxic compound conjugates with higher drug-loading capacity and enhanced tumor targeting potential.

  1. AP English language & composition crash course

    CERN Document Server

    Hogue, Dawn

    2012-01-01

    AP English Language & Composition Crash Course - Gets You a Higher Advanced Placement Score in Less Time Crash Course is perfect for the time-crunched student, the last-minute studier, or anyone who wants a refresher on the subject. AP English Language & Composition Crash Course gives you: Targeted, Focused Review - Study Only What You Need to Know Crash Course is based on an in-depth analysis of the AP English Language & Composition course description outline and actual Advanced Placement test questions. It covers only the information tested on the exam, so you can make the most of your valua

  2. Tank 241-AP-107, grab samples, 7AP-99-1, 7AP-99-3 and 7AP-99-4 analytical results for the final report

    Energy Technology Data Exchange (ETDEWEB)

    BELL, K.E.

    1999-08-12

    This document is the format IV, final report for the tank 241-AP-107 (AP-107) grab samples taken in May 1999 to address waste compatibility concerns. Chemical, radiochemical, and physical analyses on the tank AP-107 samples were performed as directed in Compatibility Grab Sampling and Analysis Plan for Fiscal year 1999. Any deviations from the instructions provided in the tank sampling and analysis plan (TSAP) were discussed in this narrative. Interim data were provided earlier to River Protection Project (RPP) personnel, however, the data presented here represent the official results. No notification limits were exceeded.

  3. [Restriction endonuclease digest - melting curve analysis: a new SNP genotyping and its application in traditional Chinese medicine authentication].

    Science.gov (United States)

    Jiang, Chao; Huang, Lu-Qi; Yuan, Yuan; Chen, Min; Hou, Jing-Yi; Wu, Zhi-Gang; Lin, Shu-Fang

    2014-04-01

    Single nucleotide polymorphisms (SNP) is an important molecular marker in traditional Chinese medicine research, and it is widely used in TCM authentication. The present study created a new genotyping method by combining restriction endonuclease digesting with melting curve analysis, which is a stable, rapid and easy doing SNP genotyping method. The new method analyzed SNP genotyping of two chloroplast SNP which was located in or out of the endonuclease recognition site, the results showed that when attaching a 14 bp GC-clamp (cggcgggagggcgg) to 5' end of the primer and selecting suited endonuclease to digest the amplification products, the melting curve of Lonicera japonica and Atractylodes macrocephala were all of double peaks and the adulterants Shan-yin-hua and A. lancea were of single peaks. The results indicated that the method had good stability and reproducibility for identifying authentic medicines from its adulterants. It is a potential SNP genotyping method and named restriction endonuclease digest - melting curve analysis.

  4. Identification of Egyptian Fasciola species by PCR and restriction endonucleases digestion of the nuclear small subunit ribosomal RNA gene.

    Science.gov (United States)

    El-Gozamy, Bothina R; Shoukry, Nahla M

    2009-08-01

    Fascioliasis is one of the familiar zoonotic health problems of worldwide distribution including Egypt. In this study, a simple and rapid polymerase chain reaction/restriction fragment length polymorphisms (PCR/RFLPs) assay, using the common restriction endonucleases Aval, EcoRI, Eael, Sac11 and Avail was applied to differentiate between both Fasciola gigantica and F. hepatica. The five restriction endonucleases were used to differentiate between the two species of Fasciola based on -1950 bp long sequence of the 18S nuclear small subunit ribosomal RNA gene. Aval and EcoRI restriction endonucleases failed to differentiate between the two Fasciola species when each restriction enzyme gave the same restriction patterns in both of them. However, F. gigantica and F. hepatica were well-differentiated when their small subunit ribosomal DNA were digested with Eael and Sac 11 restriction endonucleases.

  5. Lobular carcinoma in situ and invasive lobular breast cancer are characterized by enhanced expression of transcription factor AP-2β.

    Science.gov (United States)

    Raap, Mieke; Gronewold, Malte; Christgen, Henriette; Glage, Silke; Bentires-Alj, Mohammad; Koren, Shany; Derksen, Patrick W; Boelens, Mirjam; Jonkers, Jos; Lehmann, Ulrich; Feuerhake, Friedrich; Kuehnle, Elna; Gluz, Oleg; Kates, Ronald; Nitz, Ulrike; Harbeck, Nadia; Kreipe, Hans H; Christgen, Matthias

    2018-01-01

    Transcription factor AP-2β (TFAP2B) regulates embryonic organ development and is overexpressed in alveolar rhabdomyosarcoma, a rare childhood malignancy. Gene expression profiling has implicated AP-2β in breast cancer (BC). This study characterizes AP-2β expression in the mammary gland and in BC. AP-2β protein expression was assessed in the normal mammary gland epithelium, in various reactive, metaplastic and pre-invasive neoplastic lesions and in two clinical BC cohorts comprising >2000 patients. BCs from various genetically engineered mouse (GEM) models were also evaluated. Human BC cell lines served as functional models to study siRNA-mediated inhibition of AP-2β. The normal mammary gland epithelium showed scattered AP-2β-positive cells in the luminal cell layer. Various reactive and pre-invasive neoplastic lesions, including apocrine metaplasia, usual ductal hyperplasia and lobular carcinoma in situ (LCIS) showed enhanced AP-2β expression. Cases of ductal carcinoma in situ (DCIS) were more often AP-2β-negative (Pinvasive BC cohorts, AP-2β-positivity was associated with the lobular BC subtype (Plobular BC cell lines in vitro. In summary, AP-2β is a new mammary epithelial differentiation marker. Its expression is preferentially retained and enhanced in LCIS and invasive lobular BC and has prognostic implications. Our findings indicate that AP-2β controls tumor cell proliferation in this slow-growing BC subtype.

  6. NANCEI--Ap-riI

    Indian Academy of Sciences (India)

    structure of physical reality. Never have I made any systematic effort to ameliorate the lot of men, to fight injustice and suppression. and to improve the traditionalforms of human relations. The only thing I did was this: in long intervals. I have expressed an opinion on public issues whenever they appeared to me so bad and.

  7. Pentoxifylline Treatment in Acute Pancreatitis (AP)

    Science.gov (United States)

    2016-09-14

    Acute Pancreatitis (AP); Gallstone Pancreatitis; Alcoholic Pancreatitis; Post-ERCP/Post-procedural Pancreatitis; Trauma Acute Pancreatitis; Hypertriglyceridemia Acute Pancreatitis; Idiopathic (Unknown) Acute Pancreatitis; Medication Induced Acute Pancreatitis; Cancer Acute Pancreatitis; Miscellaneous (i.e. Acute on Chronic Pancreatitis)

  8. Spectroelectrochemical insights into structural and redox properties of immobilized endonuclease III and its catalytically inactive mutant.

    Science.gov (United States)

    Moe, Elin; Rollo, Filipe; Silveira, Célia M; Sezer, Murat; Hildebrandt, Peter; Todorovic, Smilja

    2018-01-05

    Endonuclease III is a Fe-S containing bifunctional DNA glycosylase which is involved in the repair of oxidation damaged DNA. Here we employ surface enhanced IR spectroelectrochemistry and electrochemistry to study the enzyme from the highly radiation- and desiccation-resistant bacterium Deinococcus radiodurans (DrEndoIII2). The experiments are designed to shed more light onto specific parameters that are currently proposed to govern damage search and recognition by endonucleases III. We demonstrate that electrostatic interactions required for the redox activation of DrEndoIII2 may result in high electric fields that alter its structural and thermodynamic properties. Analysis of inactive DrEndoIII2 (K132A/D150A double mutant) interacting with undamaged DNA, and the active enzyme interacting with damaged DNA also indicate that the electron transfer is modulated by subtle differences in the protein-DNA complex. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. UVI31+ is a DNA endonuclease that dynamically localizes to chloroplast pyrenoids in C. reinhardtii.

    Directory of Open Access Journals (Sweden)

    Manish Shukla

    Full Text Available UVI31+ is an evolutionarily conserved BolA family protein. In this study we examine the presence, localization and possible functions of this protein in the context of a unicellular alga, Chlamydomonas reinhardtii. UVI31+ in C. reinhardtii exhibits DNA endonuclease activity and is induced upon UV stress. Further, UVI31+ that normally localizes to the cell wall and pyrenoid regions gets redistributed into punctate foci within the whole chloroplast, away from the pyrenoid, upon UV stress. The observed induction upon UV-stress as well as the endonuclease activity suggests plausible role of this protein in DNA repair. We have also observed that UV31+ is induced in C. reinhardtii grown in dark conditions, whereby the protein localization is enhanced in the pyrenoid. Biomolecular interaction between the purified pyrenoids and UVI31+ studied by NMR demonstrates the involvement of the disordered loop domain of the protein in its interaction.

  10. Does quantum entanglement in DNA synchronize the catalytic centers of type II restriction endonucleases?

    CERN Document Server

    Kurian, P; Lindesay, J

    2014-01-01

    Several living systems have been examined for their apparent optimization of structure and function for quantum behavior at biological length scales. Orthodox type II endonucleases, the largest class of restriction enzymes, recognize four-to-eight base pair sequences of palindromic DNA, cut both strands symmetrically, and act without an external metabolite such as ATP. While it is known that these enzymes induce strand breaks by attacking phosphodiester bonds, what remains unclear is the mechanism by which cutting occurs in concert at the catalytic centers. Previous studies indicate the primacy of intimate DNA contacts made by the specifically bound enzyme in coordinating the two synchronized cuts. We propose that collective electronic behavior in the DNA helix generates coherent oscillations, quantized through boundary conditions imposed by the endonuclease, that provide the energy required to break two phosphodiester bonds. Such quanta may be preserved in the presence of thermal noise and electromagnetic in...

  11. Assembly of Francisella novicida Cpf1 endonuclease in complex with guide RNA and target DNA

    DEFF Research Database (Denmark)

    Alcón, Pablo; Montoya, Guillermo; Stella, Stefano

    2017-01-01

    a target DNA preceded by a 5'-TTN-3' protospacer-adjacent motif (PAM) complementary to the RNA guide. To obtain structural insight into the inner workings of Cpf1, the crystallization of an active complex containing the full extent of the crRNA and a 31-nucleotide dsDNA target was attempted. The gene...... encoding Cpf1 from Francisella novicida was cloned, overexpressed and purified. The crRNA was transcribed and purified in vitro. Finally, the ternary FnCpf1-crRNA-DNA complex was assembled and purified by preparative electrophoresis before crystallization. Crystals belonging to space group C2221, with unit...... into six types and 19 subtypes according to conservation of the cas gene and loci organization. Recently, a new protein with endonuclease activity belonging to class 2 type V has been identified. This endonuclease, termed Cpf1, in complex with a single CRISPR RNA (crRNA) is able to recognize and cleave...

  12. AP1000{sup TM} plant modularization

    Energy Technology Data Exchange (ETDEWEB)

    Cantarero L, C.; Demetri, K. J. [Westinghouse Electric Co., 1000 Westinghouse Drive, Cranberry Township, PA 16066 (United States); Quintero C, F. P., E-mail: cantarc@westinghouse.com [Westinghouse Electric Spain, Padilla 17, 28006 Madrid (Spain)

    2016-09-15

    The AP1000{sup TM} plant is an 1100 M We pressurized water reactor (PWR) with passive safety features and extensive plant simplifications that enhance construction, operation, maintenance and safety. Modules are used extensively in the design of the AP1000 plant nuclear island. The AP1000 plant uses modern, modular-construction techniques for plant construction. The design incorporates vendor-designed skids and equipment packages, as well as large, multi-ton structural modules and special equipment modules. Modularization allows traditionally sequential construction tasks to be completed simultaneously. Factory-built modules can be installed at the site in a planned construction schedule. The modularized AP1000 plant allows many more construction activities to proceed in parallel. This reduces plant construction calendar time, thus lowering the costs of plant financing. Furthermore, performing less work onsite significantly reduces the amount of skilled field-craft labor, which costs more than shop labor. In addition to labor cost savings, doing more welding and fabrication in a factory environment raises the quality of work, allowing more scheduling flexibility and reducing the amount of specialized tools required onsite. The site layout for the AP1000 plant has been established to support modular construction and efficient operations during construction. The plant layout is compact, using less space than previous conventional plant layouts. This paper provides and overview of the AP1000 plant modules with an emphasis on structural modules. Currently the Westinghouse AP1000 plant has four units under construction in China and four units under construction in the United States. All have shown successful fabrication and installation of various AP1000 plant modules. (Author)

  13. Zinc finger nuclease and homing endonuclease-mediated assembly of multigene plant transformation vectors.

    Science.gov (United States)

    Zeevi, Vardit; Liang, Zhuobin; Arieli, Uri; Tzfira, Tzvi

    2012-01-01

    Binary vectors are an indispensable component of modern Agrobacterium tumefaciens-mediated plant genetic transformation systems. A remarkable variety of binary plasmids have been developed to support the cloning and transfer of foreign genes into plant cells. The majority of these systems, however, are limited to the cloning and transfer of just a single gene of interest. Thus, plant biologists and biotechnologists face a major obstacle when planning the introduction of multigene traits into transgenic plants. Here, we describe the assembly of multitransgene binary vectors by using a combination of engineered zinc finger nucleases (ZFNs) and homing endonucleases. Our system is composed of a modified binary vector that has been engineered to carry an array of unique recognition sites for ZFNs and homing endonucleases and a family of modular satellite vectors. By combining the use of designed ZFNs and commercial restriction enzymes, multiple plant expression cassettes were sequentially cloned into the acceptor binary vector. Using this system, we produced binary vectors that carried up to nine genes. Arabidopsis (Arabidopsis thaliana) protoplasts and plants were transiently and stably transformed, respectively, by several multigene constructs, and the expression of the transformed genes was monitored across several generations. Because ZFNs can potentially be engineered to digest a wide variety of target sequences, our system allows overcoming the problem of the very limited number of commercial homing endonucleases. Thus, users of our system can enjoy a rich resource of plasmids that can be easily adapted to their various needs, and since our cloning system is based on ZFN and homing endonucleases, it may be possible to reconstruct other types of binary vectors and adapt our vectors for cloning on multigene vector systems in various binary plasmids.

  14. Identification of two groups of Mycobacterium paratuberculosis strains by restriction endonuclease analysis and DNA hybridization.

    OpenAIRE

    Collins, D M; Gabric, D M; de Lisle, G W

    1990-01-01

    Genomic DNA was prepared from four reference strains of Mycobacterium paratuberculosis and 46 isolates of this organism from New Zealand, Australia, Canada, and Norway and also from two mycobactin-dependent "wood pigeon" strains. The DNA was characterized by restriction endonuclease analysis, both with and without DNA hybridization, with a probe specific to a repetitive DNA sequence in M. paratuberculosis. Both techniques differentiated M. paratuberculosis strains into two groups, but DNA hyb...

  15. Endonuclease VIII-like 3 (Neil3) DNA glycosylase promotes neurogenesis induced by hypoxia-ischemia

    OpenAIRE

    Sejersted, Yngve; Hildrestrand, Gunn A.; Kunke, David; Rolseth, Veslemøy; Krokeide, Silje Z.; Neurauter, Christine G.; Suganthan, Rajikala; Atneosen-Åsegg, Monica; Fleming, Aaron M.; Saugstad, Ola D.; Burrows, Cynthia J.; Luna, Luisa; Bjørås, Magnar

    2011-01-01

    Neural stem/progenitor cell proliferation and differentiation are required to replace damaged neurons and regain brain function after hypoxic-ischemic events. DNA base lesions accumulating during hypoxic-ischemic stress are removed by DNA glycosylases in the base-excision repair pathway to prevent cytotoxicity and mutagenesis. Expression of the DNA glycosylase endonuclease VIII-like 3 (Neil3) is confined to regenerative subregions in the embryonic and perinatal brains. Here we show profound n...

  16. Tank 241-AP-106, Grab samples, 6AP-98-1, 6AP-98-2 and 6AP-98-3 Analytical results for the final report

    Energy Technology Data Exchange (ETDEWEB)

    FULLER, R.K.

    1999-02-23

    This document is the final report for tank 241-AP-106 grab samples. Three grab samples 6AP-98-1, 6AP-98-2 and 6AP-98-3 were taken from riser 1 of tank 241-AP-106 on May 28, 1998 and received by the 222-S Laboratory on May 28, 1998. Analyses were performed in accordance with the ''Compatability Grab Sampling and Analysis Plan'' (TSAP) (Sasaki, 1998) and the ''Data Quality Objectives for Tank Farms Waste Compatability Program (DQO). The analytical results are presented in the data summary report. No notification limits were exceeded. The request for sample analysis received for AP-106 indicated that the samples were polychlorinated biphenyl (PCB) suspects. The results of this analysis indicated that no PCBs were present at the Toxic Substance Control Act (TSCA) regulated limit of 50 ppm. The results and raw data for the PCB analysis are included in this document.

  17. Engineering of restriction endonucleases: using methylation activity of the bifunctional endonuclease Eco57I to select the mutant with a novel sequence specificity.

    Science.gov (United States)

    Rimseliene, Renata; Maneliene, Zita; Lubys, Arvydas; Janulaitis, Arvydas

    2003-03-21

    Type II restriction endonucleases (REs) are widely used tools in molecular biology, biotechnology and diagnostics. Efforts to generate new specificities by structure-guided design and random mutagenesis have been unsuccessful so far. We have developed a new procedure called the methylation activity-based selection (MABS) for generating REs with a new specificity. MABS uses a unique property of bifunctional type II REs to methylate DNA targets they recognize. The procedure includes three steps: (1) conversion of a bifunctional RE into a monofunctional DNA-modifying enzyme by cleavage center disruption; (2) mutagenesis and selection of mutants with altered DNA modification specificity based on their ability to protect predetermined DNA targets; (3) reconstitution of the cleavage center's wild-type structure. The efficiency of the MABS technique was demonstrated by altering the sequence specificity of the bifunctional RE Eco57I from 5'-CTGAAG to 5'-CTGRAG, and thus generating the mutant restriction endonuclease (and DNA methyltransferase) of a specificity not known before. This study provides evidence that MABS is a promising technique for generation of REs with new specificities.

  18. Quantitation and analysis of the formation of HO-endonuclease stimulated chromosomal translocations by single-strand annealing in Saccharomyces cerevisiae.

    Science.gov (United States)

    Liddell, Lauren; Manthey, Glenn; Pannunzio, Nicholas; Bailis, Adam

    2011-09-23

    Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms(1-3). The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs(4). Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases(5), as well as cancer in humans(6-9). Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange(10,11). The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents. In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-Δ3', contains a 3' truncated HIS3 allele and is located on one copy of chromosome XV at the native HIS3 locus. The second substrate, his3-Δ5', is located at the LEU2 locus on one copy of chromosome III, and contains a 5' truncated HIS3 allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for

  19. Investigation of the salicylaldehyde thiosemicarbazone scaffold for inhibition of influenza virus PA endonuclease.

    Science.gov (United States)

    Rogolino, Dominga; Bacchi, Alessia; De Luca, Laura; Rispoli, Gabriele; Sechi, Mario; Stevaert, Annelies; Naesens, Lieve; Carcelli, Mauro

    2015-10-01

    The influenza virus PA endonuclease is an attractive target for the development of novel anti-influenza virus therapeutics, which are urgently needed because of the emergence of drug-resistant viral strains. Reported PA inhibitors are assumed to chelate the divalent metal ion(s) (Mg²⁺ or Mn²⁺) in the enzyme's catalytic site, which is located in the N-terminal part of PA (PA-Nter). In the present work, a series of salicylaldehyde thiosemicarbazone derivatives have been synthesized and evaluated for their ability to inhibit the PA-Nter catalytic activity. Compounds 1-6 have been evaluated against influenza virus, both in enzymatic assays with influenza virus PA-Nter and in virus yield assays in MDCK cells. In order to establish a structure-activity relationship, the hydrazone analogue of the most active thiosemicarbazone has also been evaluated. Since chelation may represent a mode of action of such class of molecules, we studied the interaction of two of them, one with and one without biological activity versus the PA enzyme, towards Mg²⁺, the ion that is probably involved in the endonuclease activity of the heterotrimeric influenza polymerase complex. The crystal structure of the magnesium complex of the o-vanillin thiosemicarbazone ligand 1 is also described. Moreover, docking studies of PA endonuclease with compounds 1 and 2 were performed, to further analyse the possible mechanism of action of this class of inhibitors.

  20. Structure and mutagenesis of the DNA modification-dependent restriction endonuclease AspBHI.

    Science.gov (United States)

    Horton, John R; Nugent, Rebecca L; Li, Andrew; Mabuchi, Megumu Yamada; Fomenkov, Alexey; Cohen-Karni, Devora; Griggs, Rose M; Zhang, Xing; Wilson, Geoffrey G; Zheng, Yu; Xu, Shuang-yong; Cheng, Xiaodong

    2014-03-07

    The modification-dependent restriction endonuclease AspBHI recognizes 5-methylcytosine (5mC) in the double-strand DNA sequence context of (C/T)(C/G)(5mC)N(C/G) (N = any nucleotide) and cleaves the two strands a fixed distance (N12/N16) 3' to the modified cytosine. We determined the crystal structure of the homo-tetrameric AspBHI. Each subunit of the protein comprises two domains: an N-terminal DNA-recognition domain and a C-terminal DNA cleavage domain. The N-terminal domain is structurally similar to the eukaryotic SET and RING-associated (SRA) domain, which is known to bind to a hemi-methylated CpG dinucleotide. The C-terminal domain is structurally similar to classic Type II restriction enzymes and contains the endonuclease catalytic-site motif of DX20EAK. To understand how specific amino acids affect AspBHI recognition preference, we generated a homology model of the AspBHI-DNA complex, and probed the importance of individual amino acids by mutagenesis. Ser41 and Arg42 are predicted to be located in the DNA minor groove 5' to the modified cytosine. Substitution of Ser41 with alanine (S41A) and cysteine (S41C) resulted in mutants with altered cleavage activity. All 19 Arg42 variants resulted in loss of endonuclease activity.

  1. On the role of steric clashes in methylation control of restriction endonuclease activity.

    Science.gov (United States)

    Mierzejewska, Karolina; Bochtler, Matthias; Czapinska, Honorata

    2016-01-08

    Restriction-modification systems digest non-methylated invading DNA, while protecting host DNA against the endonuclease activity by methylation. It is widely believed that the methylated DNA would not 'fit' into the binding site of the endonuclease in the productive orientation, and thus steric clashes should account for most of the protection. We test this concept statistically by grafting methyl groups in silico onto non-methylated DNA in co-crystal structures with restriction endonucleases. Clash scores are significantly higher for protective than non-protective methylation (P < 0.05% according to the Wilcoxon rank sum test). Structural data alone are sufficient to distinguish between protective and non-protective DNA methylation with 90% confidence and decision thresholds of 1.1 Å and 48 Å(3) for the most severe distance-based and cumulative volume-based clash with the protein, respectively (0.1 Å was deducted from each interatomic distance to allow for coordinate errors). The most severe clashes are more pronounced for protective methyl groups attached to the nitrogen atoms (N6-methyladenines and N4-methylcytosines) than for C5-methyl groups on cytosines. Cumulative clashes are comparable for all three types of protective methylation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Restriction endonuclease AgeI is a monomer which dimerizes to cleave DNA.

    Science.gov (United States)

    Tamulaitiene, Giedre; Jovaisaite, Virginija; Tamulaitis, Gintautas; Songailiene, Inga; Manakova, Elena; Zaremba, Mindaugas; Grazulis, Saulius; Xu, Shuang-Yong; Siksnys, Virginijus

    2017-04-07

    Although all Type II restriction endonucleases catalyze phosphodiester bond hydrolysis within or close to their DNA target sites, they form different oligomeric assemblies ranging from monomers, dimers, tetramers to higher order oligomers to generate a double strand break in DNA. Type IIP restriction endonuclease AgeI recognizes a palindromic sequence 5΄-A/CCGGT-3΄ and cuts it ('/' denotes the cleavage site) producing staggered DNA ends. Here, we present crystal structures of AgeI in apo and DNA-bound forms. The structure of AgeI is similar to the restriction enzymes that share in their target sites a conserved CCGG tetranucleotide and a cleavage pattern. Structure analysis and biochemical data indicate, that AgeI is a monomer in the apo-form both in the crystal and in solution, however, it binds and cleaves the palindromic target site as a dimer. DNA cleavage mechanism of AgeI is novel among Type IIP restriction endonucleases. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. RNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing?

    Science.gov (United States)

    Gasiunas, Giedrius; Siksnys, Virginijus

    2013-11-01

    Tailor-made nucleases for precise genome modification, such as zinc finger or TALE nucleases, currently represent the state-of-the-art for genome editing. These nucleases combine a programmable protein module which guides the enzyme to the target site with a nuclease domain which cuts DNA at the addressed site. Reprogramming of these nucleases to cut genomes at specific locations requires major protein engineering efforts. RNA-guided DNA endonuclease Cas9 of the type II (clustered regularly interspaced short palindromic repeat) CRISPR-Cas system uses CRISPR RNA (crRNA) as a guide to locate the DNA target and the Cas9 protein to cut DNA. Easy programmability of the Cas9 endonuclease using customizable RNAs brings unprecedented flexibility and versatility for targeted genome modification. We highlight the potential of the Cas9 RNA-guided DNA endonuclease as a novel tool for genome surgery, and discuss possible constraints and future prospects. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. How driving endonuclease genes can be used to combat pests and disease vectors.

    Science.gov (United States)

    Godfray, H Charles J; North, Ace; Burt, Austin

    2017-09-11

    Driving endonuclease genes (DEGs) spread through a population by a non-Mendelian mechanism. In a heterozygote, the protein encoded by a DEG causes a double-strand break in the homologous chromosome opposite to where its gene is inserted and when the break is repaired using the homologue as a template the DEG heterozygote is converted to a homozygote. Some DEGs occur naturally while several classes of endonucleases can be engineered to spread in this way, with CRISPR-Cas9 based systems being particularly flexible. There is great interest in using driving endonuclease genes to impose a genetic load on insects that vector diseases or are economic pests to reduce their population density, or to introduce a beneficial gene such as one that might interrupt disease transmission. This paper reviews both the population genetics and population dynamics of DEGs. It summarises the theory that guides the design of DEG constructs intended to perform different functions. It also reviews the studies that have explored the likelihood of resistance to DEG phenotypes arising, and how this risk may be reduced. The review is intended for a general audience and mathematical details are kept to a minimum.

  5. FcgammaRII mediates platelet aggregation caused by disintegrins and GPIIb/IIIa monoclonal antibody, AP2.

    Science.gov (United States)

    Huang, Tur-Fu; Chang, Chien-Hsin; Ho, Pei-Ling; Chung, Ching-Hu

    2008-12-01

    Disintegrins, snake venom-derived Arg-Gly-Asp (RGD)-containing polypeptides, and GPIIb/IIIa antagonist (AP2) block fibrinogen binding to GPIIb/IIIa of activated platelets, however, the combination of these two agents caused platelet aggregation. We hypothesize that disintegrin initially binds to specific epitope of GPIIb/IIIa, causing conformational change, and the recruitment of FcgammaRII, which can be bound by AP2, and finally triggering platelet aggregation. We prepared human platelet suspensions and measured platelet aggregation, Ca2+ mobilization, thromboxane B2 formation, and signal transduction. Disintegrin (e.g., accutin) and AP2 (a monoclonal antibody [mAb]-raised against GPIIb/IIIa) individually inhibited human platelet aggregation caused by collagen. However, as both accutin and AP2 were sequentially added into platelet suspension, platelet aggregation occurred. Accutin/AP2 caused shape change, cytosolic Ca2+ mobilization, P-selectin expression, and thromboxane A2 formation. Tirofiban, FcgammaRII mAb, or indomethacin completely inhibited platelet aggregation caused by accutin/AP2. Accutin/AP2 also caused tyrosine phosphorylation of signal molecules. Disintegrins enhanced AP2 binding to platelets, and AP2 also promoted disintegrin binding to platelets. FcgammaRII mAb inhibited the enhanced fluorescein isothiocyanate-disintegrin binding to platelet caused by AP2. Immunoprecipitation of the lysates of disintegrin/AP2-treated platelets using FcgammaRII Ab showed complex formation of GPIIb/IIIa and FcgammaRII. FcgammaRII mediates platelet aggregation caused by disintegrin and AP2, triggering a phospholipase C, phospholipase A2, Src-, Syk kinases, and Ca2+-dependent activation process. AP2 triggers platelet aggregation via binding to accessible FcgammaRII and the conformation-altered GPIIb/IIIa caused by disintegrin.

  6. Recombinant thermostable AP exonuclease from Thermoanaerobacter tengcongensis: cloning, expression, purification, properties and PCR application

    DEFF Research Database (Denmark)

    Dabrowski, Slawomir; Brillowska-Dabrowska, Anna; Ahring, Birgitte Kiær

    2013-01-01

    and transformed into Escherichia coli. The protein product showed high identity (80%) to human Ape1 nuclease, whereas to E. coli exonuclease III - 78%. This is the first prokaryotic AP nuclease that exhibits such high identity to human Ape1 nuclease. The very high expression level (57% of total soluble proteins......Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity, especially at high temperatures. The gene encoding a homologue of AP exonuclease was cloned from the thermophilic anaerobic bacterium Thermoanaerobacter tengcongensis......) of fully active and soluble His6-tagged Tte AP enzyme with His6-tag on C-terminal end was obtained in Escherichia coli Rosetta (DE3) pLysS. The active enzyme was purified up to 98% homogeneity in one chromatographic step using metal-affinity chromatography on Ni(2+)-IDA-Sepharose resin. The yield was 90 mg...

  7. Kepler observations of rapidly oscillating Ap, δ Scuti and γ Doradus pulsations in Ap stars

    DEFF Research Database (Denmark)

    Balona, Luis A.; Cunha, Margarida S.; Kurtz, Donald W.

    2011-01-01

    Observations of the A5p star KIC 8677585 obtained during the Kepler 10-d commissioning run with 1-min time resolution show that it is a rapidly oscillating Ap (roAp) star with several frequencies with periods near 10 min. In addition, a low frequency at 3.142 d−1 is also clearly present....... Multiperiodic γ Doradus (γ Dor) and δ Scuti (δ Sct) pulsations, never before seen in any Ap star, are present in Kepler observations of at least three other Ap stars. Since γ Dor pulsations are seen in Ap stars, it is likely that the low frequency in KIC 8677585 is also a γ Dor pulsation. The simultaneous...... presence of both γ Dor and roAp pulsations and the unexpected detection of δ Sct and γ Dor pulsations in Ap stars present new opportunities and challenges for the interpretation of these stars. Since it is easy to confuse Am and Ap stars at classification dispersions, the nature of these Ap stars...

  8. Mycobacterium tuberculosis class II apurinic/apyrimidinic-endonuclease/3'-5' exonuclease III exhibits DNA regulated modes of interaction with the sliding DNA β-clamp.

    Science.gov (United States)

    Khanam, Taran; Rai, Niyati; Ramachandran, Ravishankar

    2015-10-01

    The class-II AP-endonuclease (XthA) acts on abasic sites of damaged DNA in bacterial base excision repair. We identified that the sliding DNA β-clamp forms in vivo and in vitro complexes with XthA in Mycobacterium tuberculosis. A novel 239 QLRFPKK245 motif in the DNA-binding domain of XthA was found to be important for the interactions. Likewise, the peptide binding-groove (PBG) and the C-terminal of β-clamp located on different domains interact with XthA. The β-clamp-XthA complex can be disrupted by clamp binding peptides and also by a specific bacterial clamp inhibitor that binds at the PBG. We also identified that β-clamp stimulates the activities of XthA primarily by increasing its affinity for the substrate and its processivity. Additionally, loading of the β-clamp onto DNA is required for activity stimulation. A reduction in XthA activity stimulation was observed in the presence of β-clamp binding peptides supporting that direct interactions between the proteins are necessary to cause stimulation. Finally, we found that in the absence of DNA, the PBG located on the second domain of the β-clamp is important for interactions with XthA, while the C-terminal domain predominantly mediates functional interactions in the substrate's presence. © 2015 John Wiley & Sons Ltd.

  9. Endonucleases : new tools to edit the mouse genome

    NARCIS (Netherlands)

    Wijshake, Tobias; Baker, Darren J.; van de Sluis, Bart

    2014-01-01

    Mouse transgenesis has been instrumental in determining the function of genes in the pathophysiology of human diseases and modification of genes by homologous recombination in mouse embryonic stem cells remains a widely used technology. However, this approach harbors a number of disadvantages, as it

  10. Translational independence between overlapping genes for a restriction endonuclease and its transcriptional regulator

    Directory of Open Access Journals (Sweden)

    Blumenthal Robert M

    2010-11-01

    Full Text Available Abstract Background Most type II restriction-modification (RM systems have two independent enzymes that act on the same DNA sequence: a modification methyltransferase that protects target sites, and a restriction endonuclease that cleaves unmethylated target sites. When RM genes enter a new cell, methylation must occur before restriction activity appears, or the host's chromosome is digested. Transcriptional mechanisms that delay endonuclease expression have been identified in some RM systems. A substantial subset of those systems is controlled by a family of small transcription activators called C proteins. In the PvuII system, C.PvuII activates transcription of its own gene, along with that of the downstream endonuclease gene. This regulation results in very low R.PvuII mRNA levels early after gene entry, followed by rapid increase due to positive feedback. However, given the lethal consequences of premature REase accumulation, transcriptional control alone might be insufficient. In C-controlled RM systems, there is a ± 20 nt overlap between the C termination codon and the R (endonuclease initiation codon, suggesting possible translational coupling, and in many cases predicted RNA hairpins could occlude the ribosome binding site for the endonuclease gene. Results Expression levels of lacZ translational fusions to pvuIIR or pvuIIC were determined, with the native pvuII promoter having been replaced by one not controlled by C.PvuII. In-frame pvuIIC insertions did not substantially decrease either pvuIIC-lacZ or pvuIIR-lacZ expression (with or without C.PvuII provided in trans. In contrast, a frameshift mutation in pvuIIC decreased expression markedly in both fusions, but mRNA measurements indicated that this decrease could be explained by transcriptional polarity. Expression of pvuIIR-lacZ was unaffected when the pvuIIC stop codon was moved 21 nt downstream from its WT location, or 25 or 40 bp upstream of the pvuIIR initiation codon. Disrupting

  11. Translational independence between overlapping genes for a restriction endonuclease and its transcriptional regulator.

    Science.gov (United States)

    Kaw, Meenakshi K; Blumenthal, Robert M

    2010-11-19

    Most type II restriction-modification (RM) systems have two independent enzymes that act on the same DNA sequence: a modification methyltransferase that protects target sites, and a restriction endonuclease that cleaves unmethylated target sites. When RM genes enter a new cell, methylation must occur before restriction activity appears, or the host's chromosome is digested. Transcriptional mechanisms that delay endonuclease expression have been identified in some RM systems. A substantial subset of those systems is controlled by a family of small transcription activators called C proteins. In the PvuII system, C.PvuII activates transcription of its own gene, along with that of the downstream endonuclease gene. This regulation results in very low R.PvuII mRNA levels early after gene entry, followed by rapid increase due to positive feedback. However, given the lethal consequences of premature REase accumulation, transcriptional control alone might be insufficient. In C-controlled RM systems, there is a ± 20 nt overlap between the C termination codon and the R (endonuclease) initiation codon, suggesting possible translational coupling, and in many cases predicted RNA hairpins could occlude the ribosome binding site for the endonuclease gene. Expression levels of lacZ translational fusions to pvuIIR or pvuIIC were determined, with the native pvuII promoter having been replaced by one not controlled by C.PvuII. In-frame pvuIIC insertions did not substantially decrease either pvuIIC-lacZ or pvuIIR-lacZ expression (with or without C.PvuII provided in trans). In contrast, a frameshift mutation in pvuIIC decreased expression markedly in both fusions, but mRNA measurements indicated that this decrease could be explained by transcriptional polarity. Expression of pvuIIR-lacZ was unaffected when the pvuIIC stop codon was moved 21 nt downstream from its WT location, or 25 or 40 bp upstream of the pvuIIR initiation codon. Disrupting the putative hairpins had no significant

  12. Multiplex loop-mediated isothermal amplification detection by sequence-based barcodes coupled with nicking endonuclease-mediated pyrosequencing.

    Science.gov (United States)

    Liang, Chao; Chu, Yanan; Cheng, Sijia; Wu, Haiping; Kajiyama, Tomoharu; Kambara, Hideki; Zhou, Guohua

    2012-04-17

    The loop-mediated isothermal amplification (LAMP) is a well-developed method for replicating a targeted DNA sequence with a high specificity, but multiplex LAMP detection is difficult because LAMP amplicons are very complicated in structure. To allow simultaneous detection of multiple LAMP products, a series of target-specific barcodes were designed and tagged in LAMP amplicons by FIP primers. The targeted barcodes were decoded by pyrosequencing on nicked LAMP amplicons. To enable the nicking reaction to occur just near the barcode regions, the recognition sequence of the nicking endonuclease (NEase) was also introduced into the FIP primer. After the nicking reaction, pyrosequencing started at the nicked 3' end when the added deoxyribonucleoside triphosphate (dNTP) was complementary to the non-nicked strand. To efficiently encode multiple targets, the barcodes were designed with a reporter base and two stuffer bases, so that the decoding of a target-specific barcode only required a single peak in a pyrogram. We have successfully detected the four kinds of pathogens including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Treponema pallidum (TP), which are easily infected in blood, by a 4-plex LAMP in a single tube, indicating that barcoded LAMP coupled with NEase-mediated pyrosequencing is a simple, rapid, and reliable way in multiple target identification.

  13. Structure-specific DNA endonuclease Mus81/Eme1 generates DNA damage caused by Chk1 inactivation.

    Directory of Open Access Journals (Sweden)

    Josep V Forment

    Full Text Available The DNA-damage checkpoint kinase Chk1 is essential in higher eukaryotes due to its role in maintaining genome stability in proliferating cells. CHK1 gene deletion is embryonically lethal, and Chk1 inhibition in replicating cells causes cell-cycle defects that eventually lead to perturbed replication and replication-fork collapse, thus generating endogenous DNA damage. What is the cause of replication-fork collapse when Chk1 is inactivated, however, remains poorly understood. Here, we show that generation of DNA double-strand breaks at replication forks when Chk1 activity is compromised relies on the DNA endonuclease complex Mus81/Eme1. Importantly, we show that Mus81/Eme1-dependent DNA damage--rather than a global increase in replication-fork stalling--is the cause of incomplete replication in Chk1-deficient cells. Consequently, Mus81/Eme1 depletion alleviates the S-phase progression defects associated with Chk1 deficiency, thereby increasing cell survival. Chk1-mediated protection of replication forks from Mus81/Eme1 even under otherwise unchallenged conditions is therefore vital to prevent uncontrolled fork collapse and ensure proper S-phase progression in human cells.

  14. Tank 241-AP-107, grab samples 7AP-97-1, 7AP-97-2 and 7AP-97-3 analytical results for the final report

    Energy Technology Data Exchange (ETDEWEB)

    Steen, F.H.

    1997-12-22

    This document is the final report for tank 241-AP-107 grab samples. Three grab samples were collected from riser 1 on September 11, 1997. Analyses were performed on samples 7AP-97-1, 7AP-97-2 and 7AP-97-3 in accordance with the Compatibility Grab Sampling and Analysis Plan (TSAP) (Sasaki, 1997) and the Data Quality Objectives for Tank Farms Waste Compatibility Program (DQO) (Rev. 1: Fowler, 1995; Rev. 2: Mulkey and Nuier, 1997). The analytical results are presented in the data summary report (Table 1). A notification was made to East Tank Farms Operations concerning low hydroxide in the tank and a hydroxide (caustic) demand analysis was requested. The request for sample analysis (RSA) (Attachment 2) received for AP-107 indicated that the samples were polychlorinated biphenyl (PCB) suspects. Therefore, prior to performing the requested analyses, aliquots were made to perform PCB analysis in accordance with the 222-S Laboratory administrative procedure, LAP-101-100. The results of this analysis indicated that no PCBs were present at 50 ppm and analysis proceeded as non-PCB samples. The results and raw data for the PCB analysis will be included in a revision to this document. The sample breakdown diagrams (Attachment 1) are provided as a cross-reference for relating the tank farm customer identification numbers with the 222-S Laboratory sample numbers and the portion of sample analyzed.

  15. A Flap Endonuclease (TcFEN1) Is Involved in Trypanosoma cruzi Cell Proliferation, DNA Repair, and Parasite Survival.

    Science.gov (United States)

    Ponce, Ivan; Aldunate, Carmen; Valenzuela, Lucia; Sepúlveda, Sofia; Garrido, Gilda; Kemmerling, Ulrike; Cabrera, Gonzalo; Galanti, Norbel

    2017-07-01

    FLAP endonucleases (FEN) are involved both in DNA replication and repair by processing DNA intermediaries presenting a nucleotide flap using its phosphodiesterase activity. In spite of these important functions in DNA metabolism, this enzyme was not yet studied in Trypanosomatids. Trypanosoma cruzi, the ethiological agent of Chagas disease, presents two dividing cellular forms (epimastigote and amastigote) and one non-proliferative, infective form (trypomastigote). The parasite survives DNA damage produced by reactive species generated in its hosts. The activity of a T. cruzi FLAP endonuclease (TcFEN1) was determined in the three cellular forms of the parasite using a DNA substrate generated by annealing three different oligonucleotides to form a double-stranded DNA with a 5' flap in the middle. This activity showed optimal pH and temperature similar to other known FENs. The substrate cut by the flap endonuclease activity could be ligated by the parasite generating a repaired DNA product. A DNA flap endonuclease coding sequence found in the T. cruzi genome (TcFEN1) was cloned, inserted in parasite expression vectors and transfected to epimastigotes. The purified native recombinant protein showed DNA flap endonuclease activity. This endonuclease was found located in the parasite nucleus of transfected epimastigotes and its over-expression increased both parasite proliferation and survival to H 2 O 2 . The presence of a flap endonuclease activity in T. cruzi and its nuclear location are indicative of the participation of this enzyme in DNA processing of flap fragments during DNA replication and repair in this parasite of ancient evolutive origin. J. Cell. Biochem. 118: 1722-1732, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  16. Highly mutagenic exocyclic DNA adducts are substrates for the human nucleotide incision repair pathway.

    Directory of Open Access Journals (Sweden)

    Paulina Prorok

    Full Text Available BACKGROUND: Oxygen free radicals induce lipid peroxidation (LPO that damages and breaks polyunsaturated fatty acids in cell membranes. LPO-derived aldehydes and hydroxyalkenals react with DNA leading to the formation of etheno(ε-bases including 1,N(6-ethenoadenine (εA and 3,N(4-ethenocytosine (εC. The εA and εC residues are highly mutagenic in mammalian cells and eliminated in the base excision repair (BER pathway and/or by AlkB family proteins in the direct damage reversal process. BER initiated by DNA glycosylases is thought to be the major pathway for the removal of non-bulky endogenous base damage. Alternatively, in the nucleotide incision repair (NIR pathway, the apurinic/apyrimidinic (AP endonucleases can directly incise DNA duplex 5' to a damaged base in a DNA glycosylase-independent manner. METHODOLOGY/PRINCIPAL FINDINGS: Here we have characterized the substrate specificity of human major AP endonuclease 1, APE1, towards εA, εC, thymine glycol (Tg and 7,8-dihydro-8-oxoguanine (8oxoG residues when present in duplex DNA. APE1 cleaves oligonucleotide duplexes containing εA, εC and Tg, but not those containing 8oxoG. Activity depends strongly on sequence context. The apparent kinetic parameters of the reactions suggest that APE1 has a high affinity for DNA containing ε-bases but cleaves DNA duplexes at an extremely slow rate. Consistent with this observation, oligonucleotide duplexes containing an ε-base strongly inhibit AP site nicking activity of APE1 with IC(50 values in the range of 5-10 nM. MALDI-TOF MS analysis of the reaction products demonstrated that APE1-catalyzed cleavage of εA•T and εC•G duplexes generates, as expected, DNA fragments containing 5'-terminal ε-base residue. CONCLUSIONS/SIGNIFICANCE: The fact that ε-bases and Tg in duplex DNA are recognized and cleaved by APE1 in vitro, suggests that NIR may act as a backup pathway to BER to remove a large variety of genotoxic base lesions in human cells.

  17. Center for Geometrisk Metrologi CGM ApS, Årsberetning 2001 til DANAK

    DEFF Research Database (Denmark)

    De Chiffre, Leonardo

    Denne årsberetning omfatter CGM ApS' akkrediterede virksomhed i kalenderåret 2001. Årsberetningen er udarbejdet til DANAK (Dansk Akkreditering, Erhvervsfremme Styrelsen), som led i opfyldelsen af laboratoriets informationspligt i henhold til gældende regler (Teknisk Forskrift Nr. TF4 af 2000...

  18. Tauroursodeoxycholic acid reduces bile acid-induced apoptosis by modulation of AP-1.

    Science.gov (United States)

    Pusl, Thomas; Vennegeerts, Timo; Wimmer, Ralf; Denk, Gerald U; Beuers, Ulrich; Rust, Christian

    2008-02-29

    Ursodeoxycholic acid (UDCA) is used in the therapy of cholestatic liver diseases. Apoptosis induced by toxic bile acids plays an important role in the pathogenesis of liver injury during cholestasis and appears to be mediated by the human transcription factor AP-1. We aimed to study if TUDCA can decrease taurolitholic acid (TLCA)-induced apoptosis by modulating AP-1. TLCA (20 microM) upregulated AP-1 proteins cFos (26-fold) and JunB (11-fold) as determined by quantitative real-time PCR in HepG2-Ntcp hepatoma cells. AP-1 transcriptional activity increased by 300% after exposure to TLCA. cFos and JunB expression as well as AP-1 transcriptional activity were unaffected by TUDCA (75 microM). However, TUDCA significantly decreased TLCA-induced upregulation of cFos and JunB. Furthermore, TUDCA inhibited TLCA-induced AP-1 transcriptional activity and reduced TLCA-induced apoptosis. These data suggest that reversal of bile acid-induced AP-1 activation may be relevant for the antiapoptotic effect of TUDCA in liver cells.

  19. The Role of the Clathrin Adaptor AP-1: Polarized Sorting and Beyond

    Directory of Open Access Journals (Sweden)

    Fubito Nakatsu

    2014-11-01

    Full Text Available The selective transport of proteins or lipids by vesicular transport is a fundamental process supporting cellular physiology. The budding process involves cargo sorting and vesicle formation at the donor membrane and constitutes an important process in vesicular transport. This process is particularly important for the polarized sorting in epithelial cells, in which the cargo molecules need to be selectively sorted and transported to two distinct destinations, the apical or basolateral plasma membrane. Adaptor protein (AP-1, a member of the AP complex family, which includes the ubiquitously expressed AP-1A and the epithelium-specific AP-1B, regulates polarized sorting at the trans-Golgi network and/or at the recycling endosomes. A growing body of evidence, especially from studies using model organisms and animals, demonstrates that the AP-1-mediated polarized sorting supports the development and physiology of multi-cellular units as functional organs and tissues (e.g., cell fate determination, inflammation and gut immune homeostasis. Furthermore, a possible involvement of AP-1B in the pathogenesis of human diseases, such as Crohn’s disease and cancer, is now becoming evident. These data highlight the significant contribution of AP-1 complexes to the physiology of multicellular organisms, as master regulators of polarized sorting in epithelial cells.

  20. AP1000, a nuclear central of advanced design; AP1000, una central nuclear de diseno avanzado

    Energy Technology Data Exchange (ETDEWEB)

    Hernandez M, N.; Viais J, J. [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)]. e-mail: nhm@nuclear.inin.mx

    2005-07-01

    The AP1000 is a design of a nuclear reactor of pressurized water (PWR) of 1000 M We with characteristic of safety in a passive way; besides presenting simplifications in the systems of the plant, the construction, the maintenance and the safety, the AP1000 is a design that uses technology endorsed by those but of 30 years of operational experience of the PWR reactors. The program AP1000 of Westinghouse is focused to the implementation of the plant to provide improvements in the economy of the same one and it is a design that is derived directly of the AP600 designs. On September 13, 2004 the US-NRC (for their initials in United States- Nuclear Regulatory Commission) approved the final design of the AP1000, now Westinghouse and the US-NRC are working on the whole in a complete program for the certification. (Author)

  1. Slotted Aloha with multi-AP diversity and APS transmit beamforming

    Directory of Open Access Journals (Sweden)

    Zheng Di

    2011-01-01

    Full Text Available Abstract Slotted Aloha is an effective random access protocol and can also be an important element of more advanced media access protocols. This paper investigates slotted Aloha in a radio environment with multiple access points. Specifically, we examine the impact of multi-access-point (multi-AP diversity on the performance of slotted Aloha. The paper considers both omni-directional (OM and beamforming (BF antennas at transmission nodes. This leads to the investigation and comparison of four different network scenarios, i.e., OM with multi-AP diversity, OM without multi-AP diversity, BF with multi-AP diversity and BF without multi-AP diversity. Performance evaluations and comparisons are presented in terms of throughput and average packet delay.

  2. Defective mitochondrial respiration, altered dNTP pools and reduced AP endonuclease 1 activity in peripheral blood mononuclear cells of Alzheimer's disease patients

    DEFF Research Database (Denmark)

    Maynard, Scott; Hejl, Anne-Mette; Dinh, Tran Thuan Son

    2015-01-01

    AIMS: Accurate biomarkers for early diagnosis of Alzheimer's disease (AD) are badly needed. Recent reports suggest that dysfunctional mitochondria and DNA damage are associated with AD development. In this report, we measured various cellular parameters, related to mitochondrial bioenergetics...

  3. Defective mitochondrial respiration, altered dNTP pools and reduced AP endonuclease 1 activity in peripheral blood mononuclear cells of Alzheimer's disease patients

    DEFF Research Database (Denmark)

    Maynard, Scott; Hejl, Anne-Mette; Dinh, Thuan-Son T

    2015-01-01

    AIMS: Accurate biomarkers for early diagnosis of Alzheimer's disease (AD) are badly needed. Recent reports suggest that dysfunctional mitochondria and DNA damage are associated with AD development. In this report, we measured various cellular parameters, related to mitochondrial bioenergetics...... as possible. We measured glycolysis and mitochondrial respiration fluxes using the Seahorse Bioscience flux analyzer, mitochondrial ROS production using flow cytometry, dNTP levels by way of a DNA polymerization assay, DNA strand breaks using the Fluorometric detection of Alkaline DNA Unwinding (FADU) assay...... on adjustments for gender and/or age. CONCLUSIONS: This study reveals impaired mitochondrial respiration, altered dNTP pools and reduced DNA repair activity in PBMCs of AD patients, thus suggesting that these biochemical activities may be useful as biomarkers for AD....

  4. In situ gel-forming AP-57 peptide delivery system for cutaneous wound healing.

    Science.gov (United States)

    Li, Xiaoling; Fan, Rangrang; Tong, Aiping; Yang, Meijia; Deng, Jiaojiao; Zhou, Liangxue; Zhang, Xiaoning; Guo, Gang

    2015-11-10

    In situ gel-forming system as local drug delivery system in dermal traumas has generated a great interest. Accumulating evidence shows that antimicrobial peptides play pivotal roles in the process of wound healing. Here in this study, to explore the potential application of antimicrobial peptide in wound healing, biodegradable poly(L-lactic acid)-Pluronic L35-poly(L-lactic acid) (PLLA-L35-PLLA) was developed at first. Then based on this polymer, an injectable in situ gel-forming system composed of human antimicrobial peptides 57 (AP-57) loaded nanoparticles and thermosensitive hydrogel was prepared and applied for cutaneous wound healing. AP-57 peptides were enclosed with biocompatible nanoparticles (AP-57-NPs) with high drug loading and encapsulation efficiency. AP-57-NPs were further encapsulated in a thermosensitive hydrogel (AP-57-NPs-H) to facilitate its application in cutaneous wound repair. As a result, AP-57-NPs-H released AP-57 in an extended period and exhibited quite low cytotoxicity and high anti-oxidant activity in vitro. Moreover, AP-57-NPs-H was free-flowing liquid at room temperature, and can form non-flowing gel without any crosslink agent upon applied on the wounds. In vivo wound healing assay using full-thickness dermal defect model of SD rats indicated that AP-57-NPs-H could significantly promote wound healing. At day 14 after operation, AP-57-NPs-H treated group showed nearly complete wound closure of 96.78 ± 3.12%, whereas NS, NPs-H and AP-57-NPs group recovered by about 68.78 ± 4.93%, 81.96 ± 3.26% and 87.80 ± 4.62%, respectively. Histopathological examination suggested that AP-57-NPs-H could promote cutaneous wound healing through enhancing granulation tissue formation, increasing collagen deposition and promoting angiogenesis in the wound tissue. Therefore, AP-57-NPs-H might have potential application in wound healing. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. An analysis of AP600 design features

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jong Kyoon; Jang, Moon Heui; Hwang, Yung Dong [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of)] [and others

    1994-01-01

    In the aspect of engineering, passive safety system concept has improved the safety degree of nuclear power plant. Therefore, the objective of this study is to check on the possibility of the capacity upgrade of nuclear power plant in the case of adopting the passive safety system concept of AP 600. The characteristics of AP 600 are the advanced functions in ECCS, heat removal of containment building and residual heat removal under the passive safety system concept. The result of this study will become the basic data of capacity upgrade of nuclear power plant and will be widely used in second year project. (Author).

  6. Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system.

    Science.gov (United States)

    Morozova, Natalia; Sabantsev, Anton; Bogdanova, Ekaterina; Fedorova, Yana; Maikova, Anna; Vedyaykin, Alexey; Rodic, Andjela; Djordjevic, Marko; Khodorkovskii, Mikhail; Severinov, Konstantin

    2016-01-29

    Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Crystal structure of endonuclease G in complex with DNA reveals how it nonspecifically degrades DNA as a homodimer.

    Science.gov (United States)

    Lin, Jason L J; Wu, Chyuan-Chuan; Yang, Wei-Zen; Yuan, Hanna S

    2016-12-01

    Endonuclease G (EndoG) is an evolutionarily conserved mitochondrial protein in eukaryotes that digests nucleus chromosomal DNA during apoptosis and paternal mitochondrial DNA during embryogenesis. Under oxidative stress, homodimeric EndoG becomes oxidized and converts to monomers with diminished nuclease activity. However, it remains unclear why EndoG has to function as a homodimer in DNA degradation. Here, we report the crystal structure of the Caenorhabditis elegans EndoG homologue, CPS-6, in complex with single-stranded DNA at a resolution of 2.3 Å. Two separate DNA strands are bound at the ββα-metal motifs in the homodimer with their nucleobases pointing away from the enzyme, explaining why CPS-6 degrades DNA without sequence specificity. Two obligatory monomeric CPS-6 mutants (P207E and K131D/F132N) were constructed, and they degrade DNA with diminished activity due to poorer DNA-binding affinity as compared to wild-type CPS-6. Moreover, the P207E mutant exhibits predominantly 3'-to-5' exonuclease activity, indicating a possible endonuclease to exonuclease activity change. Thus, the dimer conformation of CPS-6 is essential for maintaining its optimal DNA-binding and endonuclease activity. Compared to other non-specific endonucleases, which are usually monomeric enzymes, EndoG is a unique dimeric endonuclease, whose activity hence can be modulated by oxidation to induce conformational changes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Divalent metal ion differentially regulates the sequential nicking reactions of the GIY-YIG homing endonuclease I-BmoI.

    Directory of Open Access Journals (Sweden)

    Benjamin P Kleinstiver

    Full Text Available Homing endonucleases are site-specific DNA endonucleases that function as mobile genetic elements by introducing double-strand breaks or nicks at defined locations. Of the major families of homing endonucleases, the modular GIY-YIG endonucleases are least understood in terms of mechanism. The GIY-YIG homing endonuclease I-BmoI generates a double-strand break by sequential nicking reactions during which the single active site of the GIY-YIG nuclease domain must undergo a substantial reorganization. Here, we show that divalent metal ion plays a significant role in regulating the two independent nicking reactions by I-BmoI. Rate constant determination for each nicking reaction revealed that limiting divalent metal ion has a greater impact on the second strand than the first strand nicking reaction. We also show that substrate mutations within the I-BmoI cleavage site can modulate the first strand nicking reaction over a 314-fold range. Additionally, in-gel DNA footprinting with mutant substrates and modeling of an I-BmoI-substrate complex suggest that amino acid contacts to a critical GC-2 base pair are required to induce a bottom-strand distortion that likely directs conformational changes for reaction progress. Collectively, our data implies mechanistic roles for divalent metal ion and substrate bases, suggesting that divalent metal ion facilitates the re-positioning of the GIY-YIG nuclease domain between sequential nicking reactions.

  9. Enzymatic cleavage of type II restriction endonucleases on the 2'-O-methyl nucleotide and phosphorothioate substituted DNA.

    Directory of Open Access Journals (Sweden)

    Guojie Zhao

    Full Text Available The effects of nucleotide analogue substitution on the cleavage efficiencies of type II restriction endonucleases have been investigated. Six restriction endonucleases (EcoRV, SpeI, XbaI, XhoI, PstI and SphI were investigated respectively regarding their cleavage when substrates were substituted by 2'-O-methyl nucleotide (2'-OMeN and phosphorothioate (PS. Substitutions were made in the recognition sequence and the two nucleotides flanking the recognition sequence for each endonuclease. The endonuclease cleavage efficiencies were determined using FRET-based assay. Results demonstrated a position-dependent inhibitory effect of substitution on the cleavage efficiency for all the six endonucleases. In general, the 2'-OMeN substitutions had greater impact than the PS substitutions on the enzymatic activities. Nucleotides of optimal substitutions for protection against RE cleavage were identified. Experimental results and conclusions in this study facilitate our insight into the DNA-protein interactions and the enzymatic cleavage mechanism, particularly for those whose detailed structure information is not available. In addition, the information could benefit the development of bioengineering and synthetic biology.

  10. Enzymatic cleavage of type II restriction endonucleases on the 2'-O-methyl nucleotide and phosphorothioate substituted DNA.

    Science.gov (United States)

    Zhao, Guojie; Li, Jun; Tong, Zhaoxue; Zhao, Bin; Mu, Runqing; Guan, Yifu

    2013-01-01

    The effects of nucleotide analogue substitution on the cleavage efficiencies of type II restriction endonucleases have been investigated. Six restriction endonucleases (EcoRV, SpeI, XbaI, XhoI, PstI and SphI) were investigated respectively regarding their cleavage when substrates were substituted by 2'-O-methyl nucleotide (2'-OMeN) and phosphorothioate (PS). Substitutions were made in the recognition sequence and the two nucleotides flanking the recognition sequence for each endonuclease. The endonuclease cleavage efficiencies were determined using FRET-based assay. Results demonstrated a position-dependent inhibitory effect of substitution on the cleavage efficiency for all the six endonucleases. In general, the 2'-OMeN substitutions had greater impact than the PS substitutions on the enzymatic activities. Nucleotides of optimal substitutions for protection against RE cleavage were identified. Experimental results and conclusions in this study facilitate our insight into the DNA-protein interactions and the enzymatic cleavage mechanism, particularly for those whose detailed structure information is not available. In addition, the information could benefit the development of bioengineering and synthetic biology.

  11. Cloning and Molecular Characterization of the Schistosoma mansoni Genes RbAp48 and Histone H4

    Directory of Open Access Journals (Sweden)

    Patrícia P Souza

    2002-10-01

    Full Text Available The human nuclear protein RbAp48 is a member of the tryptophan/aspartate (WD repeat family, which binds to the retinoblastoma (Rb protein. It also corresponds to the smallest subunit of the chromatin assembly factor and is able to bind to the helix 1 of histone H4, taking it to the DNA in replication. A cDNA homologous to the human gene RbAp48 was isolated from a Schistosoma mansoni adult worm library and named SmRbAp48. The full length sequence of SmRbAp48 cDNA is 1036 bp long, encoding a protein of 308 amino acids. The transcript of SmRbAp48 was detected in egg, cercariae and schistosomulum stages. The protein shows 84% similarity with the human RbAp48, possessing four WD repeats on its C-terminus. A hypothetical tridimensional structure for the SmRbAp48 C-terminal domain was constructed by computational molecular modeling using the b-subunit of the G protein as a model. To further verify a possible interaction between SmRbAp48 and S. mansoni histone H4, the histone H4 gene was amplified from adult worm genomic DNA using degenerated primers. The gene fragment of SmH4 is 294 bp long, encoding a protein of 98 amino acids which is 100% identical to histone H4 from Drosophila melanogaster.

  12. Army and Marine Corps Active Protection System (APS) Efforts

    Science.gov (United States)

    2016-08-30

    and Marines ’ detailed plans for APS fielding, and APS adaptability to future threats . Army and Marine Corps Active Protection System (APS...but instead battlefield threats and the operational environment have changed—thereby emphasizing the need for an APS-like capability. 59 Selected...Hardening” the APS Based on the Marines ’ M-1A1 concept of employment and its anticipated operational environment , it is likely that the Trophy

  13. 77 FR 24480 - Application for New Awards; Advanced Placement (AP) Test Fee Program-Reopening the AP Test Fee...

    Science.gov (United States)

    2012-04-24

    ... Application for New Awards; Advanced Placement (AP) Test Fee Program--Reopening the AP Test Fee Fiscal Year.... ACTION: Notice reopening the AP Test Fee fiscal year 2012 competition. Catalog of Federal Domestic... 8848) a notice inviting applications for the AP Test Fee fiscal year (FY) 2012 competition (February 15...

  14. Chilo iridescent virus (CIV) ORF 012L encodes a protein with both exonuclease and endonuclease functions.

    Science.gov (United States)

    Dizman, Yesim Akturk; Muratoglu, Hacer; Sandalli, Cemal; Nalcacioglu, Remziye; Demirbag, Zihni

    2016-11-01

    Chilo iridescent virus (CIV) is the type member of the genus Iridovirus within the family Iridoviridae. The virions of CIV contain a single linear dsDNA molecule that is circularly permuted and terminally redundant. The genome of CIV contains an open reading frame (ORF 012L) encoding a protein homologous to exonuclease II of Schizosaccharomyces pombe. In this study, we focused on the characterization of CIV ORF 012L. The target ORF was cloned into the pET28a vector, expressed in E. coli strain BL21 (DE3) pLysS with an N-terminal His tag and purified to homogeneity by using Ni-NTA affinity chromatography. Biochemical characterization of the purified CIV 012L confirmed that this viral protein is a functional 5'-3' exonuclease that digests 3'-biotin-labelled oligonucleotides and linear double-stranded DNA (dsDNA) molecules from their 5' termini in a highly processive manner. CIV 012L also has a potent endonuclease activity on dsDNA in vitro. In addition, CIV 012L converted supercoiled plasmid DNA (replicative form I, RFI) into the open circular form (RFII) and then open circular form into linear form (RFIII). Endonuclease activity of CIV 012L was optimal in the presence of 10 mM Mg(2+) or 30 mM Mn(2+) ions and at 150 mM NaCl or KCl salt concentrations. The highest endonuclease activity was obtained at pH 8, and it reached a maximum at 55 °C. The CIV 012L protein showed deficiencies for both double- and single-stranded RNAs.

  15. Metal ion dependence of DNA cleavage by SepMI and EhoI restriction endonucleases.

    Science.gov (United States)

    Belkebir, Abdelkarim; Azeddoug, Houssine

    2013-02-22

    Most of type II restriction endonucleases show an absolute requirement for divalent metal ions as cofactors for DNA cleavage. While Mg(2+) is the natural cofactor other metal ions can substitute it and mediate the catalysis, however Ca(2+) (alone) only supports DNA binding. To investigate the role of Mg(2+) in DNA cleavage by restriction endonucleases, we have studied the Mg(2+) and Mn(2+) concentration dependence of DNA cleavage by SepMI and EhoI. Digestion reactions were carried out at different Mg(2+) and Mn(2+) concentrations at constant ionic strength. These enzymes showed different behavior regarding the ions requirement, SepMI reached near maximal level of activity between 10 and 20mM while no activity was detected in the presence of Mn(2+) and in the presence of Ca(2+) cleavage activity was significantly decreased. However, EhoI was more highly active in the presence of Mn(2+) than in the presence of Mg(2+) and can be activated by Ca(2+). Our results propose the two-metal ion mechanism for EhoI and the one-metal ion mechanism for SepMI restriction endonuclease. The analysis of the kinetic parameters under steady state conditions showed that SepMI had a K(m) value for pTrcHisB DNA of 6.15 nM and a V(max) of 1.79×10(-2)nM min(-1), while EhoI had a K(m) for pUC19 plasmid of 8.66 nM and a V(max) of 2×10(-2)nM min(-1). Copyright © 2012 Elsevier GmbH. All rights reserved.

  16. Purification of Restriction Endonuclease EcoRII and its Co-Crystallization

    Science.gov (United States)

    Karpova, E. A.; Chen, L.; Meehan, E.; Pusey, M.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    Restriction endonuclease EcoRII (EcoRII) is a homodimeric DNA-binding protein. It belongs to the type II family of restriction-modification enzymes (subclass IIe). EcoRII recognizes the nucleotide sequence 5'-CCWGG (W=A or T) and cleaves the phosphodiester bond preceding the first cytosine. Methylation at C5 of the second cytosine inhibits cleavage. The enzyme has a unique ability to search for the presence of two substrate sites before cleavage. To the best of our knowledge no other subclass IIe restriction endonuclease has been crystallized yet, without or with a DNA-substrate. We have recently grown and characterized the crystals of this enzyme (1) Here we report on the result of co-crystallization experiments of EcoRII with an 11 b.p. oligonucleotide substrate. The dissociation constant (Kd) EcoRII: 11 b.p. was determined earlier (unpublished results). The needle-like crystals of oligonucleotide-EcoRII protein complex were obtained with this substrate by the technique of vapor diffusion hanging drops. The crystals obtained were washed and dissolved in an aliquot of 10 mM Tris-HCl buffer, pH=7.5. Running a portion of this solution on the SDS-get indicated the presence of endonuclease in the solution. A UV-spectrophotometric test of a second portion confirmed the presence of DNA. We are now working on improvement of the DNA-EcoRII protein crystals. Results obtained from these and ongoing efforts will be reported.

  17. Molecular dynamics simulations of deoxyribonucleic acids and repair enzyme T4 endonuclease V

    Energy Technology Data Exchange (ETDEWEB)

    Pinak, Miroslav

    1999-01-01

    This report describes the results of molecular dynamics (MD) simulation of deoxyribonucleic acids (DNA) and specific repair enzyme T4 endonuclease V. Namely research described here is focused on the examination of specific recognition process, in which this repair enzyme recognizes the damaged site on the DNA molecule-thymine dimer (TD). TD is frequent DNA damage induced by UV radiation in sun light and unless properly repaired it may be mutagenic or lethal for cell, and is also considered among the major causes of skin cancer. T4 endonuclease V is a DNA specific repair enzyme from bacteriophage T4 that catalyzes the first reaction step of TD repair pathway. MD simulations of three molecules - native DNA dodecamer (12 base pairs), DNA of the same sequence of nucleotides as native one but with TD, and repair enzyme T4 endonuclease V - were performed for 1 ns individually for each molecule. Simulations were analyzed to determine the role of electrostatic interaction in the recognition process. It is found that electrostatic energies calculated for amino acids of the enzyme have positive values of around +15 kcal/mol. The electrostatic energy of TD site has negative value of approximately -9 kcal/mol, different from the nearly neutral value of the respective thymines site of the native DNA. The electrostatic interaction of TD site with surrounding water environment differs from the electrostatic interaction of other nucleotides. Differences found between TD site and respective thymines site of native DNA indicate that the electrostatic energy is an important factor contributing to proper recognition of TD site during scanning process in which enzyme scans the DNA. In addition to the electrostatic energy, the important factor in recognition process might be structural complementarity of enzyme and bent DNA with TD. There is significant kink formed around TD site, that is not observed in native DNA. (author)

  18. Structuring the AP Art History Course

    Science.gov (United States)

    Herscher, Walter R.

    2013-01-01

    While AP (Advanced Placement) Art History may be taught within the art department in many schools, social studies teachers are equally capable of teaching the course well. They have the historical background to discuss the reasons for changes in art styles. A teacher's preparation is similar to teaching a course stressing political history,…

  19. 2000 Johnston Site 1A-P

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — Underwater Site 1A-P was established at Johnston Atoll by Dr. James Maragos, U.S. Fish & Wildlife Service, on June 29, 2000. With a start point (meter 0) at...

  20. 2000 Johnston Site 3A-P

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — Underwater Site 3A-P was established at Johnston Atoll by Dr. James Maragos, U.S. Fish & Wildlife Service, on July 3, 2000. With a start point (meter 0) at...

  1. Two Successful Approaches to Teaching AP Government

    Science.gov (United States)

    Ladd, Brian; Stepp, Heidi

    2013-01-01

    Amador Valley High School, in Pleasanton, California, uses two unique approaches to teaching Advanced Placement Government and Politics. AP Government consists of six units: Constitutional Underpinnings; Political Behavior and Political Beliefs; Mass Media, Interest Groups, and Political Parties; Institutions of Government; Civil Liberties and…

  2. Barron's AP English literature and composition

    CERN Document Server

    Ehrenhaft EdD, George

    2016-01-01

    Includes five full-length practice AP exams with all questions answered and explained. Also features additional reviews on poetry, fiction, and drama, definitions of 175 literary and rhetorical terms, and more. Can be purchased alone or with an optional CD-ROM with two additional practice tests.

  3. The Promise of AP World History

    Science.gov (United States)

    Saldaña, Cristóbal T.

    2013-01-01

    AP World History is the ideal history course. It introduces students to 10,000 years of world history, and demands critical reading, critical writing, and critical thinking skills on the part of both the teacher and the students. It requires students to build their expertise in reading their textbook, and places demands on the teacher to assign…

  4. 2000 Johnston Site 2A-P

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — Underwater Site 2A-P was established at Johnston Atoll by Dr. James Maragos, U.S. Fish & Wildlife Service, on June 30, 2000. With a start point (meter 0) at...

  5. Creation of targeted inversion mutations in plants using an RNA-guided endonuclease

    Directory of Open Access Journals (Sweden)

    Congsheng Zhang

    2017-02-01

    Full Text Available Inversions are DNA rearrangements that are essential for plant gene evolution and adaptation to environmental changes. We demonstrate the creation of targeted inversions and previously reported targeted deletion mutations via delivery of a pair of RNA-guided endonucleases (RGENs of CRISPR/Cas9. The efficiencies of the targeted inversions were 2.6% and 2.2% in the Arabidopsis FLOWERING TIME (AtFT and TERMINAL FLOWER 1 (AtTFL1 loci, respectively. Thus, we successfully established an approach that can potentially be used to introduce targeted DNA inversions of interest for functional studies and crop improvement.

  6. Intermolecular interaction between a branching ribozyme and associated homing endonuclease mRNA

    DEFF Research Database (Denmark)

    Birgisdottir, Asa B; Nielsen, Henrik; Beckert, Bertrand

    2011-01-01

    Abstract RNA tertiary interactions involving docking of GNRA (N; any base; R; purine) hairpin loops into helical stem structures on other regions of the same RNA are one of the most common RNA tertiary interactions. In this study, we investigated a tertiary association between a GAAA hairpin......-like motif (UCUAAG-CAAGA) found within the HEG P1. The biological role of this interaction appears to be linked to the homing endonuclease expression by promoting post-cleavage release of the lariat capped mRNA. These findings add to our understanding of how protein-coding genes embedded in nuclear ribosomal...

  7. Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage

    DEFF Research Database (Denmark)

    Stella, Stefano; Alcón, Pablo; Montoya, Guillermo

    2017-01-01

    Cpf1 is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we provide insight into its DNA-targeting mechanism by determining the structure of Francisella novicida Cpf1 with the triple-stranded R-loop generated after DNA cleavage. The structure reveals the machine...... and the crRNA-DNA hybrid, avoiding DNA re-annealing. Mutations in key residues reveal a mechanism linking the PAM and DNA nuclease sites. Analysis of the Cpf1 structures proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1....

  8. Purification, crystallization, X-ray diffraction analysis and phasing of an engineered single-chain PvuII restriction endonuclease

    Energy Technology Data Exchange (ETDEWEB)

    Meramveliotaki, Chrysi [Department of Science, Agricultural University of Athens, Athens (Greece); Department of Biology, University of Crete, PO Box 2208, GR-71110 Heraklion, Crete (Greece); Institute of Molecular Biology and Biotechnology (IMBB), PO Box 1527, GR-71110 Heraklion, Crete (Greece); Department of Agricultural Biotechnology, Agricultural University of Athens, Athens (Greece); Kotsifaki, Dina [Institute of Molecular Biology and Biotechnology (IMBB), PO Box 1527, GR-71110 Heraklion, Crete (Greece); Androulaki, Maria [Department of Science, Agricultural University of Athens, Athens (Greece); Department of Biology, University of Crete, PO Box 2208, GR-71110 Heraklion, Crete (Greece); Institute of Molecular Biology and Biotechnology (IMBB), PO Box 1527, GR-71110 Heraklion, Crete (Greece); Department of Agricultural Biotechnology, Agricultural University of Athens, Athens (Greece); Hountas, Athanasios [Department of Science, Agricultural University of Athens, Athens (Greece); Eliopoulos, Elias [Department of Agricultural Biotechnology, Agricultural University of Athens, Athens (Greece); Kokkinidis, Michael, E-mail: kokkinid@imbb.forth.gr [Department of Biology, University of Crete, PO Box 2208, GR-71110 Heraklion, Crete (Greece); Institute of Molecular Biology and Biotechnology (IMBB), PO Box 1527, GR-71110 Heraklion, Crete (Greece); Department of Science, Agricultural University of Athens, Athens (Greece)

    2007-10-01

    PvuII is the first type II restriction endonuclease to be converted from its wild-type homodimeric form into an enzymatically active single-chain variant. The enzyme was crystallized and phasing was successfully performed by molecular replacement. The restriction endonuclease PvuII from Proteus vulgaris has been converted from its wild-type homodimeric form into the enzymatically active single-chain variant scPvuII by tandemly joining the two subunits through the peptide linker Gly-Ser-Gly-Gly. scPvuII, which is suitable for the development of programmed restriction endonucleases for highly specific DNA cleavage, was purified and crystallized. The crystals diffract to a resolution of 2.35 Å and belong to space group P4{sub 2}, with unit-cell parameters a = b = 101.92, c = 100.28 Å and two molecules per asymmetric unit. Phasing was successfully performed by molecular replacement.

  9. Interdomain communication in the endonuclease/motor subunit of type I restriction-modification enzyme EcoR124I.

    Science.gov (United States)

    Sinha, Dhiraj; Shamayeva, Katsiaryna; Ramasubramani, Vyas; Řeha, David; Bialevich, Vitali; Khabiri, Morteza; Guzanová, Alena; Milbar, Niv; Weiserová, Marie; Csefalvay, Eva; Carey, Jannette; Ettrich, Rüdiger

    2014-07-01

    Restriction-modification systems protect bacteria from foreign DNA. Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA-cleavage and ATP-dependent DNA translocation activities located on endonuclease/motor subunit HsdR. The recent structure of the first intact motor subunit of the type I restriction enzyme from plasmid EcoR124I suggested a mechanism by which stalled translocation triggers DNA cleavage via a lysine residue on the endonuclease domain that contacts ATP bound between the two helicase domains. In the present work, molecular dynamics simulations are used to explore this proposal. Molecular dynamics simulations suggest that the Lys-ATP contact alternates with a contact with a nearby loop housing the conserved QxxxY motif that had been implicated in DNA cleavage. This model is tested here using in vivo and in vitro experiments. The results indicate how local interactions are transduced to domain motions within the endonuclease/motor subunit.

  10. Genome of orf virus. Restriction endonuclease analysis of viral DNA isolated from lesions of orf in sheep

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, A.J.; Ellis, G.; Balassu, T. (Massay Univ., Palmerston North (New Zealand). Dept. of Veterinary Pathology and Public Health)

    1982-01-01

    The purification of orf virus directly from scab material from clinical cases of orf in sheep and restriction endonuclease analysis of the viral DNA is described. Between 7 x 10/sup 9/ and 1.6 x 10/sup 11/ virus particles, and 0.7 to 22.8 ..mu..g of viral DNA could be recovered from lg of scab material. Considerable heterogeneity was observed between different field isolates when restriction endonuclease digests of orf DNA were compared by gel electrophoresis. It was also shown, for two isolates that these fragment patterns did not change after plaque purification and passage in cell culture. It is suggested that restriction endonuclease analysis of viral DNA offers a convenient method of identification of isolates of orf virus. The molecular weight of orf DNA was determined and found to be 88.8 x 10/sup 6/.

  11. Cocaine and the AP-1 transcription factor complex.

    Science.gov (United States)

    Hope, B T

    1998-05-30

    Cocaine addition in humans develops gradually with repeated administrations and persists long after cocaine has cleared the body. The mechanisms underlying this persistent form of neuroplasticity are not understood and can involve both structural and biochemical mechanisms. The long time course for cocaine addiction in humans and for development of cocaine self-administration in animal models suggest the involvement of alterations in gene expression leading to altered signaling in the brain. In the striatum (Str) and nucleus accumbens (NAc) of rats. Pretreatment with repeated cocaine administrations downregulates the induction of various immediate early genes (IEGs) by a subsequent acute challenge with cocaine. Some of these downregulated IEGs encode Fos-related components of the activator protein-1 (AP-1) complex, which is likely re regulate a number of genes important for neuronal function. Interestingly, repeated cocaine administration induces novel delta FosB-related proteins (called chronic Fos-related antigens (Fras)) in the NAc and Str that replace the downregulated isoforms of Fos. Unlike the acutely induced, short-lasting isoforms of Fos and FosB, the chronic Fras persist long after the last cocaine administration. The known form of delta FosB per se lacks the domain required to activate transcription. If the chronic Fras are similar in structure to delta FosB, then the induction of chronic Fras likely leads to a blockade of AP-1-dependent transcription resulting in altered gene expression. We presently purifying the chronic Fras to obtain amino acid sequence in order to directly examine our hypothesis about the effects of repeated cocaine administration on AP-1 dependent transcription and gene expression in the brain

  12. The adipocyte fatty acid–binding protein aP2 is required in allergic airway inflammation

    Science.gov (United States)

    Shum, Bennett O.V.; Mackay, Charles R.; Gorgun, Cem Z.; Frost, Melinda J.; Kumar, Rakesh K.; Hotamisligil, Gökhan S.; Rolph, Michael S.

    2006-01-01

    The adipocyte fatty acid–binding protein aP2 regulates systemic glucose and lipid metabolism. We report that aP2, in addition to being abundantly expressed by adipocytes, is also expressed by human airway epithelial cells and shows a striking upregulation following stimulation of epithelial cells with the Th2 cytokines IL-4 and IL-13. Regulation of aP2 mRNA expression by Th2 cytokines was highly dependent on STAT6, a transcription factor with a major regulatory role in allergic inflammation. We examined aP2-deficient mice in a model of allergic airway inflammation and found that infiltration of leukocytes, especially eosinophils, into the airways was highly dependent on aP2 function. T cell priming was unaffected by aP2 deficiency, suggesting that aP2 was acting locally within the lung, and analysis of bone marrow chimeras implicated non-hematopoietic cells, most likely bronchial epithelial cells, as the site of action of aP2 in allergic airway inflammation. Thus, aP2 regulates allergic airway inflammation and may provide a link between fatty acid metabolism and asthma. PMID:16841093

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

    African Journals Online (AJOL)

    Correct orientation was verified by restriction endonuclease digestion. Human full length TIMP-2 gene was ligated into a plasmid, which was then transfected into PA317 cell line. G418-resistant individual clones were selected to transfect human SGC-7901 cell line. Cell proliferation, cell electrophoresis, soft agar colony ...

  14. Molecular diversity of Pasteurella multocida isolated from cattle and buffaloes in East Azerbaijan province based on restriction endonuclease analysis

    Directory of Open Access Journals (Sweden)

    jalal shayegh

    2014-05-01

    Full Text Available In order to increase information about the molecular diversity of Pasteurella multocida isolated from cattle and buffalo, 2 buffalo and 8 cattle isolates were investigated by Restriction Endonuclease Analysis (REA. REA was performed with Hha-I Endonuclease which established 2 distinct profiles: I and II.  Cattle and buffalo isolates fell into both REA profiles. Contrary to previous studies, the genetic diversity of the isolates was negligible. Considering the similarity of cattle and buffalo isolates is the present study, further studies witch larger samples should be carried out to investigate the possibility of inter-species transmission.

  15. A model of EcoRII restriction endonuclease action: the active complex is most likely formed by one protein subunit and one DNA recognition site

    Science.gov (United States)

    Karpova, E. A.; Kubareva, E. A.; Shabarova, Z. A.

    1999-01-01

    To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.

  16. Repair of DNA damaged by ionizing radiation and other oxidative agents in yeast and human

    Energy Technology Data Exchange (ETDEWEB)

    Louisek Prakash

    2000-01-15

    OAK B202 Treatment of cells with oxidative DNA damaging agents such as ionizing radiation and hydrogen peroxide produces .OH radicals which attack DNA, producing single strand breaks and double strand breaks that have a 3'-blocked terminus with a phosphoglycolate or a phosphate group attached to the 3'-terminus. While DNA strand breaks with 3'-blocked termini are the hallmark of oxidative DNA damage, the mechanisms by which such blocked 3'-termini are removed in eukaryotes remain poorly understood. The goals of this project were to identify the various genes that function in cleaning the blocked 3'ends from DNA strand breaks generated by treatments with ionizing radiation and hydrogen peroxide, to purify the proteins encoded by these genes and to characterize their biochemical activities, and to determine the biological consequences when such damage is not repaired. Because of the high degree of conservation of DNA repair proteins between yeast and humans, and because of the ease of genetic manipulations, initial studies were to be carried out in Saccharomyces cerevisiae. The homologous genes and proteins would then be studied in humans. One aspect of our proposed research was to purify the Apn2 protein from yeast cells and to examine its AP endonuclease and 3'-phosphodiesterase activities. Apn2-like proteins have been identified in eukaryotes other than yeast, including humans, and these proteins form a distinct subfamily within the ExoIII/Ape1/Apn2 family of proteins. We purified the Apn2 protein from yeast and showed that it is a class II AP endonuclease. (Class II AP endonucleases cleave the phosphodiester backbone on the 5'-side of the AP site and produce a 3'-OH group and a 5'-baseless deoxyribose 5'-phosphate residue). Yeast Apn2 and its orthologs in higher eukaryotes differ from E. coli ExoIII and human Ape1 in possessing a C terminus that is absent from the ExoIII/Ape1 subfamily. We found that deletion of

  17. Repair of DNA damaged by ionizing radiation and other oxidative agents in yeast and human

    Energy Technology Data Exchange (ETDEWEB)

    Louise Prakash

    2000-01-15

    Treatment of cells with oxidative DNA damaging agents such as ionizing radiation and hydrogen peroxide produces .OH radicals which attack DNA, producing single strand breaks and double strand breaks that have a 3'-blocked terminus with a phosphoglycolate or a phosphate group attached to the 3'-terminus. While DNA strand breaks with 3'-blocked termini are the hallmark of oxidative DNA damage, the mechanisms by which such blocked 3'-termini are removed in eukaryotes remain poorly understood. The goals of this project were to identify the various genes that function in cleaning the blocked 3'-ends from DNA strand breaks generated by treatments with ionizing radiation and hydrogen peroxide, to purify the proteins encoded by these genes and to characterize their biochemical activities, and to determine the biological consequences when such damage is not repaired. Because of the high degree of conservation of DNA repair proteins between yeast and humans, and because of the ease of genetic manipulations, initial studies were to be carried out in Saccharomyces cerevisiae. The homologous genes and proteins would then be studied in humans. One aspect of our proposed research was to purify the Apn2 protein from yeast cells and to examine its AP endonuclease and 3'-phosphodiesterase activities. Apn2-like proteins have been identified in eukaryotes other than yeast, including humans, and these proteins form a distinct subfamily within the ExoIII/Ape1/Apn2 family of proteins. We purified the Apn2 protein from yeast and showed that it is a class II AP endonuclease. (Class II AP endonucleases cleave the phosphodiester backbone on the 5'-side of the AP site and produce a 3'-OH group and a 5'-baseless deoxyribose 5'-phosphate residue). Yeast Apn2 and its orthologs in higher eukaryotes differ from E. coli ExoIII and human Ape1 in possessing a C terminus that is absent from the ExoIII/Ape1 subfamily. We found that deletion of the

  18. Polymerase-endonuclease amplification reaction (PEAR for large-scale enzymatic production of antisense oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Xiaolong Wang

    Full Text Available Antisense oligonucleotides targeting microRNAs or their mRNA targets prove to be powerful tools for molecular biology research and may eventually emerge as new therapeutic agents. Synthetic oligonucleotides are often contaminated with highly homologous failure sequences. Synthesis of a certain oligonucleotide is difficult to scale up because it requires expensive equipment, hazardous chemicals and a tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR, for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves over 100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation. PEAR has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs.

  19. Expression, purification and crystallization of two endonuclease III enzymes from Deinococcus radiodurans.

    Science.gov (United States)

    Sarre, Aili; Ökvist, Mats; Klar, Tobias; Moe, Elin; Timmins, Joanna

    2014-12-01

    Endonuclease III is a bifunctional DNA glycosylase that removes a wide range of oxidized bases in DNA. Deinococcus radiodurans is an extreme radiation-resistant and desiccation-resistant bacterium and possesses three genes encoding endonuclease III enzymes in its genome: DR2438 (EndoIII-1), DR0289 (EndoIII-2) and DR0982 (EndoIII-3). Here, EndoIII-1 and an N-terminally truncated form of EndoIII-3 (EndoIII-3Δ76) have been expressed, purified and crystallized, and preliminary X-ray crystallographic analyses have been performed to 2.15 and 1.31 Å resolution, respectively. The EndoIII-1 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 181.38, b = 38.56, c = 37.09 Å, β = 89.34° and one molecule per asymmetric unit. The EndoIII-3Δ76 crystals also belonged to the monoclinic space group C2, but with unit-cell parameters a = 91.47, b = 40.53, c = 72.47 Å, β = 102.53° and one molecule per asymmetric unit. The EndoIII-1 structure was determined by molecular replacement, while the truncated EndoIII-3Δ76 structure was determined by single-wavelength anomalous dispersion phasing. Refinement of the structures is in progress.

  20. RPA activates the XPF‐ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks

    KAUST Repository

    Abdullah, Ummi B

    2017-06-13

    During replication‐coupled DNA interstrand crosslink (ICL) repair, the XPF‐ERCC1 endonuclease is required for the incisions that release, or “unhook”, ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL. Here, we report that while purified XPF‐ERCC1 incises simple ICL‐containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single‐stranded DNA (ssDNA)‐binding replication protein A (RPA) selectively restores XPF‐ERCC1 endonuclease activity on this structure. The 5′–3′ exonuclease SNM1A can load from the XPF‐ERCC1‐RPA‐induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF‐ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.

  1. In vitro Inactivation of Latent HSV by Targeted Mutagenesis Using an HSV-specific Homing Endonuclease

    Directory of Open Access Journals (Sweden)

    Martine Aubert

    2014-01-01

    Full Text Available Following acute infection, herpes simplex virus (HSV establishes latency in sensory neurons, from which it can reactivate and cause recurrent disease. Available antiviral therapies do not affect latent viral genomes; therefore, they do not prevent reactivation following therapy cessation. One possible curative approach involves the introduction of DNA double strand breaks in latent HSV genomes by rare-cutting endonucleases, leading to mutagenesis of essential viral genes. We tested this approach in an in vitro HSV latency model using the engineered homing endonuclease (HE HSV1m5, which recognizes a sequence in the HSV-1 gene UL19, encoding the virion protein VP5. Coexpression of the 3′-exonuclease Trex2 with HEs increased HE-mediated mutagenesis frequencies up to sixfold. Following HSV1m5/Trex2 delivery with adeno-associated viral (AAV vectors, the target site was mutated in latent HSV genomes with no detectable cell toxicity. Importantly, HSV production by latently infected cells after reactivation was decreased after HSV1m5/Trex2 exposure. Exposure to histone deacetylase inhibitors prior to HSV1m5/Trex2 treatment increased mutagenesis frequencies of latent HSV genomes another two- to fivefold, suggesting that chromatin modification may be a useful adjunct to gene-targeting approaches. These results support the continuing development of HEs and other nucleases (ZFNs, TALENs, CRISPRs for cure of chronic viral infections.

  2. In vitro Inactivation of Latent HSV by Targeted Mutagenesis Using an HSV-specific Homing Endonuclease.

    Science.gov (United States)

    Aubert, Martine; Boyle, Nicole M; Stone, Daniel; Stensland, Laurence; Huang, Meei-Li; Magaret, Amalia S; Galetto, Roman; Rawlings, David J; Scharenberg, Andrew M; Jerome, Keith R

    2014-02-04

    Following acute infection, herpes simplex virus (HSV) establishes latency in sensory neurons, from which it can reactivate and cause recurrent disease. Available antiviral therapies do not affect latent viral genomes; therefore, they do not prevent reactivation following therapy cessation. One possible curative approach involves the introduction of DNA double strand breaks in latent HSV genomes by rare-cutting endonucleases, leading to mutagenesis of essential viral genes. We tested this approach in an in vitro HSV latency model using the engineered homing endonuclease (HE) HSV1m5, which recognizes a sequence in the HSV-1 gene UL19, encoding the virion protein VP5. Coexpression of the 3'-exonuclease Trex2 with HEs increased HE-mediated mutagenesis frequencies up to sixfold. Following HSV1m5/Trex2 delivery with adeno-associated viral (AAV) vectors, the target site was mutated in latent HSV genomes with no detectable cell toxicity. Importantly, HSV production by latently infected cells after reactivation was decreased after HSV1m5/Trex2 exposure. Exposure to histone deacetylase inhibitors prior to HSV1m5/Trex2 treatment increased mutagenesis frequencies of latent HSV genomes another two- to fivefold, suggesting that chromatin modification may be a useful adjunct to gene-targeting approaches. These results support the continuing development of HEs and other nucleases (ZFNs, TALENs, CRISPRs) for cure of chronic viral infections.Molecular Therapy-Nucleic Acids (2014) 3, e1; doi:10.1038/mtna.2013.75; published online 4 February 2014.

  3. Telomere-associated endonuclease-deficient Penelope-like retroelements in diverse eukaryotes

    Science.gov (United States)

    Gladyshev, Eugene A.; Arkhipova, Irina R.

    2007-01-01

    The evolutionary origin of telomerases, enzymes that maintain the ends of linear chromosomes in most eukaryotes, is a subject of debate. Penelope-like elements (PLEs) are a recently described class of eukaryotic retroelements characterized by a GIY-YIG endonuclease domain and by a reverse transcriptase domain with similarity to telomerases and group II introns. Here we report that a subset of PLEs found in bdelloid rotifers, basidiomycete fungi, stramenopiles, and plants, representing four different eukaryotic kingdoms, lack the endonuclease domain and are located at telomeres. The 5′ truncated ends of these elements are telomere-oriented and typically capped by species-specific telomeric repeats. Most of them also carry several shorter stretches of telomeric repeats at or near their 3′ ends, which could facilitate utilization of the telomeric G-rich 3′ overhangs to prime reverse transcription. Many of these telomere-associated PLEs occupy a basal phylogenetic position close to the point of divergence from the telomerase-PLE common ancestor and may descend from the missing link between early eukaryotic retroelements and present-day telomerases. PMID:17483479

  4. Expression of flap endonuclease-1 during meiosis in a basidiomycete, Coprinus cinereus.

    Science.gov (United States)

    Yamaguchi, Taiki; Namekawa, Satoshi H; Hamada, Fumika N; Kasai, Nobuyuki; Nara, Takayuki; Watanabe, Kei; Iwabata, Kazuki; Ishizaki, Takashi; Ishii, Satomi; Koshiyama, Akiyo; Inagaki, Sachiyo; Kimura, Seisuke; Sakaguchi, Kengo

    2004-05-01

    In the basidiomycete Coprinus cinereus (C. cinereus), which shows a highly synchronous meiotic cell cycle, the meiotic prophase I cells demonstrate flap endonuclease-1 activity. To investigate its role during meiosis, we isolated a C. cinereus cDNA homolog of flap endonuclease-1 (CcFEN-1), 1377bp in length with the open reading frame (ORF) encoding a predicted molecular mass of 51 kDa. At amino-acid residues Glu276-Pro345, a specific inserted sequence composed of 70 amino acids rich in polar forms was found to exist, without sequence identity to other eukaryotic FEN-1 or the polar amino acid rich sequences found in C. cinereus PCNA and C. cinereus DNA ligase IV, although the lengths and percentages of polar amino acids were similar. Northern hybridization analysis indicated CcFEN-1 to be expressed not only in the pre-meiotic S phase but also in meiotic prophase I. The roles of CcFEN-1 during meiosis are discussed.

  5. Atypical myxomatosis--virus isolation, experimental infection of rabbits and restriction endonuclease analysis of the isolate.

    Science.gov (United States)

    Psikal, I; Smíd, B; Rodák, L; Valícek, L; Bendová, J

    2003-08-01

    Atypical form of myxomatosis, which caused non-lethal and clinically mild disease in domestic rabbits 1 month after immunization with a commercially available vaccine MXT, is described. The isolated myxoma virus designated as Litovel 2 (Li-2) did not induce systemic disease following subcutaneous and intradermal applications in susceptible experimental rabbits but led to the immune response demonstrated by ELISA. No severe disease was induced in those Li-2 inoculated rabbits by challenge with the virulent strains Lausanne (Lu) or Sanar (SA), while the control animals showed nodular form of myxomatosis with lethal course of the illness. Restriction fragment length polymorphism (RFLP) of genomic DNA with KpnI and BamHI endonucleases was used for genetic characterization of the Li-2 isolate, the vaccine strain MXT and both virulent strains Lu and SA, respectively. In general, RFLP analysis has shown to be informative for inferring genetic relatedness between myxoma viruses. Based on restriction endonuclease DNA fragment size distribution, it was evident that the pathogenic strain SA is genetically related to the reference strain Lu and the isolate Li-2 is more related, but not identical, to the vaccination strain MXT.

  6. Assembly of Francisella novicida Cpf1 endonuclease in complex with guide RNA and target DNA.

    Science.gov (United States)

    Alcón, Pablo; Montoya, Guillermo; Stella, Stefano

    2017-07-01

    Bacteria and archaea use the CRISPR-Cas system as an adaptive response against infection by foreign nucleic acids. Owing to its remarkable flexibility, this mechanism has been harnessed and adopted as a powerful tool for genome editing. The CRISPR-Cas system includes two classes that are subdivided into six types and 19 subtypes according to conservation of the cas gene and loci organization. Recently, a new protein with endonuclease activity belonging to class 2 type V has been identified. This endonuclease, termed Cpf1, in complex with a single CRISPR RNA (crRNA) is able to recognize and cleave a target DNA preceded by a 5'-TTN-3' protospacer-adjacent motif (PAM) complementary to the RNA guide. To obtain structural insight into the inner workings of Cpf1, the crystallization of an active complex containing the full extent of the crRNA and a 31-nucleotide dsDNA target was attempted. The gene encoding Cpf1 from Francisella novicida was cloned, overexpressed and purified. The crRNA was transcribed and purified in vitro. Finally, the ternary FnCpf1-crRNA-DNA complex was assembled and purified by preparative electrophoresis before crystallization. Crystals belonging to space group C2221, with unit-cell parameters a = 85.2, b = 137.6, c = 320.5 Å, were obtained and subjected to preliminary diffraction experiments.

  7. Nuclease escape elements protect messenger RNA against cleavage by multiple viral endonucleases.

    Science.gov (United States)

    Muller, Mandy; Glaunsinger, Britt A

    2017-08-01

    During lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection, the viral endonu- clease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs, including the transcript encoding interleukin-6 (IL-6), escape SOX-induced cleavage. IL-6 escape is mediated through a 3' UTR RNA regulatory element that overrides the SOX targeting mechanism. Here, we reveal that this protective RNA element functions to broadly restrict cleavage by a range of homologous and non-homologous viral endonucleases. However, it does not impede cleavage by cellular endonucleases. The IL-6 protective sequence may be representative of a larger class of nuclease escape elements, as we identified a similar protective element in the GADD45B mRNA. The IL-6 and GADD45B-derived elements display similarities in their sequence, putative structure, and several associated RNA binding proteins. However, the overall composition of their ribonucleoprotein complexes appears distinct, leading to differences in the breadth of nucleases restricted. These findings highlight how RNA elements can selectively control transcript abundance in the background of widespread virus-induced mRNA degradation.

  8. A unique family of Mrr-like modification-dependent restriction endonucleases.

    Science.gov (United States)

    Zheng, Yu; Cohen-Karni, Devora; Xu, Derrick; Chin, Hang Gyeong; Wilson, Geoffrey; Pradhan, Sriharsa; Roberts, Richard J

    2010-09-01

    Mrr superfamily of homologous genes in microbial genomes restricts modified DNA in vivo. However, their biochemical properties in vitro have remained obscure. Here, we report the experimental characterization of MspJI, a remote homolog of Escherichia coli's Mrr and show it is a DNA modification-dependent restriction endonuclease. Our results suggest MspJI recognizes (m)CNNR (R = G/A) sites and cleaves DNA at fixed distances (N(12)/N(16)) away from the modified cytosine at the 3' side (or N(9)/N(13) from R). Besides 5-methylcytosine, MspJI also recognizes 5-hydroxymethylcytosine but is blocked by 5-glucosylhydroxymethylcytosine. Several other close homologs of MspJI show similar modification-dependent endonuclease activity and display substrate preferences different from MspJI. A unique feature of these modification-dependent enzymes is that they are able to extract small DNA fragments containing modified sites on genomic DNA, for example ∼32 bp around symmetrically methylated CG sites and ∼31 bp around methylated CNG sites. The digested fragments can be directly selected for high-throughput sequencing to map the location of the modification on the genomic DNA. The MspJI enzyme family, with their different recognition specificities and cleavage properties, provides a basis on which many future methods can build to decode the epigenomes of different organisms.

  9. Probing the dynamics of restriction endonuclease NgoMIV-DNA interaction by single-molecule FRET.

    Science.gov (United States)

    Tutkus, Marijonas; Sasnauskas, Giedrius; Rutkauskas, Danielis

    2017-12-01

    Many type II restriction endonucleases require two copies of their recognition sequence for optimal activity. Concomitant binding of two DNA sites by such an enzyme produces a DNA loop. Here we exploit single-molecule Förster resonance energy transfer (smFRET) of surface-immobilized DNA fragments to study the dynamics of DNA looping induced by tetrameric endonuclease NgoMIV. We have employed a DNA fragment with two NgoMIV recognition sites and a FRET dye pair such that upon protein-induced DNA looping the dyes are brought to close proximity resulting in a FRET signal. The dynamics of DNA-NgoMIV interactions proved to be heterogeneous, with individual smFRET trajectories exhibiting broadly different average looped state durations. Distinct types of the dynamics were attributed to different types of DNA-protein complexes, mediated either by one NgoMIV tetramer simultaneously bound to two specific sites ("slow" trajectories) or by semi-specific interactions of two DNA-bound NgoMIV tetramers ("fast" trajectories), as well as to conformational heterogeneity of individual NgoMIV molecules. © 2017 Wiley Periodicals, Inc.

  10. Natural transformation of an engineered Helicobacter pylori strain deficient in type II restriction endonucleases.

    Science.gov (United States)

    Zhang, Xue-Song; Blaser, Martin J

    2012-07-01

    Restriction-modification (RM) systems are important for bacteria to limit foreign DNA invasion. The naturally competent bacterium Helicobacter pylori has highly diverse strain-specific type II systems. To evaluate the roles of strain-specific restriction in H. pylori natural transformation, a markerless type II restriction endonuclease-deficient (REd) mutant was constructed. We deleted the genes encoding all four active type II restriction endonucleases in H. pylori strain 26695 using sacB-mediated counterselection. Transformation by donor DNA with exogenous cassettes methylated by Escherichia coli was substantially (1.7 and 2.0 log(10) for cat and aphA, respectively) increased in the REd strain. There also was significantly increased transformation of the REd strain by donor DNA from other H. pylori strains, to an extent corresponding to their shared type II R-M system strain specificity with 26695. Comparison of the REd and wild-type strains indicates that restriction did not affect the length of DNA fragment integration during natural transformation. There also were no differentials in cell growth or susceptibility to DNA damage. In total, the data indicate that the type II REd mutant has enhanced competence with no loss of growth or repair facility compared to the wild type, facilitating H. pylori mutant construction and other genetic engineering.

  11. Comparative analysis between radiographic views for knee osteoarthrosis (bipedal AP versus monopedal AP

    Directory of Open Access Journals (Sweden)

    Rodrigo Pires e Albuquerque

    2013-08-01

    Full Text Available OBJECTIVE: A comparative analysis by applying the criteria of the original classification Ahlbäck in the anteroposterior (AP bipedal knee in extension and anteroposterior (AP monopodal knee in symptomatic knee arthrosis. With this analysis we intend to observe the agreement, any advantage or difference between the incidence and degree of joint involvement between the orthopedic surgeons and radiologists with the referring physician. METHODS: From January 2012 to March 2012, was a prospective study of 60 symptomatic arthrosis knees (60 patients, clinically selected group of outpatient knee and radiographic proposals submitted to the search. Of the 60 patients, 39 were female and 21 male, mean age 64 years (ranging from 50 to 84 years. Of the 60 knees studied, 37 corresponded to the right side and 23 on the left side. Statistical analysis was performed by Kappa statistics, which evaluates the interobserver agreement for qualitative data. RESULTS: According to the scale of Ahlbäck, there was a significant agreement (p < 0.0001 intra-observer in the classification of knee osteoarthritis among the five evaluators. There was a significant agreement (p < 0.0001 with inter-observer referring physician in the incidence of AP monopodal and AP bipedal for the four raters. CONCLUSION: The study found no difference between the incidence in the AP monopodal versus AP bipedal in osteoarthritis of the knee.

  12. Isoform-specific degradation of PR-B by E6-AP is critical for normal mammary gland development.

    Science.gov (United States)

    Ramamoorthy, Sivapriya; Dhananjayan, Sarath C; Demayo, Francesco J; Nawaz, Zafar

    2010-11-01

    E6-associated protein (E6-AP), which was originally identified as an ubiquitin-protein ligase, also functions as a coactivator of estrogen (ER-α) and progesterone (PR) receptors. To investigate the in vivo role of E6-AP in mammary gland development, we generated transgenic mouse lines that either overexpress wild-type (WT) human E6-AP (E6-AP(WT)) or ubiquitin-protein ligase-defective E6-AP (E6-AP(C833S)) in the mammary gland. Here we show that overexpression of E6-AP(WT) results in impaired mammary gland development. In contrast, overexpression of E6-AP(C833S) or loss of E6-AP (E6-AP(KO)) increases lateral branching and alveolus-like protuberances in the mammary gland. We also show that the mammary phenotypes observed in the E6-AP transgenic and knockout mice are due, in large part, to the alteration of PR-B protein levels. We also observed alteration in ER-α protein level, which might contribute to the observed mammary phenotype by regulating PR expression. Furthermore, E6-AP regulates PR-B protein levels via the ubiquitin-proteasome pathway. Additionally, we also show that E6-AP impairs progesterone-induced Wnt-4 expression by decreasing the steady state level of PR-B in both mice and in human breast cancer cells. In conclusion, we present the novel observation that E6-AP controls mammary gland development by regulating PR-B protein turnover via the ubiquitin proteasome pathway. For the first time, we show that the E3-ligase activity rather than the coactivation function of E6-AP plays an important role in the mammary gland development, and the ubiquitin-dependent PR-B degradation is not required for its transactivation functions. This mechanism appears to regulate normal mammogenesis, and dysregulation of this process may be an important contributor to mammary cancer development and progression.

  13. Radiation effects on active pixel sensors (APS); Effets de l'irradiation sur les capteurs a pixels actifs (APS)

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, M.; David, J.P. [ONERA-CERT/, 31 - Toulouse (France)

    1999-07-01

    Active pixel sensor (APS) is a new generation of image sensors which presents several advantages relatively to charge coupled devices (CCDs) particularly for space applications (APS requires only 1 voltage to operate which reduces considerably current consumption). Irradiation was performed using {sup 60}Co gamma radiation at room temperature and at a dose rate of 150 Gy(Si)/h. 2 types of APS have been tested: photodiode-APS and photoMOS-APS. The results show that photoMOS-APS is more sensitive to radiation effects than photodiode-APS. Important parameters of image sensors like dark currents increase sharply with dose levels. Nevertheless photodiode-APS sensitivity is one hundred time lower than photoMOS-APS sensitivity.

  14. Mutation screening of NOS1AP gene in a large sample of psychiatric patients and controls

    Directory of Open Access Journals (Sweden)

    Nygren Gudrun

    2010-07-01

    Full Text Available Abstract Background The gene encoding carboxyl-terminal PDZ ligand of neuronal nitric oxide synthase (NOS1AP is located on chromosome 1q23.3, a candidate region for schizophrenia, autism spectrum disorders (ASD and obsessive-compulsive disorder (OCD. Previous genetic and functional studies explored the role of NOS1AP in these psychiatric conditions, but only a limited number explored the sequence variability of NOS1AP. Methods We analyzed the coding sequence of NOS1AP in a large population (n = 280, including patients with schizophrenia (n = 72, ASD (n = 81 or OCD (n = 34, and in healthy volunteers controlled for the absence of personal or familial history of psychiatric disorders (n = 93. Results Two non-synonymous variations, V37I and D423N were identified in two families, one with two siblings with OCD and the other with two brothers with ASD. These rare variations apparently segregate with the presence of psychiatric conditions. Conclusions Coding variations of NOS1AP are relatively rare in patients and controls. Nevertheless, we report the first non-synonymous variations within the human NOS1AP gene that warrant further genetic and functional investigations to ascertain their roles in the susceptibility to psychiatric disorders.

  15. Analysis of recessive CD2AP and ACTN4 mutations in steroid-resistant nephrotic syndrome.

    Science.gov (United States)

    Benoit, Geneviève; Machuca, Eduardo; Nevo, Fabien; Gribouval, Olivier; Lepage, David; Antignac, Corinne

    2010-03-01

    Mutations in podocyte genes have been identified in patients with steroid-resistant nephrotic syndrome (SRNS). Point mutations in the ACTN4 gene cause an autosomal dominant form of human focal segmental glomerular sclerosis (FSGS); however, reports of CD2AP mutations remain scarce. Based on the phenotype of Actn4 and Cd2ap null mice, we aimed to define the role of recessive CD2AP and ACTN4 mutations in a cohort of children with SRNS for which NPHS1, NPHS2, and PLCE1 mutations had been previously excluded. CD2AP and ACTN4 mutational analysis was performed in 42 children from 35 unrelated families. The median age of disease onset was 20 (range 0-102) months. Sixteen patients reached end-stage kidney disease at a median age of 84 (range 4-161) months. Renal histology showed FSGS lesions and minimal glomerular changes in 49% and 20% of patients, respectively. Microsatellite marker analysis excluded linkage to the CD2AP locus in 26 families and to the ACTN4 locus in 31 families. No disease-causing mutations were identified in the remaining families. Recessive CD2AP and ACTN4 mutations are rare in children with SRNS. The absence of mutations in this study suggests that there are other genetic causes of SRNS that still need to be identified.

  16. CLEARING MAGNET DESIGN FOR APS-U

    Energy Technology Data Exchange (ETDEWEB)

    Abliz, M.; Grimmer, J.; Jaski, Y.; Westferro, F.; Ramanathan, M.

    2017-06-25

    The Advanced Photon Source is in the process of developing an upgrade (APS-U) of the storage ring. The upgrade will be converting the current double bend achromat (DBA) lattice to a multi-bend achromat (MBA) lattice. In addition, the storage ring will be operated at 6 GeV and 200 mA with regular swap-out injection to keep the stored beam current constant [1]. The swap-out injection will take place with beamline shutters open. For radiation safety to ensure that no electrons can exit the storage ring, a passive method of protecting the beamline and containing the electrons inside the storage ring is proposed. A clearing magnet will be located in all beamline front ends inside the storage ring tunnel. This article will discuss the features and design of the clearing magnet scheme for APS-U.

  17. St. Ap. Thomas and steps of faith

    Directory of Open Access Journals (Sweden)

    Nicolae Popescu

    2015-11-01

    Full Text Available There is, in folk tradition, an expression, “Doubting Thomas”, making an obvious allusion to doubts shown by St. Ap. Thomas against the other apostles’ testimonials of the resurrection of Jesus. But we prefer to say that St. Ap. Thomas doubted because he recognizes that the Saviour Himself “believed” only because he saw (Jn 20, 27. But pure faith denies doubt, (evidence shall rebuke the disciples of John, St. Ap. Thomas and the other apostles, who demanded an over-confirmation, and without guile, (showing the condemnation of the Pharisees, scribes and wicked generation, which together “sky sign”, and with aspects which the Saviour added:” Seeing, blessed are those who hear the word of God and obey it “(Lk 11: 28. The Beatitudes are the blessings of the Law of Love as the Commandments are applications of the Law of Moses. The doubt of St. Ap. Thomas, strikingly approaches the existential doubt of the whole being,(while the methodical doubt of Descartes was intellectual, scientists considered this the only legitimate way to access complete certainty. It is thrilling that Descartes wanted to be like a man, who moving through the darkness, and compelled to go slow and be circumspect in all things, as you would do to keep from falling. But he refused to accept any idea before it passed through his reason and without having sought simultaneously, a way to get to know everything that his spirit is capable of, and being like Adam, who wanted to reach deification by his own powers, without the gift of God.

  18. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, Edward M.; Cullen, Bryan R., E-mail: bryan.cullen@duke.edu

    2015-05-15

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  19. [Epidemiological characteristics of road traffic injuries in AP Vojvodina].

    Science.gov (United States)

    Durić, Predrag; Miladinov-Mikov, Marica

    2009-01-01

    Traffic accidents are one of the leading epidemiological problems in developed countries as well as in developing ones. It is estimated that every day in the world 1308 person die in car accidents. Causes of traffic accidents are factors of road, vehicle and human factors, the latter one being the cause of more than 90% car accidents, isolated or linked with other factors. Data from the Ministry of Internal Affairs - Department in Novi Sad were collected for the period 1992-2001 and analyzed. All road traffic accidents were divided into those with and without injured persons. All injured were divided in three categories: slightly injured, severely injured and killed. Categorization was done by the Ministry of Internal Affairs (police). Average number of road traffic accidents in AP Vojvodina is 13,191, and 3502 with injured persons per year. Avarage incidence is 174 accidents per 100,000 population, and 301 injured per 100,000 population. Mortality rate is 30.6 per 100,000 population. Number of killed in road traffic accidents per 10,000 vehicles is 6.91. During the 1992-2001 period, morbidity and mortality rates of traffic accidents in AP Vojvodina were high. Participating of bicycle drivers, car passengers and pedestrians is higher than in developed countries. Injured persons are mostly 20-29 y.o., but rates of killed persons are the same in all age groups older than 19. Men are more in danger than women. Majority of accidents happen during the early autumn months, on Fridays and Saturdays, in early evening hours. Most persons have been injured or killed in city accidents, along Subotica-Belgrade highway. AP Vojvodina had the highest mortality rate of road traffic injuries per ten thousand vehicles in Europe -- 6.91. Effective preventive measures are needed to decrease rates of road traffic accidents.

  20. AIRE variations in Addison's disease and autoimmune polyendocrine syndromes (APS)

    DEFF Research Database (Denmark)

    Bøe Wolff, A S; Oftedal, B; Johansson, S

    2008-01-01

    Autoimmune Addison's disease (AAD) is often associated with other components in autoimmune polyendocrine syndromes (APS). Whereas APS I is caused by mutations in the AIRE gene, the susceptibility genes for AAD and APS II are unclear. In the present study, we investigated whether polymorphisms...... or copy number variations in the AIRE gene were associated with AAD and APS II. First, nine SNPs in the AIRE gene were analyzed in 311 patients with AAD and APS II and 521 healthy controls, identifying no associated risk. Second, in a subgroup of 25 of these patients, AIRE sequencing revealed three novel...... polymorphisms. Finally, the AIRE copy number was determined by duplex quantitative PCR in 14 patients with APS I, 161 patients with AAD and APS II and in 39 healthy subjects. In two Scandinavian APS I patients previously reported to be homozygous for common AIRE mutations, we identified large deletions...

  1. The 2009 Version of the Aeroprediction Code: The AP09

    National Research Council Canada - National Science Library

    Moore, Frank G; Moore, Linda Y

    2008-01-01

    The AP05 code was evaluated when applied to configurations with boattails. Results of the evaluation indicated the AP05 predictions for normal force, center of pressure, pitch and roll damping moments needed improvement...

  2. Recruitment and positioning determine the specific role of the XPF-ERCC1 endonuclease in interstrand crosslink repair

    NARCIS (Netherlands)

    Klein Douwel, Daisy; Hoogenboom, Wouter S; Boonen, Rick Acm; Knipscheer, Puck

    2017-01-01

    XPF-ERCC1 is a structure-specific endonuclease pivotal for several DNA repair pathways and, when mutated, can cause multiple diseases. Although the disease-specific mutations are thought to affect different DNA repair pathways, the molecular basis for this is unknown. Here we examine the function of

  3. Recruitment of the nucleotide excision repair endonuclease XPG to sites of UV-induced DNA damage depends on functional TFIIH

    NARCIS (Netherlands)

    A. Zotter (Angelika); A.B. Houtsmuller (Adriaan); M.S. Luijsterburg (Martijn); D.O. Warmerdam (Daniël); S.M. Ibrahim (Shehu); A.L. Nigg (Alex); W.A. van Cappellen (Gert); J.H.J. Hoeijmakers (Jan); R. van Driel; W. Vermeulen (Wim)

    2006-01-01

    textabstractThe structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3′ side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5′ incision by the

  4. RECQ5 Helicase Cooperates with MUS81 Endonuclease in Processing Stalled Replication Forks at Common Fragile Sites during Mitosis

    DEFF Research Database (Denmark)

    Di Marco, Stefano; Hasanova, Zdenka; Kanagaraj, Radhakrishnan

    2017-01-01

    The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent o...

  5. A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease

    Science.gov (United States)

    The Cas9 endonuclease of the Type II-a clustered regularly interspersed short palindromic repeats (CRISPR), of Streptococcus pyogenes (SpCas9) has been adapted as a widely used tool for genome editing and genome engineering. Herein, we describe a gene encoding a novel Cas9 ortholog (BpsuCas9) and th...

  6. The structure-specific endonuclease Ercc1-Xpf is required for targeted gene replacement in embryonic stem cells

    NARCIS (Netherlands)

    L.J. Niedernhofer (Laura); J. Essers (Jeroen); G. Weeda (Geert); H.B. Beverloo (Berna); J. de Wit (Jan); M. Muijtjens (Manja); H. Odijk (Hanny); J.H.J. Hoeijmakers (Jan); R. Kanaar (Roland)

    2001-01-01

    textabstractThe Ercc1-Xpf heterodimer, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair and homologous recombination. Erccl-Xpf incises

  7. Genetic location of a mutant of bacteriophage T4 deficient in the ability to induce endonuclease II.

    Science.gov (United States)

    Ray, P; Sinha, N K; Warner, H R; Snustad, D P

    1972-01-01

    Reciprocal three-factor crosses and the use of a partial revertant of a putative ribonucleotide reductase mutant of Escherichia coli B/5 as indicator have made it possible to map denA (deficient in endonuclease II) between nrd-11 (ribonucleotide reductase gene B) and amM69 (gene 63) on the bacteriophage T4 chromosome.

  8. Crystal Structure of the Homing Endonuclease I-CvuI Provides a New Template for Genome Modification

    DEFF Research Database (Denmark)

    Molina, Rafael; Redondo, Pilar; López-Méndez, Blanca

    2015-01-01

    Homing endonucleases recognize and generate a DNA double-strand break, which has been used to promote gene targeting. These enzymes recognize long DNA stretches; they are highly sequence-specific enzymes and display a very low frequency of cleavage even in complete genomes. Although a large numbe...

  9. A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells.

    Science.gov (United States)

    Dai, Feihan F; Bhattacharjee, Alpana; Liu, Ying; Batchuluun, Battsetseg; Zhang, Ming; Wang, Xinye Serena; Huang, Xinyi; Luu, Lemieux; Zhu, Dan; Gaisano, Herbert; Wheeler, Michael B

    2015-10-09

    GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid assays to identify novel GLP1R interactors in both mouse and human islets. Among these, ATP6ap2 (ATPase H(+)-transporting lysosomal accessory protein 2) was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion; however, siRNA knockdown decreased both glucose-stimulated and GLP1-induced insulin secretion. Decreases in GLP1-induced insulin secretion were accompanied by attenuated GLP1 stimulated cAMP accumulation. Because ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knockdown but paradoxically increased proinsulin secretion. Importantly, as a GLP1R interactor, ATP6ap2 was required for GLP1-induced Ca(2+) influx, in part explaining decreased insulin secretion in ATP6ap2 knockdown cells. Taken together, our findings identify a group of proteins that interact with the GLP1R. We further show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signaling and insulin processing to affect insulin secretion. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Interaction of apurinic/apyrimidinic endonucleases Nfo and ExoA with the DNA integrity scanning protein DisA in the processing of oxidative DNA damage during Bacillus subtilis spore outgrowth.

    Science.gov (United States)

    Campos, Silvia S; Ibarra-Rodriguez, Juan R; Barajas-Ornelas, Rocío C; Ramírez-Guadiana, Fernando H; Obregón-Herrera, Armando; Setlow, Peter; Pedraza-Reyes, Mario

    2014-02-01

    Oxidative stress-induced damage, including 8-oxo-guanine and apurinic/apyrimidinic (AP) DNA lesions, were detected in dormant and outgrowing Bacillus subtilis spores lacking the AP endonucleases Nfo and ExoA. Spores of the Δnfo exoA strain exhibited slightly slowed germination and greatly slowed outgrowth that drastically slowed the spores' return to vegetative growth. A null mutation in the disA gene, encoding a DNA integrity scanning protein (DisA), suppressed this phenotype, as spores lacking Nfo, ExoA, and DisA exhibited germination and outgrowth kinetics very similar to those of wild-type spores. Overexpression of DisA also restored the slow germination and outgrowth phenotype to nfo exoA disA spores. A disA-lacZ fusion was expressed during sporulation but not in the forespore compartment. However, disA-lacZ was expressed during spore germination/outgrowth, as was a DisA-green fluorescent protein (GFP) fusion protein. Fluorescence microscopy revealed that, as previously shown in sporulating cells, DisA-GFP formed discrete globular foci that colocalized with the nucleoid of germinating and outgrowing spores and remained located primarily in a single cell during early vegetative growth. Finally, the slow-outgrowth phenotype of nfo exoA spores was accompanied by a delay in DNA synthesis to repair AP and 8-oxo-guanine lesions, and these effects were suppressed following disA disruption. We postulate that a DisA-dependent checkpoint arrests DNA replication during B. subtilis spore outgrowth until the germinating spore's genome is free of damage.

  11. Book Eyes Impact of AP Classes and Exams

    Science.gov (United States)

    Viadero, Debra

    2010-01-01

    At a time of mushrooming interest in Advanced Placement (AP) tests, a new book, "AP: A Critical Examination of the Advanced Placement Program," assembles studies on how capable the program is of meeting the increasingly diverse expectations held up for it. Growing out of a symposium held at Harvard in 2007, the book focuses on AP science…

  12. Teaching Materials and Strategies for the AP Music Theory Exam

    Science.gov (United States)

    Lively, Michael T.

    2017-01-01

    Each year, many students take the Advanced Placement (AP) Music Theory Exam, and the majority of these students enroll in specialized AP music theory classes as part of the preparation process. For the teachers of these AP music theory classes, a number of challenges are presented by the difficulty and complexity of the exam subject material as…

  13. Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease

    NARCIS (Netherlands)

    G.P. van Nierop (Gijs); A.A.F. de Vries (Antoine); M. Holkers (Maarten); K.R. Vrijsen (Krijn); M.A.F.V. Gonçalves (Manuel)

    2009-01-01

    textabstractHomologous recombination (HR) is a highly accurate mechanism of DNA repair that can be exploited for homology-directed gene targeting. Since in most cell types HR occurs very infrequently (̃10.-6to 10.-8), its practical application has been largely restricted to specific experimental

  14. Tauroursodeoxycholic acid reduces bile acid-induced apoptosis by modulation of AP-1

    NARCIS (Netherlands)

    Pusl, Thomas; Vennegeerts, Timo; Wimmer, Ralf; Denk, Gerald U.; Beuers, Ulrich; Rust, Christian

    2008-01-01

    Ursodeoxycholic acid (UDCA) is used in the therapy of cholestatic liver diseases. Apoptosis induced by toxic bile acids plays an important role in the pathogenesis of liver injury during cholestasis and appears to be mediated by the human transcription factor AP-1. We aimed to study if TUDCA can

  15. Another Strategy for Teaching Histology to A&P Students: Classification versus Memorization.

    Science.gov (United States)

    Bavis, Ryan W.; Seveyka, Jerred; Shigeoka, Cassie A.

    2000-01-01

    Defines dichotomous keys as common learning tools based on identification rather than memorization. Provides an example of a dichotomous key developed for introducing histology in human anatomy and physiology (A&P) courses and explains how students can use the dichotomous key. Discusses the goals of the exercises and the process of…

  16. Tumores de apéndice cecal

    Directory of Open Access Journals (Sweden)

    Rubén Bembilbre Taboada

    1998-08-01

    Full Text Available Se realizó un estudio descriptivo-retrospectivo de 8 pacientes con tumores de apéndice cecal, en el período comprendido entre el 1 de enero de 1990 y el 1 de enero de 1997, los cuales fueron intervenidos quirúrgicamente en el Hospital Provincial Clinicoquirúrgico Docente "Dr. Gustavo Aldereguía". Se revisaron todos los libros de biopsias del Departamento de Anatomía Patológica correspondientes al período analizado, para obtener aquellos casos con diagnóstico de afección tumoral de apéndice. Se estudiaron las historias clínicas y se recogieron datos de interés como sexo, manifestaciones clínicas, diagnóstico presuntivo, diagnóstico anatomopatológico y tipo de intervención. La afección tumoral de apéndice cecal es infrecuente y constituyó el 0,38% del total de 20057 apéndices examinadas. No se hallaron diferencias respecto al sexo. Hubo un ligero predominio en pacientes con edades de más de 60 años. Los hallazgos clínicos más frecuentes fueron dolor agudo en fosa inguinal derecha y fiebre, con predominio del adenocarcinoma. Los principales resultados se exponen en tablasA retrospective-descriptive study of 8 patients with appendix ceci tumors attending the hospital from January 1st, 1990 to January 1st 1997 was made. These patients were operated at "Dr. Gustavo Aldereguía" clinical surgical teaching hospital in Cienfuegos. All the biopsy records of the analyzed period were checked in the Pathological Anatomy Department so as to collect those cases diagnosed with appendix ceci tumors. Medical records were examined and interesting data were collected as follows: sex, clinical symptoms, presumptive diagnosis, anatomopathological diagnosis and type of surgery. Tumors in appendix ceci are unusual and represented 0.38 % of 20 057 analyzed appendixes. Sex was not a determining factor. The disease was slightly predominant in patients over 60. The most frequent clinical findings were: nagging pain in the right inguinal fosa and

  17. Artificial pancreas (AP) clinical trial participants' acceptance of future AP technology.

    Science.gov (United States)

    Bevier, Wendy C; Fuller, Serena M; Fuller, Ryan P; Rubin, Richard R; Dassau, Eyal; Doyle, Francis J; Jovanovič, Lois; Zisser, Howard C

    2014-09-01

    Artificial pancreas (AP) systems are currently an active field of diabetes research. This pilot study examined the attitudes of AP clinical trial participants toward future acceptance of the technology, having gained firsthand experience. After possible influencers of AP technology adoption were considered, a 34-question questionnaire was developed. The survey assessed current treatment satisfaction, dimensions of clinical trial participant motivation, and variables of the technology acceptance model (TAM). Forty-seven subjects were contacted to complete the survey. The reliability of the survey scales was tested using Cronbach's α. The relationship of the factors to the likelihood of AP technology adoption was explored using regression analysis. Thirty-six subjects (76.6%) completed the survey. Of the respondents, 86.1% were either highly likely or likely to adopt the technology once available. Reliability analysis of the survey dimensions revealed good internal consistency, with scores of >0.7 for current treatment satisfaction, convenience (motivation), personal health benefit (motivation), perceived ease of use (TAM), and perceived usefulness (TAM). Linear modeling showed that future acceptance of the AP was significantly associated with TAM and the motivation variables of convenience plus the individual item benefit to others (R(2)=0.26, P=0.05). When insulin pump and continuous glucose monitor use were added, the model significance improved (R(2)=0.37, P=0.02). This pilot study demonstrated that individuals with direct AP technology experience expressed high likelihood of future acceptance. Results support the factors of personal benefit, convenience, perceived usefulness, and perceived ease of use as reliable scales that suggest system adoption in this highly motivated patient population.

  18. Crystallization and preliminary X-ray analysis of flap endonuclease 1 (FEN1) from Desulfurococcus amylolyticus.

    Science.gov (United States)

    Mase, Tomoko; Kubota, Keiko; Miyazono, Ken-ichi; Kawarabayasi, Yutaka; Tanokura, Masaru

    2009-09-01

    Flap endonuclease 1 (FEN1) is a structure-specific nuclease that removes 5'-overhanging flaps in DNA repair and removes the RNA/DNA primer during maturation of the Okazaki fragment in lagging-strand DNA replication. FEN1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with monoammonium dihydrogen phosphate as the precipitant at pH 8.3. X-ray diffraction data were collected to 2.00 A resolution. The space group of the crystal was determined as the primitive hexagonal space group P321, with unit-cell parameters a = b = 103.76, c = 84.58 A. The crystal contained one molecule in the asymmetric unit.

  19. Structure of flap endonuclease 1 from the hyperthermophilic archaeon Desulfuro­coccus amylolyticus

    Science.gov (United States)

    Mase, Tomoko; Kubota, Keiko; Miyazono, Ken-ichi; Kawarabayasi, Yutaka; Tanokura, Masaru

    2011-01-01

    Flap endonuclease 1 (FEN1) is a key enzyme in DNA repair and DNA replication. It is a structure-specific nuclease that removes 5′-overhanging flaps and the RNA/DNA primer during maturation of the Okazaki fragment. Homologues of FEN1 exist in a wide range of bacteria, archaea and eukaryotes. In order to further understand the structural basis of the DNA recognition, binding and cleavage mechanism of FEN1, the structure of FEN1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus (DaFEN1) was determined at 2.00 Å resolution. The overall fold of DaFEN1 was similar to those of other archaeal FEN1 proteins; however, the helical clamp and the flexible loop exhibited a putative substrate-binding pocket with a unique conformation. PMID:21301087

  20. Structure and dynamics of mesophilic variants from the homing endonuclease I-DmoI

    DEFF Research Database (Denmark)

    Alba, Josephine; Marcaida, Maria Jose; Prieto, Jesus

    2017-01-01

    this particular application, many efforts have been made to generate mesophilic variants of I-DmoI that function at lower temperatures than the wild-type. Here, we report a structural and computational analysis of two I-DmoI mesophilic mutants. Despite very limited structural variations between the crystal......I-DmoI, from the hyperthermophilic archaeon Desulfurococcus mobilis, belongs to the LAGLIDADG homing endonuclease protein family. Its members are highly specific enzymes capable of recognizing long DNA target sequences, thus providing potential tools for genome manipulation. Working towards...... structures of these variants and the wild-type, a different dynamical behaviour near the cleavage sites is observed. In particular, both the dynamics of the water molecules and the protein perturbation effect on the cleavage site correlate well with the changes observed in the experimental enzymatic activity....

  1. Insights on copper coordination and reactivity of endonuclease EcoRI by ESR spectroscopy and modeling

    Science.gov (United States)

    Ji, Ming

    2009-03-01

    The cleavage of DNA by restriction endonuclease EcoRI is catalyzed by metal ions such as Mg^2+. However, Cu^2+ does not catalyze the cleavage of DNA by EcoRI. In order to understand the functional difference between Cu^2+ and Mg^2+, coordination of Cu^2+ in the EcoRI--DNA complex was clarified by ESR and MD simulation. There are two Cu^2+ components in the specific EcoRI-DNA complex. Each component has one N atom from histidine imidazole and one oxygen atom from the phosphate backbone of DNA coordinate to Cu^2+ based on the ESR experimental results. MD simulation further confirmed that the Nδ atom of His114 imidazole and one oxygen atom from the phosphate backbone of DNA coordinate to Cu^2+. Difference in the coordination of Cu^2+ and Mg^2+ explains their different functional behaviors.

  2. A web-based restriction endonuclease tool for mycobacteriophage cluster prediction.

    Science.gov (United States)

    Gissendanner, Chris R; Wiedemeier, Allison M D; Wiedemeier, Paul D; Minton, Russell L; Bhuiyan, Swapan; Harmson, Jeremy S; Findley, Ann M

    2014-10-01

    A recent explosion in the amount of genomic data has revealed a large genetic diversity in the bacteriophages that infect Mycobacterium smegmatis. In an effort to assess the novelty of newly described mycobacteriophage isolates and provide a preliminary determination of their probable cluster assignment prior to full genome sequencing, we have developed a systematic approach that relies on restriction endonuclease analysis. We demonstrate that a web-based tool, the Phage Enzyme Tool (or PET), is capable of rapidly facilitating this analysis and exhibits reliability in the putative placement of mycobacteriophages into specific clusters of previously sequenced phages. We propose that this tool represents a useful analytical step in the initial study of phage genomes and that this tool will increase the efficiency of phage genome characterization and enhance the educational activities involving mycobacteriophage discovery. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Cleavage of DNA containing 5-fluorocytosine or 5-fluorouracil by type II restriction endonucleases.

    Science.gov (United States)

    Olszewska, Agata; Dadová, Jitka; Mačková, Michaela; Hocek, Michal

    2015-11-01

    A systematic study of the cleavage of DNA sequences containing 5-fluorocytosine or 5-fluorouracil by type II restriction endonucleases (REs) was performed and the results compared with the same sequences containing natural pyrimidine bases, uracil or 5-methylcytosine. The results show that some REs recognize fluorine as a hydrogen on cytosine and cleave the corresponding sequences where the presence of m5dC leads to blocking of the cleavage. However, on uracil, the same REs recognize the F as a methyl surrogate and cleave the sequences which are not cleaved if uracil is incorporated instead of thymine. These results are interesting for understanding the recognition of DNA sequences by REs and for manipulation of the specific DNA cutting. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Role of Magnesium Ions in DNA Recognition by the EcoRV Restriction Endonuclease

    Energy Technology Data Exchange (ETDEWEB)

    Zahran, Mai [ORNL; Berezniak, Tomasz [University of Heidelberg; Imhof, Petra [University of Heidelberg; Smith, Jeremy C [ORNL

    2011-01-01

    The restriction endonuclease EcoRV binds two magnesium ions. One of these ions, Mg2+A, binds to the phosphate group where the cleavage occurs and is required for catalysis, but the role of the other ion, Mg2+B is debated. Here, multiple independent molecular dynamics simulations suggest that Mg2+B is crucial for achieving a tightly bound protein DNA complex and stabilizing a conformation that allows cleavage. In the absence of Mg2+B in all simulations the protein DNA hydrogen bond network is significantly disrupted and the sharp kink at the central base pair step of the DNA, which is observed in the two-metal complex, is not present. Also, the active site residues rearrange in such a way that the formation of a nucleophile, required for DNA hydrolysis, is unlikely.

  5. Structure and dynamics of mesophilic variants from the homing endonuclease I-DmoI

    Science.gov (United States)

    Alba, Josephine; Marcaida, Maria Jose; Prieto, Jesus; Montoya, Guillermo; Molina, Rafael; D'Abramo, Marco

    2017-12-01

    I-DmoI, from the hyperthermophilic archaeon Desulfurococcus mobilis, belongs to the LAGLIDADG homing endonuclease protein family. Its members are highly specific enzymes capable of recognizing long DNA target sequences, thus providing potential tools for genome manipulation. Working towards this particular application, many efforts have been made to generate mesophilic variants of I-DmoI that function at lower temperatures than the wild-type. Here, we report a structural and computational analysis of two I-DmoI mesophilic mutants. Despite very limited structural variations between the crystal structures of these variants and the wild-type, a different dynamical behaviour near the cleavage sites is observed. In particular, both the dynamics of the water molecules and the protein perturbation effect on the cleavage site correlate well with the changes observed in the experimental enzymatic activity.

  6. Crystallization and preliminary X-ray diffraction analysis of the small subunit of the heterodimeric restriction endonuclease R.BspD6I

    Energy Technology Data Exchange (ETDEWEB)

    Kachalova, Galina S. [Max-Planck Unit for Structural Molecular Biology, Hamburg 22607 (Germany); Yunusova, Alfiya K.; Artyukh, Rimma I.; Rogulin, Eugeny A.; Perevyazova, Tatyana A.; Zheleznaya, Ludmila A. [Institute of Theoretical and Experimental Biophysics, Pushchino 142290 (Russian Federation); Matvienko, Nickolay I. [Institute of Protein Research, Pushchino 14229 (Russian Federation); Bartunik, Hans D., E-mail: bartunik@mpghdb.desy.de [Max-Planck Unit for Structural Molecular Biology, Hamburg 22607 (Germany)

    2007-09-01

    The crystallization of the small subunit of the heterodimeric restriction endonuclease R.BspD6I and diffraction data collection to 1.5 Å resolution are reported. The heterodimeric restriction endonuclease R.BspD6I is composed of a small subunit with a cleavage site and a large subunit, containing a recognition domain and a cleavage domain, that may function separately as a monomeric nicking endonuclease. Here, the crystallization of the small subunit and diffraction data collection to 1.5 Å resolution are reported.

  7. AE8/AP8 Implementations in AE9/AP9, IRBEM, and SPENVIS

    Science.gov (United States)

    2014-02-18

    1970 [Cain et al., 1967]; IGRF (no extrapolation of field beyond 01 Jan 2015) [IAGA, 2010]; Olsen-Pfitzer [1977] • The interpolation methods applied...from the integral flux values using a power-law method , instead of a linear method . This correction is implemented in AE9/AP9/SPM V1.1. Approved for...Geomag. Geoelectr ., 19, p. 335. Ginet, G. P., et al. (2013), AE9/AP9/SPM, New models for specifying the trapped energetic particle and space plasma

  8. [Progresses on plant AP2/ERF transcription factors].

    Science.gov (United States)

    Zhang, Ji-Yu; Wang, Qing-Ju; Guo, Zhong-Ren

    2012-07-01

    Plant AP2/ERF transcription factor with AP2/ERF domain containing 60-70 amino acids is a huge gene family present in all plant. AP2/ERF transcriptional factors are involved in various biological functions such as plant development, flower development, fruit and seed maturation, wounding, pathogen defense, high salty, drought, and so on. AP2/ERF transcription factor are involved in salicylic acid, jasmonic acid, ethylene, abscisic acid signal transduction pathways and among them. The transcription factors are cross-talk factor in stress signal pathway. This paper summarizes the most advanced researches on types, biological functions, and gene regulations of plant AP2/ERF transcription factors.

  9. CgII cleaves DNA using a mechanism distinct from other ATP-dependent restriction endonucleases.

    Science.gov (United States)

    Toliusis, Paulius; Zaremba, Mindaugas; Silanskas, Arunas; Szczelkun, Mark D; Siksnys, Virginijus

    2017-08-21

    The restriction endonuclease CglI from Corynebacterium glutamicum recognizes an asymmetric 5'-GCCGC-3' site and cleaves the DNA 7 and 6/7 nucleotides downstream on the top and bottom DNA strands, respectively, in an NTP-hydrolysis dependent reaction. CglI is composed of two different proteins: an endonuclease (R.CglI) and a DEAD-family helicase-like ATPase (H.CglI). These subunits form a heterotetrameric complex with R2H2 stoichiometry. However, the R2H2·CglI complex has only one nuclease active site sufficient to cut one DNA strand suggesting that two complexes are required to introduce a double strand break. Here, we report studies to evaluate the DNA cleavage mechanism of CglI. Using one- and two-site circular DNA substrates we show that CglI does not require two sites on the same DNA for optimal catalytic activity. However, one-site linear DNA is a poor substrate, supporting a mechanism where CglI complexes must communicate along the one-dimensional DNA contour before cleavage is activated. Based on experimental data, we propose that adenosine triphosphate (ATP) hydrolysis by CglI produces translocation on DNA preferentially in a downstream direction from the target, although upstream translocation is also possible. Our results are consistent with a mechanism of CglI action that is distinct from that of other ATP-dependent restriction-modification enzymes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Three structure-selective endonucleases are essential in the absence of BLM helicase in Drosophila.

    Directory of Open Access Journals (Sweden)

    Sabrina L Andersen

    2011-10-01

    Full Text Available DNA repair mechanisms in mitotically proliferating cells avoid generating crossovers, which can contribute to genome instability. Most models for the production of crossovers involve an intermediate with one or more four-stranded Holliday junctions (HJs, which are resolved into duplex molecules through cleavage by specialized endonucleases. In vitro studies have implicated three nuclear enzymes in HJ resolution: MUS81-EME1/Mms4, GEN1/Yen1, and SLX4-SLX1. The Bloom syndrome helicase, BLM, plays key roles in preventing mitotic crossover, either by blocking the formation of HJ intermediates or by removing HJs without cleavage. Saccharomyces cerevisiae mutants that lack Sgs1 (the BLM ortholog and either Mus81-Mms4 or Slx4-Slx1 are inviable, but mutants that lack Sgs1 and Yen1 are viable. The current view is that Yen1 serves primarily as a backup to Mus81-Mms4. Previous studies with Drosophila melanogaster showed that, as in yeast, loss of both DmBLM and MUS81 or MUS312 (the ortholog of SLX4 is lethal. We have now recovered and analyzed mutations in Drosophila Gen. As in yeast, there is some redundancy between Gen and mus81; however, in contrast to the case in yeast, GEN plays a more predominant role in responding to DNA damage than MUS81-MMS4. Furthermore, loss of DmBLM and GEN leads to lethality early in development. We present a comparison of phenotypes occurring in double mutants that lack DmBLM and either MUS81, GEN, or MUS312, including chromosome instability and deficiencies in cell proliferation. Our studies of synthetic lethality provide insights into the multiple functions of DmBLM and how various endonucleases may function when DmBLM is absent.

  11. Interplay between structure-specific endonucleases for crossover control during Caenorhabditis elegans meiosis.

    Directory of Open Access Journals (Sweden)

    Takamune T Saito

    Full Text Available The number and distribution of crossover events are tightly regulated at prophase of meiosis I. The resolution of Holliday junctions by structure-specific endonucleases, including MUS-81, SLX-1, XPF-1 and GEN-1, is one of the main mechanisms proposed for crossover formation. However, how these nucleases coordinately resolve Holliday junctions is still unclear. Here we identify both the functional overlap and differences between these four nucleases regarding their roles in crossover formation and control in the Caenorhabditis elegans germline. We show that MUS-81, XPF-1 and SLX-1, but not GEN-1, can bind to HIM-18/SLX4, a key scaffold for nucleases. Analysis of synthetic mitotic defects revealed that MUS-81 and SLX-1, but not XPF-1 and GEN-1, have overlapping roles with the Bloom syndrome helicase ortholog, HIM-6, supporting their in vivo roles in processing recombination intermediates. Taking advantage of the ease of genetic analysis and high-resolution imaging afforded by C. elegans, we examined crossover designation, frequency, distribution and chromosomal morphology in single, double, triple and quadruple mutants of the structure-specific endonucleases. This revealed that XPF-1 functions redundantly with MUS-81 and SLX-1 in executing crossover formation during meiotic double-strand break repair. Analysis of crossover distribution revealed that SLX-1 is required for crossover suppression at the center region of the autosomes. Finally, analysis of chromosome morphology in oocytes at late meiosis I stages uncovered that SLX-1 and XPF-1 promote meiotic chromosomal stability by preventing formation of chromosomal abnormalities. We propose a model in which coordinate action between structure-specific nucleases at different chromosome domains, namely MUS-81, SLX-1 and XPF-1 at the arms and SLX-1 at the center region, exerts positive and negative regulatory roles, respectively, for crossover control during C. elegans meiosis.

  12. Enxerto bovino liofilizado: comportamento histológico após seguimento de 49 meses em seres humanos Bovine lyophilized graft (BLG: histological analysis on behavior in humans after 49 months

    Directory of Open Access Journals (Sweden)

    Carlos Roberto Galia

    2012-01-01

    Full Text Available OBJETIVO: Analisar o comportamento histológico do ELB, produzido conforme protocolo desenvolvido pelo autor principal, em seres humanos no decorrer de 49 meses através da aferição da proporção enxerto/osso neoformado em relação ao total de área mineralizada. MÉTODOS: Série de casos com 12 pacientes, oito femininos (66% e quatro masculinos (34%, totalizando 13 biópsias, nos quais utilizou-se ELB e que posteriormente houve necessidade de reintervenção cirúrgica, no período de 2000 a 2011. As lâminas produzidas, coradas com hematoxilina-eosina (HE, foram analisadas por patologista e digitalizadas para a avaliação proposta. RESULTADOS: A média etária foi de 57 anos e o tempo médio de seguimento de 49 meses (6-115. A proporção média de ELB foi de 42% (13-85 e de osso neoformado de 58% (15-87 em relação ao total de área mineralizada. CONCLUSÕES: O presente estudo demonstra que o ELB utilizado apresenta característica osteocondutora e biocompatibilidade. O ELB apresentado é opção terapêutica a ser utilizada em cirurgias ortopédicas que necessitem preenchimento de deficiências ósseas.OBJECTIVE: To analyze the histological behavior of bovine lyophilized grafts (BLG produced according to a protocol developed by the first author, in humans over a 49-month period by measuring the graft/bone neoformation ratio in relation to the total mineralized area. METHODS: This was a case series involving 12 patients: eight females (66% and four males (34%, totaling 13 biopsies. BLG was used, and surgical reintervention was subsequently required during the period 2000 to 2011. The slides produced were stained with hematoxylin-eosin (HE, were analyzed by a pathologist and were digitized for the proposed evaluation. RESULTS: The mean age was 57 years and the mean follow-up was 49 months (range: 6-115. The average proportion of BLG was 42% range: 13-85 and neoformed bone, 58% (range: 15-87 in relation to the total area mineralized

  13. Diagnostic utility of aP2/FABP4 expression in soft tissue tumours.

    Science.gov (United States)

    Kashima, T G; Turley, H; Dongre, A; Pezzella, F; Athanasou, N A

    2013-04-01

    Adipocyte P2 (aP2), also known as fatty acid-binding protein 4 (FABP4), is a fatty acid-binding protein found in the cytoplasm of cells of adipocyte differentiation. In this study, we examined a large number of soft tissue tumours with a commercial polyclonal anti-aP2/FABP4 antibody and a newly developed mouse monoclonal antibody raised against this protein to determine the diagnostic utility of aP2/FABP4 as a marker of tumours of adipose differentiation. A mouse monoclonal antibody, clone 175d, was raised against a mixture of synthetic peptides corresponding to the amino acid sequence of residues 10-28 and 121-132 of the human aP2/FABP4 protein. Antigen expression with polyclonal and monoclonal antibodies was immunohistochemically determined in paraffin sections of normal adipose tissue and a wide range of benign and malignant primary soft tissue tumours (n = 200). aP2/FABP4 was expressed around the cytoplasmic lipid vacuole in white and brown fat cells in benign lipomas and hibernomas. Immature fat cells and lipoblasts in spindle cell/pleomorphic lipoma, atypical lipomatous tumour/well-differentiated liposarcoma, myxoid/round cell liposarcoma and pleomorphic liposarcoma also reacted strongly for aP2/FABP4. No specific staining was seen in non-adipose benign and malignant mesenchymal and non-mesenchymal tumours. aP2/FABP4 is expressed by mature and immature fat cells and is a marker of tumours of adipose differentiation. Immunophenotypic aP2/FABP4 expression is useful in identifying lipoblasts, and immunohistochemistry with polyclonal/monoclonal anti-aP2/FABP4 antibodies should be useful in distinguishing liposarcoma from other malignancies, particularly round cell, myxoid and pleomorphic soft tissue sarcomas.

  14. Westinghouse AP1000 advanced passive plant: design features and benefits

    Energy Technology Data Exchange (ETDEWEB)

    Walls, S.J. [British Nuclear Fuels plc, Seascale, Cumbria (United Kingdom); Cummins, W.E. [Westinghouse Electric Company, Pittsburgh, Pennsylvania (United States)

    2003-07-01

    The Westinghouse AP1000 Program is aimed at implementing the AP1000 plant to provide a further major improvement in plant economics while maintaining the passive safety advantages established by the AP600. An objective is to retain to the maximum extent possible the plant design of the AP600 so as to retain the licensing basis, cost estimate, construction schedule, modularization scheme, and the detailed design from the AP600 program. Westinghouse and the US Nuclear Regulatory Commission staff have embarked on a program to complete Design Certification for the AP1000 by 2004. A pre-certification review phase was completed in March 2002 and was successful in establishing the applicability of the AP600 test program and AP600 safety analysis codes to the AP1000 Design Certification. On March 28, 2002, Westinghouse submitted to US NRC the AP1000 Design Control Document and Probabilistic Risk Assessment, thereby initiating the formal design certification review process. The results presented in these documents verify the safety performance of the API 000 and conformance with US NRC licensing requirements. Plans are being developed for implementation of a series of AP1000 plants in the US. Key factors in this planning are the economics of AP1000, and the associated business model for licensing, constructing and operating these new plants. Similarly plans are being developed to get the AP1000 design reviewed for use in the UK. Part of this planning has been to examine the AP1000 design relative to anticipated UK safety and licensing issues. (author)

  15. Bivalent Motif-Ear Interactions Mediate the Association of the Accessory Protein Tepsin with the AP-4 Adaptor Complex.

    Science.gov (United States)

    Mattera, Rafael; Guardia, Carlos M; Sidhu, Sachdev S; Bonifacino, Juan S

    2015-12-25

    The heterotetrameric (ϵ-β4-μ4-σ4) complex adaptor protein 4 (AP-4) is a component of a non-clathrin coat involved in protein sorting at the trans-Golgi network (TGN). Considerable interest in this complex has arisen from the recent discovery that mutations in each of its four subunits are the cause of a congenital intellectual disability and movement disorder in humans. Despite its physiological importance, the structure and function of this coat remain poorly understood. To investigate the assembly of the AP-4 coat, we dissected the determinants of interaction of AP-4 with its only known accessory protein, the ENTH/VHS-domain-containing protein tepsin. Using a variety of protein interaction assays, we found that tepsin comprises two phylogenetically conserved peptide motifs, [GS]LFXG[ML]X[LV] and S[AV]F[SA]FLN, within its C-terminal unstructured region, which interact with the C-terminal ear (or appendage) domains of the β4 and ϵ subunits of AP-4, respectively. Structure-based mutational analyses mapped the binding site for the [GS]LFXG[ML]X[LV] motif to a conserved, hydrophobic surface on the β4-ear platform fold. Both peptide-ear interactions are required for efficient association of tepsin with AP-4, and for recruitment of tepsin to the TGN. The bivalency of the interactions increases the avidity of tepsin for AP-4 and may enable cross-linking of multiple AP-4 heterotetramers, thus contributing to the assembly of the AP-4 coat. In addition to revealing critical aspects of this coat, our findings extend the paradigm of peptide-ear interactions, previously established for clathrin-AP-1/AP-2 coats, to a non-clathrin coat. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. AP-1/KIF13A Blocking Peptides Impair Melanosome Maturation and Melanin Synthesis.

    Science.gov (United States)

    Campagne, Cécile; Ripoll, Léa; Gilles-Marsens, Floriane; Raposo, Graça; Delevoye, Cédric

    2018-02-14

    Melanocytes are specialized cells that generate unique organelles called melanosomes in which melanin is synthesized and stored. Melanosome biogenesis and melanocyte pigmentation require the transport and delivery of melanin synthesizing enzymes, such as tyrosinase and related proteins (e.g., TYRP1), from endosomes to maturing melanosomes. Among the proteins controlling endosome-melanosome transport, AP-1 together with KIF13A coordinates the endosomal sorting and trafficking of TYRP1 to melanosomes. We identify here β1-adaptin AP-1 subunit-derived peptides of 5 amino acids that block the interaction of KIF13A with AP-1 in cells. Incubating these peptides with human MNT-1 cells or 3D-reconstructed pigmented epidermis decreases pigmentation by impacting the maturation of melanosomes in fully pigmented organelles. This study highlights that peptides targeting the intracellular trafficking of melanocytes are candidate molecules to tune pigmentation in health and disease.

  17. AP-1/KIF13A Blocking Peptides Impair Melanosome Maturation and Melanin Synthesis

    Directory of Open Access Journals (Sweden)

    Cécile Campagne

    2018-02-01

    Full Text Available Melanocytes are specialized cells that generate unique organelles called melanosomes in which melanin is synthesized and stored. Melanosome biogenesis and melanocyte pigmentation require the transport and delivery of melanin synthesizing enzymes, such as tyrosinase and related proteins (e.g., TYRP1, from endosomes to maturing melanosomes. Among the proteins controlling endosome-melanosome transport, AP-1 together with KIF13A coordinates the endosomal sorting and trafficking of TYRP1 to melanosomes. We identify here β1-adaptin AP-1 subunit-derived peptides of 5 amino acids that block the interaction of KIF13A with AP-1 in cells. Incubating these peptides with human MNT-1 cells or 3D-reconstructed pigmented epidermis decreases pigmentation by impacting the maturation of melanosomes in fully pigmented organelles. This study highlights that peptides targeting the intracellular trafficking of melanocytes are candidate molecules to tune pigmentation in health and disease.

  18. Bifunctional TaqII restriction endonuclease: redefining the prototype DNA recognition site and establishing the Fidelity Index for partial cleaving

    Directory of Open Access Journals (Sweden)

    Żylicz-Stachula Agnieszka

    2011-12-01

    Full Text Available Abstract Background The TaqII enzyme is a member of the Thermus sp. enzyme family that we propounded previously within Type IIS restriction endonucleases, containing related thermophilic bifunctional endonucleases-methyltransferases from various Thermus sp.: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI and TsoI. These enzymes show significant nucleotide and amino acid sequence similarities, a rare phenomenon among restriction endonucleases, along with similarities in biochemical properties, molecular size, DNA recognition sequences and cleavage sites. They also feature some characteristics of Types I and III. Results Barker et al. reported the Type IIS/IIC restriction endonuclease TaqII as recognizing two distinct cognate site variants (5'-GACCGA-3' and 5'-CACCCA-3' while cleaving 11/9 nucleotides downstream. We used four independent methods, namely, shotgun cloning and sequencing, restriction pattern analysis, digestion of particular custom substrates and GeneScan analysis, to demonstrate that the recombinant enzyme recognizes only 5'-GACCGA-3' sites and cleaves 11/9 nucleotides downstream. We did not observe any 5'-CACCCA-3' cleavage under a variety of conditions and site arrangements tested. We also characterized the enzyme biochemically and established new digestion conditions optimal for practical enzyme applications. Finally, we developed and propose a new version of the Fidelity Index - the Fidelity Index for Partial Cleavage (FI-PC. Conclusions The DNA recognition sequence of the bifunctional prototype TaqII endonuclease-methyltransferase from Thermus aquaticus has been redefined as recognizing only 5'-GACCGA-3' cognate sites. The reaction conditions (pH and salt concentrations were designed either to minimize (pH = 8.0 and 10 mM ammonium sulphate or to enhance star activity (pH = 6.0 and no salt. Redefinition of the recognition site and reaction conditions makes this prototype endonuclease a useful tool for DNA manipulation; as yet, this

  19. Bifunctional TaqII restriction endonuclease: redefining the prototype DNA recognition site and establishing the Fidelity Index for partial cleaving.

    Science.gov (United States)

    Zylicz-Stachula, Agnieszka; Zołnierkiewicz, Olga; Sliwińska, Katarzyna; Jeżewska-Frąckowiak, Joanna; Skowron, Piotr M

    2011-12-05

    The TaqII enzyme is a member of the Thermus sp. enzyme family that we propounded previously within Type IIS restriction endonucleases, containing related thermophilic bifunctional endonucleases-methyltransferases from various Thermus sp.: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI and TsoI. These enzymes show significant nucleotide and amino acid sequence similarities, a rare phenomenon among restriction endonucleases, along with similarities in biochemical properties, molecular size, DNA recognition sequences and cleavage sites. They also feature some characteristics of Types I and III. Barker et al. reported the Type IIS/IIC restriction endonuclease TaqII as recognizing two distinct cognate site variants (5'-GACCGA-3' and 5'-CACCCA-3') while cleaving 11/9 nucleotides downstream. We used four independent methods, namely, shotgun cloning and sequencing, restriction pattern analysis, digestion of particular custom substrates and GeneScan analysis, to demonstrate that the recombinant enzyme recognizes only 5'-GACCGA-3' sites and cleaves 11/9 nucleotides downstream. We did not observe any 5'-CACCCA-3' cleavage under a variety of conditions and site arrangements tested. We also characterized the enzyme biochemically and established new digestion conditions optimal for practical enzyme applications. Finally, we developed and propose a new version of the Fidelity Index - the Fidelity Index for Partial Cleavage (FI-PC). The DNA recognition sequence of the bifunctional prototype TaqII endonuclease-methyltransferase from Thermus aquaticus has been redefined as recognizing only 5'-GACCGA-3' cognate sites. The reaction conditions (pH and salt concentrations) were designed either to minimize (pH = 8.0 and 10 mM ammonium sulphate) or to enhance star activity (pH = 6.0 and no salt). Redefinition of the recognition site and reaction conditions makes this prototype endonuclease a useful tool for DNA manipulation; as yet, this enzyme has no practical applications. The extension of

  20. Matrix proteins of Nipah and Hendra viruses interact with beta subunits of AP-3 complexes.

    Science.gov (United States)

    Sun, Weina; McCrory, Thomas S; Khaw, Wei Young; Petzing, Stephanie; Myers, Terrell; Schmitt, Anthony P

    2014-11-01

    Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people

  1. Creating Memorable Learning Experiences with Foldables in AP Human Geography

    Science.gov (United States)

    Purcell, Jane

    2014-01-01

    Many teachers struggle with helping their students simultaneously comprehend and retain the information that they read. These classroom educators have students take notes (copious amounts of notes) that neither produces the intended learning effect nor actively engages the student in the learning process. One way to increase retention is through…

  2. Epigenetic Segregation of Microbial Genomes from Complex Samples Using Restriction Endonucleases HpaII and McrB.

    Science.gov (United States)

    Liu, Guohong; Weston, Christopher Q; Pham, Long K; Waltz, Shannon; Barnes, Helen; King, Paula; Sphar, Dan; Yamamoto, Robert T; Forsyth, R Allyn

    2016-01-01

    We describe continuing work to develop restriction endonucleases as tools to enrich targeted genomes of interest from diverse populations. Two approaches were developed in parallel to segregate genomic DNA based on cytosine methylation. First, the methyl-sensitive endonuclease HpaII was used to bind non-CG methylated DNA. Second, a truncated fragment of McrB was used to bind CpG methylated DNA. Enrichment levels of microbial genomes can exceed 100-fold with HpaII allowing improved genomic detection and coverage of otherwise trace microbial genomes from sputum. Additionally, we observe interesting enrichment results that correlate with the methylation states not only of bacteria, but of fungi, viruses, a protist and plants. The methods presented here offer promise for testing biological samples for pathogens and global analysis of population methylomes.

  3. Epigenetic Segregation of Microbial Genomes from Complex Samples Using Restriction Endonucleases HpaII and McrB.

    Directory of Open Access Journals (Sweden)

    Guohong Liu

    Full Text Available We describe continuing work to develop restriction endonucleases as tools to enrich targeted genomes of interest from diverse populations. Two approaches were developed in parallel to segregate genomic DNA based on cytosine methylation. First, the methyl-sensitive endonuclease HpaII was used to bind non-CG methylated DNA. Second, a truncated fragment of McrB was used to bind CpG methylated DNA. Enrichment levels of microbial genomes can exceed 100-fold with HpaII allowing improved genomic detection and coverage of otherwise trace microbial genomes from sputum. Additionally, we observe interesting enrichment results that correlate with the methylation states not only of bacteria, but of fungi, viruses, a protist and plants. The methods presented here offer promise for testing biological samples for pathogens and global analysis of population methylomes.

  4. The Cryptosporidium parvum ApiAP2 gene family: insights into the evolution of apicomplexan AP2 regulatory systems.

    Science.gov (United States)

    Oberstaller, Jenna; Pumpalova, Yoanna; Schieler, Ariel; Llinás, Manuel; Kissinger, Jessica C

    2014-07-01

    We provide the first comprehensive analysis of any transcription factor family in Cryptosporidium, a basal-branching apicomplexan that is the second leading cause of infant diarrhea globally. AP2 domain-containing proteins have evolved to be the major regulatory family in the phylum to the exclusion of canonical regulators. We show that apicomplexan and perkinsid AP2 domains cluster distinctly from other chromalveolate AP2s. Protein-binding specificity assays of C. parvum AP2 domains combined with motif conservation upstream of co-regulated gene clusters allowed the construction of putative AP2 regulons across the in vitro life cycle. Orthologous Apicomplexan AP2 (ApiAP2) expression has been rearranged relative to the malaria parasite P. falciparum, suggesting ApiAP2 network rewiring during evolution. C. hominis orthologs of putative C. parvum ApiAP2 proteins and target genes show greater than average variation. C. parvum AP2 domains display reduced binding diversity relative to P. falciparum, with multiple domains binding the 5'-TGCAT-3', 5'-CACACA-3' and G-box motifs (5'-G[T/C]GGGG-3'). Many overrepresented motifs in C. parvum upstream regions are not AP2 binding motifs. We propose that C. parvum is less reliant on ApiAP2 regulators in part because it utilizes E2F/DP1 transcription factors. C. parvum may provide clues to the ancestral state of apicomplexan transcriptional regulation, pre-AP2 domination. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Quantum Entanglement in the Genome? The Role of Quantum Effects in Catalytic Synchronization of Type II Restriction Endonucleases

    Science.gov (United States)

    Kurian, P.

    Several living systems have been examined for their exhibition of macroscopic quantum effects, showcasing biology's apparent optimization of structure and function for quantum behavior. Prevalent in lower organisms with analogues in eukaryotes, type II restriction endonucleases are the largest class of restriction enzymes. Orthodox type II endonucleases recognize four-to-eight base pair sequences of palindromic DNA, cut both strands symmetrically, and act without an external metabolite such as ATP. While it is known that these enzymes induce strand breaks by nucleophilic attack on opposing phosphodiester bonds of the DNA helix, what remains unclear is the mechanism by which cutting occurs in concert at the catalytic centers. Previous studies indicate the primacy of intimate DNA contacts made by the specifically bound enzyme in coordinating the two synchronized cuts. We propose that collective electronic behavior in the DNA helix generates coherent oscillations---quantized through boundary conditions imposed by the endonuclease---that provide the energy required to break two phosphodiester bonds. Such quanta may be preserved in the presence of thermal noise and electromagnetic interference through the specific complex's exclusion of water and ions surrounding the helix, with the enzyme serving as a decoherence shield. Clamping energy imparted by the decoherence shield is comparable with zero-point modes of the dipole-dipole oscillations in the DNA recognition sequence. The palindromic mirror symmetry of this sequence should conserve parity during the process. Experimental data corroborate that symmetric bond-breaking ceases when the symmetry of the endonuclease complex is violated, or when environmental parameters are perturbed far from biological optima. Persistent correlation between states in DNA sequence across spatial separations of any length---a characteristic signature of quantum entanglement---may be explained by such a physical mechanism.

  6. Role of endonucleases XPF and XPG in nucleotide excision repair of platinated DNA and cisplatin/oxaliplatin cytotoxicity

    OpenAIRE

    Graf, Nora; Ang, Wee Han; Zhu, Guangyu; Myint, MyatNoeZin; Lippard, Stephen J.

    2011-01-01

    Resistance of tumor cells to platinum anticancer agents poses a major problem in cancer chemotherapy. One of the mechanisms associated with platinum-based drug resistance is the enhanced capacity of the cell to carry out nucleotide excision repair (NER) on platinum-damaged DNA. Endonucleases XPF and XPG are critical components of NER, responsible for excising the damaged DNA strand to remove the DNA lesion. Here, we investigated possible consequences of down-regulation of XPF and XPG gene exp...

  7. Apurinic/Apyrimidinic Endonuclease 1 Upregulation Reduces Oxidative DNA Damage and Protects Hippocampal Neurons from Ischemic Injury

    OpenAIRE

    Leak, Rehana K.; Li, Peiying; Zhang, Feng; Sulaiman, Hassan H.; Weng, Zhongfang; Wang, Guohua; Stetler, R. Anne; Shi, Yejie; Cao, Guodong; Gao, Yanqin; Chen, Jun

    2015-01-01

    Aims: Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme that participates in base-excision repair of oxidative DNA damage and in the redox activation of transcription factors. We tested the hypothesis that APE1 upregulation protects neuronal structure and function against transient global cerebral ischemia (tGCI). Results: Upregulation of APE1 by low-dose proton irradiation (PI) or by transgene overexpression protected hippocampal CA1 neurons against tGCI-induced cell lo...

  8. Biological significance of facilitated diffusion in protein-DNA interactions. Applications to T4 endonuclease V-initiated DNA repair

    Energy Technology Data Exchange (ETDEWEB)

    Dowd, D.R.; Lloyd, R.S. (Vanderbilt Univ. School of Medicine, Nashville, TN (USA))

    1990-02-25

    Facilitated diffusion along nontarget DNA is employed by numerous DNA-interactive proteins to locate specific targets. Until now, the biological significance of DNA scanning has remained elusive. T4 endonuclease V is a DNA repair enzyme which scans nontarget DNA and processively incises DNA at the site of pyrimidine dimers which are produced by exposure to ultraviolet (UV) light. In this study we tested the hypothesis that there exists a direct correlation between the degree of processivity of wild type and mutant endonuclease V molecules and the degree of enhanced UV resistance which is conferred to repair-deficient Eshcerichia coli. This was accomplished by first creating a series of endonuclease V mutants whose in vitro catalytic activities were shown to be very similar to that of the wild type enzyme. However, when the mechanisms by which these enzymes search nontarget DNA for its substrate were analyzed in vitro and in vivo, the mutants displayed varying degrees of nontarget DNA scanning ranging from being nearly as processive as wild type to randomly incising dimers within the DNA population. The ability of these altered endonuclease V molecules to enhance UV survival in DNA repair-deficient E. coli then was assessed. The degree of enhanced UV survival was directly correlated with the level of facilitated diffusion. This is the first conclusive evidence directly relating a reduction of in vivo facilitated diffusion with a change in an observed phenotype. These results support the assertion that the mechanisms which DNA-interactive proteins employ in locating their target sites are of biological significance.

  9. Biological significance of facilitated diffusion in protein-DNA interactions. Applications to T4 endonuclease V-initiated DNA repair.

    Science.gov (United States)

    Dowd, D R; Lloyd, R S

    1990-02-25

    Facilitated diffusion along nontarget DNA is employed by numerous DNA-interactive proteins to locate specific targets. Until now, the biological significance of DNA scanning has remained elusive. T4 endonuclease V is a DNA repair enzyme which scans nontarget DNA and processively incises DNA at the site of pyrimidine dimers which are produced by exposure to ultraviolet (UV) light. In this study we tested the hypothesis that there exists a direct correlation between the degree of processivity of wild type and mutant endonuclease V molecules and the degree of enhanced UV resistance which is conferred to repair-deficient Eshcerichia coli. This was accomplished by first creating a series of endonuclease V mutants whose in vitro catalytic activities were shown to be very similar to that of the wild type enzyme. However, when the mechanisms by which these enzymes search nontarget DNA for its substrate were analyzed in vitro and in vivo, the mutants displayed varying degrees of nontarget DNA scanning ranging from being nearly as processive as wild type to randomly incising dimers within the DNA population. The ability of these altered endonuclease V molecules to enhance UV survival in DNA repair-deficient E. coli then was assessed. The degree of enhanced UV survival was directly correlated with the level of facilitated diffusion. This is the first conclusive evidence directly relating a reduction of in vivo facilitated diffusion with a change in an observed phenotype. These results support the assertion that the mechanisms which DNA-interactive proteins employ in locating their target sites are of biological significance.

  10. Small Molecule Inhibitors Targeting Activator Protein 1 (AP-1)

    OpenAIRE

    Ye, Na; Ding, Ye; Wild, Christopher; Shen, Qiang; Zhou, Jia

    2014-01-01

    Activator protein 1 (AP-1) is a pivotal transcription factor that regulates a wide range of cellular processes including proliferation, apoptosis, differentiation, survival, cell migration, and transformation. Accumulating evidence supports that AP-1 plays an important role in several severe disorders including cancer, fibrosis, and organ injury, as well as inflammatory disorders such as asthma, psoriasis, and rheumatoid arthritis. AP-1 has emerged as an actively pursued drug discovery target...

  11. ONLINE MINIMIZATION OF VERTICAL BEAM SIZES AT APS

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Yipeng

    2017-06-25

    In this paper, online minimization of vertical beam sizes along the APS (Advanced Photon Source) storage ring is presented. A genetic algorithm (GA) was developed and employed for the online optimization in the APS storage ring. A total of 59 families of skew quadrupole magnets were employed as knobs to adjust the coupling and the vertical dispersion in the APS storage ring. Starting from initially zero current skew quadrupoles, small vertical beam sizes along the APS storage ring were achieved in a short optimization time of one hour. The optimization results from this method are briefly compared with the one from LOCO (Linear Optics from Closed Orbits) response matrix correction.

  12. Structural and functional analysis of the symmetrical Type I restriction endonuclease R.EcoR124I(NT.

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    James E Taylor

    Full Text Available Type I restriction-modification (RM systems are comprised of two multi-subunit enzymes, the methyltransferase (∼160 kDa, responsible for methylation of DNA, and the restriction endonuclease (∼400 kDa, responsible for DNA cleavage. Both enzymes share a number of subunits. An engineered RM system, EcoR124I(NT, based on the N-terminal domain of the specificity subunit of EcoR124I was constructed that recognises the symmetrical sequence GAAN(7TTC and is active as a methyltransferase. Here, we investigate the restriction endonuclease activity of R. EcoR124I(NTin vitro and the subunit assembly of the multi-subunit enzyme. Finally, using small-angle neutron scattering and selective deuteration, we present a low-resolution structural model of the endonuclease and locate the motor subunits within the multi-subunit enzyme. We show that the covalent linkage between the two target recognition domains of the specificity subunit is not required for subunit assembly or enzyme activity, and discuss the implications for the evolution of Type I enzymes.

  13. Structural basis for the substrate selectivity of PvuRts1I, a 5-hydroxymethylcytosine DNA restriction endonuclease.

    Science.gov (United States)

    Shao, Chen; Wang, Chengliang; Zang, Jianye

    2014-09-01

    5-Hydroxymethylation is a curious modification of cytosine that was discovered some decades ago, but its functional role in eukaryotes still awaits elucidation. 5-Hydroxymethylcytosine is an epigenetic marker that is crucial for multiple biological processes. The profile is altered under certain disease conditions such as cancer, Huntington's disease and Alzheimer's disease. Using the DNA-modification-dependent restriction endonuclease AbaSI coupled with sequencing (Aba-seq), the hydroxymethylome can be deciphered at the resolution of individual bases. The method is based on the enzymatic properties of AbaSI, a member of the PvuRts1I family of endonucleases. PvuRts1I is a modification-dependent endonuclease with high selectivity for 5-hydroxymethylcytosine over 5-methylcytosine and cytosine. In this study, the crystal structure of PvuRts1I was determined in order to understand and improve the substrate selectivity. A nuclease domain and an SRA-like domain are located at the N- and C-termini, respectively. Through comparison with other SRA-domain structures, the SRA-like domain was proposed to be the 5-hmC recognition module. Several mutants of PvuRts1I with enzymatic activity restricted to 5-hydroxymethylcytosine only were generated based on the structural analysis, and these enzyme variants are appropriate for separating the hydroxymethylome from the wider methylome.

  14. Characterization of LlaKI, a New Metal Ion-Independent Restriction Endonuclease from Lactococcus lactis KLDS4.

    Science.gov (United States)

    Belkebir, Abdelkarim; Azeddoug, Houssine

    2012-01-01

    Requirement of divalent cations for DNA cleavage is a general feature of type II restriction enzymes with the exception of few members of this group. A new type II restriction endonuclease has been partially purified from Lactococcus lactis KLDS4. The enzyme was denoted as LlaKI and showed to recognize and cleave the same site as FokI. The enzyme displayed a denatured molecular weight of 50 kDa and behaved as a dimer in solution as evidenced by the size exclusion chromatography. To investigate the role of divalent cations in DNA cleavage by LlaKI, digestion reactions were carried out at different Mg(2+), Mn(2+), and Ca(2+) concentrations. Unlike most of type II restriction endonucleases, LlaKI did not require divalent metal ions to cleave DNA and is one of the few metal-independent restriction endonucleases found in bacteria. The enzyme showed near-maximal levels of activity in 10 mM Tris-HCl pH 7.9, 50 mM NaCl, 10 mM MgCl2, and 1 mM dithiothreitol at 30°C. The presence of DNA modification was also determined and was correlated with the correspondent restriction enzyme.

  15. Quantifying DNA double-strand breaks induced by site-specific endonucleases in living cells by ligation-mediated purification.

    Science.gov (United States)

    Chailleux, Catherine; Aymard, François; Caron, Pierre; Daburon, Virginie; Courilleau, Céline; Canitrot, Yvan; Legube, Gaëlle; Trouche, Didier

    2014-03-01

    Recent advances in our understanding of the management and repair of DNA double-strand breaks (DSBs) rely on the study of targeted DSBs that have been induced in living cells by the controlled activity of site-specific endonucleases, usually recombinant restriction enzymes. Here we describe a protocol for quantifying these endonuclease-induced DSBs; this quantification is essential to an interpretation of how DSBs are managed and repaired. A biotinylated double-stranded oligonucleotide is ligated to enzyme-cleaved genomic DNA, allowing the purification of the cleaved DNA on streptavidin beads. The extent of cleavage is then quantified either by quantitative PCR (qPCR) at a given site or at multiple sites by genome-wide techniques (e.g., microarrays or high-throughput sequencing). This technique, named ligation-mediated purification, can be performed in 2 d. It is more accurate and sensitive than existing alternative methods, and it is compatible with genome-wide analysis. It allows the amount of endonuclease-mediated breaks to be precisely compared between two conditions or across the genome, thereby giving insight into the influence of a given factor or of various chromatin contexts on local repair parameters.

  16. Promotion of Homologous Recombination and Genomic Stability byRAD51AP1 via RAD51 Recombinase Enhancement

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    Wiese, Claudia; Dray, Eloise; Groesser, Torsten; San Filippo,Joseph; Shi, Idina; Collins, David W.; Tsai, Miaw-Sheue; Williams,Gareth; Rydberg, Bjorn; Sung, Patrick; Schild, David

    2007-04-11

    Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand pairing step in HR. RAD51AP1 (RAD51 Associated Protein 1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA damaging treatment. Purified RAD51AP1 binds dsDNA and RAD51, and it greatly stimulates the RAD51-mediated D-loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide the first evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.

  17. Genome-Wide Identification of the Target Genes of AP2-O, a Plasmodium AP2-Family Transcription Factor.

    Directory of Open Access Journals (Sweden)

    Izumi Kaneko

    2015-05-01

    Full Text Available Stage-specific transcription is a fundamental biological process in the life cycle of the Plasmodium parasite. Proteins containing the AP2 DNA-binding domain are responsible for stage-specific transcriptional regulation and belong to the only known family of transcription factors in Plasmodium parasites. Comprehensive identification of their target genes will advance our understanding of the molecular basis of stage-specific transcriptional regulation and stage-specific parasite development. AP2-O is an AP2 family transcription factor that is expressed in the mosquito midgut-invading stage, called the ookinete, and is essential for normal morphogenesis of this stage. In this study, we identified the genome-wide target genes of AP2-O by chromatin immunoprecipitation-sequencing and elucidate how this AP2 family transcription factor contributes to the formation of this motile stage. The analysis revealed that AP2-O binds specifically to the upstream genomic regions of more than 500 genes, suggesting that approximately 10% of the parasite genome is directly regulated by AP2-O. These genes are involved in distinct biological processes such as morphogenesis, locomotion, midgut penetration, protection against mosquito immunity and preparation for subsequent oocyst development. This direct and global regulation by AP2-O provides a model for gene regulation in Plasmodium parasites and may explain how these parasites manage to control their complex life cycle using a small number of sequence-specific AP2 transcription factors.

  18. Genome-Wide Identification of the Target Genes of AP2-O, a Plasmodium AP2-Family Transcription Factor.

    Science.gov (United States)

    Kaneko, Izumi; Iwanaga, Shiroh; Kato, Tomomi; Kobayashi, Issei; Yuda, Masao

    2015-05-01

    Stage-specific transcription is a fundamental biological process in the life cycle of the Plasmodium parasite. Proteins containing the AP2 DNA-binding domain are responsible for stage-specific transcriptional regulation and belong to the only known family of transcription factors in Plasmodium parasites. Comprehensive identification of their target genes will advance our understanding of the molecular basis of stage-specific transcriptional regulation and stage-specific parasite development. AP2-O is an AP2 family transcription factor that is expressed in the mosquito midgut-invading stage, called the ookinete, and is essential for normal morphogenesis of this stage. In this study, we identified the genome-wide target genes of AP2-O by chromatin immunoprecipitation-sequencing and elucidate how this AP2 family transcription factor contributes to the formation of this motile stage. The analysis revealed that AP2-O binds specifically to the upstream genomic regions of more than 500 genes, suggesting that approximately 10% of the parasite genome is directly regulated by AP2-O. These genes are involved in distinct biological processes such as morphogenesis, locomotion, midgut penetration, protection against mosquito immunity and preparation for subsequent oocyst development. This direct and global regulation by AP2-O provides a model for gene regulation in Plasmodium parasites and may explain how these parasites manage to control their complex life cycle using a small number of sequence-specific AP2 transcription factors.

  19. Ten ERK-related proteins in three distinct classes associate with AP-1 proteins and/or AP-1 DNA.

    Science.gov (United States)

    Kumar, N V; Bernstein, L R

    2001-08-24

    We have identified seven ERK-related proteins ("ERPs"), including ERK2, that are stably associated in vivo with AP-1 dimers composed of diverse Jun and Fos family proteins. These complexes have kinase activity. We designate them as "class I ERPs." We originally hypothesized that these ERPs associate with DNA along with AP-1 proteins. We devised a DNA affinity chromatography-based analytical assay for DNA binding, the "nucleotide affinity preincubation specificity test recognition" (NAPSTER) assay. In this assay, class I ERPs do not associate with AP-1 DNA. However, several new "class II" ERPs do associate with DNA. p41 and p44 are ERK1/2-related ERPs that lack kinase activity and associate along with AP-1 proteins with AP-1 DNA. Class I ERPs and their associated kinase activity thus appear to bind AP-1 dimers when they are not bound to DNA and then disengage and are replaced by class II ERPs to form higher order complexes when AP-1 dimers bind DNA. p97 is a class III ERP, related to ERK3, that associates with AP-1 DNA without AP-1 proteins. With the exception of ERK2, none of the 10 ERPs appear to be known mitogen-activated protein kinase superfamily members.

  20. Polymorphism of the Flap Endonuclease 1 Gene in Keratoconus and Fuchs Endothelial Corneal Dystrophy

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    Katarzyna A. Wojcik

    2014-08-01

    Full Text Available Oxidative stress is implicated in the pathogenesis of many diseases, including serious ocular diseases, keratoconus (KC and Fuchs endothelial corneal dystrophy (FECD. Flap endonuclease 1 (FEN1 plays an important role in the repair of oxidative DNA damage in the base excision repair pathway. We determined the association between two single nucleotide polymorphisms (SNPs, c.–441G>A (rs174538 and g.61564299G>T (rs4246215, in the FEN1 gene and the occurrence of KC and FECD. This study involved 279 patients with KC, 225 patients with FECD and 322 control individuals. Polymerase chain reaction (PCR and length polymorphism restriction fragment analysis (RFLP were applied. The T/T genotype of the g.61564299G>T polymorphism was associated with an increased occurrence of KC and FECD. There was no association between the c.–441G>A polymorphism and either disease. However, the GG haplotype of both polymorphisms was observed more frequently and the GT haplotype less frequently in the KC group than the control. The AG haplotype was associated with increased FECD occurrence. Our findings suggest that the g.61564299G>T and c.–441G>A polymorphisms in the FEN1 gene may modulate the risk of keratoconus and Fuchs endothelial corneal dystrophy.

  1. Restriction endonuclease triggered bacterial apoptosis as a mechanism for long time survival.

    Science.gov (United States)

    Nagamalleswari, Easa; Rao, Sandhya; Vasu, Kommireddy; Nagaraja, Valakunja

    2017-08-21

    Programmed cell death (PCD) under certain conditions is one of the features of bacterial altruism. Given the bacterial diversity and varied life style, different PCD mechanisms must be operational that remain largely unexplored. We describe restriction endonuclease (REase) mediated cell death by an apoptotic pathway, beneficial for isogenic bacterial communities. Cell death is pronounced in stationary phase and when the enzyme exhibits promiscuous DNA cleavage activity. We have elucidated the molecular mechanism of REase mediated cell killing and demonstrate that released nutrients from dying cells support the growth of the remaining cells in the population. These findings illustrate a new intracellular moonlighting role for REases which are otherwise established host defence arsenals. REase induced PCD appears to be a cellular design to replenish nutrients for cells undergoing starvation stress and the phenomenon could be wide spread in bacteria, given the abundance of restriction-modification (R-M) systems in the microbial population. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Mitochondrial Targeted Endonuclease III DNA Repair Enzyme Protects against Ventilator Induced Lung Injury in Mice

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    Masahiro Hashizume

    2014-08-01

    Full Text Available The mitochondrial targeted DNA repair enzyme, 8-oxoguanine DNA glycosylase 1, was previously reported to protect against mitochondrial DNA (mtDNA damage and ventilator induced lung injury (VILI. In the present study we determined whether mitochondrial targeted endonuclease III (EndoIII which cleaves oxidized pyrimidines rather than purines from damaged DNA would also protect the lung. Minimal injury from 1 h ventilation at 40 cmH2O peak inflation pressure (PIP was reversed by EndoIII pretreatment. Moderate lung injury due to ventilation for 2 h at 40 cmH2O PIP produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio, and marked increases in MIP-2 and IL-6. Oxidative mtDNA damage and decreases in the total tissue glutathione (GSH and the GSH/GSSH ratio also occurred. All of these indices of injury were attenuated by mitochondrial targeted EndoIII. Massive lung injury caused by 2 h ventilation at 50 cmH2O PIP was not attenuated by EndoIII pretreatment, but all untreated mice died prior to completing the two hour ventilation protocol, whereas all EndoIII-treated mice lived for the duration of ventilation. Thus, mitochondrial targeted DNA repair enzymes were protective against mild and moderate lung damage and they enhanced survival in the most severely injured group.

  3. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

    Science.gov (United States)

    Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

    2014-03-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

  4. How quantum entanglement in DNA synchronizes double-strand breakage by type II restriction endonucleases.

    Science.gov (United States)

    Kurian, P; Dunston, G; Lindesay, J

    2016-02-21

    Macroscopic quantum effects in living systems have been studied widely in pursuit of fundamental explanations for biological energy transport and sensing. While it is known that type II endonucleases, the largest class of restriction enzymes, induce DNA double-strand breaks by attacking phosphodiester bonds, the mechanism by which simultaneous cutting is coordinated between the catalytic centers remains unclear. We propose a quantum mechanical model for collective electronic behavior in the DNA helix, where dipole-dipole oscillations are quantized through boundary conditions imposed by the enzyme. Zero-point modes of coherent oscillations would provide the energy required for double-strand breakage. Such quanta may be preserved in the presence of thermal noise by the enzyme's displacement of water surrounding the DNA recognition sequence. The enzyme thus serves as a decoherence shield. Palindromic mirror symmetry of the enzyme-DNA complex should conserve parity, because symmetric bond-breaking ceases when the symmetry of the complex is violated or when physiological parameters are perturbed from optima. Persistent correlations in DNA across longer spatial separations-a possible signature of quantum entanglement-may be explained by such a mechanism. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication.

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    Eveline Kindler

    2017-02-01

    Full Text Available Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I. This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU activity is key to prevent early induction of double-stranded RNA (dsRNA host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis-within the replicase complex-suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses.

  6. Reverse transcriptase and endonuclease activities encoded by Penelope-like retroelements

    Science.gov (United States)

    Pyatkov, Konstantin I.; Arkhipova, Irina R.; Malkova, Natalia V.; Finnegan, David J.; Evgen'ev, Michael B.

    2004-01-01

    Penelope-like elements are a class of retroelement that have now been identified in >50 species belonging to at least 10 animal phyla. The Penelope element isolated from Drosophila virilis is the only transpositionally active representative of this class isolated so far. The single ORF of Penelope and its relatives contains regions homologous to a reverse transcriptase of atypical structure and to the GIY-YIG, or Uri, an endonuclease (EN) domain not previously found in retroelements. We have expressed the single ORF of Penelope in a baculovirus expression system and have shown that it encodes a polyprotein with reverse transcriptase activity that requires divalent cations (Mn2+ and Mg2+). We have also expressed and purified the EN domain in Escherichia coli and have demonstrated that it has EN activity in vitro. Mutations in the conserved residues of the EN catalytic module abolish its nicking activity, whereas the DNA-binding properties of the mutant proteins remain unaffected. Only one strand of the target sequence is cleaved, and there is a certain degree of cleavage specificity. We propose that the Penelope EN cleaves the target DNA during transposition, generating a primer for reverse transcription. Our results show that an active Uri EN has been adopted by a retrotransposon. PMID:15465912

  7. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

    Science.gov (United States)

    Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

    2014-01-01

    The CRISPR-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA:DNA base-pairing to target foreign DNA in bacteria. Cas9:guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9:RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9:RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9:RNA. DNA strand separation and RNA:DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 employs PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate dsDNA scission. PMID:24476820

  8. Comparative Structural and Functional Analysis of Bunyavirus and Arenavirus Cap-Snatching Endonucleases.

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    Juan Reguera

    2016-06-01

    Full Text Available Segmented negative strand RNA viruses of the arena-, bunya- and orthomyxovirus families uniquely carry out viral mRNA transcription by the cap-snatching mechanism. This involves cleavage of host mRNAs close to their capped 5' end by an endonuclease (EN domain located in the N-terminal region of the viral polymerase. We present the structure of the cap-snatching EN of Hantaan virus, a bunyavirus belonging to hantavirus genus. Hantaan EN has an active site configuration, including a metal co-ordinating histidine, and nuclease activity similar to the previously reported La Crosse virus and Influenza virus ENs (orthobunyavirus and orthomyxovirus respectively, but is more active in cleaving a double stranded RNA substrate. In contrast, Lassa arenavirus EN has only acidic metal co-ordinating residues. We present three high resolution structures of Lassa virus EN with different bound ion configurations and show in comparative biophysical and biochemical experiments with Hantaan, La Crosse and influenza ENs that the isolated Lassa EN is essentially inactive. The results are discussed in the light of EN activation mechanisms revealed by recent structures of full-length influenza virus polymerase.

  9. Structure-guided sequence specificity engineering of the modification-dependent restriction endonuclease LpnPI.

    Science.gov (United States)

    Sasnauskas, Giedrius; Zagorskaitė, Evelina; Kauneckaitė, Kotryna; Tamulaitiene, Giedre; Siksnys, Virginijus

    2015-07-13

    The eukaryotic Set and Ring Associated (SRA) domains and structurally similar DNA recognition domains of prokaryotic cytosine modification-dependent restriction endonucleases recognize methylated, hydroxymethylated or glucosylated cytosine in various sequence contexts. Here, we report the apo-structure of the N-terminal SRA-like domain of the cytosine modification-dependent restriction enzyme LpnPI that recognizes modified cytosine in the 5'-C(mC)DG-3' target sequence (where mC is 5-methylcytosine or 5-hydroxymethylcytosine and D = A/T/G). Structure-guided mutational analysis revealed LpnPI residues involved in base-specific interactions and demonstrated binding site plasticity that allowed limited target sequence degeneracy. Furthermore, modular exchange of the LpnPI specificity loops by structural equivalents of related enzymes AspBHI and SgrTI altered sequence specificity of LpnPI. Taken together, our results pave the way for specificity engineering of the cytosine modification-dependent restriction enzymes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Enhancement of PCR Detection Limit by Single-Tube Restriction Endonuclease-PCR (RE-PCR).

    Science.gov (United States)

    Datta, Sibnarayan; Budhauliya, Raghvendra; Chatterjee, Soumya; Vanlalhmuaka; Veer, Vijay; Chakravarty, Runu

    2016-06-01

    Polymerase chain reaction (PCR) is widely used in biological research and diagnostics because of its high sensitivity and specificity. However, the sensitivity of PCR is strongly influenced by topological characteristics of the template. Supercoiled templates are known to inhibit PCR, whereas linearized forms of the same supercoiled templates facilitate PCR. This study was conducted to compare the PCR efficiency of circular supercoiled DNA templates to their restriction endonuclease (RE)-mediated linearized forms. Additionally, we also evaluated the possibility of RE digestion of the circular supercoiled templates within the complete PCR buffer. Following a systematic approach, we demonstrated that circular supercoiled templates could be efficiently linearized by RE in the complete PCR buffer itself. This allowed linearization of circular supercoiled templates and their subsequent amplification in the PCR buffer in a single-tube format. Using this extremely simple RE-PCR approach, we documented up to tenfold increases in detection efficiency of PCR with two different circular supercoiled templates of clinical origin, including an international calibration standard. This inexpensive and easy approach to increasing PCR sensitivity can be easily adapted to any standard PCR protocol aimed at amplifying circular supercoiled genomes. Apart from its application in the development of sensitive clinical diagnostic PCR assays for a large number of organisms, this method could also prove to be very useful in simplifying the existing protocols for other applications where pre-PCR restriction digestion is required, such as mutation detection, genotyping, and selective template amplification.

  11. High pressure activation of the Mrr restriction endonuclease in Escherichia coli involves tetramer dissociation.

    Science.gov (United States)

    Bourges, Anaïs C; Torres Montaguth, Oscar E; Ghosh, Anirban; Tadesse, Wubishet M; Declerck, Nathalie; Aertsen, Abram; Royer, Catherine A

    2017-05-19

    A sub-lethal hydrostatic pressure (HP) shock of ∼100 MPa elicits a RecA-dependent DNA damage (SOS) response in Escherichia coli K-12, despite the fact that pressure cannot compromise the covalent integrity of DNA. Prior screens for HP resistance identified Mrr (Methylated adenine Recognition and Restriction), a Type IV restriction endonuclease (REase), as instigator for this enigmatic HP-induced SOS response. Type IV REases tend to target modified DNA sites, and E. coli Mrr activity was previously shown to be elicited by expression of the foreign M.HhaII Type II methytransferase (MTase), as well. Here we measured the concentration and stoichiometry of a functional GFP-Mrr fusion protein using in vivo fluorescence fluctuation microscopy. Our results demonstrate that Mrr is a tetramer in unstressed cells, but shifts to a dimer after HP shock or co-expression with M.HhaII. Based on the differences in reversibility of tetramer dissociation observed for wild-type GFP-Mrr and a catalytic mutant upon HP shock compared to M.HhaII expression, we propose a model by which (i) HP triggers Mrr activity by directly pushing inactive Mrr tetramers to dissociate into active Mrr dimers, while (ii) M.HhaII triggers Mrr activity by creating high affinity target sites on the chromosome, which pull the equilibrium from inactive tetrameric Mrr toward active dimer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Insights on the Structural Details of Endonuclease EcoRI-DNA Complexes by Electron Spin Resonance

    Science.gov (United States)

    Sarver, Jessica

    2009-03-01

    Pulsed electron spin resonance (ESR) was used to probe the binding specificity of EcoRI, a restriction endonuclease. Using site-directed spin labeling, a nitroxide side chain was incorporated into the protein, enabling the use of ESR to study structural details of EcoRI. Distance measurements were performed on EcoRI mutants when bound to varying sequences of DNA using the Double Electron-Electron Resonance experiment. These distances demonstrated that the average structure in the arm regions of EcoRI, thought to play a major role in binding specificity, is the same when the protein binds to different sequences of DNA. Also, it was determined that the arms exhibit higher flexibility when bound to sequences other than the specific sequence due to the larger distance distributions acquired from these spin labeled complexes. Molecular dynamics (MD) simulations were performed on the spin-label-modified specific EcoRI-DNA crystal structure to model the average nitroxide orientation. The distance distributions from MD were found to be narrower than experiment, indicating the need for a more rigorous sampling of the nitroxide conformers in silico.

  13. The dynamics of the monomeric restriction endonuclease BcnI during its interaction with DNA.

    Science.gov (United States)

    Kostiuk, Georgij; Dikic, Jasmina; Schwarz, Friedrich W; Sasnauskas, Giedrius; Seidel, Ralf; Siksnys, Virginijus

    2017-06-02

    Endonucleases that generate DNA double strand breaks often employ two independent subunits such that the active site from each subunit cuts either DNA strand. Restriction enzyme BcnI is a remarkable exception. It binds to the 5΄-CC/SGG-3΄ (where S = C or G, '/' designates the cleavage position) target as a monomer forming an asymmetric complex, where a single catalytic center approaches the scissile phosphodiester bond in one of DNA strands. Bulk kinetic measurements have previously shown that the same BcnI molecule cuts both DNA strands at the target site without dissociation from the DNA. Here, we analyse the BcnI DNA binding and target recognition steps at the single molecule level. We find, using FRET, that BcnI adopts either 'open' or 'closed' conformation in solution. Next, we directly demonstrate that BcnI slides over long distances on DNA using 1D diffusion and show that sliding is accompanied by occasional jumping events, where the enzyme leaves the DNA and rebinds immediately at a distant site. Furthermore, we quantify the dynamics of the BcnI interactions with cognate and non-cognate DNA, and determine the preferred binding orientation of BcnI to the target site. These results provide new insights into the intricate dynamics of BcnI-DNA interactions. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Crystallization and preliminary X-ray diffraction analysis of restriction endonuclease EcoRII

    Science.gov (United States)

    Karpova, E. A.; Meehan, E.; Pusey, M. L.; Chen, L.

    1999-01-01

    Crystals of the restriction endonuclease EcoRII have been obtained by the vapor-diffusion technique in the presence of ammonium sulfate or polyethylene glycol. The best crystals were grown with ammonium sulfate as a precipitant. Crystals with dimensions of up to 0.6 x 0. 6 x 0.6 mm have been observed. The crystals diffract to about 4.0 A resolution at a cryo-temperature of 100 K using a rotating-anode X-ray source and a Rigaku R-AXIS IV imaging-plate detector. The space group has been determined to be either I23 or I2(1)3, with unit-cell parameters a = b = c = 160.3 A, alpha = beta = gamma = 90 degrees. The crystal asymmetric unit contains two protein molecules, and self-rotation function analysis shows a pseudo-twofold symmetry relating the two monomers. Attempts to improve the resolution of crystal diffraction and to search for heavy-atom derivatives are under way.

  15. Primary processing of CRISPR RNA by the endonuclease Cas6 in Staphylococcus epidermidis.

    Science.gov (United States)

    Wakefield, Noelle; Rajan, Rakhi; Sontheimer, Erik J

    2015-10-07

    In many bacteria and archaea, an adaptive immune system (CRISPR-Cas) provides immunity against foreign genetic elements. This system uses CRISPR RNAs (crRNAs) derived from the CRISPR array, along with CRISPR-associated (Cas) proteins, to target foreign nucleic acids. In most CRISPR systems, endonucleolytic processing of crRNA precursors (pre-crRNAs) is essential for the pathway. Here we study the Cas6 endonuclease responsible for crRNA processing in the Type III-A CRISPR-Cas system from Staphylococcus epidermidis RP62a, a model for Type III-A CRISPR-Cas systems, and define substrate requirements for SeCas6 activity. We find that SeCas6 is necessary and sufficient for full-length crRNA biogenesis in vitro, and that it relies on both sequence and stem-loop structure in the 3' half of the CRISPR repeat for recognition and processing. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  16. Nicking endonuclease and target recycles signal amplification assisted quantum dots for fluorescence detection of DNA

    Energy Technology Data Exchange (ETDEWEB)

    Niu Shuyan; Li Quanyi; Qu Lijing; Wang Wei [Key Lab of Eco-chemical Engineering, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042 (China)

    2010-11-08

    An ultrasensitive fluorescence detection method for DNA based on nicking endonuclease (NEase) and target recycles assisted with CdTe quantum dots (QDs) is reported. In the detection system, when the target DNA is present, it hybridizes with a linker strand to from a duplex, in which the NEase recognizes specific nucleotide sequences and cleaves the linker strand. After nicking, the fragments of the linker strand spontaneously dissociate from the target DNA and another linker strand hybridizes to the target to trigger another strand-scission cycle. On the other hand, when the target was absent, no duplex is formed and no fragment of linker strand is produced. Then CdTe QDs and magnetic beads (MBs), which were all modified with DNA sequences complementary to that of the linker strands are added to the solution to detect the presence of a target DNA. The signal was generated through the difference in Foerster resonance energy transfer (FRET) between the MB and CdTe QDs. This method indicates that one target DNA leads to cleavage of hundreds of linker DNA, increasing detection sensitivity by nearly three orders of magnitude. This method should be applicable whenever there is a requirement to detect a specific DNA sequence and can also be used for multicomponent detection.

  17. Sensitive fluorescent detection of DNA methyltransferase using nicking endonuclease-mediated multiple primers-like rolling circle amplification.

    Science.gov (United States)

    Huang, Juan; Li, Xiao-Yu; Du, Yi-Chen; Zhang, Li-Na; Liu, Ke-Ke; Zhu, Li-Na; Kong, De-Ming

    2017-05-15

    Sensitive and reliable detection of DNA methyltransferase (MTase) is of great significance for both early tumor diagnosis and therapy. In this study, a simple, label-free and sensitive DNA MTase-sensing method was developed on the basis of a nicking endonuclease-mediated multiple primers-like rolling circle amplification (RCA) strategy. In this method, a dumbbell RCA template was prepared by blunt-end ligation of two molecules of hairpin DNA. In addition to the primer-binding sequence, the dumbbell template contained another three important parts: 5'-CCGG-3' sequences in double-stranded stems, nicking endonuclease recognition sites and C-rich sequences in single-stranded loops. The introduction of 5'-CCGG-3' sequences allows the dumbbell template to be destroyed by the restriction endonuclease, HpaII, but is not destroyed in the presence of the target MTase-M.SssI MTase. The introduction of nicking endonuclease recognition sites makes the M.SssI MTase-protected dumbbell template-mediated RCA proceed in a multiple primers-like exponential mode, thus providing the RCA with high amplification efficiency. The introduction of C-rich sequences may promote the folding of amplification products into a G-quadruplex structure, which is specifically recognized by the commercially available fluorescent probe thioflavin T. Improved RCA amplification efficiency and specific fluorescent recognition of RCA products provide the M.SssI MTase-sensing platform with high sensitivity. When a dumbbell template containing four nicking endonuclease sites is used, highly specific M.SssI MTase activity detection can be achieved in the range of 0.008-50U/mL with a detection limit as low as 0.0011U/mL. Simple experimental operation and mix-and-detection fluorescent sensing mode ensures that M.SssI MTase quantitation works well in a real-time RCA mode, thus further simplifying the sensing performance and making high throughput detection possible. The proposed MTase-sensing strategy was also

  18. Transcription factor TFIIH and DNA endonuclease Rad2 constitute yeast nucleotide excision repair factor 3: implications for nucleotide excision repair and Cockayne syndrome.

    Science.gov (United States)

    Habraken, Y; Sung, P; Prakash, S; Prakash, L

    1996-10-01

    Nucleotide excision repair (NER) of ultraviolet light-damaged DNA in eukaryotes requires a large number of highly conserved protein factors. Recent studies in yeast have suggested that NER involves the action of distinct protein subassemblies at the damage site rather than the placement there of a "preformed repairosome" containing all the essential NER factors. Neither of the two endonucleases, Rad1-Rad10 and Rad2, required for dual incision, shows any affinity for ultraviolet-damaged DNA. Rad1-Rad10 forms a ternary complex with the DNA damage recognition protein Rad14, providing a means for targeting this nuclease to the damage site. It has remained unclear how the Rad2 nuclease is targeted to the DNA damage site and why mutations in the human RAD2 counterpart, XPG, result in Cockayne syndrome. Here we examine whether Rad2 is part of a higher order subassembly. Interestingly, we find copurification of Rad2 protein with TFIIH, such that TFIIH purified from a strain that overexpresses Rad2 contains a stoichiometric amount of Rad2. By several independent criteria, we establish that Rad2 is tightly associated with TFIIH, exhibiting an apparent dissociation constant Cockayne syndrome.

  19. AP: A Critical Examination of the Advanced Placement Program

    Science.gov (United States)

    Sadler, Philip M.; Sonnert, Gerhard; Tai, Robert; Klopfenstein, Kirstin

    2016-01-01

    The Advanced Placement (AP) program was created to enhance the experience of gifted students as they transition from high school to college. "AP: A Critical Examination of the Advanced Placement Program," edited by Philip M. Sadler, Gerhard Sonnert, Robert Tai, and Kirstin Klopfenstein (2010, Harvard Education Press), questions the…

  20. APS extends open access to all its journals

    CERN Multimedia

    Thomas, Kim

    2006-01-01

    "Physics research promoter and publisher the American Physical Society (APS) is to extend open access to all its journals. Th APS previously made its five print journals available through subscriptions, and its two e-journals (Physical Review Special Topics and Physics Educatoin Research) on an open access basis." (1/2 page)

  1. 76 FR 82079 - AP1000 Design Certification Amendment

    Science.gov (United States)

    2011-12-30

    ... AP1000 DCD to correct spelling, ] punctuation, grammar, designations, and references. None of these... Commission. ACTION: Final rule. SUMMARY: The U.S. Nuclear Regulatory Commission (NRC or Commission) is... regulation (AP1000 design certification rule (DCR)), and need not demonstrate in their applications the...

  2. Building an SDN enterprise WLAN based on virtual APs

    NARCIS (Netherlands)

    Sequeira, L.; Cruz, J.L. de la; Ruiz-Mas, J.; Saldana, J.; Fernandez-Navajas, J.; Almodovar, J.

    2017-01-01

    In this letter, the development and testing of an open enterprise Wi-Fi solution based on virtual access points (APs), managed by a central WLAN controller is presented. It allows seamless handovers between APs in different channels, maintaining the QoS of real-time services. The potential

  3. Army and Marine Corps Active Protection System (APS) Efforts

    Science.gov (United States)

    2016-08-30

    as catalysts for intensifying U.S. APS efforts. Technical and operational challenges to APS include being able to work under extremely demanding...system employs the multi-layered defenses, comprising electro-optical jammers, Instantaneous smoke screens and, if necessary, an interceptor-based

  4. AP@home: The Artificial Pancreas Is Now at Home

    NARCIS (Netherlands)

    Heinemann, Lutz; Benesch, Carsten; DeVries, J. Hans

    2016-01-01

    In the past years the development of an artificial pancreas (AP) has made great progress and many activities are ongoing in this area of research. The major step forward made in the last years was moving the evaluation of AP systems from highly controlled experimental conditions to daily life

  5. High-resolution structure of the N-terminal endonuclease domain of the Lassa virus L polymerase in complex with magnesium ions.

    Directory of Open Access Journals (Sweden)

    Gregor D Wallat

    Full Text Available Lassa virus (LASV causes deadly hemorrhagic fever disease for which there are no vaccines and limited treatments. LASV-encoded L polymerase is required for viral RNA replication and transcription. The functional domains of L-a large protein of 2218 amino acid residues-are largely undefined, except for the centrally located RNA-dependent RNA polymerase (RdRP motif. Recent structural and functional analyses of the N-terminal region of the L protein from lymphocytic choriomeningitis virus (LCMV, which is in the same Arenaviridae family as LASV, have identified an endonuclease domain that presumably cleaves the cap structures of host mRNAs in order to initiate viral transcription. Here we present a high-resolution crystal structure of the N-terminal 173-aa region of the LASV L protein (LASV L173 in complex with magnesium ions at 1.72 Å. The structure is highly homologous to other known viral endonucleases of arena- (LCMV NL1, orthomyxo- (influenza virus PA, and bunyaviruses (La Crosse virus NL1. Although the catalytic residues (D89, E102 and K122 are highly conserved among the known viral endonucleases, LASV L endonuclease structure shows some notable differences. Our data collected from in vitro endonuclease assays and a reporter-based LASV minigenome transcriptional assay in mammalian cells confirm structural prediction of LASV L173 as an active endonuclease. The high-resolution structure of the LASV L endonuclease domain in complex with magnesium ions should aid the development of antivirals against lethal Lassa hemorrhagic fever.

  6. Properties of LuAP: CE scintillator containing intentional impurities

    CERN Document Server

    Petrosyan, A G; Ovanesyan, K; Lecoq, Paul; Auffray, Etiennette; Trummer, Julia; Kronberger, Matthias; Pédrini, C; Dujardin, C; Anfre, P

    2007-01-01

    Single crystals of LuAP:Ce and LuYAP(Lu*70%):Ce co-doped with tetravalent (Hf and Zr) and pentavalent (Ta) ions were grown from melts by the Bridgman process. Underlying absorption, slope of the optical edge and transmission in the range of emission were compared to those of LuAP:Ce crystals. Absorption coefficients at 260 nm less than 2 cm−1 have been recorded in LuAP:Ce crystals containing tetravalent ions that are lower than the corresponding figures (5–6 cm−1) measured in undoped LuAP. At high concentrations of added impurities, despite of suppression of the parasitic underlying absorption below 300 nm, the slope of the optical edge and transmission in the range of emission are seriously damaged. Scintillation parameters of crystals with added impurities are compared to those of LuAP:Ce.

  7. Detecção do DNA do papilomavírus humano após excisão da zona de transformação com alça diatérmica para tratamento de neoplasia intra-epitelial cervical Human papillomavirus DNA detection after large loop excision of the transformation zone for the treatment of cervical intraepithelial neoplasia

    Directory of Open Access Journals (Sweden)

    Priscila Garcia Figueirêdo

    2003-02-01

    Full Text Available OBJETIVO: avaliar a presença do DNA do papilomavírus humano (HPV de alto risco oncológico antes e quatro meses após excisão da zona de transformação com alça diatérmica em mulheres com neoplasia intra-epitelial cervical (NIC. MÉTODOS: neste estudo clínico prospectivo foram incluídas 78 mulheres submetidas à excisão da zona de transformação tratadas no período de fevereiro a dezembro de 2001. Todas foram submetidas a colposcopia, citologia oncológica e captura híbrida II (CH II antes da cirurgia e após 4±1,25 meses. Para análise estatística utilizou-se o cálculo do odds ratio (OR com intervalo de confiança de 95% (IC 95%. RESULTADOS: antes da excisão, 67 (86% mulheres apresentavam CH II positiva para DNA-HPV de alto risco oncológico e destas, apenas 22 (33% mantiveram a CH II positiva quatro meses após. A detecção do DNA-HPV após o tratamento não se relacionou com a carga viral prévia, presença de doença nas margens da peça cirúrgica ou idade da mulher. Após quatro meses, a detecção do DNA-HPV associou-se significativamente com a presença de alterações citológicas (OR = 4,8; IC 95% = 1,7-13,7, porém não se relacionou com doença residual ou recidiva histológica (OR = 6,0; IC 95% = 0,8-52,3. CONCLUSÃO: após o tratamento da NIC, a detecção do DNA-HPV diminuiu significativamente porém não se observou relação com a presença de doença residual ou recidiva histológica.PURPOSE: to evaluate the detection of high oncogenic risk human papillomavirus DNA (HPV-DNA immediately before and 4±1.25 months after large loop excision of the transformation zone (LLETZ in the treatment of cervical intraepithelial neoplasia (CIN. METHODS: in this clinical prospective study, 78 patients submitted to LLETZ from February to December 2001 were enrolled. All patients were submitted to colposcopic evaluation and had Pap smear and hybrid capture II (HC II specimens collected immediately before LLETZ and four months

  8. Assessment of Combined Ascorbyl Palmitate (AP) and Sodium Ascorbyl Phosphate (SAP) on Facial Skin Sebum Control in Female Healthy Volunteers.

    Science.gov (United States)

    Khan, H; Akhtar, N; Ali, A

    2017-01-01

    The skin is fortified with a setup of lipophilic and hydrophilic, enzymatic and non-enzymatic antioxidant systems. Ascorbyl palmitate (AP) and sodium ascorbyl phosphate (SAP) are reported as lipophilic and hydrophilic antioxidants, respectively used for skin care. Present study was aimed to assess the combined AP (in oil phase) and SAP (in aqueous phase) via multiple emulsion (ME1) for controlling sebum secretions in healthy human females. FTIR analysis of AP and SAP was performed for identification. Multiple emulsions (ME1 and control) were prepared and analyzed for physical stability. Antioxidant activities of AP, SAP as well as ME1 (with combination of these compounds) were determined by DPPH method. 11 female volunteers were included in a single-blinded, placebo-controlled, split-face comparative study. Volunteers were instructed to apply ME1 on left cheek while control (without AP and SAP) on right cheek, for a period of 90 days. A non-invasive photometric device (Sebumeter®) was used for the measurement of sebum secretions on both sides of the face with subsequent time intervals. A good antioxidant activity of ME1 was observed. ME1 treatments reduced significant facial sebum secretions as compared with control/placebo treatments. It was concluded that combined AP and SAP supplementations to skin proved a promising choice for controlling facial sebum secretions and could be evaluated for undesired oily skin and acne reductions for beautifying the facial appearance. © Georg Thieme Verlag KG Stuttgart · New York.

  9. Thermal Decomposition Characteristics of Orthorhombic Ammonium Perchlorate (o-AP) and an 0-AP/HTPB-Based Propellant

    Energy Technology Data Exchange (ETDEWEB)

    BEHRENS JR.,RICHARD; MINIER,LEANNA M.G.

    1999-10-25

    A study to characterize the low-temperature reactive processes for o-AP and an AP/HTPB-based propellant (class 1.3) is being conducted in the laboratory using the techniques of simultaneous thermogravimetric modulated beam mass spectrometry (STMBMS) and scanning electron microscopy (SEM). The results presented in this paper are a follow up of the previous work that showed the overall decomposition to be complex and controlled by both physical and chemical processes. The decomposition is characterized by the occurrence of one major event that consumes up to {approx}35% of the AP, depending upon particle size, and leaves behind a porous agglomerate of AP. The major gaseous products released during this event include H{sub 2}O, O{sub 2}, Cl{sub 2}, N{sub 2}O and HCl. The recent efforts provide further insight into the decomposition processes for o-AP. The temporal behaviors of the gas formation rates (GFRs) for the products indicate that the major decomposition event consists of three chemical channels. The first and third channels are affected by the pressure in the reaction cell and occur at the surface or in the gas phase above the surface of the AP particles. The second channel is not affected by pressure and accounts for the solid-phase reactions characteristic of o-AP. The third channel involves the interactions of the decomposition products with the surface of the AP. SEM images of partially decomposed o-AP provide insight to how the morphology changes as the decomposition progresses. A conceptual model has been developed, based upon the STMBMS and SEM results, that provides a basic description of the processes. The thermal decomposition characteristics of the propellant are evaluated from the identities of the products and the temporal behaviors of their GFRs. First, the volatile components in the propellant evolve from the propellant as it is heated. Second, the hot AP (and HClO{sub 4}) at the AP-binder interface oxidize the binder through reactions that

  10. Dominant mutations in S. cerevisiae PMS1 identify the Mlh1-Pms1 endonuclease active site and an exonuclease 1-independent mismatch repair pathway.

    Directory of Open Access Journals (Sweden)

    Catherine E Smith

    2013-10-01

    Full Text Available Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.

  11. Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage.

    Science.gov (United States)

    Stella, Stefano; Alcón, Pablo; Montoya, Guillermo

    2017-06-22

    Cpf1 is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we provide insight into its DNA-targeting mechanism by determining the structure of Francisella novicida Cpf1 with the triple-stranded R-loop generated after DNA cleavage. The structure reveals the machinery involved in DNA unwinding to form a CRISPR RNA (crRNA)-DNA hybrid and a displaced DNA strand. The protospacer adjacent motif (PAM) is recognized by the PAM-interacting domain. The loop-lysine helix-loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner into the double-stranded DNA. Unzipping of the double-stranded DNA occurs in a cleft arranged by acidic and hydrophobic residues facilitating the crRNA-DNA hybrid formation. The PAM single-stranded DNA is funnelled towards the nuclease site through a mixed hydrophobic and basic cavity. In this catalytic conformation, the PAM-interacting domain and the helix-loop-helix motif in the REC1 domain adopt a 'rail' shape and 'flap-on' conformations, respectively, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms the 'septum', separating the displaced PAM strand and the crRNA-DNA hybrid, avoiding DNA re-annealing. Mutations in key residues reveal a mechanism linking the PAM and DNA nuclease sites. Analysis of the Cpf1 structures proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1.

  12. Total sequence decomposition distinguishes functional modules, "molegos" in apurinic/apyrimidinic endonucleases

    Directory of Open Access Journals (Sweden)

    Braun Werner

    2002-11-01

    Full Text Available Abstract Background Total sequence decomposition, using the web-based MASIA tool, identifies areas of conservation in aligned protein sequences. By structurally annotating these motifs, the sequence can be parsed into individual building blocks, molecular legos ("molegos", that can eventually be related to function. Here, the approach is applied to the apurinic/apyrimidinic endonuclease (APE DNA repair proteins, essential enzymes that have been highly conserved throughout evolution. The APEs, DNase-1 and inositol 5'-polyphosphate phosphatases (IPP form a superfamily that catalyze metal ion based phosphorolysis, but recognize different substrates. Results MASIA decomposition of APE yielded 12 sequence motifs, 10 of which are also structurally conserved within the family and are designated as molegos. The 12 motifs include all the residues known to be essential for DNA cleavage by APE. Five of these molegos are sequentially and structurally conserved in DNase-1 and the IPP family. Correcting the sequence alignment to match the residues at the ends of two of the molegos that are absolutely conserved in each of the three families greatly improved the local structural alignment of APEs, DNase-1 and synaptojanin. Comparing substrate/product binding of molegos common to DNase-1 showed that those distinctive for APEs are not directly involved in cleavage, but establish protein-DNA interactions 3' to the abasic site. These additional bonds enhance both specific binding to damaged DNA and the processivity of APE1. Conclusion A modular approach can improve structurally predictive alignments of homologous proteins with low sequence identity and reveal residues peripheral to the traditional "active site" that control the specificity of enzymatic activity.

  13. Induction of Apurinic Endonuclease 1 Overexpression by Endoplasmic Reticulum Stress in Hepatoma Cells

    Directory of Open Access Journals (Sweden)

    Tsung-Lin Cheng

    2014-07-01

    Full Text Available Hepatocellular carcinoma (HCC is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. Previous studies have noted the induction of endoplasmic reticulum stress or apurinic endonuclease 1 (APE1 expression in many tumors. Therefore, the aim of this study was to investigate the relationship between endoplasmic reticulum (ER stress and APE1 in hepatocellular carcinoma. Here we investigate the expression of APE1 during ER stress in HepG2 and Huh-7 cell lines. Tunicamycin or brefeldin A, two ER stress inducers, increased APE1 and GRP78, an ER stress marker, expression in HepG2 and Huh-7 cells. Induction of APE1 expression was observed through transcription level in response to ER stress. APE1 nuclear localization during ER stress was determined using immunofluorescence assays in HepG2 cells. Furthermore, expression of Hepatitis B virus pre-S2∆ large mutant surface protein (pre-S2∆, an ER stress-induced protein, also increased GRP78 and APE1 expression in the normal hepatocyte NeHepLxHT cell line. Similarly, tumor samples showed higher expression of APE1 in ER stress-correlated liver cancer tissue in vivo. Our results demonstrate that ER stress and HBV pre-S2∆ increased APE1 expression, which may play an important role in resistance to chemotherapeutic agents or tumor development. Therefore, these data provide an important chemotherapeutic strategy in ER stress and HBV pre-S2∆-associated tumors.

  14. Análises morfométrica e morfológica das alterações cutâneas após uso do laser Nd-YAG em tecidos palpebrais humanos Cutaneous changes morphometrical and histological analysis after the use of laser Nd-YAG in human eyelid tissue

    Directory of Open Access Journals (Sweden)

    Lúcia de Noronha

    2004-06-01

    Full Text Available O uso do laser neodymium-yttrium-aluminium-garnet (Nd-YAG como instrumento auxiliar para o rejuvenescimento propiciou um refinamento da técnica tradicional. Tal procedimento mostra resultados satisfatórios com relação ao aprimoramento técnico e à recuperação pós-operatória. OBJETIVO: Evidenciar as alterações histológicas e morfométricas encontradas na pálpebra de humanos após aplicação do laser Nd-YAG e comparar com a pele normal. MATERIAL E MÉTODO: Nove pacientes do sexo feminino foram submetidas à aplicação do laser Nd-YAG em região palpebral direita. A pálpebra esquerda correspondeu à área controle, livre de qualquer tipo de procedimento. Foram realizadas várias aplicações do laser Nd-YAG, sendo que, no último dia, foi realizada biópsia palpebral bilateralmente. Os critérios morfométricos avaliados foram: medida da espessura da epiderme, da área subepidérmica livre de lesão, da zona de degeneração do colágeno e da espessura total da derme. RESULTADOS: Nas áreas submetidas à aplicação do laser Nd-YAG, a espessura média da epiderme foi de 33,53 micrômetros (µm e a da pele normal foi de 29,61µm (p = 0,1099. A média da medida da área subepidérmica livre de lesão, no lado direito, foi de 40,93µm, e de 36,27µm (p = 0,1373 no esquerdo. A espessura da região de degeneração do colágeno na pálpebra que recebeu tratamento com laser teve média de 293,54µm, enquanto que na pálpebra normal ela foi de 292,22µm (p = 0,4835. No lado onde se aplicou o laser Nd-YAG, a espessura total da derme teve uma média de 681,48µm e, no lado livre de procedimento, 664,14µm (p = 0,3492. DISCUSSÃO E CONCLUSÕES: Apesar da diferença clínica observada após tratamento de lesões de envelhecimento em pálpebra de humanos com o uso do laser Nd-YAG, nota-se que não houve diferença histológica ou morfométrica após tal procedimento.The use of laser Neodymium-Yttrium-Aluminium-Garnet (Nd-YAG as an auxiliary

  15. MULTI-OBJECTIVE ONLINE OPTIMIZATION OF BEAM LIFETIME AT APS

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Yipeng

    2017-06-25

    In this paper, online optimization of beam lifetime at the APS (Advanced Photon Source) storage ring is presented. A general genetic algorithm (GA) is developed and employed for some online optimizations in the APS storage ring. Sextupole magnets in 40 sectors of the APS storage ring are employed as variables for the online nonlinear beam dynamics optimization. The algorithm employs several optimization objectives and is designed to run with topup mode or beam current decay mode. Up to 50\\% improvement of beam lifetime is demonstrated, without affecting the transverse beam sizes and other relevant parameters. In some cases, the top-up injection efficiency is also improved.

  16. AP2 adaptor complex mediates bile salt export pump internalization and modulates its hepatocanalicular expression and transport function.

    Science.gov (United States)

    Hayashi, Hisamitsu; Inamura, Kaori; Aida, Kensuke; Naoi, Sotaro; Horikawa, Reiko; Nagasaka, Hironori; Takatani, Tomozumi; Fukushima, Tamio; Hattori, Asami; Yabuki, Takashi; Horii, Ikuo; Sugiyama, Yuichi

    2012-06-01

    The bile salt export pump (BSEP) mediates the biliary excretion of bile salts and its dysfunction induces intrahepatic cholestasis. Reduced canalicular expression of BSEP resulting from the promotion of its internalization is one of the causes of this disease state. However, the molecular mechanism underlying BSEP internalization from the canalicular membrane (CM) remains unknown. We have shown previously that 4-phenylbutyrate (4PBA), a drug used for ornithine transcarbamylase deficiency (OTCD), inhibited internalization and subsequent degradation of cell-surface-resident BSEP. The current study found that 4PBA treatment decreased significantly the expression of α- and μ2-adaptin, both of which are subunits of the AP2 adaptor complex (AP2) that mediates clathrin-dependent endocytosis, in liver specimens from rats and patients with OTCD, and that BSEP has potential AP2 recognition motifs in its cytosolic region. Based on this, the role of AP2 in BSEP internalization was explored further. In vitro analysis with 3×FLAG-human BSEP-expressing HeLa cells and human sandwich-culture hepatocytes indicates that the impairment of AP2 function by RNA interference targeting of α-adaptin inhibits BSEP internalization from the plasma membrane and increases its cell-surface expression and transport function. Studies using immunostaining, coimmunoprecipitation, glutathione S-transferase pulldown assay, and time-lapse imaging show that AP2 interacts with BSEP at the CM through a tyrosine motif at the carboxyl terminus of BSEP and mediates BSEP internalization from the CM of hepatocytes. AP2 mediates the internalization and subsequent degradation of CM-resident BSEP through direct interaction with BSEP and thereby modulates the canalicular expression and transport function of BSEP. This information should be useful for understanding the pathogenesis of severe liver diseases associated with intrahepatic cholestasis. Copyright © 2012 American Association for the Study of Liver

  17. Small molecule activation of apurinic/apyrimidinic endonuclease 1 reduces DNA damage induced by cisplatin in cultured sensory neurons.

    Science.gov (United States)

    Georgiadis, Millie M; Chen, Qiujia; Meng, Jingwei; Guo, Chunlu; Wireman, Randall; Reed, April; Vasko, Michael R; Kelley, Mark R

    2016-05-01

    Although chemotherapy-induced peripheral neuropathy (CIPN) affects approximately 5-60% of cancer patients, there are currently no treatments available in part due to the fact that the underlying causes of CIPN are not well understood. One contributing factor in CIPN may be persistence of DNA lesions resulting from treatment with platinum-based agents such as cisplatin. In support of this hypothesis, overexpression of the base excision repair (BER) enzyme, apurinic/apyrimidinic endonuclease 1 (APE1), reduces DNA damage and protects cultured sensory neurons treated with cisplatin. Here, we address stimulation of APE1's endonuclease through a small molecule, nicorandil, as a means of mimicking the beneficial effects observed for overexpression of APE1. Nicorandil, was identified through high-throughput screening of small molecule libraries and found to stimulate APE1 endonuclease activity by increasing catalytic efficiency approximately 2-fold. This stimulation is primarily due to an increase in kcat. To prevent metabolism of nicorandil, an approved drug in Europe for the treatment of angina, cultured sensory neurons were pretreated with nicorandil and daidzin, an aldehyde dehydrogenase 2 inhibitor, resulting in decreased DNA damage but not altered transmitter release by cisplatin. This finding suggests that activation of APE1 by nicorandil in cisplatin-treated cultured sensory neurons does not imbalance the BER pathway in contrast to overexpression of the kinetically faster R177A APE1. Taken together, our results suggest that APE1 activators can be used to reduce DNA damage induced by cisplatin in cultured sensory neurons, although further studies will be required to fully assess their protective effects. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Exploring both sequence detection and restriction endonuclease cleavage kinetics by recognition site via single-molecule microfluidic trapping.

    Science.gov (United States)

    Xu, Weilin; Muller, Susan J

    2011-02-07

    We demonstrate the feasibility of a single-molecule microfluidic approach to both sequence detection and obtaining kinetic information for restriction endonucleases on dsDNA. In this method, a microfluidic stagnation point flow is designed to trap, hold, and linearize double-stranded (ds) genomic DNA to which a restriction endonuclease has been pre-bound sequence-specifically. By introducing the cofactor magnesium, we determine the binding location of the enzyme by the cleavage process of dsDNA as in optical restriction mapping, however here the DNA need not be immobilized on a surface. We note that no special labeling of the enzyme is required, which makes it simpler than our previous scheme using stagnation point flows for sequence detection. Our accuracy in determining the location of the recognition site is comparable to or better than other single molecule techniques due to the fidelity with which we can control the linearization of the DNA molecules. In addition, since the cleavage process can be followed in real time, information about the cleavage kinetics, and subtle differences in binding and cleavage frequencies among the recognition sites, may also be obtained. Data for the five recognition sites for the type II restriction endonuclease EcoRI on λ-DNA are presented as a model system. While the roles of the varying fluid velocity and tension along the chain backbone on the measured kinetics remain to be determined, we believe this new method holds promise for a broad range of studies of DNA-protein interactions, including the kinetics of other DNA cleavage processes, the dissociation of a restriction enzyme from the cleaved substrate, and other macromolecular cleavage processes.

  19. Development of a therapeutic monoclonal antibody that targets secreted fatty acid-binding protein aP2 to treat type 2 diabetes.

    Science.gov (United States)

    Burak, M Furkan; Inouye, Karen E; White, Ariel; Lee, Alexandra; Tuncman, Gurol; Calay, Ediz S; Sekiya, Motohiro; Tirosh, Amir; Eguchi, Kosei; Birrane, Gabriel; Lightwood, Daniel; Howells, Louise; Odede, Geofrey; Hailu, Hanna; West, Shauna; Garlish, Rachel; Neale, Helen; Doyle, Carl; Moore, Adrian; Hotamisligil, Gökhan S

    2015-12-23

    The lipid chaperone aP2/FABP4 has been implicated in the pathology of many immunometabolic diseases, including diabetes in humans, but aP2 has not yet been targeted for therapeutic applications. aP2 is not only an intracellular protein but also an active adipokine that contributes to hyperglycemia by promoting hepatic gluconeogenesis and interfering with peripheral insulin action. Serum aP2 levels are markedly elevated in mouse and human obesity and strongly correlate with metabolic complications. These observations raise the possibility of a new strategy to treat metabolic disease by targeting serum aP2 with a monoclonal antibody (mAb) to aP2. We evaluated mAbs to aP2 and identified one, CA33, that lowered fasting blood glucose, improved systemic glucose metabolism, increased systemic insulin sensitivity, and reduced fat mass and liver steatosis in obese mouse models. We examined the structure of the aP2-CA33 complex and resolved the target epitope by crystallographic studies in comparison to another mAb that lacked efficacy in vivo. In hyperinsulinemic-euglycemic clamp studies, we found that the antidiabetic effect of CA33 was predominantly linked to the regulation of hepatic glucose output and peripheral glucose utilization. The antibody had no effect in aP2-deficient mice, demonstrating its target specificity. We conclude that an aP2 mAb-mediated therapeutic constitutes a feasible approach for the treatment of diabetes. Copyright © 2015, American Association for the Advancement of Science.

  20. Using Group II Introns for Attenuating the In Vitro and In Vivo Expression of a Homing Endonuclease

    OpenAIRE

    Tuhin Kumar Guha; Georg Hausner

    2016-01-01

    In Chaetomium thermophilum (DSM 1495) within the mitochondrial DNA (mtDNA) small ribosomal subunit (rns) gene a group IIA1 intron interrupts an open reading frame (ORF) encoded within a group I intron (mS1247). This arrangement offers the opportunity to examine if the nested group II intron could be utilized as a regulatory element for the expression of the homing endonuclease (HEase). Constructs were generated where the codon-optimized ORF was interrupted with either the native group IIA1 in...

  1. Peculiarities of the interaction of the restriction endonuclease BspD6I with DNA containing its recognition site.

    Science.gov (United States)

    Abrosimova, Liudmila A; Kubareva, Elena A; Migur, Anzhela Yu; Gavshina, Aleksandra V; Ryazanova, Aleksandra Yu; Norkin, Maxim V; Perevyazova, Tatiana A; Wende, Wolfgang; Hianik, Tibor; Zheleznaya, Liudmila A; Oretskaya, Tatiana S

    2016-09-01

    Nicking endonucleases are enzymes that recognize specific sites in double-stranded DNA and cleave only one strand at a predetermined position. These enzymes are involved in DNA replication and repair; they can also function as subunits of bacterial heterodimeric restriction endonucleases. One example of such a proteins is the restriction endonuclease BspD6I (R.BspD6I) from Bacillus species strain D6, which consists of the large subunit - nicking endonuclease BspD6I (Nt.BspD6I), and the small subunit (ss.BspD6I). Nt.BspD6I can function independently. Similar enzymes are now widely used in numerous biotechnological applications. The aim of this study was to investigate the fundamental properties of two subunits of R.BspD6I and their interdependence in the course of R.BspD6I activity. The binding and hydrolysis of DNA duplexes by R.BspD6I are primary analyzed by gel electrophoresis. To elucidate the difference between Nt.BspD6I interaction with the substrate and product of hydrolysis, the thickness shear mode acoustic method is used. The thermodynamic and kinetic parameters of the Nt.BspD6I interaction with DNA are determined. For the first time we demonstrated that Nt.BspD6I bends the DNA during complex formation. Nt.BspD6I is able to form complexes with the product nicked in the top strand and ss.BspD6I cleaves the bottom strand of the DNA consecutively. Furthermore, the influence of dA methylation in the R.BspD6I recognition site on ss.BspD6I activity is analyzed. The obtained results provide evidence that Nt.BspD6I coordinates the activity of R.BspD6I by strictly coupling of the bottom strand cleavage by ss.BspD6I to the top strand cleavage. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Genomic DNA restriction endonuclease from Pasteurella multocida isolated from Indonesia, katha strain and reference strains and analysed by PFGE

    Directory of Open Access Journals (Sweden)

    Supar

    2003-10-01

    Full Text Available Pasteurella multocida strains are the causative disease agents of wide range of domestic and wild animals in Indonesia. The most important serotypes are associated with Hemorrhagic septicaemic (HS diseases in cattle and buffaloes, cholera in ducks and chickens. The HS disease associated with P. multocia in large ruminants in Indonesia is controled by killed whole cell vaccines produced by the use of P. multocida Katha strains. There is no discriminatory data of the molecular biology technique has been applied to investigate P. multocida isolates from different geographic locations in Indonesia. The purpose of this studies were to observe the genetic diversity among P. multocida isolated from various geograpic locations and compared with Katha vaccine strain and other reference strains. A total samples of 38 isolates and strains of P. multocida were analysed by means of pulsed-field gel electrophoresis (PFGE. Each sample was grown in nutrient broth, cells were separeted by centrifugation. Whole cell pellet was mixed with agarose and then prepared agarose plugs. The genomic DNA of each sample was digested in situ (plug with either restriction endonuclease of ApaI and/or BamHI. The digested genomic DNA of each sample was analysed by PFGE, the genomic DNA restricted profile of each sample was compared with others. The use of ApaI restriction endonuclease digestion and analysed by PFGE, demonstrated that 34 out of 38 P. multocia samples could be differentiated into 16 ApaI types, whereas based on the BamHI digestion of these samples were differentiated into 20 BamHI types. Genomic DNA restriction pattern of Indonesian P. multocida isolates originated from cattle and buffaloes associated with haemorrhagic septicaemic diseases demonstrated different pattern to those of vaccine Katha strain, poultry strains as well as the reference strains currenly kept at Balitvet Culture Collection (BCC unit. Two P. multocida isolates derived from ducks with cholera

  3. HIGLE is a bifunctional homing endonuclease that directly interacts with HYL1 and SERRATE in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Cho, Seok Keun; Ryu, Moon Young; Poulsen, Christian

    2017-01-01

    A highly coordinated complex known as the microprocessor precisely processes primary transcripts of MIRNA genes into mature miRNAs. In plants, the microprocessor minimally consists of three components: Dicer-like protein 1 (DCL1), HYPONASTIC LEAF 1 (HYL1), and SERRATE (SE). To precisely modulate mi......RNA maturation, the microprocessor cooperates with at least 12 proteins in plants. In addition, we here show the involvement of a novel gene, HYL1-interacting GIY-YIG-like endonuclease (HIGLE). The encoded protein has a GIY-YIG domain that is generally found within a class of homing endonucleases. HIGLE directly...... interacts with the microprocessor components HYL1 and SE. Unlike the functions of other GIY-YIG endonucleases, the catalytic core of HIGLE has both DNase and RNase activities that sufficiently processes miRNA precursors into short fragments in vitro....

  4. Isolation of β-globin related genes from a human cosmid library.

    NARCIS (Netherlands)

    F.G. Grosveld (Frank); H.H.M. Dahl; R.A. Flavell (Richard); E. de Boer (Ernie)

    1981-01-01

    textabstractA human gene library was constructed using an improved cloning technique for cosmid vectors. Human placental DNA was partially digested with restriction endonuclease MboI; size-fractionated and ligated to BamHI-cut and phosphatase-treated cosmid vector pJB8. After packaging in lambda

  5. Engaging Cuban Physicists Through the APS/CPS Partnership

    Science.gov (United States)

    Lerch, Irving A.; Lerch, Irving A.

    In his reflections on Cuban physics, Marcelo Alonso urges APS to take steps to promote interactions between Cuban and US physicists. As an introduction to Marcello's essay, this note will summarize past and current activities.

  6. Asteroseismic Theory of Rapidly Oscillating Ap Stars Margarida S ...

    Indian Academy of Sciences (India)

    roAp) stars depends strongly on our ability to understand their oscillation spectra. Questions like: which modes are excited and why, what is the expected spacing between eigenfrequencies, how many components are expected to be found in ...

  7. Lumbar pedicle screw placement: Using only AP plane imaging

    Directory of Open Access Journals (Sweden)

    Anil Sethi

    2012-01-01

    Conclusion: Placement of pedicle screws under fluoroscopic guidance using AP plane imaging alone with tactile guidance is safe, fast, and reliable. However, a good understanding of the radiographic landmarks is a prerequisite.

  8. Autoscreening of restriction endonucleases for PCR-restriction fragment length polymorphism identification of fungal species, with Pleurotus spp. as an example.

    Science.gov (United States)

    Yang, Zhi-Hui; Huang, Ji-Xiang; Yao, Yi-Jian

    2007-12-01

    A molecular method based on PCR-restriction fragment length polymorphism (RFLP) analysis of internal transcribed spacer (ITS) ribosomal DNA sequences was designed to rapidly identify fungal species, with members of the genus Pleurotus as an example. Based on the results of phylogenetic analysis of ITS sequences from Pleurotus, a PCR-RFLP endonuclease autoscreening (PRE Auto) program was developed to screen restriction endonucleases for discriminating multiple sequences from different species. The PRE Auto program analyzes the endonuclease recognition sites and calculates the sizes of the fragments in the sequences that are imported into the program in groups according to species recognition. Every restriction endonuclease is scored through the calculation of the average coefficient for the sequence groups and the average coefficient for the sequences within a group, and then virtual electrophoresis maps for the selected restriction enzymes, based on the results of the scoring system, are displayed for the rapid determination of the candidate endonucleases. A total of 85 haplotypes representing 151 ITS sequences were used for the analysis, and 2,992 restriction endonucleases were screened to find the candidates for the identification of species. This method was verified by an experiment with 28 samples representing 12 species of Pleurotus. The results of the digestion by the restriction enzymes showed the same patterns of DNA fragments anticipated by the PRE Auto program, apart from those for four misidentified samples. ITS sequences from 14 samples (of which nine sequences were obtained in this study), including four originally misidentified samples, confirmed the species identities revealed by the PCR-RFLP analysis. The method developed here can be used for the identification of species of other living microorganisms.

  9. Roles of AP-2 in clathrin-mediated endocytosis.

    Directory of Open Access Journals (Sweden)

    Emmanuel Boucrot

    Full Text Available BACKGROUND: The notion that AP-2 clathrin adaptor is an essential component of an endocytic clathrin coat appears to conflict with recent observations that substantial AP-2 depletion, using RNA interference with synthesis of AP-2 subunits, fails to block uptake of certain ligands known to internalize through a clathrin-based pathway. METHODOLOGY/PRINCIPAL FINDINGS: We report here the use of in vivo imaging data obtained by spinning-disk confocal microscopy to study the formation of clathrin-coated structures at the plasma membranes of BSC1 and HeLa cells depleted by RNAi of the clathrin adaptor, AP-2. Very few clathrin coats continue to assemble after AP-2 knockdown. Moreover, there is a total absence of clathrin-containing structures completely lacking AP-2 while all the remaining coats still contain a small amount of AP-2. These observations suggest that AP-2 is essential for endocytic coated-pit and coated-vesicle formation. We also find that AP-2 knockdown strongly inhibits light-density lipoprotein (LDL receptor-mediated endocytosis, as long as cells are maintained in complete serum and at 37 degrees C. If cells are first incubated with LDL at 4 degrees C, followed by warming, there is little or no decrease in LDL uptake with respect to control cells. LDL uptake at 37 degrees C is also not affected in AP-2 depleted cells first deprived of LDL by incubation with either serum-starved or LDL-starved cells for 24 hr. The LDL-deprived cells display a significant increase in endocytic structures enriched on deeply invaginated tubes that contain LDL and we suggest that under this condition of stress, LDL might enter through this alternative pathway. CONCLUSIONS/SIGNIFICANCE: These results suggest that AP-2 is essential for endocytic clathrin coated-pit and coated-vesicle formation. They also indicate that under normal conditions, functional endocytic clathrin coated pits are required for LDL internalization. We also show that under certain

  10. Transcription factor complex AP-1 mediates inflammation initiated by Chlamydia pneumoniae infection.

    Science.gov (United States)

    Wang, Anyou; Al-Kuhlani, Mufadhal; Johnston, S Claiborne; Ojcius, David M; Chou, Joyce; Dean, Deborah

    2013-05-01

    Chlamydia pneumoniae is responsible for a high prevalence of respiratory infections worldwide and has been implicated in atherosclerosis. Inflammation is regulated by transcription factor (TF) networks. Yet, the core TF network triggered by chlamydiae remains largely unknown. Primary human coronary artery endothelial cells were mock-infected or infected with C. pneumoniae to generate human transcriptome data throughout the chlamydial developmental cycle. Using systems network analysis, the predominant TF network involved receptor, binding and adhesion and immune response complexes. Cells transfected with interfering RNA against activator protein-1 (AP-1) members FOS, FOSB, JUN and JUNB had significantly decreased expression and protein levels of inflammatory mediators interleukin (IL)6, IL8, CD38 and tumour necrosis factor compared with controls. These mediators have been shown to be associated with C. pneumoniae disease. Expression of AP-1 components was regulated by MAPK3K8, a MAPK pathway component. Additionally, knock-down of JUN and FOS showed significantly decreased expression of Toll-like receptor (TLR)3 during infection, implicating JUN and FOS in TLR3 regulation. TLR3 stimulation led to elevated IL8. These findings suggest that C. pneumoniae initiates signalling via TLR3 and MAPK that activate AP-1, a known immune activator in other bacteria not previously shown for chlamydiae, triggering inflammation linked to C. pneumoniae disease. © 2012 Blackwell Publishing Ltd.

  11. The Dushak–Erekdag Survey of roAp Stars

    Indian Academy of Sciences (India)

    The search of roAp stars at Mt. Dushak–Erekdag Observatory was started in 1992 using the 0.8m Odessa telescope equipped with a two-star high-speed photometer. We have observed more than a dozen stars so far and discovered HD 99563 as roAp star while BD+8087 is suspected to have rapid oscillations. Negative ...

  12. The (Very) Slow Rotation of Magnetic Ap Stars

    Science.gov (United States)

    Mathys, Gautier

    2015-08-01

    To this date, 34 magnetic Ap stars that have periods of variation longer than 30 days are known. They represent a considerable fraction of the total number of Ap stars whose period has been reliably determined. All the available evidence unambiguously indicates that the observed variations of those long-period Ap stars result from the changing aspect of their visible hemisphere as they rotate, thus that the oblique rotator model is applicable throughout the whole range of periods of variation of the Ap stars. We show that the periods of the most slowly rotating Ap stars must be of the order of 300 years, and that some may even be longer, possibly up to 1000 years. The 5 to 6 orders of magnitude spanned by the rotation periods of the Ap stars present a major challenge for the understanding of their origin and their evolution. To guide the theo- retical developments, observational hints may be found in possible differences between the magnetic properties of stars that have rotation periods in different ranges. Such differences are starting to emerge from the existing data. To increase their significance level, study of the longest-period stars must be continued over their full rotation cycle. Failure to secure observations now may leave critical data missing for several decades, or even centuries.

  13. Naturally-occurring, dually-functional fusions between restriction endonucleases and regulatory proteins.

    Science.gov (United States)

    Liang, Jixiao; Blumenthal, Robert M

    2013-10-02

    Restriction-modification (RM) systems appear to play key roles in modulating gene flow among bacteria and archaea. Because the restriction endonuclease (REase) is potentially lethal to unmethylated new host cells, regulation to ensure pre-expression of the protective DNA methyltransferase (MTase) is essential to the spread of RM genes. This is particularly true for Type IIP RM systems, in which the REase and MTase are separate, independently-active proteins. A substantial subset of Type IIP RM systems are controlled by an activator-repressor called C protein. In these systems, C controls the promoter for its own gene, and for the downstream REase gene that lacks its own promoter. Thus MTase is expressed immediately after the RM genes enter a new cell, while expression of REase is delayed until sufficient C protein accumulates. To study the variation in and evolution of this regulatory mechanism, we searched for RM systems closely related to the well-studied C protein-dependent PvuII RM system. Unexpectedly, among those found were several in which the C protein and REase genes were fused. The gene for CR.NsoJS138I fusion protein (nsoJS138ICR, from the bacterium Niabella soli) was cloned, and the fusion protein produced and partially purified. Western blots provided no evidence that, under the conditions tested, anything other than full-length fusion protein is produced. This protein had REase activity in vitro and, as expected from the sequence similarity, its specificity was indistinguishable from that for PvuII REase, though the optimal reaction conditions were different. Furthermore, the fusion was active as a C protein, as revealed by in vivo activation of a lacZ reporter fusion to the promoter region for the nsoJS138ICR gene. Fusions between C proteins and REases have not previously been characterized, though other fusions have (such as between REases and MTases). These results reinforce the evidence for impressive modularity among RM system proteins, and raise

  14. Use of an rRNA probe and restriction endonuclease analysis to fingerprint Pasteurella multocida isolated from turkeys and wildlife.

    Science.gov (United States)

    Snipes, K P; Hirsh, D C; Kasten, R W; Hansen, L M; Hird, D W; Carpenter, T E; McCapes, R H

    1989-01-01

    Twenty-five isolates of the bacterium Pasteurella multocida were characterized (fingerprinted) phenotypically and genotypically in order to compare the abilities of various techniques to differentiate strains for epidemiologic studies of fowl cholera. Isolates were obtained over a 16-month period from turkeys dying from fowl cholera (six outbreak flocks) and from wildlife captured on premises with a history of the disease. The characteristics compared included (i) serotype, (ii) subspecies, (iii) antibiogram, (iv) presence of plasmid DNA, (v) restriction endonuclease analysis patterns of whole-cell DNA, and (vi) ribotype. Ribotyping, a method of highlighting DNA restriction site heterogeneity by using an rRNA probe, worked well for differentiating the strains of P. multocida when the majority of the other techniques could not. Ribotyping results correlated directly with and confirmed results obtained from restriction endonuclease analysis. Ribotyping demonstrated the presence of up to three strains of P. multocida in one outbreak flock, recurrence of a single strain in five of the flocks over an 11-month period, and the presence of common strains in turkeys and wildlife on the premises. Images PMID:2768471

  15. Resolution of the EcoRII restriction endonuclease-DNA complex structure in solution using fluorescence spectroscopy.

    Science.gov (United States)

    Subach, Fedor; Kirsanova, Olga; Liquier, Jean; Gromova, Elizaveta S

    2008-12-01

    The X-ray structure for the type IIE EcoRII restriction endonuclease has been resolved [X.E. Zhou, Y. Wang, M. Reuter, M. Mucke, D.H. Kruger, E.J. Meehan and L. Chen. Crystal structure of type IIE restriction endonuclease EcoRII reveals an autoinhibition mechanism by a novel effector-binding fold. J. Mol. Biol. 335 (2004) 307-319.], but the structure of the R.EcoRII-DNA complex is still unknown. The aim of this article was to examine the structure of the pre-reactive R.EcoRII-DNA complex in solution by fluorescence spectroscopy. The structure for the R.EcoRII-DNA complex was resolved by determining the fluorescence resonance energy transfer (FRET) between two fluorescent dyes, covalently attached near the EcoRII recognition sites, that were located at opposite ends of a lengthy two-site DNA molecule. Analysis of the FRET data from the two-site DNA revealed a likely model for the arrangement of the two EcoRII recognition sites relative to each other in the R.EcoRII-DNA complex in the presence of Ca(2+) ions. According to this model, the R.EcoRII binds the two-site DNA and forms a DNA loop in which the EcoRII recognition sites are 20+/-10 A distant to each other and situated at an angle of 70+/-10 degrees.

  16. Functional significance of protein assemblies predicted by the crystal structure of the restriction endonuclease BsaWI.

    Science.gov (United States)

    Tamulaitis, Gintautas; Rutkauskas, Marius; Zaremba, Mindaugas; Grazulis, Saulius; Tamulaitiene, Giedre; Siksnys, Virginijus

    2015-09-18

    Type II restriction endonuclease BsaWI recognizes a degenerated sequence 5'-W/CCGGW-3' (W stands for A or T, '/' denotes the cleavage site). It belongs to a large family of restriction enzymes that contain a conserved CCGG tetranucleotide in their target sites. These enzymes are arranged as dimers or tetramers, and require binding of one, two or three DNA targets for their optimal catalytic activity. Here, we present a crystal structure and biochemical characterization of the restriction endonuclease BsaWI. BsaWI is arranged as an 'open' configuration dimer and binds a single DNA copy through a minor groove contacts. In the crystal primary BsaWI dimers form an indefinite linear chain via the C-terminal domain contacts implying possible higher order aggregates. We show that in solution BsaWI protein exists in a dimer-tetramer-oligomer equilibrium, but in the presence of specific DNA forms a tetramer bound to two target sites. Site-directed mutagenesis and kinetic experiments show that BsaWI is active as a tetramer and requires two target sites for optimal activity. We propose BsaWI mechanism that shares common features both with dimeric Ecl18kI/SgrAI and bona fide tetrameric NgoMIV/SfiI enzymes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Cleavage of phosphorothioated DNA and methylated DNA by the type IV restriction endonuclease ScoMcrA.

    Directory of Open Access Journals (Sweden)

    Guang Liu

    2010-12-01

    Full Text Available Many taxonomically diverse prokaryotes enzymatically modify their DNA by replacing a non-bridging oxygen with a sulfur atom at specific sequences. The biological implications of this DNA S-modification (phosphorothioation were unknown. We observed that simultaneous expression of the dndA-E gene cluster from Streptomyces lividans 66, which is responsible for the DNA S-modification, and the putative Streptomyces coelicolor A(32 Type IV methyl-dependent restriction endonuclease ScoA3McrA (Sco4631 leads to cell death in the same host. A His-tagged derivative of ScoA3McrA cleaved S-modified DNA and also Dcm-methylated DNA in vitro near the respective modification sites. Double-strand cleavage occurred 16-28 nucleotides away from the phosphorothioate links. DNase I footprinting demonstrated binding of ScoA3McrA to the Dcm methylation site, but no clear binding could be detected at the S-modified site under cleavage conditions. This is the first report of in vitro endonuclease activity of a McrA homologue and also the first demonstration of an enzyme that specifically cleaves S-modified DNA.

  18. A homing endonuclease and the 50-nt ribosomal bypass sequence of phage T4 constitute a mobile DNA cassette

    Science.gov (United States)

    Bonocora, Richard P.; Zeng, Qinglu; Abel, Ethan V.; Shub, David A.

    2011-01-01

    Since its initial description more than two decades ago, the ribosome bypass (or “hop”) sequence of phage T4 stands out as a uniquely extreme example of programmed translational frameshifting. The gene for a DNA topoisomerase subunit of T4 has been split by a 1-kb insertion into two genes that retain topoisomerase function. A second 50-nt insertion, beginning with an in-phase stop codon, is inserted near the start of the newly created downstream gene 60. Instead of terminating at this stop codon, approximately half of the ribosomes skip 50 nucleotides and continue translation in a new reading frame. However, no functions, regulatory or otherwise, have been imputed for the truncated peptide that results from termination at codon 46 or for the bypass sequence itself. Moreover, how this unusual mRNA organization arose and why it is maintained have never been explained. We show here that a homing endonuclease (MobA) is encoded in the insertion that created gene 60, and the mobA gene together with the bypass sequence constitute a mobile DNA cassette. The bypass sequence provides protection against self-cleavage by the nuclease, whereas the nuclease promotes horizontal spread of the entire cassette to related bacteriophages. Group I introns frequently provide protection against self-cleavage by associated homing endonucleases. We present a scenario by which the bypass sequence, which is otherwise a unique genetic element, might have been derived from a degenerate group I intron. PMID:21930924

  19. Computational study of hydration at the TD damaged site of DNA in complex with repair enzyme T4 endonuclease V

    Energy Technology Data Exchange (ETDEWEB)

    Pinak, Miroslav [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    2000-02-01

    An analysis of the distribution of water around DNA surface focusing on the role of the distribution of water molecules in the proper recognition of damaged site by repair enzyme T4 Endonuclease V was performed. The native DNA dodecamer, dodecamer with the thymine dimer (TD) and complex of DNA and part of repair enzyme T4 Endonuclease V were examined throughout the 500 ps of molecular dynamics simulation. During simulation the number of water molecules close to the DNA atoms and the residence time were calculated. There is an increase in number of water molecules lying in the close vicinity to TD if compared with those lying close to two native thymines (TT). Densely populated area with water molecules around TD is one of the factors detected by enzyme during scanning process. The residence time was found higher for molecule of the complex and the six water molecules were found occupying the stabile positions between the TD and catalytic center close to atoms P, C3' and N3. These molecules originate water mediated hydrogen bond network that contribute to the stability of complex required for the onset of repair process. (author)

  20. Di-Adenosine Tetraphosphate (Ap4A) Metabolism Impacts Biofilm Formation by Pseudomonas fluorescens via Modulation of c-di-GMP-Dependent Pathways▿

    OpenAIRE

    Monds, Russell D.; Newell, Peter D.; Wagner, Jeffrey C.; Schwartzman, Julia A.; Lu, Wenyun; Rabinowitz, Joshua D.; O'Toole, George A.

    2010-01-01

    Dinucleoside tetraphosphates are common constituents of the cell and are thought to play diverse biological roles in organisms ranging from bacteria to humans. In this study we characterized two independent mechanisms by which di-adenosine tetraphosphate (Ap4A) metabolism impacts biofilm formation by Pseudomonas fluorescens. Null mutations in apaH, the gene encoding nucleoside tetraphosphate hydrolase, resulted in a marked increase in the cellular level of Ap4A. Concomitant with this increase...

  1. Identification of restriction endonucleases sensitive to 5-cytosine methylation at non-CpG sites, including expanded (CAG)n/(CTG)n repeats.

    Science.gov (United States)

    López Castel, Arturo; Nakamori, Masayuki; Thornton, Charles A; Pearson, Christopher E

    2011-04-01

    Most epigenetic studies assess methylation of 5'-CpG-3' sites but recent evidence indicates that non-CpG cytosine methylation occurs at high levels in humans and other species. This is most prevalent at 5'-CHG-3', where H = A, C or T, and it preferentially occurs at 5'-CpA-3' and 5'-CpT-3' sites. With the goal of facilitating the detection of non-CpG methylation, the restriction endonucleases ApeKI, BbvI, EcoP15I, Fnu 4HI, MwoI and TseI were assessed for their sensitivity to 5-methylcytosine at GpCpA, GpCpT, GpCpC or GpCpG sites, where methylation is catalyzed by the DNA 5-cytosine 5'-GpC-3' methyltransferase M.CviPI. We tested a variety of sequences including various plasmid-based sites, a cloned disease-associated (CAG)83•(CTG)83 repeat and in vitro synthesized tracts of only (CAG)500•(CTG)500 or (CAG)800•(CTG)800. The repeat tracts are enriched for the preferred CpA and CpT motifs. We found that none of the tested enzymes can cleave their recognition sequences when they are 5'-GpC-3' methylated. A genomic site known to convert its non-CpG methylation levels upon C2C12 differentiation was confirmed through the use of these enzymes. These enzymes can be useful in rapidly and easily determining the most common non-CpG methylation status in various sequence contexts, as well as at expansions of (CAG)n•(CTG)n repeat tracts associated with diseases like myotonic dystrophy and Huntington disease.

  2. BEACON{sup TM} Core Monitoring and Analysis for Operations of the Westinghouse AP1000

    Energy Technology Data Exchange (ETDEWEB)

    Skidmore, Sam A.; Krieg, David J. [Westinghouse Electric Company, Nuclear Fuel Division, P.O.Box 355, Pittsburgh, PA, 15230 (United States)

    2009-06-15

    provide continuous monitoring of Operating License (Technical Specifications) limits of peak linear heat rate, departure from nucleate boiling and shutdown margin. BEACON integration with AP1000 operations starts with the specific data needed to comply with license requirements associated with power distribution and shutdown reactivity. The Online Power Distribution Monitoring System (OPDMS) will be the plant system providing the necessary information from BEACON to the reactor operator. The information displays will be integrated with the control room alarm and display systems to be consistent with the other systems requiring interaction with the reactor operator. The Human/System Interface requirements and design that is utilized in every other system is utilized with the display of the BEACON data. Operations integration continues with the core information needs of the reactor operator and BEACON functions designed to provide the information. Present reactors often utilize 'Core Design Reports' or 'Station Curve Books' for transmittal of reactor information to the operator. These will not suffice for the AP1000 and BEACON will be utilized when the reactor operator needs nuclear information. BEACON will also provide analysis functions tailored for the reactor operator and engineer for use in the control room to support day to day operations decision making and planning specifically designed for the unique needs of the AP1000. The MShim control system allows for significant automation and simplicity in reactor operations. These systems will be modeled with BEACON allowing prediction functions that in today's plants are most commonly utilized by a specialist or reactor engineer to be used directly by control room operations personnel. The AP1000 is the leader of the nuclear renaissance in the United States. Unlike present PWRs, the BEACON system is not just a desirable optional feature but will be incorporated in to plant design and integral to

  3. AP2 hemicomplexes contribute independently to synaptic vesicle endocytosis.

    Science.gov (United States)

    Gu, Mingyu; Liu, Qiang; Watanabe, Shigeki; Sun, Lin; Hollopeter, Gunther; Grant, Barth D; Jorgensen, Erik M

    2013-03-05

    The clathrin adaptor complex AP2 is thought to be an obligate heterotetramer. We identify null mutations in the α subunit of AP2 in the nematode Caenorhabditis elegans. α-adaptin mutants are viable and the remaining μ2/β hemicomplex retains some function. Conversely, in μ2 mutants, the alpha/sigma2 hemicomplex is localized and is partially functional. α-μ2 double mutants disrupt both halves of the complex and are lethal. The lethality can be rescued by expression of AP2 components in the skin, which allowed us to evaluate the requirement for AP2 subunits at synapses. Mutations in either α or μ2 subunits alone reduce the number of synaptic vesicles by about 30%; however, simultaneous loss of both α and μ2 subunits leads to a 70% reduction in synaptic vesicles and the presence of large vacuoles. These data suggest that AP2 may function as two partially independent hemicomplexes. DOI:http://dx.doi.org/10.7554/eLife.00190.001.

  4. Turning CALM into excitement: AP180 and CALM in endocytosis and disease.

    Science.gov (United States)

    Maritzen, Tanja; Koo, Seong Joo; Haucke, Volker

    2012-10-01

    Dynamic flux of membrane between intracellular compartments is a key feature of all eukaryotic cells. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a crucial role in membrane dynamics by facilitating membrane fusion, for example at synapses where small synaptic vesicles (SVs) undergo activity-regulated neuroexocytosis, followed by the endocytic re-cycling of SV proteins and lipids. Recent work shows that the assembly protein 180 (AP180) N-terminal homology (ANTH) domain containing proteins AP180 and clathrin assembly lymphoid myeloid leukaemia (CALM) not only regulate the assembly of the endocytic machinery but also act as sorters for a subset of SNAREs, the vesicle-associated membrane proteins (VAMPs), most notably VAMP/synaptobrevin 2 at synapses. In this review, we summarise the current state of knowledge about the roles of AP180 and CALM family members in clathrin-dependent membrane traffic, the molecular mechanistic basis for their activities and their potential involvement in human disease. Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.

  5. Continuous monitoring of restriction endonuclease cleavage activity by universal molecular beacon light quenching coupled with real-time polymerase chain reaction.

    Science.gov (United States)

    Li, Xiaomin; Song, Chen; Zhao, Meiping; Li, Yuanzong

    2008-10-01

    We describe a method for sensitive monitoring of restriction endonuclease kinetics and activity by use of a universal molecular beacon (U-MB) coupled with real-time polymerase chain reaction (PCR). The method is used to monitor the progress of DNA cleavage in a sealed reaction tube and offers more accurate and high-throughput detection. The template has a universal tail hybridized with the U-MB and the remaining sequence is complementary to one of the restriction endonuclease digestion products. The U-MB is replaced by the extension of digested product and the fluorescence quenches. With this concept, one universal fluorescence probe can be used in different enzyme analytical systems. In the work described here, homogenous assays were performed with the restriction endonucleases AluI, EcoRI, XhoI, and SacI at smoothly controlled temperature. Cleavage efficiencies were determined, and the potential applications of this method were discussed. Furthermore, the AluI and EcoRI cleavage reactions were monitored online at varying substrate concentrations at the molecular level, and K(m), V(max), and K(cat) values were calculated. The results suggest that U-MB monitoring of restriction endonuclease assays based on real-time PCR will be very useful for high-throughput, sensitive, and precise assays for enzyme activity screening and evolutionary biotechnology analysis.

  6. Microinjection of Micrococcus luteus UV-endonuclease restores UV-induced unscheduled DNA synthesis in cells of 9 xeroderma pigmentosum complementation groups.

    NARCIS (Netherlands)

    A.J.R. de Jonge; W. Vermeulen (Wim); W. Keijzer; J.H.J. Hoeijmakers (Jan); D. Bootsma (Dirk)

    1985-01-01

    textabstractThe UV-induced unscheduled DNA synthesis (UDS) in cultured cells of excision-deficient xeroderma pigmentosum (XP) complementation groups A through I was assayed after injection of Micrococcus luteus UV-endonuclease using glass microneedles. In all complementation groups a restoration of

  7. Comparison of the mismatch-specific endonuclease method and denaturing high-performance liquid chromatography for the identification of HBB gene mutations

    Science.gov (United States)

    Hung, Chia-Cheng; Su, Yi-Ning; Lin, Chia-Yun; Chang, Yin-Fei; Chang, Chien-Hui; Cheng, Wen-Fang; Chen, Chi-An; Lee, Chien-Nan; Lin, Win-Li

    2008-01-01

    Background Beta-thalassemia is a common autosomal recessive hereditary disease in the Meditertanean, Asia and African areas. Over 600 mutations have been described in the beta-globin (HBB), of which more than 200 are associated with a beta-thalassemia phenotype. Results We used two highly-specific mutation screening methods, mismatch-specific endonuclease and denaturing high-performance liquid chromatography, to identify mutations in the HBB gene. The sensitivity and specificity of these two methods were compared. We successfully distinguished mutations in the HBB gene by the mismatch-specific endonuclease method without need for further assay. This technique had 100% sensitivity and specificity for the study sample. Conclusion Compared to the DHPLC approach, the mismatch-specific endonuclease method allows mutational screening of a large number of samples because of its speed, sensitivity and adaptability to semi-automated systems. These findings demonstrate the feasibility of using the mismatch-specific endonuclease method as a tool for mutation screening. PMID:18694524

  8. Comparison of the mismatch-specific endonuclease method and denaturing high-performance liquid chromatography for the identification of HBB gene mutations

    Directory of Open Access Journals (Sweden)

    Cheng Wen-Fang

    2008-08-01

    Full Text Available Abstract Background Beta-thalassemia is a common autosomal recessive hereditary disease in the Meditertanean, Asia and African areas. Over 600 mutations have been described in the beta-globin (HBB, of which more than 200 are associated with a beta-thalassemia phenotype. Results We used two highly-specific mutation screening methods, mismatch-specific endonuclease and denaturing high-performance liquid chromatography, to identify mutations in the HBB gene. The sensitivity and specificity of these two methods were compared. We successfully distinguished mutations in the HBB gene by the mismatch-specific endonuclease method without need for further assay. This technique had 100% sensitivity and specificity for the study sample. Conclusion Compared to the DHPLC approach, the mismatch-specific endonuclease method allows mutational screening of a large number of samples because of its speed, sensitivity and adaptability to semi-automated systems. These findings demonstrate the feasibility of using the mismatch-specific endonuclease method as a tool for mutation screening.

  9. Design of the influenza virus inhibitors targeting the PA endonuclease using 3D-QSAR modeling, side-chain hopping, and docking.

    Science.gov (United States)

    Yan, Zhihui; Zhang, Lijie; Fu, Haiyang; Wang, Zhonghua; Lin, Jianping

    2014-01-15

    With the emergence of drug resistance and the structural determination of the PA N-terminal domain (PAN), influenza endonucleases have become an attractive target for antiviral therapies for influenza infection. Here, we combined 3D-QSAR with side-chain hopping and molecular docking to produce novel structures as endonuclease inhibitors. First, a new molecular library was generated with side-chain hopping on an existing template molecule, L-742001, using an in-house fragment library that targets bivalent-cation-binding proteins. Then, the best 3D-QSAR model (AAAHR.500), with q(2)=0.76 and r(2)=0.97 from phase modeling, was constructed from 23 endonuclease inhibitors and validated with 17 test compounds. The AAAHR.500 model was then used to select effective candidates from the new molecular library. Combining 3D-QSAR with docking using Glide and Autodock, 13 compounds were considered the most likely candidate inhibitors. Docking studies showed that the binding modes of these compounds were consistent with the crystal structures of known inhibitors. These compounds could serve as potential endonuclease inhibitors for further biological activity tests. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. DNA recognition by the SwaI restriction endonuclease involves unusual distortion of an 8 base pair A:T-rich target.

    Science.gov (United States)

    Shen, Betty W; Heiter, Daniel F; Lunnen, Keith D; Wilson, Geoffrey G; Stoddard, Barry L

    2017-02-17

    R.SwaI, a Type IIP restriction endonuclease, recognizes a palindromic eight base pair (bp) symmetric sequence, 5΄-ATTTAAAT-3΄, and cleaves that target at its center to generate blunt-ended DNA fragments. Here, we report three crystal structures of SwaI: unbound enzyme, a DNA-bound complex with calcium ions; and a DNA-bound, fully cleaved complex with magnesium ions. We compare these structures to two structurally similar ‘PD-D/ExK’ restriction endonucleases (EcoRV and HincII) that also generate blunt-ended products, and to a structurally distinct enzyme (the HNH endonuclease PacI) that also recognizes an 8-bp target site consisting solely of A:T base pairs. Binding by SwaI induces an extreme bend in the target sequence accompanied by un-pairing and re-ordering of its central A:T base pairs. This result is reminiscent of a more dramatic target deformation previously described for PacI, implying that long A:T-rich target sites might display structural or dynamic behaviors that play a significant role in endonuclease recognition and cleavage.

  11. Type III restriction endonuclease EcoP15I is a heterotrimeric complex containing one Res subunit with several DNA-binding regions and ATPase activity.

    Science.gov (United States)

    Wyszomirski, Karol H; Curth, Ute; Alves, Jürgen; Mackeldanz, Petra; Möncke-Buchner, Elisabeth; Schutkowski, Mike; Krüger, Detlev H; Reuter, Monika

    2012-04-01

    For efficient DNA cleavage, the Type III restriction endonuclease EcoP15I communicates with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of methylation (Mod) and restriction (Res) subunits forming a multifunctional enzyme complex able to methylate or to cleave DNA. In this study, we determined by different analytical methods that EcoP15I contains a single Res subunit in a Mod(2)Res stoichiometry. The Res subunit comprises a translocase (Tr) domain carrying functional motifs of superfamily 2 helicases and an endonuclease domain with a PD..D/EXK motif. We show that the isolated Tr domain retains ATP-hydrolyzing activity and binds single- and double-stranded DNA in a sequence-independent manner. To localize the regions of DNA binding, we screened peptide arrays representing the entire Res sequence for their ability to interact with DNA. We discovered four DNA-binding regions in the Tr domain and two DNA-binding regions in the endonuclease domain. Modelling of the Tr domain shows that these multiple DNA-binding regions are located on the surface, free to interact with DNA. Interestingly, the positions of the DNA-binding regions are conserved among other Type III restriction endonucleases.

  12. CHROMOSOME BANDING IN CRUSTACEA. I. KARYOTYPE, Ag-NORs, C BANDING AND TREATMENT WITH EcoRI, PstI and KpnI RESTRICTION ENDONUCLEASES IN Artemia franciscana

    Directory of Open Access Journals (Sweden)

    Ingrid Vilar Accioly

    2014-08-01

    Full Text Available Características cariotípicas do microcrustáceo Artemia franciscana Kellog, 1906, introduzida nas salinas do litoral nordeste do Brasil, na década de 70, foram investigadas através de coloração convencional, bandamento C, endonucleases de restrição (EcoRI, PstI e KpnI e Ag-NORs. O cariótipo consiste de 42 cromossomos, onde se individualiza sobre alguns pares a presença de constrições secundárias. Grandes blocos heterocromáticos encontram-se distribuídos nas porções teloméricas da maioria dos cromossomos. A digestão com PstI e KpnI revelou um padrão similar ao obtido pelo bandamento C. Preparações tratadas com EcoRI apresentam digestão das regiões heterocromáticas indicando a presença de sítios de restrição nestas regiões. Ag-NORs múltiplas estão associadas a blocos heterocromáticos. Os dados apresentados representam passo inicial para identificação de possíveis modificações ocorridas após o isolamento geográfico desta amostra, assim como no entendimento das modificações evolutivas ocorridas no cariótipo deste grupo. Palavras-chave: bandamento cromossômico, camarão de água salgada, citogenética de crustáceos. DOI: http://dx.doi.org/10.18561/2179-5746/biotaamazonia.v4n2p15-19

  13. Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage.

    Science.gov (United States)

    Wang, Yan; Mao, Lili; Kankanala, Jayakanth; Wang, Zhengqiang; Geraghty, Robert J

    2017-02-01

    The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease. Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus

  14. Curcumin and EGCG Suppress Apurinic/Apyrimidinic Endonuclease 1 and Induce Complete Remission in B-cell Non-Hodgkin's lymphoma Patients

    Directory of Open Access Journals (Sweden)

    Hashem M. Neenaa

    2011-12-01

    Full Text Available ABSTRACT:Background: Follicular lymphoma (FL is the most common subtype of indolent lymphoma. FL is still considered to be an incurable disease and palliation of symptoms is an acceptable approach to the expected pattern of repeated relapses due to developing resistance to chemotherapy agents. Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1 is a multifunctional protein involved in DNA base excision repair (BER of oxidative DNA damage and in redox regulation of a number of transcription factors. It was observed that cytoplasmic APE1 induced COX-2 expression through NF-êB activation. It has been shown that chemopreventive agents potentiate the efficacy of chemotherapy through the regulation of multiple signaling pathways, including NF-êB, c-Myc, cyclooxygenase-2, apoptosis, and others, suggesting a multitargeted nature of chemopreventive agents. We hypothesized that curcumin, a polyphenolic antioxidant derived from the spice turmeric, and epigallocatechin gallate (EGCG from green tea would potentiate the effect of chemotherapy in B-cell lymphoma.Objective: We examined the role of human apurinic/apyrimidinic endonuclease 1 (APE1 in resistance and prognosis in patients with FL. Our major objective was to update the safety and efficacy results of the antitumor effect of combination of curcumin and EGCG therapy in relapsed or resistant indolent or transformed non-Hodgkin follicular lymphoma patients and their peripheral blood mononuclear cells (PBMCs compared with healthy donors’ controls.Methods: Thirty patients with FL with over-expression of constitutive active NF-êB in their PBMCs received regular CHOP and consumed capsules compatible with curcumin doses between 0.9 and 5.4 g daily for up to 9 months and 9.0 g/day green tea whole extract "1000 mg tablets of green tea whole extract containing 200 mg EGCG. We designed a dose-escalation Functional Foods in Health and Disease 2011, 1(12:525-544 study to explore the efficacy of CHOP

  15. Experience with the EPICS PV Gateway at the APS

    CERN Document Server

    Evans, Kenneth

    2005-01-01

    The EPICS PV Gateway has become a stable, high-performance application that provides access to process variables while minimizing the impact on critical IOCs and implementing additional access security. The additional access security typically prevents write access but is highly configurable. The Advanced Photon Source (APS) currently uses 40 Gateways running on 11 machines to provide access to the machine network from the offices and for the individual experimental teams. These include reverse Gateways that allow administration of all 40 APS Gateways from a single MEDM screen, even though the Gateways are running on separate networks. This administration includes starting, stopping, making and viewing reports, and viewing and editing access security files. There is one Gateway that provides process variable renaming. This paper provides an overview of the Gateways at the APS and describes the procedures that have been set up to use and administer them.

  16. APS-U LATTICE DESIGN FOR OFF-AXIS ACCUMULATION

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Yipeng; Borland, M.; Lindberg, R.; Sajaev, V.

    2017-06-25

    A 67-pm hybrid-seven-bend achromat (H7BA) lattice is being proposed for a future Advanced Photon Source (APS) multi-bend-achromat (MBA) upgrade project. This lattice design pushes for smaller emittance and requires use of a swap-out (on-axis) injection scheme due to limited dynamic acceptance. Alternate lattice design work has also been performed for the APS upgrade to achieve better beam dynamics performance than the nominal APS MBA lattice, in order to allow off-axis accumulation. Two such alternate H7BA lattice designs, which target a still-low emittance of 90 pm, are discussed in detail in this paper. Although the single-particle-dynamics performance is good, simulations of collective effects indicate that surprising difficulty would be expected accumulating high single-bunch charge in this lattice. The brightness of the 90-pm lattice is also a factor of two lower than the 67-pm H7BA lattice.

  17. Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity.

    Science.gov (United States)

    Yeh, Hsuan-Heng; Tseng, Yu-Fen; Hsu, Yu-Chiao; Lan, Sheng-Hui; Wu, Shan-Ying; Raghavaraju, Giri; Cheng, Da-En; Lee, Ying-Ray; Chang, Tsuey-Yu; Chow, Nan-Haw; Hung, Wen-Chun; Liu, Hsiao-Sheng

    2015-03-25

    Mutant Ras plays multiple functions in tumorigenesis including tumor formation and metastasis. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a metastasis inhibitor gene, suppresses matrix metalloproteinase (MMP) activity in the metastatic cascade. Clarifying the relationship between Ras and RECK and understanding the underlying molecular mechanism may lead to the development of better treatment for Ras-related tumors. Suppression subtractive hybridization PCR (SSH PCR) was conducted to identify Ha-ras (val12) up-regulated genes in bladder cancer cells. Stable cell lines of human breast cancer (MCF-7-ras) and mouse NIH3T3 fibroblasts (7-4) harboring the inducible Ha-ras (val12) oncogene, which could be induced by isopropylthio-β-D-galactoside (IPTG), were used to clarify the relationship between Ras and the up-regulated genes. Chromatin immunoprecipitation (ChIP) assay, DNA affinity precipitation assay (DAPA) and RECK reporter gene assay were utilized to confirm the complex formation and binding with promoters. Retinoblastoma binding protein-7 (RbAp46) was identified and confirmed as a Ha-ras (val12) up-regulated gene. RbAp46 could bind with histone deacetylase (HDAC1) and Sp1, followed by binding to RECK promoter at the Sp1 site resulting in repression of RECK expression. High expression of Ras protein accompanied with high RbAp46 and low RECK expression were detected in 75% (3/4) of the clinical bladder cancer tumor tissues compared to the adjacent normal parts. Ras induced RbAp46 expression increases invasion of the bladder cancer T24 cells and MMP-9 activity was increased, which was confirmed by specific lentiviral shRNAs inhibitors against Ras and RbAp46. Similarly, knockdown of RbAp46 expression in the stable NIH3T3 cells "7-4" by shRNA decreased Ras-related lung metastasis using a xenograft nude mice model. We confirmed that RbAp46 is a Ha-ras (val12) up-regulated gene and binds with HDAC1 and Sp1. Furthermore, RbAp46 binds to the RECK

  18. Introduction. Teaching Advanced Placement Human Geography.

    Science.gov (United States)

    Murphy, Alexander B.

    2000-01-01

    Introduces this special issue of "Journal of Geography" focusing on the teaching of Advanced Placement (AP) human geography. States that essays were developed by members of the AP Human Geography Development Committee focusing on areas in the human geography course outline which are included in the appendix. (CMK)

  19. Modified 16S-23S rRNA intergenic region restriction endonuclease analysis for species identification of Enterococcus strains isolated from pigs, compared with identification using classical methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    Nowakiewicz, Aneta; Ziółkowska, Grażyna; Zięba, Przemysław; Trościańczyk, Aleksandra; Banach, Tomasz; Kowalski, Cezary

    2015-03-01

    Fast and reliable identification of bacteria to at least the species level is currently the basis for correct diagnosis and appropriate treatment of infections. This is particularly important in the case of bacteria of the genus Enterococcus, whose resistance profile is often correlated with their species (e.g. resistance to vancomycin). In this study, we evaluated restriction endonuclease analysis of the 16S-23S rRNA gene intergenic transcribed spacer (ITS) region for species identification of Enterococcus. The utility of the method was compared with that of phenotypic methods [biochemical profile evaluation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)]. Identification was based on 21 Enterococcus reference strains, of the species E. faecalis, E. faecium, E. hirae, E. durans, E. casseliflavus, E. gallinarum, E. avium, E. cecorum and E. columbae, and 47 Enterococcus field strains isolated from pigs. Restriction endonuclease analysis of the ITS-PCR product using HinfI, RsaI and MboI, in the order specified, enabled species differentiation of the Enterococcus reference and field strains, and in the case of the latter, the results of species identification were identical (47/47) to those obtained by MALDI-TOF MS. Moreover, as a result of digestion with MboI, a unique restriction profile was also obtained for the strains (3/3) identified by MALDI-TOF MS as E. thailandicus. In our opinion, restriction endonuclease analysis of the 16S-23S rRNA gene ITS region of Enterococcus may be a simple and relatively fast (less than 4 h) alternative method for identifying the species occurring most frequently in humans and animals. © 2015 The Authors.

  20. ION EFFECTS IN THE APS PARTICLE ACCUMULATOR RING

    Energy Technology Data Exchange (ETDEWEB)

    Calvey, J.; Harkay, K.; Yao, CY.

    2017-06-25

    Trapped ions in the APS Particle Accumulator Ring (PAR) lead to a positive coherent tune shift in both planes, which increases along the PAR cycle as more ions accumulate. This effect has been studied using an ion simulation code developed at SLAC. After modifying the code to include a realistic vacuum profile, multiple ionization, and the effect of shaking the beam to measure the tune, the simulation agrees well with our measurements. This code has also been used to evaluate the possibility of ion instabilities at the high bunch charge needed for the APS-Upgrade.

  1. On the abundance of Europium. [in Ap and Am stars

    Science.gov (United States)

    Hartoog, M. R.; Cowley, C. R.; Adelman, S. J.

    1974-01-01

    The inclusion of the effects of hyperfine splitting can significantly lower the abundance estimate of Eu from singly ionized lines which lie on the flat portion of the curve of growth. In the 21 cool Ap stars studied by Adelman and the five Am stars studied by Smith, the Eu abundance was reduced by 0.4 dex on the average. In individual cases, the reductions were as great as 0.9 dex. This makes the Eu abundance comparable to that of its neighboring rare earths Sm and Gd in the Ap stars and less than Sm and Gd in the Am stars, but still substantially overabundant with respect to solar values.

  2. Past, Present, and Future of AP Chemistry: A Brief History of Course and Exam Alignment Efforts

    Science.gov (United States)

    Magrogan, Serena

    2014-01-01

    As part of the Advanced Placement (AP) Program's commitment to continually enhance alignment with current best practices in college-level learning, the AP Program is currently evaluating and redesigning courses and exams, one of which launched during the 2013-2014 academic school year: AP chemistry. The history of the AP chemistry course and…

  3. Cross-linking of bromodeoxyuridine-substituted oligonucleotides to the EcoRI and EcoRV restriction endonucleases.

    Science.gov (United States)

    Wolfes, H; Fliess, A; Winkler, F; Pingoud, A

    1986-09-01

    We have synthesized several self-complementary oligodeoxynucleotides which contain bromodeoxyuridine in various positions within and outside of the recognition sequence for the EcoRI and EcoRV restriction endonucleases. These oligodeoxynucleotides are cleaved in the presence of Mg2+ by their respective enzyme. Upon irradiation by long-wavelength ultraviolet light and in the absence of Mg2+ they are cross-linked in low yield to their enzymes, forming 1:1 and 1:2 (oligodeoxynucleotide:enzyme subunit) adducts. Cross-linking occurs with both specific and non-specific complexes. With EcoRI the site of cross-linking was determined to be at or close to Met-137, i.e. in a region of the molecule implicated by other studies from our laboratory [Scholtissek et al. (1986) J. Biol. Chem. 261, 2228-2234] in the binding and cleavage of the substrate.

  4. I-ApeI: a novel intron-encoded LAGLIDADG homing endonuclease from the archaeon, Aeropyrum pernix K1

    Science.gov (United States)

    Nomura, Norimichi; Morinaga, Yayoi; Shirai, Nobuaki; Sako, Yoshihiko

    2005-01-01

    Over 50 introns have been reported in archaeal rRNA genes (rDNAs), a subset of which nests putative homing endonuclease (HEase) genes. Here, we report the identification and characterization of a novel archaeal LAGLIDADG-type HEase, I-ApeI, encoded by the ApeK1.S908 intron within the 16S rDNA of Aeropyrum pernix K1. I-ApeI consists of 222 amino acids and harbors two LAGLIDADG-like sequences. It recognizes the 20 bp non-palindromic sequence 5′-GCAAGGCTGAAAC↓TTAAAGG and cleaves target DNA to produce protruding tetranucleotide 3′ ends. Either Mn2+ or Co2+ can be substituted for Mg2+ as a cofactor in the cleavage reaction. Of the 20 bases within the minimal recognition site, 7 are essential for cleavage and are located at positions proximal to the cleavage sites. PMID:16049020

  5. I-ApeKI [corrected]: a novel intron-encoded LAGLIDADG homing endonuclease from the archaeon, Aeropyrum pernix K1.

    Science.gov (United States)

    Nomura, Norimichi; Morinaga, Yayoi; Shirai, Nobuaki; Sako, Yoshihiko

    2005-07-26

    Over 50 introns have been reported in archaeal rRNA genes (rDNAs), a subset of which nests putative homing endonuclease (HEase) genes. Here, we report the identification and characterization of a novel archaeal LAGLIDADG-type HEase, I-ApeKI [corrected], encoded by the ApeK1.S908 intron within the 16S rDNA of Aeropyrum pernix K1. I-ApeKI [corrected] consists of 222 amino acids and harbors two LAGLIDADG-like sequences. It recognizes the 20 bp non-palindromic sequence 5'-GCAAGGCTGAAAC downward arrowTTAAAGG and cleaves target DNA to produce protruding tetranucleotide 3' ends. Either Mn2+ or Co2+ can be substituted for Mg2+ as a cofactor in the cleavage reaction. Of the 20 bases within the minimal recognition site, 7 are essential for cleavage and are located at positions proximal to the cleavage sites.

  6. MmoSTI restriction endonuclease, isolated from Morganella morganii infecting a tropical moth, Actias selene, cleaving 5'-|CCNGG-3' sequences.

    Science.gov (United States)

    Skowron, Marta A; Zebrowska, Joanna; Wegrzyn, Grzegorz; Skowron, Piotr M

    2016-02-01

    A type II restriction endonuclease, MmoSTI, from the pathogenic bacterium Morganella morganii infecting a tropical moth, Actias selene, has been detected and biochemically characterized, as a potential etiological differentiation factor. The described REase recognizes interrupted palindromes, i.e., 5'-CCNGG-3' sequences and cleaves DNA leaving 5-nucleotide (nt) long, single-stranded (ss), 5'-cohesive ends, which was determined by three complementary methods: (i) cleavage of custom and standard DNA substrates, (ii) run-off sequencing of cleavage products, and (iii) shotgun cloning and sequencing of bacteriophage lambda (λ) DNA digested with MmoSTI. MmoSTI, the first 5'-CCNGG-3' REase characterized from M. morganii, is a neoschizomer of ScrFI, which cleaves DNA leaving 1-nt long, ss, 5'-cohesive ends. It is a high-frequency cutter and can be isolated from easily cultured bacteria, thus it can potentially serve as a tool for DNA manipulations.

  7. Polymerase synthesis of DNAs bearing vinyl groups in the major groove and their cleavage by restriction endonucleases.

    Science.gov (United States)

    Mačková, Michaela; Pohl, Radek; Hocek, Michal

    2014-10-13

    DNA molecules containing 5-vinyluracil, 5-vinylcytosine, or 7-deaza-7-vinyladenine were prepared by polymerase incorporation of the corresponding vinyl-modified 2'-deoxyribonucleoside triphosphates, and the influence of the vinyl group in the major groove of DNA on the cleavage by diverse type II restriction endonucleases (REs) was studied. The presence of 5-vinyluracil was tolerated by most of the REs, whereas only some REs were able to cleave sequences containing 7-deaza-7-vinyladenine. The enzyme ScaI was found to cleave DNA containing 5-vinylcytosine efficiently but not DNA containing the related 5-ethynylcytosine. All other REs failed to cleave sequences containing any cytosine modifications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Autosomal recessive spastic tetraplegia caused by AP4M1 and AP4B1 gene mutation: expansion of the facial and neuroimaging features.

    Science.gov (United States)

    Tüysüz, Beyhan; Bilguvar, Kaya; Koçer, Naci; Yalçınkaya, Cengiz; Çağlayan, Okay; Gül, Ece; Sahin, Sezgin; Çomu, Sinan; Günel, Murat

    2014-07-01

    Adaptor protein complex-4 (AP4) is a component of intracellular transportation of proteins, which is thought to have a unique role in neurons. Recently, mutations affecting all four subunits of AP4 (AP4M1, AP4E1, AP4S1, and AP4B1) have been found to cause similar autosomal recessive phenotype consisting of tetraplegic cerebral palsy and intellectual disability. The aim of this study was analyzing AP4 genes in three new families with this phenotype, and discussing their clinical findings with an emphasis on neuroimaging and facial features. Using homozygosity mapping followed by whole-exome sequencing, we identified two novel homozygous mutations in AP4M1 and a homozygous deletion in AP4B1 in three pairs of siblings. Spastic tetraplegia, microcephaly, severe intellectual disability, limited speech, and stereotypic laughter were common findings in our patients. All patients also had similar facial features consisting of coarse and hypotonic face, bitemporal narrowing, bulbous nose with broad nasal ridge, and short philtrum which were not described in patients with AP4M1 and AP4B1 mutations previously. The patients presented here and previously with AP4M1, AP4B1, and AP4E1 mutations shared brain abnormalities including asymmetrical ventriculomegaly, thin splenium of the corpus callosum, and reduced white matter volume. The patients also had hippocampal globoid formation and thin hippocampus. In conclusion, disorders due to mutations in AP4 complex have similar neurological, facial, and cranial imaging findings. Thus, these four genes encoding AP4 subunits should be screened in patients with autosomal recessive spastic tetraplegic cerebral palsy, severe intellectual disability, and stereotypic laughter, especially with the described facial and cranial MRI features. © 2014 Wiley Periodicals, Inc.

  9. Transcriptional Regulation of the AP-2α Promoter by BTEB-1 and AP-2rep, a Novel wt-1/egr-Related Zinc Finger Repressor

    OpenAIRE

    Imhof, Axel; Schuierer, Marion; Werner, Oliver; Moser, Markus; Roth, Christina; Bauer, Reinhard; Buettner, Reinhard

    1999-01-01

    AP-2 transcription factors have been suggested to exert key regulatory functions in vertebrate embryonic development, in tumorigenicity of various cancer cell types, and in controlling cell cycle and apoptotic effector genes. In this study, we investigated transcriptional regulation of the AP-2α gene promoter mediated by an autoregulatory element (referred to as A32) with a core consensus AP-2 binding site at position −336 relative to the mRNA initiation site. AP-2 and multiple different nucl...

  10. The DNA repair endonuclease XPG interacts directly and functionally with the WRN helicase defective in Werner syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Trego, Kelly S.; Chernikova, Sophia B.; Davalos, Albert R.; Perry, J. Jefferson P.; Finger, L. David; Ng, Cliff; Tsai, Miaw-Sheue; Yannone, Steven M.; Tainer, John A.; Campisi, Judith; Cooper, Priscilla K.

    2011-04-20

    XPG is a structure-specific endonuclease required for nucleotide excision repair (NER). XPG incision defects result in the cancer-prone syndrome xeroderma pigmentosum, whereas truncating mutations of XPG cause the severe postnatal progeroid developmental disorder Cockayne syndrome. We show that XPG interacts directly with WRN protein, which is defective in the premature aging disorder Werner syndrome, and that the two proteins undergo similar sub-nuclear redistribution in S-phase and co-localize in nuclear foci. The co-localization was observed in mid- to late-S-phase, when WRN moves from nucleoli to nuclear foci that have been shown to contain protein markers of both stalled replication forks and telomeric proteins. We mapped the interaction between XPG and WRN to the C-terminal domains of each and show that interaction with the C-terminal domain of XPG strongly stimulates WRN helicase activity. WRN also possesses a competing DNA single-strand annealing activity that, combined with unwinding, has been shown to coordinate regression of model replication forks to form Holliday junction/chicken foot intermediate structures. We tested whether XPG stimulated WRN annealing activity and found that XPG itself has intrinsic strand annealing activity that requires the unstructured R- and C-terminal domains, but not the conserved catalytic core or endonuclease activity. Annealing by XPG is cooperative, rather than additive, with WRN annealing. Taken together, our results suggest a novel function for XPG in S-phase that is at least in part carried out coordinately with WRN, and which may contribute to the severity of the phenotypes that occur upon loss of XPG.

  11. Crystal structure of the R-protein of the multisubunit ATP-dependent restriction endonuclease NgoAVII.

    Science.gov (United States)

    Tamulaitiene, Giedre; Silanskas, Arunas; Grazulis, Saulius; Zaremba, Mindaugas; Siksnys, Virginijus

    2014-12-16

    The restriction endonuclease (REase) NgoAVII is composed of two proteins, R.NgoAVII and N.NgoAVII, and shares features of both Type II restriction enzymes and Type I/III ATP-dependent restriction enzymes (see accompanying paper Zaremba et al., 2014). Here we present crystal structures of the R.NgoAVII apo-protein and the R.NgoAVII C-terminal domain bound to a specific DNA. R.NgoAVII is composed of two domains: an N-terminal nucleolytic PLD domain; and a C-terminal B3-like DNA-binding domain identified previously in BfiI and EcoRII REases, and in plant transcription factors. Structural comparison of the B3-like domains of R.NgoAVII, EcoRII, BfiI and the plant transcription factors revealed a conserved DNA-binding surface comprised of N- and C-arms that together grip the DNA. The C-arms of R.NgoAVII, EcoRII, BfiI and plant B3 domains are similar in size, but the R.NgoAVII N-arm which makes the majority of the contacts to the target site is much longer. The overall structures of R.NgoAVII and BfiI are similar; however, whilst BfiI has stand-alone catalytic activity, R.NgoAVII requires an auxiliary cognate N.NgoAVII protein and ATP hydrolysis in order to cleave DNA at the target site. The structures we present will help formulate future experiments to explore the molecular mechanisms of intersubunit crosstalk that control DNA cleavage by R.NgoAVII and related endonucleases. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Effect of AP102, a subtype 2 and 5 specific somatostatin analog, on glucose metabolism in rats.

    Science.gov (United States)

    Tarasco, Erika; Seebeck, Petra; Pfundstein, Svende; Daly, Adrian F; Eugster, Philippe J; Harris, Alan G; Grouzmann, Eric; Lutz, Thomas A; Boyle, Christina N

    2017-10-01

    Somatostatin analogs are widely used to treat conditions associated with hormonal hypersecretion such as acromegaly and metastatic neuroendocrine tumors. First generation somatostatin analogs, such as octreotide and lanreotide, have high affinity for somatostatin receptor subtype 2 (SSTR2), but have incomplete efficacy in many patients. Pasireotide targets multiple SSTRs, having the highest affinity for SSTR5, but causes hyperglycemia and diabetes mellitus in preclinical and clinical studies. AP102 is a new somatostatin analogs with high affinity at both SSTR2 and SSTR5. We aimed to characterize the effects of AP102 vs. pasireotide on random and dynamic glucose levels, glucoregulatory hormone concentrations and growth axis measures in healthy Sprague-Dawley rats. Three doses of each compound were evaluated under acute conditions (1, 10, and 30 µg/kg s.c.), and two doses during a chronic (4-week) infusion (3 and 10 µg/kg/h s.c.). Neither acute nor chronic AP102 administration altered blood glucose concentrations or dynamic responses following an intraperitoneal glucose tolerance test. In contrast, acute and chronic pasireotide dosing increased random and post-intraperitoneal glucose tolerance test blood glucose measures, compared to vehicle-treated controls. Both AP102 and pasireotide acutely suppressed growth hormone levels, although insulin-like growth factor-1 and somatic growth was suppressed to a greater extent with pasireotide. AP102 is a new dual SSTR2/SSTR5-specific somatostatin analog that acutely reduces growth hormone but does not cause hyperglycemia during acute or chronic administration in a healthy rat model. Further studies in diabetic animals and in humans are necessary to determine the potential utility of AP102 in the clinical setting.

  13. Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering.

    Science.gov (United States)

    Raza, M Hashim; Mattera, Rafael; Morell, Robert; Sainz, Eduardo; Rahn, Rachel; Gutierrez, Joanne; Paris, Emily; Root, Jessica; Solomon, Beth; Brewer, Carmen; Basra, M Asim Raza; Khan, Shaheen; Riazuddin, Sheikh; Braun, Allen; Bonifacino, Juan S; Drayna, Dennis

    2015-11-05

    Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  14. 76 FR 10269 - AP1000 Design Certification Amendment

    Science.gov (United States)

    2011-02-24

    ... Comments and Accessing Information Comments submitted in writing or in electronic form will be posted on... discussed below: Editorial Changes Westinghouse requested changes to the AP1000 DCD to correct spelling... reduced use of operator actions. In other words, the applicant or licensee must continue to show, with the...

  15. APS undulator and wiggler sources: Monte-Carlo simulation

    Energy Technology Data Exchange (ETDEWEB)

    Xu, S.L.; Lai, B.; Viccaro, P.J.

    1992-02-01

    Standard insertion devices will be provided to each sector by the Advanced Photon Source. It is important to define the radiation characteristics of these general purpose devices. In this document,results of Monte-Carlo simulation are presented. These results, based on the SHADOW program, include the APS Undulator A (UA), Wiggler A (WA), and Wiggler B (WB).

  16. Nuclear Reactor Safety--The APS Submits its Report

    Science.gov (United States)

    Physics Today, 1975

    1975-01-01

    Presents the summary section of the American Physical Society (APS) report on the safety features of the light-water reactor, reviews the design, construction, and operation of a reactor and outlines the primary engineered safety features. Summarizes the major recommendations of the study group. (GS)

  17. Tank 241AP104 Grab Sampling and Analysis Plan

    Energy Technology Data Exchange (ETDEWEB)

    TEMPLETON, A.M.

    2000-11-09

    This sampling and analysis plan (SAP) identifies characterization objectives pertaining to sample collection, laboratory analytical evaluation, and reporting requirements for samples obtained from tank 241-AP-104. The purpose of this sampling event is to obtain information about the characteristics of the contents of 241-AP-104 required to provide sample material to the Waste Treatment Contractor. Grab samples will be obtained from riser 001 to provide sufficient material for the chemical analyses and tests required to satisfy these data quality objectives and ICD-23. The 222-S Laboratory will receive samples; composite the samples; perform chemical analyses on composite samples; and provide samples to the Waste Treatment Contractor and the Process Chemistry Laboratory. The Process Chemistry Laboratory at the 222-S Laboratory Complex will perform process tests to evaluate the behavior of the 241-AP-104 waste undergoing the retrieval and treatment scenarios defined in the applicable DQOs. The Waste Treatment Contractor will perform process verification and waste form qualification tests. Requirements for analyses of samples originating in the L & H DQO process tests will be documented in the corresponding test plan (Person 2000) and are not within the scope of this SAP. This report provides the general methodology and procedures to be used in the preparation, retrieval, transport, analysis, and reporting of results from grab samples retrieved from tank 241-AP-104.

  18. Integrating Particulate Representations into AP Chemistry and Introductory Chemistry Courses

    Science.gov (United States)

    Prilliman, Stephen G.

    2014-01-01

    The College Board's recently revised curriculum for advanced placement (AP) chemistry places a strong emphasis on conceptual understanding, including representations of particle phenomena. This change in emphasis is informed by years of research showing that students could perform algorithmic calculations but not explain those calculations…

  19. Arbitrarily primed sequence-related amplified polymorphism (AP ...

    African Journals Online (AJOL)

    Additionally, 80 SRAP primers were used to screen markers in seven plant species (Chinese cabbage, Chinese kale, eggplant, pepper, cucumber, rose and lily), which indicated obvious polymorphism. The primers of AP-SRAP combine simply and reliably. It can overcome the limitation of the number of standard SRAP ...

  20. Effective Teaching Strategies for Open Enrollment Honors and AP Classes

    Science.gov (United States)

    Winebrenner, Susan

    2006-01-01

    A trend is emerging to open enrollment for honors and AP classes to all students who wish to take them. Teachers of these open enrollment classes may be facing several dilemmas. How can the high standards and academic rigor of the course be maintained? How can students who struggle to learn be supported in their endeavors to keep up with the…

  1. Penetration dynamics of AP8 in thin ceramic tiles

    NARCIS (Netherlands)

    Abadjieva, E.; Khoe, Y.S.

    2013-01-01

    The interaction of thin ceramic tiles with AP8 (WC core, 7,62 mm) at 1000 m/s velocity has been studied experimentally and numerically. “Thin” ceramic tiles refers here to ratio of the tile thickness (t) to the projectile diameter, (d), t/d@ 1, as they are both in the same order. The method applied

  2. A process definition repository based on step AP 213

    Energy Technology Data Exchange (ETDEWEB)

    Butler, J.W.

    1997-09-01

    Over the years, in the context of numerically controlled machined part manufacturing, the loss, misinterpretation, and redundancy of re-inputting manufacturing instructions and data during the evolution of a product design into the finished product has been a resource depleting and costly endeavor. It is the intent of this project to utilize the emerging standards from the International Standards Organization, ISO 10303 Standard for the Exchange of Product Model Data, commonly referred to as STEP, to store and retrieve process planning information for a set of production work instructions. The project focuses on the utilization of the 1995 version of the Draft International Standard ISO/DIS 10303-213:1995 (E) Application protocol: Numerical control process plans for machined parts (AP213). This project illustrates the methodologies used to build an object-oriented Process Definition Repository (PDR), describes both the benefits and shortcomings experienced in implementing AP213, and recommends enhancements to AP213 for process planning information. The deliverable will be a Part 21 data file, based on the application-interpreted model for AP213 and integrated with product design data.

  3. Multipliers of Ap((0 ,((0 ,((0,∞)) with order convolution

    Indian Academy of Sciences (India)

    R. Narasimhan (Krishtel eMaging) 1461 1996 Oct 15 13:05:22

    Introduction. The algebra Ap(G) of elements in L1(G) whose Fourier transforms belong to Lp( ˆG) and the multipliers for these algebras have been studied by various authors [1,7–9]. Let. I = (0, ∞) be the locally compact idempotent commutative topological semigroup with the usual topology and max multiplication and ˆI be ...

  4. SEPTUM MAGNET DESIGN FOR THE APS-U

    Energy Technology Data Exchange (ETDEWEB)

    Abliz, M.; Jaski, M.; Xiao, A.; Wienands, U.; Cease, H.; Borland, M.; Decker, G.; Kerby, J.

    2017-06-25

    The Advanced Photon Source is in the process of upgrading its storage ring from a double-bend to a multi-bend lattice as part of the APS Upgrade Project (APS-U). A swap-out injection scheme is planned for the APS-U to keep a constant beam current and to enable a small dynamic aperture. A septum magnet with a minimum thickness of 2 mm and an injection field of 1.06 T has been designed, delivering the required total deflecting angle is 89 mrad with a ring energy of 6 GeV. The stored beam chamber has an 8 mm x 6 mm super-ellipsoidal aperture. The magnet is straight; however, it is tilted in yaw, roll, and pitch from the stored beam chamber to meet the on axis swap out injection requirements for the APS-U lattice. In order to minimize the leakage field inside the stored beam chamber, four different techniques were utilized in the design. As a result, the horizontal deflecting angle of the stored beam was held to only 5 µrad, and the integrated skew quadrupole inside the stored beam chamber was held to 0.09 T. The detailed techniques that were applied to the design, field multipoles, and resulting trajectories of the injected and stored beams are reported.

  5. Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

    Science.gov (United States)

    Szeberenyi, Jozsef

    2013-01-01

    The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

  6. Control of sulfate concentration by miR395-targeted APS genes in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Qin Ai

    2016-04-01

    Full Text Available Sulfur nutrition is crucial for plant growth and development, as well as crop yield and quality. Inorganic sulfate in the soil is the major sulfur source for plants. After uptake, sulfate is activated by ATP sulfurylase, and then gets assimilated into sulfur-containing metabolites. However, the mechanism of regulation of sulfate levels by ATP sulfurylase is unclear. Here, we investigated the control of sulfate levels by miR395-mediated regulation of APS1/3/4. Sulfate was over-accumulated in the shoots of miR395 over-expression plants in which the expression of the APS1, APS3, and APS4 genes was suppressed. Accordingly, reduced expression of miR395 caused a decline of sulfate concentration. In agreement with these results, over-expression of the APS1, APS3, and APS4 genes led to the reduction of sulfate levels. Differential expression of these three APS genes in response to sulfate starvation implied that they have different functions. Further investigation revealed that the regulation of sulfate levels mediated by miR395 depends on the repression of its APS targets. Unlike the APS1, APS3, and APS4 genes, which encode plastid-localized ATP sulfurylases, the APS2 gene encodes a cytosolic version of ATP sulfurylase. Genetic analysis indicated that APS2 has no significant effect on sulfate levels. Our data suggest that miR395-targeted APS genes are key regulators of sulfate concentration in leaves.

  7. PREFACE: Asia-Pacific Interdisciplinary Research Conference 2011 (AP-IRC 2011)

    Science.gov (United States)

    Sandhu, Adarsh; Okada, Hiroshi; Maekawa, Toru; Okano, Ken

    2012-03-01

    AP-IRC Logo Scientists, engineers, entrepreneurs and policymakers gather at the first truly interdisciplinary conference held in Asia-Pacific http://www.apirc.jp/ The inaugural Asia-Pacific Interdisciplinary Research Conference 2011 (AP-IRC 2011) was held at Toyohashi University of Technology (Toyohashi Tech) on 17-18 November 2011. The conference is a forum for enhancing mutual understanding between scientists, engineers, policymakers and experts from a wide spectrum of pure and applied sciences, to resolve the daunting global issues facing mankind. The conference attracted approximately 300 participants including delegates from France, Germany, India, Indonesia, Korea, Malaysia, Russia, Sweden, United Kingdom, USA and Vietnam. AP-IRC 2011 was chaired by Dr Yoshiyuki Sakaki, President of Toyohashi Tech, who opened the proceedings by stressing the importance of an interdisciplinary approach to research, to resolve global scientific and technical issues. Recalling his own experience as the leader of Japan's efforts in the Human Genome Project, Sakaki also encouraged participants to make an effort to try to understand the sometimes difficult concepts and terminology of other areas of research. The presentations at AP-IRC 2011 were divided into three focus sessions: innovative mechano-magneto-electronic systems, life sciences, and green science and technology. A total of 174 papers were presented over the two-day conference including eight by invited speakers. Highlights of AP-IRC 2011 included a first-hand account of the damage caused by the massive earthquake in March 2011 to experimental facilities at Tohoku University by Masayoshi Esashi; the fascinating world of bees and the inborn numerical competence of humans and animals by Hans J Gross; research on robots and cognition-enabled technical systems at Technische Universität München by Sandra Hirche; the history of events leading to the invention of the world's strongest NdFeB permanent magnet by Masato Sagawa

  8. Cerebellar ataxia induced by 3-AP affects immunological function.

    Science.gov (United States)

    Jiang, Yong-Ying; Cao, Bei-Bei; Wang, Xiao-Qin; Peng, Yu-Ping; Qiu, Yi-Hua

    2015-01-01

    We previously showed that the cerebellum modulates the immune system. Here we determined whether cerebellar ataxia alters immunological function to further demonstrate an involvement of the cerebellum in immune modulation. Neurotoxin 3-acetylpyridine (3-AP) was intraperitoneally injected in rats to induce cerebellar ataxia. Behavior and motor coordination were tested on day 7 following 3-AP injection. Nissl staining and high-performance liquid chromatography (HPLC) were used to determine neuronal loss and neurotransmitter contents, respectively, in all the three cerebellar nuclei, fastigial nucleus (FN), interposed nucleus (IN) and dentate nucleus (DN). T and B lymphocyte differentiation and function were measured by flow cytometry, Western blot and ELISA. 3-AP induced motor discoordination and locomotor reduction. In all the three cerebellar nuclei, FN, IN and DN, there was a neuronal loss and a decrease in contents of glutamate and GABA (but not glycine) after 3-AP injection. Importantly, CD4+ T cells, but not CD8+ T cells, were increased by the 3-AP treatment. Moreover, interferon (IFN)-γ-producing cells and interleukin (IL)-17-producing cells were decreased in cerebellar ataxia rats, but IL-4-producing cells and CD25-expressing cells were increased. Expression of the T helper (Th)1- and Th17-related cytokines, IFN-γ, IL-2, IL-17 and IL-22, was downregulated in CD4+ cells in cerebellar ataxia rats, while expression of the Th2 and regulatory T (Treg)-related cytokines, IL-4, IL-5, IL-10 and transforming growth factor (TGF)-β, was upregulated. Furthermore, B lymphocyte number and anti-bovine serum albumin (BSA) IgM and IgG antibody levels were elevated in cerebellar ataxia. Cerebellar ataxia alters cellular and humoral immunity.

  9. Application of modular construction techniques to the AP1000

    Energy Technology Data Exchange (ETDEWEB)

    Winters, James W.; Cummins, W. E. [Westinghouse Electric Company, Windsor (United States)

    2002-04-15

    The new Westinghouse pressurized water reactor, AP1000, has been designed to fully utilize the benefits of modular construction. AP1000 uses passive safety systems and simplified plant support systems. This simplification results in a low overnight cost. To further reduce overall owner costs, the plant has been designed for modular construction. This allows parallel fabrication and construction of large portions of the plant, reducing construction time, thus reducing interest burden during construction. AP1000 uses the same modular approach as the AP600, which has received its Design Certification from the United States Nuclear Regulatory Commission. The standard AP1000 construction schedule is 60 months long from plant order to commercial operation. It consists of 18 months after plant order for site preparation, equipment and module procurement, and excavation for the nuclear island. This is followed by 36 months from pouring of the basemat concrete until fuel load, then 6 months for startup and commissioning testing. This short construction schedule incorporates the early and fabrication of modules. Modules are fabricated using standard shipyard and fabrication shop techniques. The large, building size, structural modules are based on the parallel plate approach used in many double hull ships. The module structure is sufficiently strong to support its interior equipment and outfitting. Concrete is then poured between the plates after module placement to provide shielding and additional structure. This technique eliminates the need for reinforcing bar in the walls and floors and the need to retest installed equipment and systems at site. The smaller, room size, modules are fabricated around conventional steel frames. Equipment and piping is installed and tested prior to shipment. This approach allows considerable parallel work to continue on the plant, shortening the schedule. When considering the overall reduction of cost to the plant investor, modular

  10. Complement-mediated opsonization of invasive group A Streptococcus pyogenes strain AP53 is regulated by the bacterial two-component cluster of virulence responder/sensor (CovRS) system.

    Science.gov (United States)

    Agrahari, Garima; Liang, Zhong; Mayfield, Jeffrey A; Balsara, Rashna D; Ploplis, Victoria A; Castellino, Francis J

    2013-09-20

    Group A Streptococcus pyogenes (GAS) strain AP53 is a primary isolate from a patient with necrotizing fasciitis. These AP53 cells contain an inactivating mutation in the sensor component of the cluster of virulence (cov) responder (R)/sensor (S) two-component gene regulatory system (covRS), which enhances the virulence of the primary strain, AP53/covR(+)S(-). However, specific mechanisms by which the covRS system regulates the survival of GAS in humans are incomplete. Here, we show a key role for covRS in the regulation of opsonophagocytosis of AP53 by human neutrophils. AP53/covR(+)S(-) cells displayed potent binding of host complement inhibitors of C3 convertase, viz. Factor H (FH) and C4-binding protein (C4BP), which concomitantly led to minimal C3b deposition on AP53 cells, further showing that these plasma protein inhibitors are active on GAS cells. This resulted in weak killing of the bacteria by human neutrophils and a corresponding high death rate of mice after injection of these cells. After targeted allelic alteration of covS(-) to wild-type covS (covS(+)), a dramatic loss of FH and C4BP binding to the AP53/covR(+)S(+) cells was observed. This resulted in elevated C3b deposition on AP53/covR(+)S(+) cells, a high level of opsonophagocytosis by human neutrophils, and a very low death rate of mice infected with AP53/covR(+)S(+). We show that covRS is a critical transcriptional regulator of genes directing AP53 killing by neutrophils and regulates the levels of the receptors for FH and C4BP, which we identify as the products of the fba and enn genes, respectively.

  11. Functional analysis of the two Brassica AP3 genes involved in apetalous and stamen carpelloid phenotypes.

    Directory of Open Access Journals (Sweden)

    Yanfeng Zhang

    Full Text Available The Arabidopsis homeotic genes APETALA3 (AP3 and PISTILLATA (PI are B genes which encode MADS-box transcription factors and specify petal and stamen identities. In the current study, the stamen carpelloid (SC mutants, HGMS and AMS, of B. rapa and B. napus were investigated and two types of AP3 genes, B.AP3.a and B.AP3.b, were functional characterized. B.AP3.a and B.AP3.b share high similarity in amino acid sequences except for 8 residues difference located at the C-terminus. Loss of this 8 residues in B.AP3.b led to the change of PI-derived motifs. Meanwhile, B.AP3.a specified petal and stamen development, whereas B.AP3.b only specified stamen development. In B. rapa, the mutations of both genes generated the SC mutant HGMS. In B. napus that contained two B.AP3.a and two B.AP3.b, loss of the two B.AP3.a functions was the key reason for the apetalous mutation, however, the loss-of-function in all four AP3 was related to the SC mutant AMS. We inferred that the 8 residues or the PI-derived motif in AP3 gene probably relates to petal formation.

  12. The AP2-like gene OitaAP2 is alternatively spliced and differentially expressed in inflorescence and vegetative tissues of the orchid Orchis italica.

    Science.gov (United States)

    Salemme, Marinella; Sica, Maria; Iazzetti, Giovanni; Gaudio, Luciano; Aceto, Serena

    2013-01-01

    The AP2/ERF proteins are plant-specific transcription factors involved in multiple regulatory pathways, from plant organ development to response to various environmental stresses. One of the mechanisms that regulates the AP2-like genes involves the microRNA miR172, which controls their activity at the post-transcriptional level. Extensive studies on AP2-like genes are available in many different species; however, in orchids, one of the largest plant families, studies are restricted to a few species, all belonging to the Epidendroideae subfamily. In the present study, we report the isolation of an AP2-like gene in the Mediterranean orchid Orchis italica (Orchidoideae). The OitaAP2 locus includes 10 exons and 9 introns, and its transcript is alternatively spliced, resulting in the long OitaAP2 and the short OitaAP2_ISO isoforms, with the latter skipping exon 9. Both isoforms contain the conserved target site for miR172, whose action is demonstrated by the presence of cleaved OitaAP2 mRNA. The OitaAP2 and OitaAP2_ISO mRNAs are present in the tepals and lip before and after anthesis at different expression levels. In addition, the OitaAP2_ISO isoform is expressed in the ovary before pollination and in the root and stem. The isoform-specific expression pattern suggests a functional differentiation of the OitaAP2 alternatively spliced transcripts. The expression profile of miR172 is complementary to that of the OitaAP2 isoforms in inflorescence tissues before anthesis, whereas after anthesis and in ovary tissue before and after pollination, this relationship disappears, suggesting the existence of OitaAP2 inhibitory mechanisms in these tissues that differ from that involving miR172.

  13. Time resolved spectroscopy of the cool Ap star HD 213637*

    Science.gov (United States)

    Elkin, V. G.; Kurtz, D. W.; Mathys, G.

    2015-02-01

    We present an analysis of high time resolution spectra of the chemically peculiar Ap star HD 213637. The star shows rapid radial velocity variations with a period close to the photometric pulsation period. Radial velocity pulsation amplitudes vary significantly for different rare earth elements. The highest pulsation amplitudes belong to lines of Tb III (˜360 m s-1), Pr II (˜250 m s-1) and Pr III (˜230 m s-1). We did not detect any pulsations from spectral lines of Eu II and in Hα, in contrast to many other roAp stars. We also did not find radial velocity pulsations using spectral lines of other chemical elements, including Mg, Si, Ca, Sc, Cr, Fe, Ni, Y and Ba. There are phase shifts between the maxima of pulsation amplitudes of different rare earth elements and ions, which is evidence of an outwardly running magneto-acoustic wave propagating through the upper stellar atmosphere.

  14. Impedance studies - Part 4: The APS impedance budget

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1988-07-01

    This note will wrap up the numerical results that were obtained in our calculations of the wake potentials, the loss factors, and the impedances for a variety of structures in the APS storage ring. It consists of five sections and one appendix. Section 1 is an introduction. Section 2 summarizes the hand calculations. The computer calculations are the subject 1 of Section 3. Section 4 discusses several tests in our numerical methods. Section 5 presents the APS impedance budget, along with some discussion. The appendix contains the figures of the structures, the longitudinal/transverse wake potentials and the real/imaginary part of the impedances of various sorts of geometries that have been included in the budget.

  15. Validation of the new trapped environment AE9/AP9/SPM at low Earth orbit

    Science.gov (United States)

    Badavi, Francis F.

    2014-09-01

    The completion of the international space station (ISS) in 2011 has provided the space research community an ideal proving ground for future long duration human activities in space. Ionizing radiation measurements in ISS form the ideal tool for the validation of radiation environmental models, nuclear transport codes and nuclear reaction cross sections. Indeed, prior measurements on the space transportation system (STS; shuttle) provided vital information impacting both the environmental models and the nuclear transport code developments by indicating the need for an improved dynamic model of the low Earth orbit (LEO) trapped environment. Additional studies using thermo-luminescent detector (TLD), tissue equivalent proportional counter (TEPC) area monitors, and computer aided design (CAD) model of earlier ISS configurations, confirmed STS observations that, as input, computational dosimetry requires an environmental model with dynamic and directional (anisotropic) behavior, as well as an accurate six degree of freedom (DOF) definition of the vehicle attitude and orientation along the orbit of ISS. At LEO, a vehicle encounters exposure from trapped particles and attenuated galactic cosmic rays (GCR). Within the trapped field, a challenge arises from properly estimating the amount of exposure acquired. There exist a number of models to define the intensities of the trapped particles during the solar quiet and active times. At active times, solar energetic particles (SEP) generated by solar flare or coronal mass ejection (CME) also contribute to the exposure at high northern and southern latitudes. Among the more established trapped models are the historic and popular AE8/AP8, dating back to the 1980s, the historic and less popular CRRES electron/proton, dating back to 1990s and the recently released AE9/AP9/SPM. The AE9/AP9/SPM model is a major improvement over the older AE8/AP8 and CRRES models. This model is derived from numerous measurements acquired over four

  16. UBV photometry of the Ap variable UZ Psc = HD 10783

    Science.gov (United States)

    Hardie, Robert H.; Reichmann, Edwin J.; Burke, Edward W., Jr.; Hall, Douglas S.

    1990-01-01

    The photometric variability of the Ap star HD 10783 = UZ Psc is discussed. New UBV photometry, obtained between late 1965 and early 1969, is presented and it is combined with existing published photometry to derive an improved ephemeris for times of maximum brightness: 2439758.00 + 4d.1328 n. It is concluded that the results are not very sensitive to the small shifts applied to the blue photometry.

  17. Evolutionary and biogeographical implications of degraded LAGLIDADG endonuclease functionality and group I intron occurrence in stony corals (Scleractinia) and mushroom corals (Corallimorpharia).

    Science.gov (United States)

    Celis, Juan Sebastián; Edgell, David R; Stelbrink, Björn; Wibberg, Daniel; Hauffe, Torsten; Blom, Jochen; Kalinowski, Jörn; Wilke, Thomas

    2017-01-01

    Group I introns and homing endonuclease genes (HEGs) are mobile genetic elements, capable of invading target sequences in intron-less genomes. LAGLIDADG HEGs are the largest family of endonucleases, playing a key role in the mobility of group I introns in a process known as 'homing'. Group I introns and HEGs are rare in metazoans, and can be mainly found inserted in the COXI gene of some sponges and cnidarians, including stony corals (Scleractinia) and mushroom corals (Corallimorpharia). Vertical and horizontal intron transfer mechanisms have been proposed as explanations for intron occurrence in cnidarians. However, the central role of LAGLIDADG motifs in intron mobility mechanisms remains poorly understood. To resolve questions regarding the evolutionary origin and distribution of group I introns and HEGs in Scleractinia and Corallimorpharia, we examined intron/HEGs sequences within a comprehensive phylogenetic framework. Analyses of LAGLIDADG motif conservation showed a high degree of degradation in complex Scleractinia and Corallimorpharia. Moreover, the two motifs lack the respective acidic residues necessary for metal-ion binding and catalysis, potentially impairing horizontal intron mobility. In contrast, both motifs are highly conserved within robust Scleractinia, indicating a fully functional endonuclease capable of promoting horizontal intron transference. A higher rate of non-synonymous substitutions (Ka) detected in the HEGs of complex Scleractinia and Corallimorpharia suggests degradation of the HEG, whereas lower Ka rates in robust Scleractinia are consistent with a scenario of purifying selection. Molecular-clock analyses and ancestral inference of intron type indicated an earlier intron insertion in complex Scleractinia and Corallimorpharia in comparison to robust Scleractinia. These findings suggest that the lack of horizontal intron transfers in the former two groups is related to an age-dependent degradation of the endonuclease activity. Moreover

  18. 17β-Estradiol Increases Expression of the Oxidative Stress Response and DNA Repair Protein Apurinic Endonuclease (Ape1) in the Cerebral Cortex of Female Mice Following Hypoxia

    OpenAIRE

    Alicia K., Dietrich; Gwendolyn I Humphreys; Nardulli, Ann M.

    2013-01-01

    While it is well established that 17β-estradiol (E2) protects the rodent brain from ischemia-induced damage, it has been unclear how this neuroprotective effect is mediated. Interestingly, convincing evidence has also demonstrated that maintaining or increasing the expression of the oxidative stress response and DNA repair protein apurinic endonuclease 1 (Ape1) is instrumental in reducing ischemiainduced damage in the brain. Since E2 increases expression of the oxidative stress response prote...

  19. BPM STABILTIY STUDIES FOR THE APS MBA UPGRADE

    Energy Technology Data Exchange (ETDEWEB)

    Lill, R.; Sereno, N.; Yang, B.

    2017-03-25

    The Advanced Photon Source (APS) is currently in the preliminary design phase for the multi-bend achromat (MBA) lattice upgrade. Beam stability is critical for the MBA and will require long term drift defined as beam mo-tion over a seven-day timescale to be no more than 1 mi-cron at the insertion device locations and beam angle change no more than 0.25 micro-radian. Mechanical stabil-ity of beam position monitor (BPM) pickup electrodes mounted on insertion device vacuum chambers place a fun-damental limitation on long-term beam stability for inser-tion device beamlines. We present the design and imple-mentation of prototype mechanical motion system (MMS) instrumentation for quantifying this type of motion specif-ically in the APS accelerator tunnel and experiment hall floor under normal operating conditions. The MMS pres-ently provides critical position information on the vacuum chamber and BPM support systems. Initial results of the R&D prototype systems have demonstrated that the cham-ber movements far exceed the long-term drift tolerance specified for the APS Upgrade MBA storage ring.

  20. Analysis of gymnosperm two-AP2-domain-containing genes.

    Science.gov (United States)

    Shigyo, Mikao; Ito, Motomi

    2004-03-01

    AINTEGUMENTA (ANT) and APETALA2 (AP2) are transcription factors that are involved in several developmental processes in Arabidopsis thaliana. They are similar in structure, containing two AP2 domains, and have partially redundant functions in reproductive organ development. Expression and functional analyses of ANT and AP2 homologs have been performed previously in almost all angiosperms. In this study, one ANT homolog and two AP2 homologs were isolated from the gymnosperm Pinus thunbergii and were named PtANTL1, PtAP2L1, and PtAP2L2. PtANTL1 is the first reported gymnosperm ANT homologous gene. Based on a gene tree constructed with sequences of all A. thaliana two-AP2-domain-containing genes, it is likely that PtANTL1 and ANT, and likewise PtAP2L1 and AP2, are orthologs. The expression patterns of PtANTL1/PtAP2L1/PtAP2L2 were examined with Southern hybridization of the quantitative RT-PCR products and in situ hybridization. PtANTL1 and PtAP2L1 had almost identical expression patterns in the analyzed organs, and PtANTL1/PtAP2L1/PtAP2L2 were continually expressed in the developing female cone. Our analysis suggests that gymnosperms have orthologs to both ANT and AP2, and that the most recent common ancestor of extant seed plants has two type AP2 subfamily genes, ANT-like and AP2-like, involved in the development of female reproductive organs.