WorldWideScience

Sample records for bethlehem dnab intein

  1. Künstlich gespaltene Inteine für die Protein-Semisynthese - Charakterisierung des Ssp DnaB Inteins und Modifikation des Ionenkanals OmpF mit Hilfe des Psp-GBD Pol Inteins

    OpenAIRE

    Brenzel, Steffen

    2010-01-01

    Gespaltene Inteine sind leistungsfähige Werkzeuge zur Knüpfung nativer Peptidbindungen zwischen Polypeptidsequenzen. Die von ihnen vermittelte Protein trans-Splei߬reaktion beginnt zunächst mit der Assoziation der Fragmente des gespaltenen Inteins, die Teile von zwei separaten Fusionsproteinen sind. Der gebildete spleißkompetente Inteinkomplex schnei¬det sich anschließend aus dem Vorläufer¬protein heraus und verbindet dab...

  2. Kepler and the Star of Bethlehem

    Science.gov (United States)

    Hansen, Rahlf

    Johannes Kepler (1571-1630) was a famous astronomer. But like other astronomers he had a problem to find work that would guarantee a regular income. So he was lucky to get work as "Styrian landscape mathematician" in Graz. One of his tasks was to write an annual calendar of weather forecasts and policital developments on the basis of astrological facts. He correctly predicted a conflict with the Osmanic Empire, although it is not clear whether the stars or the newspapers were the cause for that. Both his horoscope for Wallenstein and his book "Warnung an die Gegner der Astrologie" are well known. Kepler believed in some aspects of astrology, the influence of the planets for example. He deduced this front his ideas about physics. He neglected other aspects of astrology. e.g. the significance of the zodiac. In 1604 Kepler observed a new star and believed in a connection to a special and very rare planetary conjunction. After a Jupiter-Saturn-conjunction Jupiter met Mars. Kepler speculated that the star of Bethlehem might be a new star which was generated after a similar conjunction and recalculated it for 6/7 BC. Nowadays examples of both astronomical (and astrological) interpretations of the star of Bethlehem exist. The best known is the three time conjunction of 6/7 BC. But the interpretation of Martin (1980) for 213 BC seems equally excellent. Vardaman (1989) takes the Halley comet of 12 BC to be the star of Bethlehem. Other speculations arise from two Novae in the years 5 and 4 BC, tabulated in sources from the Far East. But historians tell us that there is no need fo a real star. The text in Matthew, book 2 is a legend. What is important in regard to the understanding of the star of Bethlehem is the "sidus Julium" the comet which could be seen in the sky during Caesar's funeral and the match of the King of Armenia Tiridates to Nero in Rome during. There was no real star over Bethlehem. All we have are interesting speculations, like those by Kepler.

  3. A new example of viral intein in Mimivirus

    Directory of Open Access Journals (Sweden)

    Claverie Jean-Michel

    2005-02-01

    Full Text Available Abstract Background Inteins are "protein introns" that remove themselves from their host proteins through an autocatalytic protein-splicing. After their discovery, inteins have been quickly identified in all domains of life, but only once to date in the genome of a eukaryote-infecting virus. Results Here we report the identification and bioinformatics characterization of an intein in the DNA polymerase PolB gene of amoeba infecting Mimivirus, the largest known double-stranded DNA virus, the origin of which has been proposed to predate the emergence of eukaryotes. Mimivirus intein exhibits canonical sequence motifs and clearly belongs to a subclass of archaeal inteins always found in the same location of PolB genes. On the other hand, the Mimivirus PolB is most similar to eukaryotic Polδ sequences. Conclusions The intriguing association of an extremophilic archaeal-type intein with a mesophilic eukaryotic-like PolB in Mimivirus is consistent with the hypothesis that DNA viruses might have been the central reservoir of inteins throughout the course of evolution.

  4. Synthesis of an Intein-mediated Artificial Protein Hydrogel

    OpenAIRE

    Ramirez, Miguel A.; Chen, Zhilei

    2014-01-01

    We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, y...

  5. Design of a Split Intein with Exceptional Protein Splicing Activity.

    Science.gov (United States)

    Stevens, Adam J; Brown, Zachary Z; Shah, Neel H; Sekar, Giridhar; Cowburn, David; Muir, Tom W

    2016-02-24

    Protein trans-splicing (PTS) by split inteins has found widespread use in chemical biology and biotechnology. Herein, we describe the use of a consensus design approach to engineer a split intein with enhanced stability and activity that make it more robust than any known PTS system. Using batch mutagenesis, we first conduct a detailed analysis of the difference in splicing rates between the Npu (fast) and Ssp (slow) split inteins of the DnaE family and find that most impactful residues lie on the second shell of the protein, directly adjacent to the active site. These residues are then used to generate an alignment of 73 naturally occurring DnaE inteins that are predicted to be fast. The consensus sequence from this alignment (Cfa) demonstrates both rapid protein splicing and unprecedented thermal and chaotropic stability. Moreover, when fused to various proteins including antibody heavy chains, the N-terminal fragment of Cfa exhibits increased expression levels relative to other N-intein fusions. The durability and efficiency of Cfa should improve current intein based technologies and may provide a platform for the development of new protein chemistry techniques. PMID:26854538

  6. Inteins in pathogenic fungi: a phylogenetic tool and perspectives for therapeutic applications

    OpenAIRE

    Raquel Cordeiro Theodoro; Eduardo Bagagli

    2009-01-01

    Inteins or "internal proteins" are coding sequences that are transcribed and translated with flanking sequences (exteins). After translation, the inteins are excised by an autocatalytic process and the host protein assumes its normal conformation and develops its expected function. These parasitic genetic elements have been found in important, conserved proteins in all three domains of life. Most of the eukaryotic inteins are present in the fungi kingdom and the PRP8 intein is one of the most...

  7. Mechanism of protein splicing of the Pyrococcus abyssi lon protease intein

    International Nuclear Information System (INIS)

    Research highlights: → The Pyrococcus abyssi lon protease intein promotes efficient protein splicing. → Inteins with mutations that interfere with individual steps of splicing do not promote unproductive side reactions. → The intein splices with Lys in place of the highly conserved penultimate His. → The intein is flanked by a Gly-rich region at its C terminus that may increase the efficiency of the third step of splicing, Asn cyclization coupled to peptide bond cleavage. -- Abstract: Protein splicing is a post-translational process by which an intervening polypeptide, the intein, excises itself from the flanking polypeptides, the exteins, coupled to ligation of the exteins. The lon protease of Pyrococcus abyssi (Pab) is interrupted by an intein. When over-expressed as a fusion protein in Escherichia coli, the Pab lon protease intein can promote efficient protein splicing. Mutations that block individual steps of splicing generally do not lead to unproductive side reactions, suggesting that the intein tightly coordinates the splicing process. The intein can splice, although it has Lys in place of the highly conserved penultimate His, and mutants of the intein in the C-terminal region lead to the accumulation of stable branched-ester intermediate.

  8. Mechanism of protein splicing of the Pyrococcus abyssi lon protease intein

    Energy Technology Data Exchange (ETDEWEB)

    O' Brien, Kevin M.; Schufreider, Ann K.; McGill, Melissa A.; O' Brien, Kathryn M.; Reitter, Julie N. [Department of Chemistry, College of the Holy Cross, Worcester, MA 01610 (United States); Mills, Kenneth V., E-mail: kmills@holycross.edu [Department of Chemistry, College of the Holy Cross, Worcester, MA 01610 (United States)

    2010-12-17

    Research highlights: {yields} The Pyrococcus abyssi lon protease intein promotes efficient protein splicing. {yields} Inteins with mutations that interfere with individual steps of splicing do not promote unproductive side reactions. {yields} The intein splices with Lys in place of the highly conserved penultimate His. {yields} The intein is flanked by a Gly-rich region at its C terminus that may increase the efficiency of the third step of splicing, Asn cyclization coupled to peptide bond cleavage. -- Abstract: Protein splicing is a post-translational process by which an intervening polypeptide, the intein, excises itself from the flanking polypeptides, the exteins, coupled to ligation of the exteins. The lon protease of Pyrococcus abyssi (Pab) is interrupted by an intein. When over-expressed as a fusion protein in Escherichia coli, the Pab lon protease intein can promote efficient protein splicing. Mutations that block individual steps of splicing generally do not lead to unproductive side reactions, suggesting that the intein tightly coordinates the splicing process. The intein can splice, although it has Lys in place of the highly conserved penultimate His, and mutants of the intein in the C-terminal region lead to the accumulation of stable branched-ester intermediate.

  9. Impact of a homing intein on recombination frequency and organismal fitness.

    Science.gov (United States)

    Naor, Adit; Altman-Price, Neta; Soucy, Shannon M; Green, Anna G; Mitiagin, Yulia; Turgeman-Grott, Israela; Davidovich, Noam; Gogarten, Johann Peter; Gophna, Uri

    2016-08-01

    Inteins are parasitic genetic elements that excise themselves at the protein level by self-splicing, allowing the formation of functional, nondisrupted proteins. Many inteins contain a homing endonuclease (HEN) domain and rely on its activity for horizontal propagation. However, successful invasion of an entire population will make this activity redundant, and the HEN domain is expected to degenerate quickly under these conditions. Several theories have been proposed for the continued existence of the both active HEN and noninvaded alleles within a population. However, to date, these models were not directly tested experimentally. Using the natural cell fusion ability of the halophilic archaeon Haloferax volcanii we were able to examine this question in vivo, by mating polB intein-positive [insertion site c in the gene encoding DNA polymerase B (polB-c)] and intein-negative cells and examining the dispersal efficiency of this intein in a natural, polyploid population. Through competition between otherwise isogenic intein-positive and intein-negative strains we determined a surprisingly high fitness cost of over 7% for the polB-c intein. Our laboratory culture experiments and samples taken from Israel's Mediterranean coastline show that the polB-c inteins do not efficiently take over an inteinless population through mating, even under ideal conditions. The presence of the HEN/intein promoted recombination when intein-positive and intein-negative cells were mated. Increased recombination due to HEN activity contributes not only to intein dissemination but also to variation at the population level because recombination tracts during repair extend substantially from the homing site. PMID:27462108

  10. A general purification platform for toxic proteins based on intein trans-splicing.

    Science.gov (United States)

    Shi, Changhua; Tarimala, Anirudh; Meng, Qing; Wood, David W

    2014-11-01

    Many important functional proteins often exhibit toxicity when overexpressed in heterologous hosts. Unfortunately, this toxicity can complicate the production of these proteins in recombinant systems, which can slow their characterization. Although a number of engineered expression strains and plasmids have been developed to optimize toxic protein expression, many targets remain recalcitrant in these systems due to extreme toxicity to the expression host. In this work, we have developed a novel protein purification platform based on intein trans-splicing, with special relevance for proteins that are extremely toxic to recombinant host cells. The toxic protein is split into two inactive fragments, which are separately expressed in fusion to the segments of a split intein. The N-terminal intein segment is first immobilized onto an affinity column and washed, followed by addition of the C-terminal segment and purification of the complex. The assembled intein controllably splices to deliver the mature target protein, simultaneously releasing the purified target from the affinity column. To optimize this method, we generated a hybrid split intein consisting of the N-terminus of the Npu DnaE intein and the C-terminus of the Ssp DnaE intein. This hybrid intein tolerates a wider range of amino acids at the +2 site of the C-terminal splicing junction than the Npu intein alone. In the production of the highly toxic homing endonuclease I-TevI, the yield from the hybrid intein is 50 % higher than the native Npu DnaE intein, while the I-TevI protein purified from both inteins showed native activity. PMID:25296714

  11. Intein Applications: From Protein Purification and Labeling to Metabolic Control Methods*

    OpenAIRE

    Wood, David W.; Camarero, Julio A.

    2014-01-01

    The discovery of inteins in the early 1990s opened the door to a wide variety of new technologies. Early engineered inteins from various sources allowed the development of self-cleaving affinity tags and new methods for joining protein segments through expressed protein ligation. Some applications were developed around native and engineered split inteins, which allow protein segments expressed separately to be spliced together in vitro. More recently, these early applications have been expand...

  12. Deoxyribonucleic acid initiation mutation dnaB252 is suppressed by elevated dnaC+ gene dosage.

    OpenAIRE

    Sclafani, R A; Wechsler, J A

    1981-01-01

    The Escherichia coli dnaB252 allele is the only dnaB mutation which confers a deoxyribonucleic acid initiation-defective phenotype on the cell. The presence of a multicopy hybrid plasmid containing the dnaC+ gene in a dnaB252 strain completely suppressed the temperature-sensitive phenotype. It is suggested that at high temperature the dnaB252 protein has a lowered affinity for dnaC protein, and that the formation of a dnaB-dnaC complex is mandatory for initiation.

  13. An intein with genetically selectable markers provides a new approach to internally label proteins with GFP

    Directory of Open Access Journals (Sweden)

    Davis Trisha N

    2011-06-01

    Full Text Available Abstract Background Inteins are proteins that catalyze their own removal from within larger precursor proteins. In the process they splice the flanking protein sequences, termed the N-and C-terminal exteins. Large inteins frequently have a homing endonuclease that is involved in maintaining the intein in the host. Splicing and nuclease activity are independent and distinct domains in the folded structure. We show here that other biochemical activities can be incorporated into an intein in place of the endonuclease without affecting splicing and that these activities can provide genetic selection for the intein. We have coupled such a genetically marked intein with GFP as the N-terminal extein to create a cassette to introduce GFP within the interior of a targeted protein. Results The Pch PRP8 mini-intein of Penicillium chrysogenum was modified to include: 1 aminoglycoside phosphotransferase; 2 imidazoleglycerol-phosphate dehydratase, His5 from S. pombe ; 3 hygromycin B phosphotransferase; and 4 the transcriptional activator LexA-VP16. The proteins were inserted at the site of the lost endonuclease. When expressed in E. coli, all of the modified inteins spliced at high efficiency. Splicing efficiency was also greater than 96% when expressed from a plasmid in S. cerevisiae. In addition the inteins conferred either G418 or hygromycin resistance, or histidine or leucine prototropy, depending on the inserted marker and the yeast genetic background. DNA encoding the marked inteins coupled to GFP as the N-terminal extein was PCR amplified with ends homologous to an internal site in the yeast calmodulin gene CMD1. The DNA was transformed into yeast and integrants obtained by direct selection for the intein's marker. The His5-marked intein yielded a fully functional calmodulin that was tagged with GFP within its central linker. Conclusions Inteins continue to show their flexibility as tools in molecular biology. The Pch PRP8 intein can successfully

  14. Distinct evolutionary histories of the DNA-A and DNA-B components of bipartite begomoviruses

    Directory of Open Access Journals (Sweden)

    Nawaz-ul-Rehman Muhammad

    2010-04-01

    Full Text Available Abstract Background Viruses of the genus Begomovirus (family Geminiviridae have genomes consisting of either one or two genomic components. The component of bipartite begomoviruses known as DNA-A is homologous to the genomes of all geminiviruses and encodes proteins required for replication, control of gene expression, overcoming host defenses, encapsidation and insect transmission. The second component, referred to as DNA-B, encodes two proteins with functions in intra- and intercellular movement in host plants. The origin of the DNA-B component remains unclear. The study described here was initiated to investigate the relationship between the DNA-A and DNA-B components of bipartite begomoviruses with a view to unraveling their evolutionary histories and providing information on the possible origin of the DNA-B component. Results Comparative phylogenetic and exhaustive pairwise sequence comparison of all DNA-A and DNA-B components of begomoviruses demonstrates that the two molecules have very distinct molecular evolutionary histories and likely are under very different evolutionary pressures. The analysis highlights that component exchange has played a far greater role in diversification of begomoviruses than previously suspected, although there are distinct differences in the apparent ability of different groups of viruses to utilize this "sexual" mechanism of genetic exchange. Additionally we explore the hypothesis that DNA-B originated as a satellite that was captured by the monopartite progenitor of all extant bipartite begomoviruses and subsequently evolved to become the integral (essential genome component that we recognize today. The situation with present-day satellites associated with begomoviruses provides some clues to the processes and selection pressures that may have led to the "domestication" of a wild progenitor of the DNA-B component. Conclusions The analysis has highlighted the greater genetic variation of DNA-B components, in

  15. Multiple, non-allelic, intein-coding sequences in eukaryotic RNA polymerase genes

    Directory of Open Access Journals (Sweden)

    Butler Margaret I

    2006-10-01

    Full Text Available Abstract Background Inteins are self-splicing protein elements. They are translated as inserts within host proteins that excise themselves and ligate the flanking portions of the host protein (exteins with a peptide bond. They are encoded as in-frame insertions within the genes for the host proteins. Inteins are found in all three domains of life and in viruses, but have a very sporadic distribution. Only a small number of intein coding sequences have been identified in eukaryotic nuclear genes, and all of these are from ascomycete or basidiomycete fungi. Results We identified seven intein coding sequences within nuclear genes coding for the second largest subunits of RNA polymerase. These sequences were found in diverse eukaryotes: one is in the second largest subunit of RNA polymerase I (RPA2 from the ascomycete fungus Phaeosphaeria nodorum, one is in the RNA polymerase III (RPC2 of the slime mould Dictyostelium discoideum and four intein coding sequences are in RNA polymerase II genes (RPB2, one each from the green alga Chlamydomonas reinhardtii, the zygomycete fungus Spiromyces aspiralis and the chytrid fungi Batrachochytrium dendrobatidis and Coelomomyces stegomyiae. The remaining intein coding sequence is in a viral relic embedded within the genome of the oomycete Phytophthora ramorum. The Chlamydomonas and Dictyostelium inteins are the first nuclear-encoded inteins found outside of the fungi. These new inteins represent a unique dataset: they are found in homologous proteins that form a paralogous group. Although these paralogues diverged early in eukaryotic evolution, their sequences can be aligned over most of their length. The inteins are inserted at multiple distinct sites, each of which corresponds to a highly conserved region of RNA polymerase. This dataset supports earlier work suggesting that inteins preferentially occur in highly conserved regions of their host proteins. Conclusion The identification of these new inteins

  16. Development of an intein-mediated split-Cas9 system for gene therapy

    OpenAIRE

    Truong, D.J.J.; Kuehner, K.; Kuehn, R.; Werfel, S.; Engelhardt, S.; WURST, W; Ortiz, O.

    2015-01-01

    Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split–Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 pro...

  17. Investigating hexameric helicases: Single-molecule studies of DnaB and T4 gp41

    Science.gov (United States)

    Saleh, Omar; Ribeck, Noah; Berezney, John

    2011-03-01

    Hexameric, ring-shaped motor proteins serve as replicative helicases in many systems. They function by encircling and translocating along ssDNA, denaturing dsDNA in advance of its motion by sterically occluding the complementary strand to the outside of the ring. We investigate the helicase activity of two such motors using single-molecule measurements with magnetic tweezers. First, we measure the activity of the E. coli helicase DnaB complexed with the tau subunit of the Pol III holoenzyme. Tau is known from bulk measurements to stimulate DnaB activity (Kim et al., Cell, 1996); we investigate the means of this stimulation. Second, we measure helicase activity of the T4 phage helicase gp41 in multiple tethered DNA geometries. Previous work on DnaB showed a dependence of helicase activity on DNA geometry (Ribeck et al., Biophys. J., 2010); here, we test gp41 for similar behavior to see whether it is a common characteristic of hexameric helicases.

  18. Cryptic single-stranded-DNA binding activities of the phage λ P and Escherichia coli DnaC replication initiation proteins facilitate the transfer of E. coli DnaB helicase onto DNA

    OpenAIRE

    Learn, Brian A.; Um, Soo-Jong; Huang, Li; McMacken, Roger

    1997-01-01

    The bacteriophage λ P and Escherichia coli DnaC proteins are known to recruit the bacterial DnaB replicative helicase to initiator complexes assembled at the phage and bacterial origins, respectively. These specialized nucleoprotein assemblies facilitate the transfer of one or more molecules of DnaB helicase onto the chromosome; the transferred DnaB, in turn, promotes establishment of a processive replication fork apparatus. To learn more about the mechanism of the DnaB transfer reaction, we ...

  19. The Bacillus subtilis DnaD and DnaB Proteins Exhibit Different DNA Remodelling Activities

    OpenAIRE

    Zhang, Wenke; Carneiro, Maria J. V. M.; Turner, Ian J.; ALLEN, Stephanie; Roberts, Clive J.; Soultanas, Panos

    2005-01-01

    Primosomal protein cascades load the replicative helicase onto DNA. In Bacillus subtilis a putative primosomal cascade involving the DnaD-DnaB-DnaI proteins has been suggested to participate in both the DnaA and PriA-dependent loading of the replicative helicase DnaC onto the DNA. Recently we discovered that DnaD has a global remodelling DNA activity suggesting a more widespread role in bacterial nucleoid architecture. Here, we show that DnaB forms a “square-like” tetramer with a hole in the ...

  20. An Essential DnaB Helicase of Bacillus anthracis: Identification, Characterization, and Mechanism of Action▿

    OpenAIRE

    Biswas, Esther E.; Barnes, Marjorie H.; Moir, Donald T.; Biswas, Subhasis B

    2008-01-01

    We have described a novel essential replicative DNA helicase from Bacillus anthracis, the identification of its gene, and the elucidation of its enzymatic characteristics. Anthrax DnaB helicase (DnaBBA) is a 453-amino-acid, 50-kDa polypeptide with ATPase and DNA helicase activities. DnaBBA displayed distinct enzymatic and kinetic properties. DnaBBA has low single-stranded DNA (ssDNA)-dependent ATPase activity but possesses a strong 5′→3′ DNA helicase activity. The stimulation of ATPase activi...

  1. Mechanism of recruitment of DnaB helicase to the replication origin of the plasmid pSC101

    OpenAIRE

    Datta, Hirock J.; Khatri, Ghan Shyam; Bastia, Deepak

    1999-01-01

    Although many bacterial chromosomes require only one replication initiator protein, e.g., DnaA, most plasmid replicons depend on dual initiators: host-encoded DnaA and plasmid-encoded Rep initiator protein for replication initiation. Using the plasmid pSC101 as a model system, this work investigates the biological rationale for the requirement for dual initiators and shows that the plasmid-encoded RepA specifically interacts with the replicative helicase DnaB. Mutations in DnaB or RepA that d...

  2. Intein-mediated purification of cytotoxic endonuclease I-TevI by insertional inactivation and pH-controllable splicing.

    Science.gov (United States)

    Wu, Wei; Wood, David W; Belfort, Georges; Derbyshire, Victoria; Belfort, Marlene

    2002-11-15

    An intein-mediated approach was developed for expression and affinity purification of a protein that is lethal to Escherichia coli. The protein, I-TevI, is an intron-encoded endonuclease. The approach involved the insertional inactivation of I-TevI with a controllable mini-intein placed in front of a cysteine required for splicing (an I-TevI::intein fusion). The purification was facilitated by a chitin-binding domain inserted into the mini-intein. Affinity purification of the I-TevI::intein fusion precursor on a chitin column was followed by pH-controllable splicing to restore the structure and function of I-TevI. To study the impact of the insertion context on I-TevI inactivation, the chimeric intein was inserted independently in front of seven cysteines of I-TevI. One of the seven intein integrants yielded I-TevI of high activity. This technique is, in principle, generalizable to the expression and purification of other cytotoxic proteins and is amenable to scale-up. PMID:12433989

  3. Chimeric spider silk proteins mediated by intein result in artificial hybrid silks.

    Science.gov (United States)

    Lin, Senzhu; Chen, Gefei; Liu, Xiangqin; Meng, Qing

    2016-07-01

    Hybrid silks hold a great potential as specific biomaterials due to its controlled mechanical properties. To produce fibers with tunable properties, here we firstly made chimeric proteins in vitro, called W2C4CT and W2C8CT, with ligation of MaSp repetitive modules (C) with AcSp modules (W) by intein trans splicing technology from smaller precursors without final yield reduction. Intein mediated chimeric proteins form fibers at a low concentration of 0.4 mg/mL in 50 mM K3 PO4 pH 7.5 just drawn by hand. Hybrid fibers show smoother surface, and also have stronger chemical resistance as compared with fibers from W2CT (W fibers) and mixture of W2CT/C8CT (MHF8 fibers). Fibers from chimeric protein W2C4CT (HFH4) have improved mechanical properties than W fibers; however, with more C modules W2C8CT fibers (HFH8) properties decreased, indicates the length proportion of various modules is very important and should be optimized for fibers with specific properties. Generally, hybrid silks generated via chimeric proteins, which can be simplified by intein trans splicing, has greater potential to produce fibers with tunable properties. Our research shows that intein mediated directional protein ligation is a novel way to make large chimeric spider silk proteins and hybrid silks. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 385-392, 2016. PMID:26948769

  4. Intein-mediated site-specific conjugation of Quantum Dots to proteins in vivo

    Directory of Open Access Journals (Sweden)

    Skourides Paris A

    2009-12-01

    Full Text Available Abstract We describe an intein based method to site-specifically conjugate Quantum Dots (QDs to target proteins in vivo. This approach allows the covalent conjugation of any nanostructure and/or nanodevice to any protein and thus the targeting of such material to any intracellular compartment or signalling complex within the cells of the developing embryo. We genetically fused a pleckstrin-homology (PH domain with the N-terminus half of a split intein (IN. The C-terminus half (IC of the intein was conjugated to QDs in vitro. IC-QD's and RNA encoding PH-IN were microinjected into Xenopus embryos. In vivo intein-splicing resulted in fully functional QD-PH conjugates that could be monitored in real time within live embryos. Use of Near Infra Red (NIR-emitting QDs allowed monitoring of QD-conjugates within the embryo at depths where EGFP is undetectable demonstrating the advantages of QD's for this type of experiment. In conclusion, we have developed a novel in vivo methodology for the site-specific conjugation of QD's and other artificial structures to target proteins in different intracellular compartments and signaling complexes.

  5. Intein-mediated Rapid Purification of Recombinant Human Pituitary Adenylate Cyclase Activating Polypeptide

    Institute of Scientific and Technical Information of China (English)

    Rong-jie YU; An HONG; Yun DAI; Yuan GAO

    2004-01-01

    In order to obtain the recombinant human PACAP efficiently by intein-mediated single column purification, a gene encoding human PACAP was synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-PAC was transferred into E. coli ER2566 cells and the target protein was over-expressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the PACAPintein-CBD fusion protein was purified by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by DTT and the rhPACAP was released from the chitin-bound intein tag. The activity of the rhPACAP to stimulate cyclic AMP accumulation was detected using the human pancreas carcinoma cells SW1990. Twenty-two milligrams of rhPACAP with the purity over 98% was obtained by single column purification from 1 liter of induced culture. The preliminary biological assay indicated that the rhPACAP, which has an extra Met at its N-terminus compared with the native human PACAP, had the similar activity of stimulating cAMP accumulation with the standard PACAP38 in the SW1990 cells. A new efficient production procedure of the active recombinant human PACAP was established.

  6. Primosomal Proteins DnaD and DnaB Are Recruited to Chromosomal Regions Bound by DnaA in Bacillus subtilis▿

    OpenAIRE

    Smits, Wiep Klaas; Merrikh, Houra; Bonilla, Carla Yaneth; Grossman, Alan D.

    2010-01-01

    The initiation of DNA replication requires the binding of the initiator protein, DnaA, to specific binding sites in the chromosomal origin of replication, oriC. DnaA also binds to many sites around the chromosome, outside oriC, and acts as a transcription factor at several of these. In low-G+C Gram-positive bacteria, the primosomal proteins DnaD and DnaB, in conjunction with loader ATPase DnaI, load the replicative helicase at oriC, and this depends on DnaA. DnaD and DnaB also are required to...

  7. KRESS INDIRECT DRY COOLING SYSTEM, BETHLEHEM STEEL'S COKE PLANT DEMONSTRATION AT SPARROWS POINT, MARYLAND - VOLUME 2. APPENDICES G-N

    Science.gov (United States)

    The report evaluates the Kress Indirect Dry Cooling (KIDC) process, an innovative system for handling and cooling coke produced from a slot-type by-product coke oven battery. The report is based on the test work and demonstration of the system at Bethlehem Steel Corporation's Sp...

  8. Association of tomato leaf curl New Delhi virus DNA-B with bhendi yellow vein mosaic virus in okra showing yellow vein mosaic disease symptoms.

    Science.gov (United States)

    Venkataravanappa, V; Lakshminarayana Reddy, C N; Jalali, S; Krishna Reddy, M

    2015-06-01

    Okra samples showing yellow vein mosaic, vein twisting and bushy appearance were collected from different locations of India during the surveys conducted between years 2005-2009. The dot blot and PCR detection revealed that 75.14% of the samples were associated with monopartite begomovirus and remaining samples with bipartite virus. Whitefly transmission was established for three samples representing widely separated geographical locations which are negative to betasatellites and associated with DNA-B. Genome components of these three representative isolates were cloned and sequenced. The analysis of DNA-A-like sequence revealed that three begomovirus isolates shared more than 93% nucleotide sequence identity with bhendi yellow vein mosaic virus from India (BYVMV), a monopartite begomovirus species that was reported previously as causative agent of bhendi yellow mosaic disease in association of bhendi yellow vein mosaic betasatellite. Further, the DNA-B-like sequences associated with the three virus isolates shared no more than 90% sequence identity with tomato leaf curl New Delhi virus (ToLCNDV). Analyses of putative iteron-binding sequence required for trans-replication suggests that begomovirus sequences shared compatible rep-binding iterons with DNA-B of ToLCNDV. Our data suggest that the monopartite begomovirus associated with okra yellow vein disease has captured DNA-B of ToLCNDV to infect okra. Widespread distribution of the complex shows the increasing trend of the capturing of DNA-B of ToLCNDV by monopartite begomoviruses in the Indian subcontinent. The recombination analysis showed that the DNA-A might have been derived from the inter-specific recombination of begomoviruses, while DNA-B was derived from the ToLCNDV infecting different hosts. PMID:26104329

  9. 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

  10. The Sustainable Viability of Adaptive Reuse of Historic Buildings : the experiences of Two World Heritage Old Cities; Bethlehem in Palestine and Visby in Sweden

    OpenAIRE

    Ijla, Akram; Broström, Tor

    2015-01-01

    The paper aims at investigating the viability of adaptive reuse of abandoned buildings (religious, Nobel Architecture, residential, commercial, and other) and the impact it has on the sustainability of existing environment in Bethlehem and Visby. There are many historic buildings in Bethlehem and Visby that are unique in their history, architecture, and built environment. This paper explores the importance of adaptive reuse by looking at several examples of reused historic buildings in both c...

  11. Resolució de l'estructura tridimensional de l'helicasa hexamètrica DnaB

    OpenAIRE

    Arribas Bosacoma, Raquel

    2009-01-01

    Es presenta el model atòmic a 4.5 Å de DnaB, la principal helicasa replicativa bacteriana, d'Aquifex aeolicus. És un anell hexamèric de 100 Å d'amplada i 80 Å d'alçada amb dues capes de simetria diferenciada, la dels dominis N-terminals en C3 i la dels C-terminals propera a C6. El diàmetre central és de 25 Å al llarg d'ambdues capes, principal diferència amb les estructures prèvies, on era 25 Å més estret a la capa N-terminal. L'estretament s'origina pel trencament d'una de les dues superfíci...

  12. Cloning, expression, purification, crystallization and preliminary X-ray diffraction data of the Pyrococcus horikoshii RadA intein

    International Nuclear Information System (INIS)

    Cloning, expression, purification and X-ray data collection of the RadA intein from the hyperthermophilic archaebacterium P. horikoshii are reported. The RadA intein from the hyperthermophilic archaebacterium Pyrococcus horikoshii was cloned, expressed and purified for subsequent structure determination. The protein crystallized rapidly in several conditions. The best crystals, which diffracted to 1.75 Å resolution, were harvested from drops consisting of 0.1 M HEPES pH 7.5, 3.0 M NaCl and were cryoprotected with Paratone-N before flash-cooling. The collected data were processed in the orthorhombic space group P212121, with unit-cell parameters a = 58.1, b = 67.4, c = 82.9 Å. Molecular replacement with Rosetta using energy- and density-guided structure optimization provided the initial solution, which is currently under refinement

  13. Cost effective purification of intein based syntetic cationic antimicrobial peptide expressed in cold shock expression system using salt inducible E. coli GJ1158

    Directory of Open Access Journals (Sweden)

    Seetha Ram Kotra

    2014-03-01

    Full Text Available Objective:Synthetic cationic antimicrobial peptide (SC-AMP is an important and upcoming therapeutic molecule against onventional antibiotics. In this study, an attempt was made to purify the SC-AMP without the enzymatic cleavage of the affinity tag, by using an intein-based system. Methods:The intein sequence was amplified from pTYB11 vector using PCR methodologies and the N-terminal of intein was ligated with SC-AMP. The designed construct, intein-SC-AMP was cloned into MCS region of cold shock expression vector, pCOLDI and the recombinant peptide was purified on a chitin affinity column by cleaving intein with 50 mM DTT without applying enzymatic cleavage. Later the peptide was quantified and its antibacterial activity of the purified peptide was studied using well diffusion method. Results: Initially, intein-SC-AMP was expressed as a fusion protein in both IPTG inducible E. coli BL21(DE3 and salt inducible E. coli GJ1158. Single step purification using CBD (chitin binding domain - intein tag in salt inducible E. coli GJ1158, yields the SC-AMP in the soluble form at a oncentration of 208 mg/L. The antibacterial activity and minimal inhibitory concentration (MIC of the purified SC-AMP was studied against both Gram positive and Gram negative microorganisms. Conclusion: For the first time, single step purification of soluble SC-AMP was carried out using chitin-binding domain affinity tag in salt inducible E. coli GJ1158 without an application of enzymatic cleavage. J Microbiol Infect Dis 2014;4(1:13-19

  14. Three-dimensional structure of N-terminal domain of DnaB helicase and helicase-primase interactions in Helicobacter pylori.

    Directory of Open Access Journals (Sweden)

    Tara Kashav

    Full Text Available Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD of H. pylori DnaB (HpDnaB helicase at 2.2 A resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria.

  15. Composition of cuticular waxes coating flag leaf blades and peduncles of Triticum aestivum cv. Bethlehem.

    Science.gov (United States)

    Racovita, Radu C; Hen-Avivi, Shelly; Fernandez-Moreno, Josefina-Patricia; Granell, Antonio; Aharoni, Asaph; Jetter, Reinhard

    2016-10-01

    The work herein presents comprehensive analyses of the cuticular wax mixtures covering the flag leaf blade and peduncle of bread wheat (Triticum aestivum) cv. Bethlehem. Overall, Gas Chromatography-Mass Spectrometry and Flame Ionization Detection revealed a wax coverage of flag leaf blades (16 μg/cm(2)) a third that of peduncles (49 μg/cm(2)). Flag leaf blade wax was dominated by 1-alkanols, while peduncle wax contained primarily β-diketone and hydroxy-β-diketones, thus suggesting differential regulation of the acyl reduction and β-diketone biosynthetic pathways in the two analyzed organs. The characteristic chain length distributions of the various wax compound classes are discussed in light of their individual biosynthetic pathways and biosynthetic relationships between classes. Along with previously reported wheat wax compound classes (fatty acids, 1-alkanols, 1-alkanol esters, aldehydes, alkanes, β-diketone, hydroxy-β-diketones, alkylresorcinols and methyl alkylresorcinols), esters of 2-alkanols and three types of aromatic esters (benzyl, phenethyl and p-hydroxyphenethyl) are also reported. In particular, 2-heptanol esters were identified. Detailed analyses of the isomer distributions within 1-alkanol and 2-alkanol ester homologs revealed distinct patterns of esterified acids and alcohols, suggesting several wax ester synthases with very different substrate preferences in both wheat organs. Terpenoids, including two terpenoid esters, were present only in peduncle wax. PMID:27264640

  16. Crystallographic and Mutational Studies of Mycobacterium Tuberculosis recA Mini-inteins Suggest a Pivotal Role for a Highly Conserved Aspartate Residue

    International Nuclear Information System (INIS)

    The 440 amino acid Mtu recA intein consists of independent protein-splicing and endonuclease domains. Previously, removal of the central endonuclease domain of the intein, and selection for function, generated a 168 residue mini-intein, ΔI-SM, that had splicing activity similar to that of the full-length, wild-type protein. A D422G mutation (ΔI-CM) increased C-terminal cleavage activity. Using the ΔI-SM mini-intein structure (presented here) as a guide, we previously generated a highly active 139 residue mini-intein, ΔΔIhh-SM, by replacing 36 amino acid residues in the residual endonuclease loop with a seven-residue β-turn from the autoprocessing domain of Hedgehog protein. The three-dimensional structures of ΔI-SM, ΔΔIhh-SM, and two variants, ΔΔIhh-CM and ΔΔIhh, have been determined to evaluate the effects of the minimization on intein integrity and to investigate the structural and functional consequences of the D422G mutation. These structural studies show that Asp422 is capable of interacting with both the N and C termini. These interactions are lacking in the CM variant, but are replaced by contacts with water molecules. Accordingly, additional mutagenesis of residue 422, combined with mutations that isolate N-terminal and C-terminal cleavage, showed that the side-chain of Asp422 plays a role in both N and C-terminal cleavage, thereby suggesting that this highly conserved residue regulates the balance between the two reactions

  17. 基于内含肽技术的生物传感器%Intein-based Biosensor

    Institute of Scientific and Technical Information of China (English)

    赵仲麟; 马鑫; 李燕; 李淑英; 袁超

    2012-01-01

    开发用于分析复杂生物学问题的生物传感器,对于研究人类疾病和发现潜在药物分子十分重要.内含肽介导的蛋白质剪接技术的应用推动了新型生物传感器发展,基于内含肽技术的生物传感器可以对酶活性、激素受体、维生素和蛋白质与蛋白质间的相互作用等一些重要的生物学过程进行高效精准的分析和研究.重点阐述基于内含肽技术的生物传感器相关研究进展,并对其进行了展望.%The development of biosensors for the analysis of complex biological problems is essential for both the study of human diseases and validation of potential drug molecules. A novel application of intein-rnediated protein splicing technologies has led to the development of a biosensor. Inteins have played critical roles in design of many assays and biosensors enabling efficient and precise analysis of important biological processes,such as enzyme activity,hormone receptor,vitamins and protein-protein interactions. In this paper,we review the application and development of intein-based biosensors.

  18. Large-Scale Evaluation of Nickel Aluminide Rools In A Heat-Treat Furnace at Bethlehem Steel's (now ISG) Burns Harbor Plate Mill

    Energy Technology Data Exchange (ETDEWEB)

    John Mengel; Anthony Martocci; Larry Fabina; RObert Petrusha; Ronald Chango

    2003-09-01

    At Bethlehem Steel Burns Harbor Plate Division (now ISG Burns Harbor Plate Inc.)'s annealing furnace, new nickel aluminide intermetallic alloy rolls provide greater high-temperature strength and wear resistance compared to the conventional H series cast austenitic alloys currently used in the industry, Oak Ridge National Laboratory and Bethlehem (ISG) partnered under a U.S. Department of Energy, Office of Industrial Technology's Emerging Technology Deployment Program to demonstrate and evaluate the nickel aluminide intermetallic alloy rolls as part of an updated energy efficient large commercial annealing furnace system.

  19. Project Bethlehem - Training Educators and Health Workers in the Therapeutic Use of Music in the West Bank

    OpenAIRE

    Elizabeth Coombes

    2011-01-01

    This article describes Project Bethlehem, a music therapy initiative which is taking place   in the West Bank.  It’s aim is to train teachers and health care workers in the therapeutic use of music with children.

    The background to the Project is described, and the music therapy work is then detailed with references to the theoretical thinking behind it. An Appendix containing the booklet prepared as a teaching resource for staff involved in the Project is attached t...

  20. Intein-mediated rapid purification of recombinant maxadilan and M65 and their acute effects on plasma glucose

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Maxadilan is a potent vasodilatory peptide present in the sali-vary glands of the sand fly. Maxadilan and M65, a deletion variation of maxadilan, are agonist- and antagonist-specific for the PAC1 receptor. In order to obtain the recombinant maxadilan and M65 efficiently by intein-mediated single col-umn purification, the genes encoding maxadilan and M65 were designed, synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant maxadilan and M65 with homogeneity over 95% were released from the chitin-bound intein tag by β-mercaptoethanol. Intraperitoneal in-jection of the recombinant maxadilan caused an acute eleva-tion of plasma glucose, imitating pituitary adenylate cyclase-activating polypeptide (PACAP) 27, in NIH mice, while the VPACl-agonist and VPAC2-agonist had no significant ef-fects on the levels of plasma glucose. M65 alone had no effect on the plasma glucose, but blocked the glucose excursion caused by maxadilan by 12.7% and blocked the glucose ex-cursion caused by the PACAP 27 by 11.6%. The acute ef-fects of the recombinant maxadilan and M65 on the plasma glucose indicated that they had the characteristics as the agonist and antagonist for PAC1.

  1. The Star of Bethlehem is Not the Nova DO Aquilae (Nor Any Other Nova, Supernova, or Comet)

    CERN Document Server

    Schaefer, Bradley E

    2013-01-01

    The Star of Bethlehem is only known from a few verses in the Gospel of Matthew, with the Star inspiring and leading the Magi (i.e., Persian astrologers) to Jerusalem and ultimately worshipping the young Jesus Christ in Bethlehem. In the last four centuries, astronomers have put forth over a dozen greatly different naturalistic explanations, all involving astronomical events, often a bright nova, supernova, or comet. This paper will evaluate one prominent recent proposal, that the Star was a 'recurrent nova' now catalogued as DO Aquilae, and provide three refutations. In particular, (1) DO Aql is certainly not a recurrent nova, but rather an ordinary nova with a recurrence time scale of over a million years, (2) in its 1925 eruption, DO Aql certainly never got brighter than 8.5 mag, and the physics of the system proves that it could never get to the required luminosity of a supernova, and (3) the Magi were astrologers who had no recognition or interpretation for novae (or supernovae or comets) so any such even...

  2. KRESS INDIRECT DRY COOLING SYSTEM, BETHLEHEM STEEL'S COKE PLANT DEMONSTRATION AT SPARROWS POINT, MARYLAND - VOLUME 1. TECHNICAL REPORT AND APPENDICES A-F

    Science.gov (United States)

    The report evaluates the Kress Indirect Dry Cooling (KIDC) process, an innovative system for handling and cooling coke produced from a slot-type by-product coke oven battery. he report is based on the test work and demonstration of the system at Bethlehem Steel Corporation's Spar...

  3. Survey on infant hearing loss at Caritas Baby Hospital in Bethlehem-Palestine

    Directory of Open Access Journals (Sweden)

    Lucia Corradin

    2014-03-01

    Full Text Available This study describes the epidemiology of infants’ hearing loss (IHL among patients under 3 months of age at Caritas Baby Hospital, the only pediatric hospital in Palestine. It was aimed to demonstrate that IHL is a major health problem in Palestine and to assess the first available data of the newborn hearing screening program conducted between September 25, 2006 and December 31, 2011. Data was uploaded and analyzed using Microsoft Excel and the Statistical Package for the Social Sciences software (SPSS version 21. A total of 8144 infants were tested, 4812 (59% were males and 3332 (41% were females. As to their origin, 72% (5886 came from the Bethlehem district, 25% (2044 from the Hebron district, while 3% (214 from the other Palestinian districts (Jericho, Ramallah, Nablus, Jenin and Jerusalem. The transient evoked otoacoustic emissions (TEOAEs and the automated auditory brainstem response were used according to the manufacturer guidelines. The results were interpreted according to the indications of the American Academy of Pediatrics, the National Institutes of Health, and the European Consensus Development Conference on Neonatal Hearing Screening. Out of the 8144 infants tested, 1507 (14.6% did not pass the 1st test, 477 (32.8% of these 1507 infants failed retesting, while 498 (33% patients were lost to follow-up. Only 152 (31.9% patients that failed retesting went to an audiologist. The audiologist evaluation revealed that 101 (66.4% patients presented with a mild-moderate or profound hearing loss according to the Bureau International of Audiophonologie standards, 44 (28.9% patients had otitis media, whereas 7 cases (4.7% had no hearing disorders. The overall unadjusted percentage of hearing loss was 1.24%, and the adjusted overall percentage was 1.85%. The chart review showed that jaundice, sepsis, prematurity, lung disease were more common among the affected patients. The high prevalence of childhood deafness in Palestine is of utmost

  4. Survey on Infant Hearing Loss at Caritas Baby Hospital in Bethlehem-Palestine.

    Science.gov (United States)

    Corradin, Lucia; Hindiyeh, Musa; Khaled, Rasha; Rishmawi, Fadi; Zidan, Marwan; Marzouqa, Hiyam

    2014-03-01

    This study describes the epidemiology of infants' hearing loss (IHL) among patients under 3 months of age at Caritas Baby Hospital, the only pediatric hospital in Palestine. It was aimed to demonstrate that IHL is a major health problem in Palestine and to assess the first available data of the newborn hearing screening program conducted between September 25, 2006 and December 31, 2011. Data was uploaded and analyzed using Microsoft Excel and the Statistical Package for the Social Sciences software (SPSS version 21). A total of 8144 infants were tested, 4812 (59%) were males and 3332 (41%) were females. As to their origin, 72% (5886) came from the Bethlehem district, 25% (2044) from the Hebron district, while 3% (214) from the other Palestinian districts (Jericho, Ramallah, Nablus, Jenin and Jerusalem). The transient evoked otoacoustic emissions (TEOAEs) and the automated auditory brainstem response were used according to the manufacturer guidelines. The results were interpreted according to the indications of the American Academy of Pediatrics, the National Institutes of Health, and the European Consensus Development Conference on Neonatal Hearing Screening. Out of the 8144 infants tested, 1507 (14.6%) did not pass the 1(st) test, 477 (32.8%) of these 1507 infants failed retesting, while 498 (33%) patients were lost to follow-up. Only 152 (31.9%) patients that failed retesting went to an audiologist. The audiologist evaluation revealed that 101 (66.4%) patients presented with a mild-moderate or profound hearing loss according to the Bureau International of Audiophonologie standards, 44 (28.9%) patients had otitis media, whereas 7 cases (4.7%) had no hearing disorders. The overall unadjusted percentage of hearing loss was 1.24%, and the adjusted overall percentage was 1.85%. The chart review showed that jaundice, sepsis, prematurity, lung disease were more common among the affected patients. The high prevalence of childhood deafness in Palestine is of utmost

  5. Expression and purification of ELP-intein-tagged target proteins in high cell density E. coli fermentation

    OpenAIRE

    Wood David W; Fong Baley A

    2010-01-01

    Abstract Background Elastin-like polypeptides (ELPs) are useful tools that can be used to non-chromatographically purify proteins. When paired with self-cleaving inteins, they can be used as economical self-cleaving purification tags. However, ELPs and ELP-tagged target proteins have been traditionally expressed using highly enriched media in shake flask cultures, which are generally not amenable to scale-up. Results In this work, we describe the high cell-density expression of self-cleaving ...

  6. Development of the intein-mediated method for production of recombinant thymosin β4 from the acetylated in vivo fusion protein.

    Science.gov (United States)

    Esipov, Roman S; Makarov, Dmitry A; Stepanenko, Vasily N; Miroshnikov, Anatoly I

    2016-06-20

    Thymosin β4 is a 43 amino acid long peptide with an acetylated N-terminal serin that has a high potential as a remedy for healing ulcers, wounds and burns. Although protein biosynthesis offers attractive opportunities in terms of a large-scale production, currently thymosin β4 is mainly produced by chemical synthesis. The problems that hinder the successful commercialization of the biotechnological approach are associated with the small peptides expression and N-terminal acetylation. This work presents an innovative biotechnological method for thymosin β4 production that employs the peptide acetylation in vivo. A genetically engineered construct was created, where the Tβ4 coding sequence fused with the intein Mxe GyrA sequence and chitin-binding domain was combined with the acetyltransferase coding sequence to form a polycistronic construct under a stringent control of T7 promoter. This plasmid construct provided for the expression of the Tβ4-intein fusion protein. In the process of the post-translational modification in vivo formyl methionine was completely removed from the target peptide N-terminus and followed by the Tβ4 precursor N-terminal acetylation. The use of the intein-mediated expression system made it possible to extract thymosin β4 in only 2 chromatographic runs. The method is straightforward to implement and scale up. PMID:27015974

  7. Quantitative analysis of G-protein-coupled receptor internalization using DnaE intein-based assay.

    Science.gov (United States)

    Lu, Bin; Chen, Linjie; Zhang, Yaping; Shi, Ying; Zhou, Naiming

    2016-01-01

    G-protein-coupled receptors (GPCRs), the largest family of cell surface receptors, are involved in many physiological processes. They represent highly important therapeutic targets for drug discovery. Currently, there are numerous cell-based assays developed for the pharmacological profiling of GPCRs and the identification of novel agonists and antagonists. However, the development of new, faster, easier, and more cost-effective approaches to detect GPCR activity remains highly desirable. β-arrestin-dependent internalization has been demonstrated to be a common mechanism for most GPCRs. Here we describe a novel assay for quantitative analysis of GPCR internalization based on DnaE intein-mediated reconstitution of fragmented Renilla luciferase or Firefly luciferase when activated GPCRs interact with β-arrestin2 or Rab5. Further validation, using functionally divergent GPCRs, showed that EC50 values obtained for the known agonists and antagonists were in close agreement with the results of previous reports. This suggests that this assay is sensitive enough to permit quantification of GPCR internalization. Compared with conventional assays, this novel assay system is cost-effective, rapid, and easy to manipulate. These advantages may allow this assay to be used universally as a functional cell-based system for GPCR characterization and in the screening process of drug discovery. PMID:26928549

  8. Mapping and identification of cassava mosaic geminivirus DNA-A and DNA-B genome sequences for efficient siRNA expression and RNAi based virus resistance by transient agro-infiltration studies.

    Science.gov (United States)

    Patil, Basavaprabhu L; Bagewadi, Basavaraj; Yadav, Jitender S; Fauquet, Claude M

    2016-02-01

    Geminiviruses are among the most serious pathogens of many economically important crop plants and RNA interference (RNAi) is an important strategy for their control. Although any fragment of a viral genome can be used to generate a double stranded (ds) RNA trigger, the precursor for generation of siRNAs, the exact sequence and size requirements for efficient gene silencing and virus resistance have so far not been investigated. Previous efforts to control geminiviruses by gene silencing mostly targeted AC1, the gene encoding replication-associated protein. In this study we made RNAi constructs for all the genes of both the genomic components (DNA-A and DNA-B) of African cassava mosaic virus (ACMV-CM), one of the most devastating geminiviruses causing cassava mosaic disease (CMD) in Africa. Using transient agro-infiltration studies, RNAi constructs were evaluated for their ability to trigger gene silencing against the invading virus and protection against it. The results show that the selection of the DNA target sequence is an important determinant for the amount of siRNA produced and the extent of resistance. The ACMV genes AC1, AC2, AC4 from DNA-A and BC1 from DNA-B were effective targets for RNAi-mediated resistance and their siRNA expression was higher compared to other RNAi constructs. The RNAi construct targeting AC2, the suppressor of gene silencing of ACMV-CM gave highest level of resistance in the transient studies. This is the first report of targeting DNA-B to confer resistance to a bipartite geminivirus infection. PMID:26581664

  9. Analysis of a new strain of Euphorbia mosaic virus with distinct replication specificity unveils a lineage of begomoviruses with short Rep sequences in the DNA-B intergenic region

    Directory of Open Access Journals (Sweden)

    Argüello-Astorga Gerardo R

    2010-10-01

    Full Text Available Abstract Background Euphorbia mosaic virus (EuMV is a member of the SLCV clade, a lineage of New World begomoviruses that display distinctive features in their replication-associated protein (Rep and virion-strand replication origin. The first entirely characterized EuMV isolate is native from Yucatan Peninsula, Mexico; subsequently, EuMV was detected in weeds and pepper plants from another region of Mexico, and partial DNA-A sequences revealed significant differences in their putative replication specificity determinants with respect to EuMV-YP. This study was aimed to investigate the replication compatibility between two EuMV isolates from the same country. Results A new isolate of EuMV was obtained from pepper plants collected at Jalisco, Mexico. Full-length clones of both genomic components of EuMV-Jal were biolistically inoculated into plants of three different species, which developed symptoms indistinguishable from those induced by EuMV-YP. Pseudorecombination experiments with EuMV-Jal and EuMV-YP genomic components demonstrated that these viruses do not form infectious reassortants in Nicotiana benthamiana, presumably because of Rep-iteron incompatibility. Sequence analysis of the EuMV-Jal DNA-B intergenic region (IR led to the unexpected discovery of a 35-nt-long sequence that is identical to a segment of the rep gene in the cognate viral DNA-A. Similar short rep sequences ranging from 35- to 51-nt in length were identified in all EuMV isolates and in three distinct viruses from South America related to EuMV. These short rep sequences in the DNA-B IR are positioned downstream to a ~160-nt non-coding domain highly similar to the CP promoter of begomoviruses belonging to the SLCV clade. Conclusions EuMV strains are not compatible in replication, indicating that this begomovirus species probably is not a replicating lineage in nature. The genomic analysis of EuMV-Jal led to the discovery of a subgroup of SLCV clade viruses that contain in

  10. Escherichia coli DnaB Helicase–DnaC Protein Complex: Allosteric Effects of the Nucleotides on the Nucleic Acid Binding and the Kinetic Mechanism of NTP Hydrolysis. 3†

    OpenAIRE

    Roychowdhury, Anasuya; Szymanski, Michal R.; Jezewska, Maria J.; Bujalowski, Wlodzimierz

    2009-01-01

    Allosteric interactions between the DNA- and NTP-binding sites of the Escherichia coli DnaB helicase engaged in the DnaB–DnaC complex and the mechanism of NTP hydrolysis by the complex have been examined using the fluorescence titration, analytical ultracentrifugation, and rapid quench-flow technique. Surprisingly, the ssDNA affinity of the DnaB–DnaC complex is independent of the structure of the phosphate group of the cofactor bound to the helicase. Thus, the DnaC protein eliminates the anta...

  11. Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: complexes with lambda O protein and with lambda O, lambda P, and Escherichia coli DnaB proteins.

    OpenAIRE

    Dodson, M; Roberts, J.; McMacken, R; Echols, H

    1985-01-01

    The O protein of bacteriophage lambda is required for initiation of DNA replication at the lambda replicative origin designated ori lambda. The binding sites for O protein are four direct repeats, each of which is an inverted repeat. By means of electron microscopy, we have found that phage lambda O protein utilizes these multiple binding sites to form a specific nucleoprotein structure in which the origin DNA is inferred to be folded or wound. The phage lambda O and P proteins and host DnaB ...

  12. An efficient protocol towards site-specifically clickable nanobodies in high yield: cytoplasmic expression in Escherichia coli combined with intein-mediated protein ligation.

    Science.gov (United States)

    Ta, Duy Tien; Redeker, Erik Steen; Billen, Brecht; Reekmans, Gunter; Sikulu, Josephine; Noben, Jean-Paul; Guedens, Wanda; Adriaensens, Peter

    2015-10-01

    In this study, several expression strategies were investigated in order to develop a generic, highly productive and efficient protocol to produce nanobodies modified with a clickable alkyne function at their C-terminus via the intein-mediated protein ligation (IPL) technique. Hereto, the nanobody targeting the vascular cell adhesion molecule 1 (NbVCAM1) was used as a workhorse. The highlights of the protocol can be ascribed to a cytoplasmic expression of the nanobody-intein-chitin-binding domain fusion protein in the Escherichia coli SHuffle(®) T7 cells with a C-terminal extension, i.e. LEY, EFLEY or His6 spacer peptide, in the commonly used Luria-Bertani medium. The combination of these factors led to a high yield (up to 22 mg/l of culture) and nearly complete alkynation efficiency of the C-terminally modified nanobody via IPL. This yield can even be improved to ∼45 mg/l in the EnPresso(®) growth system but this method is more expensive and time-consuming. The resulting alkynated nanobodies retained excellent binding capacity towards the recombinant human VCAM1. The presented protocol benefits from time- and cost-effectiveness, which allows a feasible production up-scaling of generic alkynated nanobodies. The production of high quantities of site-specifically modified nanobodies paves the way to new biosurface applications that demand for a homogeneously oriented nanobody coupling. Prospectively, the alkynated nanobodies can be covalently coupled to a multitude of azide-containing counterparts, e.g. contrast labeling agents, particles or surfaces for numerous innovative applications. PMID:26243885

  13. Kress indirect dry cooling system, Bethlehem Steel's Coke Plant demonstration at Sparrows Point, Maryland. Volume 2. Appendices G-N. Final report, February 1990-February 1992

    International Nuclear Information System (INIS)

    The report provides an evaluation of the Kress Indirect Dry Cooling (KIDC) process. The KIDC process is an innovative system for the handling and cooling of coke produced from a slot type by-product coke oven battery. The report is based on the test work and demonstration of the system at Bethlehem Steel Corporation's Sparrows Point facility in 1991. The report covers both environmental and operational impacts of the KIDC process. The report, Volume 2, contains appendices G-N. Volume 1, PB93-191302, contains the technical report as well as appendices A-F. Volume 2 contains appendixes on coke quality data, blast furnace balwax model report, KIDC operating cost and maintenance requirements, Kress box thickness readings, KIDC coke discharge temperature, QA/QC program, door leak data, and coal data

  14. The star of Bethlehem

    International Nuclear Information System (INIS)

    It is stated that the cause and form of the star are still uncertain. The astrologically significant triple conjunction of Saturn and Jupiter in the constellation of Pisces appears to be the most likely explanation, although the two comets of March 5 BC and April 4 BC cannot be dismissed, nor can the possibility that the 'star' was simply legendary. The conjunction occurred in 7 BC and there are indications that Jesus Christ was probably born in the Autumn of that year, around October 7 BC. (U.K.)

  15. Students’ perception of what they learn in Teaching Hotel Château Bethlehem [EuroChrie Conference (Cooperative Education and Research for Hospitality & Tourism Educators), Freiburg (D) 16-19 October 2013

    OpenAIRE

    Schoenmakers, Sylvia; Seggelen, Harpert van; Van der Klink, Marcel; Wilms, Jogien

    2013-01-01

    Bachelor students of Hotel Management School Maastricht, part of Zuyd University of Applied Sciences in the Netherlands, start their educational program with a semester of orientation on Hotel Operations in theory and practice. The teaching staff was curious about students’ perception of what they learn during their duty in the Teaching Hotel Château Bethlehem. Students were interviewed about the learning environment, the coaching and their learning outcomes. The interview findings gave insig...

  16. Intein-mediated Trans-splicing of Chloride Ion Channel CFTR%内含肽介导的氯离子通道蛋白CFTR的反式剪接

    Institute of Scientific and Technical Information of China (English)

    朱甫祥; 刘泽隆; 屈慧鸽; 迟晓艳

    2009-01-01

    研究利用内含肽(intein)的蛋白质反式剪接功能在大肠杆菌中对囊性纤维化跨膜传导调节因子(cystic fibrosis tmnsmembrane regulator,CFTR)的反式剪接作用.CFTR基因突变导致一种常染色体隐性遗传疾病囊性纤维化(cystic fibrosis,CF).将CVTR的cDNA于剪接反应所需的保守性氨基酸残基Ser660前断裂为N端和C端,分别与split mini Ssp DnaB内含肽的106个氨基酸残基的N端和48个氨基酸残基的C端编码序列融合,构建到原核表达载体pBV220.诱导表达后SDS-PAGE可见预期大小剪接形成的CVTR蛋白条带,Western印迹用CFTR特异性抗体进一步证明为剪接所产生的CFTIR蛋白,表明内舍肽可有效催化CFTR的反式剪接.

  17. An Intein-based Strategy for the Production of Tag-free Huntingtin Exon 1 Proteins Enables New Insights into the Polyglutamine Dependence of Httex1 Aggregation and Fibril Formation.

    Science.gov (United States)

    Vieweg, Sophie; Ansaloni, Annalisa; Wang, Zhe-Ming; Warner, John B; Lashuel, Hilal A

    2016-06-01

    The first exon of the Huntingtin protein (Httex1) is one of the most actively studied Htt fragments because its overexpression in R6/2 transgenic mice has been shown to recapitulate several key features of Huntington disease. However, the majority of biophysical studies of Httex1 are based on assessing the structure and aggregation of fusion constructs where Httex1 is fused to large proteins, such as glutathione S-transferase, maltose-binding protein, or thioredoxin, or released in solution upon in situ cleavage of these proteins. Herein, we report an intein-based strategy that allows, for the first time, the rapid and efficient production of native tag-free Httex1 with polyQ repeats ranging from 7Q to 49Q. Aggregation studies on these proteins enabled us to identify interesting polyQ-length-dependent effects on Httex1 oligomer and fibril formation that were previously not observed using Httex1 fusion proteins or Httex1 proteins produced by in situ cleavage of fusion proteins. Our studies revealed the inability of Httex1-7Q/15Q to undergo amyloid fibril formation and an inverse correlation between fibril length and polyQ repeat length, suggesting possible polyQ length-dependent differences in the structural properties of the Httex1 aggregates. Altogether, our findings underscore the importance of working with tag-free Httex1 proteins and indicate that model systems based on non-native Httex1 sequences may not accurately reproduce the effect of polyQ repeat length and solution conditions on Httex1 aggregation kinetics and structural properties. PMID:27002149

  18. Feasibility Study of Economics and Performance of Solar Photovoltaics at the Former Bethlehem Steel Plant Brownfield Site in Lackawanna, New York. A Study Prepared in Partnership with the Environmental Protection Agency for the RE-Powering America's Land Initiative: Siting Renewable Energy on Potentially Contaminated Land and Mine Sites

    Energy Technology Data Exchange (ETDEWEB)

    Salasovich, J.; Geiger, J.; Mosey, G.; Healey, V.

    2013-04-01

    The U.S. Environmental Protection Agency (EPA), in accordance with the RE-Powering America's Land initiative, selected the Former Bethlehem Steel Plant site in Lackawanna, New York, for a feasibility study of renewable energy production. The National Renewable Energy Laboratory (NREL) provided technical assistance for this project. The purpose of this report is to assess the site for a possible photovoltaic (PV) system installation and estimate the cost, performance, and site impacts of different PV options. In addition, the report recommends financing options that could assist in the implementation of a PV system at the site.

  19. Expression and purification of rhIL-10-RGD from Escherichia coli as a potential wound healing agent.

    Science.gov (United States)

    Yang, Fangfang; Wan, Yi; Liu, Jiaqi; Yang, Xuekang; Wang, Hongtao; Tao, Ke; Han, Juntao; Shi, Jihong; Hu, Dahai

    2016-08-01

    Various protocols for recombinant Interleukin-10 (IL-10) purification in wound healing have been reported previously. However, the therapeutic effect was not obvious. Thus, it is of great importance to find new and effective approaches for therapy. In this study, we propose that IL-10 and Arginine-Glycine-Aspartic (RGD) peptide would be a valuable therapeutic for wound healing. To explore a high-efficiency and cost-effective approach for the production of IL-10 and RGD peptide with bioactivity, a synthetic gene was cloned into a recombinant pTWIN1 vector. As a consequence, rhIL-10-RGD and the pH-induced self-cleavable Ssp DnaB mini-intein as a fusion protein was highly expressed by IPTG induction in Escherichia coli Rosetta without extra residues in a bioreactor. After Ni affinity chromatographic purification, rhIL-10-RGD was released by the Ssp DnaB intein-mediated self-cleavage that is triggered by pH shift. SDS-PAGE and silver staining showed a major band with an estimated molecular mass of 19.3kDa. Cell proliferation assay confirmed its potent proliferation activity on MC/9 murine mast cells. In conclusion, we report a novel strategy to produce rhIL-10-RGD mediated by the pH-induced self-cleavable Ssp DnaB mini-intein, and show that rhIL-10-RGD could play an effective role in wound healing of BALB/c mice. PMID:27241829

  20. Protein trans-splicing based dual-vector delivery of the coagulation factor Ⅷ gene

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A dual-vector system was explored for the delivery of the coagulation factor VIII gene,using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally.A pair of eukaryotic expression vectors,expressing Ssp DnaB intein-fused heavy and light chain genes of B-domain deleted factor VIII (BDD-FVIII),was constructed.With transient co-transfection of the two vectors into 293 and COS-7 cells,the culture supernatants contained (137±23) and (109±22) ng mL–1 spliced BDD-FVIII antigen with an activity of (1.05±0.16) and (0.79±0.23) IU mL–1 for 293 and COS-7 cells,respectively.The spliced BDD-FVIII was also detected in supernatants from a mixture of cells transfected with inteinfused heavy and light chain genes.The spliced BDD-FVIII protein bands from cell lysates were visualized by Western blotting.The data demonstrated that intein could be used to transfer the split factor VIII gene and provided valuable information on factor VIII gene delivery by dual-adeno-associated virus in hemophilia A gene therapy.

  1. Perceptions and Expectation of Palestinian Teachers towards Inclusive Education in Bethlehem District

    Science.gov (United States)

    Abu-Heran, N.; Abukhayran, A.; Domingo, J.; Perez-Garcia, M. P.

    2014-01-01

    Introduction: The study investigates about the reality of inclusive education in Palestine according to the opinion of their teachers. It is presented in the legislative and social framework of education in Palestine and compares it with current knowledge in inclusive education, from which a series of essential elements for inclusive education can…

  2. Biosynthesis of the Cyclotide MCoTI-II using an Engineered Intein

    Energy Technology Data Exchange (ETDEWEB)

    Cantor, J; Camarero, J A

    2006-08-15

    Cyclotides are an emerging family of naturally occurring circular mini-proteins ({approx}30-40 amino acids) characterized by six conserved Cys residues (forming 3 disulfide bridges) that create a topologically unique structure designated as a cyclic cysteine knot (CCK). The cysteine knot motif, which is embedded within the macrocylic backbone, is described as two disulfide bridges that form a ring that is penetrated by the third disulfide bridge. The cyclic backbone and CCK motif together confer cyclotides with a remarkable stability and resistance to proteolytic, chemical, and thermal degradation. Further, cyclotides are functionally diverse and display a wide range of functions including uterotonic activity, trypsin inhibition, cytotoxicity, neurotensin binding, anti-HIV, antimicrobial, and insecticidal activity. Together, these characteristics make cyclotides attractive candidates for both drug design and agricultural applications, both in their native forms and as molecular scaffolds for the incorporation of novel bioactivities. [1] The ability to manipulate production of cyclotides within biological systems is critical for mutagenesis studies, production of grafted products, and the mass production of cyclotides with novel activities. My adviser's hope is to achieve this capability by employing recombinant DNA expression techniques to generate large combinatorial libraries of cyclotides. The advantage in creating a biosynthetic library (containing {approx}10{sup 6}-10{sup 10} members/library vs. chemically based libraries with typical values ranging from {approx}10{sup 3}-10{sup 5} members/library) is that it can be lead to the in vivo application of biological screening and selection methodologies based on a specific clone's ability to affect certain cellular processes.

  3. Entwicklungschancen für die Region Bethlehem unter besonderer Berücksichtigung des Tourismus- und Landwirtschaftsektors

    OpenAIRE

    Abu Hashem, Yasser

    2003-01-01

    ABSTRACT Die in der letzten Zeit erarbeiteten alternativen Strategien zu einer Lösung des Palästinakonfliktes sind vor folgendem Hintergrund zu sehen: Bis Anfang der 90er Jahre wurden die seit 1967 besetzten Gebiete von Israel beansprucht. Der Konflikt wurde häufig als Palästinafrage , Palästinaproblem oder Probleme der palästinensischen Gesellschaft tituliert. Dabei betrachtete man früher die Palästinafrage als innerstaatliches Problem Israels. Unter dem geänderten Bewusstsein der Weltö...

  4. 基于蛋白质剪接的BHK细胞Ser660前断裂的CFTR基因转移%Protein Splicing Based Gene Transfer of Split Cystic Fibrosis Transmembrane Conductance Regulator Severed Before Ser660 in BHK Cells

    Institute of Scientific and Technical Information of China (English)

    朱甫祥; 刘泽隆; 缪静; 屈慧鸽; 迟晓艳

    2010-01-01

    利用内含肽(intein)的蛋白质反式剪接技术,研究双载体真核细胞转囊性纤维化跨膜电导调节体(CFTR)基因,通过翻译后连接成为完整的功能性CFTR蛋白.应用基因重组技术,将人CFTR cDNA于剪接反应所需保守残基Ser660前断裂为N端和C端两部分,分别与split Ssp DnaB intein编码序列融合,构建到真核表达载体pEGFP-N1和pEYFP-N1.用脂质体将这对载体共转染至幼年仓鼠肾细胞(BHK),48 h后Western印迹观察CFTR蛋白质的连接,并用全细胞和单通道膜片钳技术记录Cl-通道电流.基因共转染细胞可观察到明显的由蛋白质反式剪接形成的完整CFTR蛋白,膜片钳记录到较高的全细胞Cl-电流和与转野生型CFTR基因细胞相似的单Cl-通道开放活性,提示CFTR功能的恢复.内含肽可作为一种技术策略用于双栽体转CFTR基因,为应用双腺相关病毒载体(AAV)转基因的囊性纤维化疾病(CF)基因治疗提供了依据.

  5. Gene invasion in distant eukaryotic lineages: discovery of mutually exclusive genetic elements reveals marine biodiversity.

    Science.gov (United States)

    Monier, Adam; Sudek, Sebastian; Fast, Naomi M; Worden, Alexandra Z

    2013-09-01

    Inteins are rare, translated genetic parasites mainly found in bacteria and archaea, while spliceosomal introns are distinctly eukaryotic features abundant in most nuclear genomes. Using targeted metagenomics, we discovered an intein in an Atlantic population of the photosynthetic eukaryote, Bathycoccus, harbored by the essential spliceosomal protein PRP8 (processing factor 8 protein). Although previously thought exclusive to fungi, we also identified PRP8 inteins in parasitic (Capsaspora) and predatory (Salpingoeca) protists. Most new PRP8 inteins were at novel insertion sites that, surprisingly, were not in the most conserved regions of the gene. Evolutionarily, Dikarya fungal inteins at PRP8 insertion site a appeared more related to the Bathycoccus intein at a unique insertion site, than to other fungal and opisthokont inteins. Strikingly, independent analyses of Pacific and Atlantic samples revealed an intron at the same codon as the Bathycoccus PRP8 intein. The two elements are mutually exclusive and neither was found in cultured Bathycoccus or other picoprasinophyte genomes. Thus, wild Bathycoccus contain one of few non-fungal eukaryotic inteins known and a rare polymorphic intron. Our data indicate at least two Bathycoccus ecotypes exist, associated respectively with oceanic or mesotrophic environments. We hypothesize that intein propagation is facilitated by marine viruses; and, while intron gain is still poorly understood, presence of a spliceosomal intron where a locus lacks an intein raises the possibility of new, intein-primed mechanisms for intron gain. The discovery of nucleus-encoded inteins and associated sequence polymorphisms in uncultivated marine eukaryotes highlights their diversity and reveals potential sexual boundaries between populations indistinguishable by common marker genes. PMID:23635865

  6. dnaC-dependent reconstitution of replication forks in Escherichia coli lysates.

    OpenAIRE

    Nüsslein-Crystalla, V; Niedenhof, I; Rein, R.

    1982-01-01

    Lysates of Escherichia coli exhibit a DNA-synthesizing activity that depends on the presence of replication forks and of replication proteins. Replicative activity was reconstituted in vitro by mixing lysates prepared from temperature-sensitive dnaB mutants with wild-type dnaB protein. Lysates of double mutants deficient in both dnaB and dnaC genes could only be complemented by the addition of both dnaB and dnaC proteins, whereas lysates deficient in dnaC protein did not require the addition ...

  7. Facilitating practical knowledge using flexible forms of learning in the education of Occupational Therapists in Palestine : An action research approach performed in cooperation with teachers in the Occupational Therapist program at Bethlehem University

    OpenAIRE

    Jentoft, Rita

    2009-01-01

    Practical knowledge is essential knowledge in occupational therapy. It is a situated and experienced knowledge, a knowing how and from within the situation. This knowledge has been difficult to facilitate in the learning process of a group of Occupational Therapist students in Gaza. Travel restrictions and the unstable political situation separated teachers and students. Lack of clinical occupational therapists inside Gaza also had a major impact. Educational technology such as...

  8. Students’ perception of what they learn in Teaching Hotel Château Bethlehem [EuroChrie Conference (Cooperative Education and Research for Hospitality & Tourism Educators), Freiburg (D) 16-19 October 2013

    NARCIS (Netherlands)

    Schoenmakers, Sylvia; Seggelen, Harpert van; Klink, Marcel van der; Wilms, Jogien

    2013-01-01

    Bachelor students of Hotel Management School Maastricht, part of Zuyd University of Applied Sciences in the Netherlands, start their educational program with a semester of orientation on Hotel Operations in theory and practice. The teaching staff was curious about students’ perception of what they l

  9. Dar al-Kalima akadeemia kultuuri- ja konverentsikeskus "AD DAR" : Petlemm, Palestiina = Dar al-Kalima Academy Cultural and Convention Centre "AD DAR" : Bethlehem, Palestine, 1998-2003 / Juha Leviskä

    Index Scriptorium Estoniae

    Leviskä, Juha

    2004-01-01

    Projekteerija: Vilhelm Helander, Juha Leviskä Arkkitehdit. Autorid Jyha Leviskä ja Jari Heikkinen, kaasautor Pekka Kivisalo, sisekujundaja Jari Heikkinen. Projekt 1998-1999, valmis 1999-2003. 2 joon.: plaan, vaade, 8 fotot: 4 välis- ja 4 sisevaadet

  10. La Stella di Betlemme in arte e scienza

    Science.gov (United States)

    Sigismondi, Costantino

    2014-05-01

    The star of Bethlehem has been represented in many artworks, starting from II century AD in Priscilla Catacumbs in Rome. The 14 pointed silver star of 1717 which is located in the place of birth of Jesus in Bethlehem remembers the numbers of generations 14 repeated three times since Abraham to Jesus in Matthew 1: 1-17. Finally the hypotehsis of Mira Ceti as star of Bethlehem is reviewed

  11. Genetic Mapping of a Group of Temperature-Sensitive dna Initiation Mutants in BACILLUS SUBTILIS

    OpenAIRE

    Imada, Sumi; Carroll, Lynn E.; Sueoka, Noboru

    1980-01-01

    Recombination frequencies among temperature-sensitive dna mutants from various laboratories were analyzed, and eleven dna mutants were found to be closely linked. They are classified as group B dna mutants, since these are closely linked with dnaB19, originally isolated and approximately mapped near leuA8 by Karamata and Gross (1970). However, the dnaB19 mutation itself has relatively high recombination frequencies with the other mutations, thus, we propose to subdivide the dnaB group into tw...

  12. Separate Roles of Escherichia coli Replication Proteins in Synthesis and Partitioning of pSC101 Plasmid DNA

    OpenAIRE

    Miller, Christine; Cohen, Stanley N.

    1999-01-01

    We report here that the Escherichia coli replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive dnaB mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditi...

  13. An efficient on-column expressed protein ligation strategy: Application to segmental triple labeling of human apolipoprotein E3

    OpenAIRE

    Zhao, Wentao; Zhang, Yonghong; Cui, Chunxian; Li, Qianqian; Wang, Jianjun

    2008-01-01

    Expressed protein ligation (EPL) is an intein-based approach that has been used for protein engineering and biophysical studies of protein structures. One major problem of the EPL is the low yield of final ligation product, primarily due to the complex procedure of the EPL, preventing EPL from gaining popularity in the research community. Here we report an efficient on-column EPL strategy, which focuses on enhancing the expression level of the intein-fusion protein that generates thioester fo...

  14. Structural stability and endonuclease activity of a PI-SceI GFP-fusion protein

    Directory of Open Access Journals (Sweden)

    Alireza G. Senejani, J. Peter Gogarten

    2007-01-01

    Full Text Available Homing endonucleases are site-specific and rare cutting endonucleases often encoded by intron or intein containing genes. They lead to the rapid spread of the genetic element that hosts them by a process termed 'homing'; and ultimately the allele containing the element will be fixed in the population. PI-SceI, an endonuclease encoded as a protein insert or intein within the yeast V-ATPase catalytic subunit encoding gene (vma1, is among the best characterized homing endonucleases. The structures of the Sce VMA1 intein and of the intein bound to its target site are known. Extensive biochemical studies performed on the PI-SceI enzyme provide information useful to recognize critical amino acids involved in self-splicing and endonuclease functions of the protein. Here we describe an insertion of the Green Fluorescence Protein (GFP into a loop which is located between the endonuclease and splicing domains of the Sce VMA1 intein. The GFP is functional and the additional GFP domain does not prevent intein excision and endonuclease activity. However, the endonuclease activity of the newly engineered protein was different from the wild-type protein in that it required the presence of Mn2+ and not Mg2+ metal cations for activity.

  15. In vivo characterization of the homing endonuclease within the polB gene in the halophilic archaeon Haloferax volcanii.

    Directory of Open Access Journals (Sweden)

    Adit Naor

    Full Text Available Inteins are parasitic genetic elements, analogous to introns that excise themselves at the protein level by self-splicing, allowing the formation of functional non-disrupted proteins. Many inteins contain a homing endonuclease (HEN gene, and rely on its activity for horizontal propagation. In the halophilic archaeon, Haloferax volcanii, the gene encoding DNA polymerase B (polB contains an intein with an annotated but uncharacterized HEN. Here we examine the activity of the polB HEN in vivo, within its natural archaeal host. We show that this HEN is highly active, and able to insert the intein into both a chromosomal target and an extra-chromosomal plasmid target, by gene conversion. We also demonstrate that the frequency of its incorporation depends on the length of the flanking homologous sequences around the target site, reflecting its dependence on the homologous recombination machinery. Although several evolutionary models predict that the presence of an intein involves a change in the fitness of the host organism, our results show that a strain deleted for the intein sequence shows no significant changes in growth rate compared to the wild type.

  16. In vivo characterization of the homing endonuclease within the polB gene in the halophilic archaeon Haloferax volcanii.

    Science.gov (United States)

    Naor, Adit; Lazary, Rona; Barzel, Adi; Papke, R Thane; Gophna, Uri

    2011-01-01

    Inteins are parasitic genetic elements, analogous to introns that excise themselves at the protein level by self-splicing, allowing the formation of functional non-disrupted proteins. Many inteins contain a homing endonuclease (HEN) gene, and rely on its activity for horizontal propagation. In the halophilic archaeon, Haloferax volcanii, the gene encoding DNA polymerase B (polB) contains an intein with an annotated but uncharacterized HEN. Here we examine the activity of the polB HEN in vivo, within its natural archaeal host. We show that this HEN is highly active, and able to insert the intein into both a chromosomal target and an extra-chromosomal plasmid target, by gene conversion. We also demonstrate that the frequency of its incorporation depends on the length of the flanking homologous sequences around the target site, reflecting its dependence on the homologous recombination machinery. Although several evolutionary models predict that the presence of an intein involves a change in the fitness of the host organism, our results show that a strain deleted for the intein sequence shows no significant changes in growth rate compared to the wild type. PMID:21283796

  17. Intein-mediated Soluble Expression, Purification and Functional Identification of Human AR DBD in E.coli%Intein介导的重组人AR DBD的原核可溶性表达、纯化及活性鉴定

    Institute of Scientific and Technical Information of China (English)

    姚广新; 张怡轩; 胡双纲

    2011-01-01

    Androgen receptor (AR) is a transcription factor belonging to Nuclear Hormone Receptor Superfamily and its DNA binding domain ( DBD) is essential for its binding affinity and specificity to androgen response element. Traditional method to obtain the recombinant AR DBD is fusing it to glutathione S epoxide transferase or Staphylococcus aureus proteinA in order to improve the solubility and yield. The question is the fusion tag might affect the characterization of target protein, and removing of the fusion tag is usually cumbersome. In order to obtain functional AR DBD without fusion tag in an easy and convenient way, the sequence encoded human AR 520 ~ 644 amino acids which includes the DBD is amplified by PCR, and then inserted into pTWIN1 vector. After optimizing of the inducing temperature and IPTG concentration, almost all recombinant protein is expressed in a soluble manner at 20℃ and 30℃ in host E. coli strain BL21( DE3 ). The whole cell lysate is then loaded to Chitin beads and the optimal pH for self-cleavage is determined. Using this method the AR DBD without any fusion tags is purified by only one step and the product shows high purity and good performance in electrophoretic mobility shift assay.%雄激素受体通过DNA结合域与效应元件结合发挥作用,在以往的研究中多采用与GST或Protein A融合的方式对雄激素受体的DNA结合域(AR DBD)进行重组表达,但获得无融合标签的纯AR DBD的操作非常繁琐.现借助内含肽介导的自剪切作用经一步亲和层析得到纯度较高的无融合标签的AR DBD,以利于对其性质和功能的研究.通过PCR方法扩增了编码人雄激素受体520~644位氨基酸的核苷酸序列,将该序列克隆入pTWIN1融合表达载体,转化大肠杆菌BL21(DE3)后对诱导温度及IPTG浓度进行优化,重组蛋白几乎全部可溶表达.将可溶性部分吸附到几丁质亲和层析柱上,通过pH诱导的内含肽自剪切作用释放出不含融合标签的重组人ARDBD蛋白,凝胶阻滞分析证明该蛋白只特异性结合保守的雄激素响应元件(ARE),具备正常的生物学活性.

  18. Versatile protein biotinylation strategies for potential high-throughput proteomics.

    Science.gov (United States)

    Lue, Rina Y P; Chen, Grace Y J; Hu, Yi; Zhu, Qing; Yao, Shao Q

    2004-02-01

    We present intein-mediated approaches for efficient biotinylation of proteins site-specifically. The reactive C-terminal thioester generated from intein-assisted protein splicing (either in vitro or in live cells) served as an attractive and exclusive site for attaching cysteine-containing biotin. Using these novel biotinylation strategies, we were able to efficiently biotinylate many proteins from different biological sources in a potentially high-throughput, high-content fashion. Some of these proteins were subsequently immobilized, in a very simple manner, onto different avidin-functionalized solid surfaces for applications such as protein microarray and surface plasmon resonance (SPR) spectroscopy, highlighting the numerous advantages of using biotin over other tags (e.g., GST, His-tag, etc.) as the method of choice in protein purification/immobilization. In addition, our intein-mediated strategies provided critical advantages over other protein biotinylation strategies in a number of ways. For the first time, we also successfully demonstrated that intein-mediated protein biotinylation proceeded adequately inside both bacterial and mammalian living cells, as well as in a cell-free protein synthesis system. Taken together, our results indicate the versatility of these intein-mediated strategies for potential high-throughput proteomics applications. They may also serve as useful tools for various biochemical and biophysical studies of proteins both in vitro and in vivo. PMID:14746473

  19. The Bacillus subtilis Primosomal Protein DnaD Untwists Supercoiled DNA

    OpenAIRE

    Zhang, Wenke; Allen, Stephanie; Roberts, Clive J.; Soultanas, Panos

    2006-01-01

    The essential Bacillus subtilis DnaD and DnaB proteins have been implicated in the initiation of DNA replication. Recently, DNA remodeling activities associated with both proteins were discovered that could provide a link between global or local nucleoid remodeling and initiation of replication. DnaD forms scaffolds and opens up supercoiled plasmids without nicking to form open circular complexes, while DnaB acts as a lateral compaction protein. Here we show that DnaD-mediated opening of supe...

  20. Replication initiation at the Escherichia coli chromosomal origin

    OpenAIRE

    Kaguni, Jon M.

    2011-01-01

    To initiate DNA replication, DnaA recognizes and binds to specific sequences within the Escherichia coli chromosomal origin (oriC), and then unwinds a region within oriC. Next, DnaA interacts with DnaB helicase in loading the DnaB-DnaC complex on each separated strand. Primer formation by primase (DnaG) induces the dissociation of DnaC from DnaB, which involves the hydrolysis of ATP bound to DnaC. Recent evidence indicates that DnaC acts as a checkpoint in the transition from initiation to th...

  1. ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method.

    Science.gov (United States)

    Yang, Mu; Wang, Ganggang

    2016-09-15

    The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication. In this work, a non-radioactive method for measuring ATPase activity of DnaBBst helicase was described. The working parameters and conditions were optimized. Furthermore, this method was applied to investigate effects of DnaG primase, ssDNA and helicase loader protein (DnaI) on ATPase activity of DnaBBst. Our results showed this method was sensitive and efficient. Moreover, it is suitable for the investigation of functional interaction between DnaB and related factors. PMID:27372608

  2. Protein splicing and its evolution in eukaryotes

    Directory of Open Access Journals (Sweden)

    Starokadomskyy P. L.

    2010-02-01

    Full Text Available Inteins, or protein introns, are parts of protein sequences that are post-translationally excised, their flanking regions (exteins being spliced together. This process was called protein splicing. Originally inteins were found in prokaryotic or unicellular eukaryotic organisms. But the general principles of post-translation protein rearrangement are evolving yielding different post-translation modification of proteins in multicellular organisms. For clarity, these non-intein mediated events call either protein rearrangements or protein editing. The most intriguing example of protein editing is proteasome-mediated splicing of antigens in vertebrates that may play important role in antigen presentation. Other examples of protein rearrangements are maturation of Hg-proteins (critical receptors in embryogenesis as well as maturation of several metabolic enzymes. Despite a lack of experimental data we try to analyze some intriguing examples of protein splicing evolution.

  3. Protein Trans-Splicing as a Means for Viral Vector-Mediated In Vivo Gene Therapy

    OpenAIRE

    Li, Juan; Sun, Wenchang; Wang, Bing; Xiao, Xiao; Liu, Xiang-Qin

    2008-01-01

    Inteins catalyze protein splicing in a fashion similar to how self-splicing introns catalyze RNA splicing. Split-inteins catalyze precise ligation of two separate polypeptides through trans-splicing in a highly specific manner. Here we report a method of using protein trans-splicing to circumvent the packaging size limit of gene therapy vectors. To demonstrate this method, we chose a large dystrophin gene and an adeno-associated viral (AAV) vector, which has a small packaging size. A highly f...

  4. Nucleotide and partner-protein control of bacterial replicative helicase structure and function.

    Science.gov (United States)

    Strycharska, Melania S; Arias-Palomo, Ernesto; Lyubimov, Artem Y; Erzberger, Jan P; O'Shea, Valerie L; Bustamante, Carlos J; Berger, James M

    2013-12-26

    Cellular replication forks are powered by ring-shaped, hexameric helicases that encircle and unwind DNA. To better understand the molecular mechanisms and control of these enzymes, we used multiple methods to investigate the bacterial replicative helicase, DnaB. A 3.3 Å crystal structure of Aquifex aeolicus DnaB, complexed with nucleotide, reveals a newly discovered conformational state for this motor protein. Electron microscopy and small angle X-ray scattering studies confirm the state seen crystallographically, showing that the DnaB ATPase domains and an associated N-terminal collar transition between two physical states in a nucleotide-dependent manner. Mutant helicases locked in either collar state are active but display different capacities to support critical activities such as duplex translocation and primase-dependent RNA synthesis. Our findings establish the DnaB collar as an autoregulatory hub that controls the ability of the helicase to transition between different functional states in response to both nucleotide and replication initiation/elongation factors. PMID:24373746

  5. Crystallization and preliminary X-ray crystallographic studies on the parD-encoded protein Kid from Escherichia coli plasmid R1

    NARCIS (Netherlands)

    Hargreaves, D.; Giraldo, R.; Santos-Sierra, S.; Boelens, R.; Rice, D.W.; Díaz Orejas, R.; Rafferty, J.B.

    2002-01-01

    DNA replication in Escherichia coli and therefore bacterial proliferation relies upon the efficient functioning of the DnaB helicase. The toxin protein Kid from the plasmid-stability system parD encoded on plasmid R1 of E. coli is thought to target and block DnaB-dependent DNA replication. The toxic

  6. Phage Lambda P Protein: Trans-Activation, Inhibition Phenotypes and their Suppression

    Directory of Open Access Journals (Sweden)

    Connie Hayes

    2013-02-01

    Full Text Available The initiation of bacteriophage λ replication depends upon interactions between the oriλ DNA site, phage proteins O and P, and E. coli host replication proteins. P exhibits a high affinity for DnaB, the major replicative helicase for unwinding double stranded DNA. The concept of P-lethality relates to the hypothesis that P can sequester DnaB and in turn prevent cellular replication initiation from oriC. Alternatively, it was suggested that P-lethality does not involve an interaction between P and DnaB, but is targeted to DnaA. P-lethality is assessed by examining host cells for transformation by ColE1-type plasmids that can express P, and the absence of transformants is attributed to a lethal effect of P expression. The plasmid we employed enabled conditional expression of P, where under permissive conditions, cells were efficiently transformed. We observed that ColE1 replication and plasmid establishment upon transformation is extremely sensitive to P, and distinguish this effect from P-lethality directed to cells. We show that alleles of dnaB protect the variant cells from P expression. P-dependent cellular filamentation arose in ΔrecA or lexA[Ind-] cells, defective for SOS induction. Replication propagation and restart could represent additional targets for P interference of E. coli replication, beyond the oriC-dependent initiation step.

  7. Complete Genome Sequences of Bacillus subtilis subsp. subtilis Laboratory Strains JH642 (AG174) and AG1839

    OpenAIRE

    Smith, Janet L.; Goldberg, Jonathan M.; Grossman, Alan D.

    2014-01-01

    The Gram-positive bacterium Bacillus subtilis is widely used for studies of cellular and molecular processes. We announce the complete genomic sequences of strain AG174, our stock of the commonly used strain JH642, and strain AG1839, a derivative that contains a mutation in the replication initiation gene dnaB and a linked Tn917.

  8. A review of temperature measurement in the steel reheat furnace

    International Nuclear Information System (INIS)

    The incentive for conducting research and development on reheat furnaces is substantial; the domestic steel industry spent approximately one billion dollars on fuel in reheat furnaces in 1981. Bethlehem Steel Corp. spent /145 million of that total, and neither figure includes fuel consumed in soaking pits or annealing furnaces. If the authors set a goal to save 10% of these annual fuel costs, that translates into /100 million for the domestic steel industry and /14.5 million for Bethlehem Steel. These large sums of money are significant incentives. The purpose of this paper is to review the historical heating practices and equipment at steel reheat furnaces along with current practices and instrumentation

  9. Chromatin chemistry goes cellular.

    OpenAIRE

    W. Fischle; D. Schwarzer; Mootz, H.

    2015-01-01

    Analysing post-translational modifications of histone proteins as they occur within chromatin is challenging due to their large number and chemical diversity. A major step forward has now been achieved by using split intein chemistry to engineer functionalized histones within cells.

  10. Production of Nα-acetylated thymosin α1 in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Fang Hongqing

    2011-04-01

    Full Text Available Abstract Background Thymosin α1 (Tα1, a 28-amino acid Nα-acetylated peptide, has a powerful general immunostimulating activity. Although biosynthesis is an attractive means of large-scale manufacture, to date, Tα1 can only be chemosynthesized because of two obstacles to its biosynthesis: the difficulties in expressing small peptides and obtaining Nα-acetylation. In this study, we describe a novel production process for Nα-acetylated Tα1 in Escherichia coli. Results To obtain recombinant Nα-acetylated Tα1 efficiently, a fusion protein, Tα1-Intein, was constructed, in which Tα1 was fused to the N-terminus of the smallest mini-intein, Spl DnaX (136 amino acids long, from Spirulina platensis, and a His tag was added at the C-terminus. Because Tα1 was placed at the N-terminus of the Tα1-Intein fusion protein, Tα1 could be fully acetylated when the Tα1-Intein fusion protein was co-expressed with RimJ (a known prokaryotic Nα-acetyltransferase in Escherichia coli. After purification by Ni-Sepharose affinity chromatography, the Tα1-Intein fusion protein was induced by the thiols β-mercaptoethanol or d,l-dithiothreitol, or by increasing the temperature, to release Tα1 through intein-mediated N-terminal cleavage. Under the optimal conditions, more than 90% of the Tα1-Intein fusion protein was thiolyzed, and 24.5 mg Tα1 was obtained from 1 L of culture media. The purity was 98% after a series of chromatographic purification steps. The molecular weight of recombinant Tα1 was determined to be 3107.44 Da by mass spectrometry, which was nearly identical to that of the synthetic version (3107.42 Da. The whole sequence of recombinant Tα1 was identified by tandem mass spectrometry and its N-terminal serine residue was shown to be acetylated. Conclusions The present data demonstrate that Nα-acetylated Tα1 can be efficiently produced in recombinant E. coli. This bioprocess could be used as an alternative to chemosynthesis for the production

  11. Large Scale Evaluation fo Nickel Aluminide Rolls

    Energy Technology Data Exchange (ETDEWEB)

    None

    2005-09-01

    This completed project was a joint effort between Oak Ridge National Laboratory and Bethlehem Steel (now Mittal Steel) to demonstrate the effectiveness of using nickel aluminide intermetallic alloy rolls as part of an updated, energy-efficient, commercial annealing furnace system.

  12. The CARN/ARNA Inaugural Study Day Inquiry: What Happens to Action Research after the Master's Degree?

    Science.gov (United States)

    Shosh, Joseph M.; McAteer, Mary

    2016-01-01

    The Collaborative Action Research Network (CARN) held its first American study day on the east coast of the United States in conjunction with the Action Research Network of the Americas (ARNA) 2014 conference in Bethlehem, Pennsylvania, USA. Study day participants visited three American secondary schools, one each in Pennsylvania, New York, and…

  13. READ Perspectives 1997.

    Science.gov (United States)

    Porter, Rosalie Pedalino, Ed.; Thomsen, Kerri Lynne, Ed.; Kimbrell, William, W., Ed.

    1997-01-01

    This document comprises the two 1997 issues of the journal. Articles include the following: "The Importance of Learning English: A National Survey of Hispanic Parents" (Michael La Velle); "The Languages of Immigrants" (Charles L. Glenn); "Follow-Up Study on the Bethlehem, PA School District's English Acquisition Program" (Ann Goldberg); "The Cost…

  14. THREE MILE ISLAND NUCLEAR REACTOR ACCIDENT OF MARCH 1979. ENVIRONMENTAL RADIATION DATA: VOLUME III. A REPORT TO THE PRESIDENT'S COMMISSION ON THE ACCIDENT AT THREE MILE ISLAND

    Science.gov (United States)

    This report contains a listing of environmental radiation monitoring data collected in the vicinity of Three Mile Island (TMI) following the March 28, 1979 accident. These data were collected by the EPA, NRC, DOE, HHS, the Commonwealth of Pennsylvania, or the Bethlehem Steel Corp...

  15. THREE MILE ISLAND NUCLEAR REACTOR ACCIDENT OF MARCH 1979. ENVIRONMENTAL RADIATION DATA: VOLUME II. A REPORT TO THE PRESIDENT'S COMMISSION ON THE ACCIDENT AT THREE MILE ISLAND

    Science.gov (United States)

    This report contains a listing of environmental radiation monitoring data collected in the vicinity of Three Mile Island (TMI) following the March 28, 1979 accident. These data were collected by the EPA, NRC, DOE, HHS, the Commonwealth of Pennsylvania, or the Bethlehem Steel Corp...

  16. THREE MILE ISLAND NUCLEAR REACTOR ACCIDENT OF MARCH 1979. ENVIRONMENTAL RADIATION DATA: UPDATE 2, VOLUME III

    Science.gov (United States)

    The original report contains a listing of environmental radiation monitoring data collected in the vicinity of Three Mile Island (TMI) following the March 28, 1979 accident. These data were collected by the EPA, NRC, DOE, HHS, the Commonwealth of Pennsylvania, or the Bethlehem St...

  17. THREE MILE ISLAND NUCLEAR REACTOR ACCIDENT OF MARCH 1979. ENVIRONMENTAL RADIATION DATA: VOLUME VI. A REPORT TO THE PRESIDENT'S COMMISSION ON THE ACCIDENT AT THREE MILE ISLAND

    Science.gov (United States)

    This report contains a listing of environmental radiation monitoring data collected in the vicinity of Three Mile Island (TMI) following the March 28, 1979 accident. These data were collected by the EPA, NRC, DOE, HHS, the Commonwealth of Pennsylvania, or the Bethlehem Steel Corp...

  18. THREE MILE ISLAND NUCLEAR REACTOR ACCIDENT OF MARCH 1979. ENVIRONMENTAL RADIATION DATA: UPDATE 2, VOLUME I

    Science.gov (United States)

    The original report contains a listing of environmental radiation monitoring data collected in the vicinity of Three Mile Island (TMI) following the March 28, 1979 accident. These data were collected by the EPA, NRC, DOE, HHS, the Commonwealth of Pennsylvania, or the Bethlehem St...

  19. THREE MILE ISLAND NUCLEAR REACTOR ACCIDENT OF MARCH 1979. ENVIRONMENTAL RADIATION DATA: VOLUME V. A REPORT TO THE PRESIDENT'S COMMISSION ON THE ACCIDENT AT THREE MILE ISLAND

    Science.gov (United States)

    This report contains a listing of environmental radiation monitoring data collected in the vicinity of Three Mile Island (TMI) following the March 28, 1979 accident. These data were collected by the EPA, NRC, DOE, HHS, the Commonwealth of Pennsylvania, or the Bethlehem Steel Corp...

  20. THREE MILE ISLAND NUCLEAR REACTOR ACCIDENT OF MARCH 1979. ENVIRONMENTAL RADIATION DATA: VOLUME I. A REPORT TO THE PRESIDENT'S COMMISSION ON THE ACCIDENT AT THREE MILE ISLAND

    Science.gov (United States)

    This report contains a listing of environmental radiation monitoring data collected in the vicinity of Three Mile Island (TMI) following the March 28, 1979 accident. These data were collected by the EPA, NRC, DOE, HHS, the Commonwealth of Pennsylvania, or the Bethlehem Steel Corp...

  1. THREE MILE ISLAND NUCLEAR REACTOR ACCIDENT OF MARCH 1979. ENVIRONMENTAL RADIATION DATA: VOLUME IV. A REPORT TO THE PRESIDENT'S COMMISSION ON THE ACCIDENT AT THREE MILE ISLAND

    Science.gov (United States)

    This report contains a listing of environmental radiation monitoring data collected in the vicinity of Three Mile Island (TMI) following the March 28, 1979 accident. These data were collected by the EPA, NRC, DOE, HHS, the Commonwealth of Pennsylvania, or the Bethlehem Steel Corp...

  2. THREE MILE ISLAND NUCLEAR REACTOR ACCIDENT OF MARCH 1979. ENVIRONMENTAL RADIATION DATA: UPDATE 2, VOLUME II

    Science.gov (United States)

    The original report contains a listing of environmental radiation monitoring data collected in the vicinity of Three Mile Island (TMI) following the March 28, 1979 accident. These data were collected by the EPA, NRC, DOE, HHS, the Commonwealth of Pennsylvania, or the Bethlehem St...

  3. THREE MILE ISLAND NUCLEAR REACTOR ACCIDENT OF MARCH 1979. ENVIRONMENTAL RADIATION DATA: UPDATE. A REPORT TO THE PRESIDENT'S COMMISSION ON THE ACCIDENT AT THREE MILE ISLAND

    Science.gov (United States)

    This report contains a listing of environmental radiation monitoring data collected in the vicinity of Three Mile Island (TMI) following the March 28, 1979 accident. These data were collected by the EPA, NRC, DOE, HHS, the Commonwealth of Pennsylvania, or the Bethlehem Steel Corp...

  4. Autotööstuse Delphi juht ravib firmat pankrotiga / Mikk Salu

    Index Scriptorium Estoniae

    Salu, Mikk, 1975-

    2005-01-01

    Maailma suurima autoosade valmistaja Delphi tegevjuht Steve Miller, kes on kuulsaks saanud United Airlines'i ja Bethlehem Steel'i pankrotiga, kuulutas välja Delphi pankroti ning seadis ametiühingud valiku ette: kas kärbitakse palku või lõpetatakse pensionide maksmine. Lisa: Pensionikoorem. Diagramm: Delphi kuulutas välja pankroti

  5. 76 FR 69324 - Office of Hazardous Materials Safety; Notice of Delays in Processing of Special Permits Applications

    Science.gov (United States)

    2011-11-08

    ...., Frankfort, KY. 10898-M Hydac 3 03-31-2012 Corporation, Bethlehem, PA. 11670-M Schlumberger 3 03-31-2012... LLC, State College, PA. 10646-M Schlumberger 03-31-2012 Technologies Corporation, Sugar Land, TX. 9758...-2012 Defense (MSDDC), Scott AFB, IL. 10709-R Schlumberger 4 03-31-2012 Technologies Corporation,...

  6. 77 FR 2124 - Notice of Delays in Processing of Special Permits Applications

    Science.gov (United States)

    2012-01-13

    .... 10898-M Hydac 3 03-31-2012 Corporation, Bethlehem, PA. 11670-M Schlumberger 3 03-31-2012 Oilfield UK Plc... College, PA. 10646-M Schlumberger 4 03-31-2012 Technologies Corporation, Sugar Land, TX. 14457-M Amtrol... Republic 4 03-31-2012 Environmental Systems (Pennsylvania), LLC, Hatfield, PA. 10709-R Schlumberger 4...

  7. 76 FR 56493 - Notice of Delays in Processing of Special Permits Applications

    Science.gov (United States)

    2011-09-13

    .... 10898-M Hydac 3 11-30-2011 Corporation, Bethlehem, PA. 11670-M Schlumberger 3 11-30-2011 Oilfield UK Plc.... Dept. of 4 11-30-2011 Defense (MSDDC), Scott AFB, IL. 10709-R Schlumberger 4 11-30-2011 Technologies..., Bothwell, ON. 14741-R Weatherford 4 11-30-2011 International, Fort Worth, TX. 4850-R Schlumberger 4...

  8. Molecular characterization and phylogenetic relationships of Desmodium leaf distortion virus (DeLDV): a new begomovirus infecting Desmodium glabrum in Yucatan, Mexico.

    Science.gov (United States)

    Hernández-Zepeda, Cecilia; Argüello-Astorga, Gerardo; Idris, Ali M; Carnevali, Germán; Brown, Judith K; Moreno-Valenzuela, Oscar A

    2009-12-01

    The complete DNA-A component sequence of Desmodium leaf distortion virus (DeLDV, Begomovirus) isolated in Yucatan was determined to be 2569 nucleotides (nt) in length, and it was most closely related to Cotton leaf crumple virus-California (CLCrV-[Cal]), at 76%. The complete DNA-B component sequence was 2514 nt in length, and shared its highest nucleotide identity (60%) with Potato yellow mosaic Trinidad virus (PYMTV). Phylogenetic analyses group the DeLDV DNA-A component in the SLCV clade, whereas, the DeLDV DNA-B was grouped with the Abutilon mosaic virus clade, which also contains PYMV, suggesting that the DeLDV components have distinct evolutionary histories, possibly as the result of recombination and reassortment. PMID:19757008

  9. Structural Insight into the DNA-Binding Mode of the Primosomal Proteins PriA, PriB, and DnaT

    Directory of Open Access Journals (Sweden)

    Yen-Hua Huang

    2014-01-01

    Full Text Available Replication restart primosome is a complex dynamic system that is essential for bacterial survival. This system uses various proteins to reinitiate chromosomal DNA replication to maintain genetic integrity after DNA damage. The replication restart primosome in Escherichia coli is composed of PriA helicase, PriB, PriC, DnaT, DnaC, DnaB helicase, and DnaG primase. The assembly of the protein complexes within the forked DNA responsible for reloading the replicative DnaB helicase anywhere on the chromosome for genome duplication requires the coordination of transient biomolecular interactions. Over the last decade, investigations on the structure and mechanism of these nucleoproteins have provided considerable insight into primosome assembly. In this review, we summarize and discuss our current knowledge and recent advances on the DNA-binding mode of the primosomal proteins PriA, PriB, and DnaT.

  10. Replisome mechanics: lagging strand events that influence speed and processivity

    OpenAIRE

    Georgescu, Roxana E.; Yao, Nina; Indiani, Chiara; Yurieva, Olga; O'Donnell, Mike E

    2014-01-01

    The antiparallel structure of DNA requires lagging strand synthesis to proceed in the opposite direction of the replication fork. This imposes unique events that occur only on the lagging strand, such as primase binding to DnaB helicase, RNA synthesis, and SS B antigen (SSB) displacement during Okazaki fragment extension. Single-molecule and ensemble techniques are combined to examine the effect of lagging strand events on the Escherichia coli replisome rate and processivity. We find that pri...

  11. Growth of Salmonella bacteriophage P22 in Escherichia coli dna(Ts) mutants.

    OpenAIRE

    Schanda-Mulfinger, U E; Schmieger, H

    1980-01-01

    Salmonella bacteriophage P22 grows in two deoxyribonucleic acid initiation mutants of Escherichia coli under nonpermissive conditions, dnaA and dnaC. Functional products of genes dnaE, dnaZ, lig, dnaK, and dnaG are indispensable for deoxyribonucleic acid replication of P22. In 11 E. coli dnaB mutants belonging to all phenotypic groups, phage were produced at 42 degrees C.

  12. Localization of an accessory helicase at the replisome is critical in sustaining efficient genome duplication.

    Science.gov (United States)

    Atkinson, John; Gupta, Milind K; Rudolph, Christian J; Bell, Hazel; Lloyd, Robert G; McGlynn, Peter

    2011-02-01

    Genome duplication requires accessory helicases to displace proteins ahead of advancing replication forks. Escherichia coli contains three helicases, Rep, UvrD and DinG, that might promote replication of protein-bound DNA. One of these helicases, Rep, also interacts with the replicative helicase DnaB. We demonstrate that Rep is the only putative accessory helicase whose absence results in an increased chromosome duplication time. We show also that the interaction between Rep and DnaB is required for Rep to maintain rapid genome duplication. Furthermore, this Rep-DnaB interaction is critical in minimizing the need for both recombinational processing of blocked replication forks and replisome reassembly, indicating that colocalization of Rep and DnaB minimizes stalling and subsequent inactivation of replication forks. These data indicate that E. coli contains only one helicase that acts as an accessory motor at the fork in wild-type cells, that such an activity is critical for the maintenance of rapid genome duplication and that colocalization with the replisome is crucial for this function. Given that the only other characterized accessory motor, Saccharomyces cerevisiae Rrm3p, associates physically with the replisome, our demonstration of the functional importance of such an association indicates that colocalization may be a conserved feature of accessory replicative motors. PMID:20923786

  13. Evolved plasmid-host interactions reduce plasmid interference cost.

    Science.gov (United States)

    Yano, Hirokazu; Wegrzyn, Katarznya; Loftie-Eaton, Wesley; Johnson, Jenny; Deckert, Gail E; Rogers, Linda M; Konieczny, Igor; Top, Eva M

    2016-09-01

    Antibiotic selection drives adaptation of antibiotic resistance plasmids to new bacterial hosts, but the molecular mechanisms are still poorly understood. We previously showed that a broad-host-range plasmid was poorly maintained in Shewanella oneidensis, but rapidly adapted through mutations in the replication initiation gene trfA1. Here we examined if these mutations reduced the fitness cost of TrfA1, and whether this was due to changes in interaction with the host's DNA helicase DnaB. The strains expressing evolved TrfA1 variants showed a higher growth rate than those expressing ancestral TrfA1. The evolved TrfA1 variants showed a lower affinity to the helicase than ancestral TrfA1 and were no longer able to activate the helicase at the oriV without host DnaA. Moreover, persistence of the ancestral plasmid was increased upon overexpression of DnaB. Finally, the evolved TrfA1 variants generated higher plasmid copy numbers than ancestral TrfA1. The findings suggest that ancestral plasmid instability can at least partly be explained by titration of DnaB by TrfA1. Thus under antibiotic selection resistance plasmids can adapt to a novel bacterial host through partial loss of function mutations that simultaneously increase plasmid copy number and decrease unfavorably high affinity to one of the hosts' essential proteins. PMID:27121483

  14. Seed-specific expression of spider silk protein multimers causes long-term stability

    OpenAIRE

    Nicola eWeichert; Valeska eHauptmann; Christine eHelmold; Udo eConrad

    2016-01-01

    Seeds enable plants to germinate and to grow in situations of limited availability of nutrients. The stable storage of different seed proteins is a remarkable presumption for successful germination and growth. These strategies have been adapted and used in several molecular farming projects. In this study, we explore the benefits of seed-based expression to produce the high molecular weight spider silk protein FLAG using intein-based trans-splicing. Multimers larger than 460 kDa in size are r...

  15. Small Molecule-Triggered Cas9 Protein with Improved Genome-Editing Specificity

    OpenAIRE

    Davis, Kevin M.; Pattanayak, Vikram; Thompson, David B.; Zuris, John A.; Liu, David R.

    2015-01-01

    Directly modulating the activity of genome-editing proteins has the potential to increase their specificity by reducing activity following target locus modification. We developed Cas9 nucleases that are activated by the presence of a cell-permeable small molecule by inserting an evolved 4-hydroxytamoxifen (4-HT)-responsive intein at specific positions in Cas9. In human cells, conditionally active Cas9s modify target genomic sites with up to 25-fold higher specificity than wild-type Cas9.

  16. Facile expression and purification of the antimicrobial peptide histatin 1 with a cleavable self-aggregating tag (cSAT) in Escherichia coli.

    Science.gov (United States)

    Xing, Lei; Xu, Wanghui; Zhou, Bihong; Chen, Yilu; Lin, Zhanglin

    2013-04-01

    Human histatin 1 (Hst1), a member of the histatin family, possesses antimicrobial properties. In this study, we applied a previously developed cleavable self-aggregating tag (cSAT) for the expression and purification of histatin 1 to demonstrate its utility for peptide expression and purification. The tag consists of a self-cleavable intein and a self-assembling peptide ELK16 (I-ELK16). First, an active insoluble aggregate of the recombinant histatin 1-Mxe GyrA intein-ELK16 (Hst1-I-ELK16) fusion protein was produced with a yield of 28.9 μg/mg wet cell pellet. The thiol reagent dithiothreitol (DTT) was then used to induce the intein-mediated cleavage and peptide release into the soluble fraction with a yield of 2.06 μg/mg wet cell pellet and a purity of 70%. The peptide was further purified by high performance liquid chromatography. These results were comparable to the yield and purity achieved when the more conventional glutathione transferase (GST) tag was used. The antimicrobial activities of this recombinant histatin 1 were confirmed against three Candida strains. This cSAT technique offers considerable advantages in terms of its simplicity and speed, eliminating the need for an exogenous protease, and reducing the number of chromatography purification steps. This technique should also be useful for the expression and purification of other AMPs. PMID:23403143

  17. BLAST FURNACE GRANULAR COAL INJECTION SYSTEM. Final Report Volume 2: Project Performance and Economics

    Energy Technology Data Exchange (ETDEWEB)

    Unknown

    1999-10-01

    Bethlehem Steel Corporation (BSC) requested financial assistance from the Department of Energy (DOE), for the design, construction and operation of a 2,800-ton-per-day blast furnace granulated coal injection (BFGCI) system for two existing iron-making blast furnaces. The blast furnaces are located at BSC's facilities in Burns Harbor, Indiana. The demonstration project proposal was selected by the DOE and awarded to Bethlehem in November 1990. The design of the project was completed in December 1993 and construction was completed in January 1995. The equipment startup period continued to November 1995 at which time the operating and testing program began. The blast furnace test program with different injected coals was completed in December 1998.

  18. BLAST FURNACE GRANULAR COAL INJECTION SYSTEM; FINAL

    International Nuclear Information System (INIS)

    Bethlehem Steel Corporation (BSC) requested financial assistance from the Department of Energy (DOE), for the design, construction and operation of a 2,800-ton-per-day blast furnace granulated coal injection (BFGCI) system for two existing iron-making blast furnaces. The blast furnaces are located at BSC's facilities in Burns Harbor, Indiana. The demonstration project proposal was selected by the DOE and awarded to Bethlehem in November 1990. The design of the project was completed in December 1993 and construction was completed in January 1995. The equipment startup period continued to November 1995 at which time the operating and testing program began. The blast furnace test program with different injected coals was completed in December 1998

  19. Instead of Conclusion: Jan Hus as Writer and Author

    Czech Academy of Sciences Publication Activity Database

    Šmahel, František

    Leiden : Brill, 2015 - (Šmahel, F.; Pavlíček, O.), s. 370-409 ISBN 978-90-04-28055-7. - (Brill's Companions to the Christian Tradition. 54) R&D Projects: GA ČR(CZ) GBP405/12/G148 Institutional support: RVO:67985955 Keywords : Jan Hus * John Wyclif * Translation of the Bible * Bethlehem Chapel in Prague * Czech Orthography * Citation Mode Subject RIV: AB - History

  20. Three Mile Island nuclear reactor accident of March 1979. Environmental radiation data: Update 2, Volume II

    International Nuclear Information System (INIS)

    The original report contains a listing of environmental radiation monitoring data collected in the vicinity of Three Mile Island (TMI) following the March 28, 1979 accident. These data were collected by the EPA, NRC, DOE, HHS, the Commonwealth of Pennsylvania, or the Bethlehem Steel Corporation. The original report was printed in September 1979 and the update was released in December 1979. This final update consists of additional data for 1979 by the same participating organizations, which has not been previously reported

  1. Leaning Towards Agile in 21~(st) Century

    Institute of Scientific and Technical Information of China (English)

    D; Ashall; B; Parkinson

    2002-01-01

    Background: During the last fifteen years there ha s been an increased drive for organisations to reduce costs. From a production po int, this has often centred on lean manufacturing and JIT waste elimination proc esses. However, in 1991, the Iaccocca Institute Bethlehem P.A commissioned a re port specifically to analyse the changing nature of the marketplace. As a result , in the following year, the Agile Manufacturing Forum was initiated and the ter m 'agile or responsive manufacturing' was first intr...

  2. Participation and reconciliation. Preconditions of justice

    OpenAIRE

    2011-01-01

    "In order to make justice work, participation and reconciliation is needed within and between societies, peoples, and nations. In this compilation, authors - senior academics as well as students - from Bethlehem University, Palestine, and the Catholic University of Applied Sciences, Cologne, Germany, contribute to this important field. Thus, to some extent, the book in itself is an example of the subjects it deals with." (author's abstract). Table of Contents: Sami Adwan, Armin G. Wildfeuer: ...

  3. Stop Israel!

    OpenAIRE

    Reinhart, T.

    2006-01-01

    For a whole week now, The Israeli army has been terrorizing cities and villages in the West Bank. As in the darkest days at the beginning of the present Intifada, desperate voices and reports pour through the internet, telling of massive shelling, including schools, hospitals, the university and a maternity house in Bethlehem, of curfew, houses being seized or destroyed, water tanks ruined in refugee camps. In Beit Reema, the site of Israel's latest show of horror, ambulances were not allowed...

  4. Whole-brain functional hypoconnectivity as an endophenotype of autism in adolescents

    OpenAIRE

    Moseley, R.L.; Ypma, R.J.F.; Holt, R J; Floris, D.; Chura, L.R.; M.D. Spencer; Baron-Cohen, S; Suckling, J; Bullmore, E; Rubinov, M.

    2015-01-01

    The authors wish to thank the participants and their families for their participation and the autism support organisations who assisted with recruitment. We thank colleagues at the Brain Mapping Unit for methodological discussions and thank Meng-Chuan Lai, Amber Ruigrok and Richard Bethlehem for the same. Data collection was funded by a Clinical Scientist Fellowship from the UK Medical Research Council (MRC) (G0701919) to MDS. LRC was supported by the Gates Cambridge Scholarship Trust. The st...

  5. The significance of dreams and the star in Matthew's infancy narrative

    OpenAIRE

    Francois Viljoen

    2008-01-01

    The phenomena of dreams and the star of Bethlehem in Matthew’s birth narrative have intrigued scholars through the ages. Scholarship in this regard went through the stages of identifying the origin of the material and of arguing the historicity of these events. Currently scholarship is moving into a new stage of investigating the meaning of these narratives. Without engaging the arguments developed by the first two stages mentioned, I investigate the significance of these unusual forms of rev...

  6. Molecular characterization and phylogenetic relationships of two new bipartite begomovirus infecting malvaceous plants in Yucatan, Mexico.

    Science.gov (United States)

    Hernández-Zepeda, Cecilia; Idris, Ali M; Carnevali, Germán; Brown, Judith K; Moreno-Valenzuela, Oscar A

    2007-10-01

    Sida acuta and Corchorus siliquosus plants showing yellow mosaic and yellow vein symptoms, respectively, were collected in the Yucatan Peninsula, Mexico. Total DNA was isolated from both plant species and used for the amplification, cloning, and sequencing of the Begomovirus genome. Nucleotide comparison of the complete DNA-A component isolated from S. acuta and C. siliquosus confirmed the presence of two distinct begomoviruses species. Based on phenotypic symptoms observed in infected field plants, the names Sida yellow mosaic Yucatan virus (SiYMYuV) and Corchorus yellow vein Yucatan virus (CoYVYuV) were proposed. The SiYMYuV DNA-A shared the highest nucleotide identity (86%) with the Okra yellow mosaic Mexico virus (OkYMMV). The complete DNA-B component shared the highest nucleotide identity (80%) with CoYVYuV. The CoYVYuV DNA-A shared the highest nucleotide identity (84%) with SiYMYuV. The 166-nt common region (CR) sequence for the DNA-A and DNA-B components of SiYMYuV shared a high nucleotide identity of 99%, and the 151 nt of CoYVYuV CR shared 95% of nucleotide identity. The organization and the iterated sequence of the putative AC1 binding site (located within the common region) of both isolates, were similar to that of the begomoviruses of the Western Hemisphere. Phylogenetic analyses placed the DNA-A and DNA-B of SiYMYuV and CoYVYuV in the clade containing the Abutilon mosaic virus (AbMV). PMID:17638064

  7. Structure and Function of the PriC DNA Replication Restart Protein.

    Science.gov (United States)

    Wessel, Sarah R; Cornilescu, Claudia C; Cornilescu, Gabriel; Metz, Alice; Leroux, Maxime; Hu, Kaifeng; Sandler, Steven J; Markley, John L; Keck, James L

    2016-08-26

    Collisions between DNA replication complexes (replisomes) and barriers such as damaged DNA or tightly bound protein complexes can dissociate replisomes from chromosomes prematurely. Replisomes must be reloaded under these circumstances to avoid incomplete replication and cell death. Bacteria have evolved multiple pathways that initiate DNA replication restart by recognizing and remodeling abandoned replication forks and reloading the replicative helicase. In vitro, the simplest of these pathways is mediated by the single-domain PriC protein, which, along with the DnaC helicase loader, can load the DnaB replicative helicase onto DNA bound by the single-stranded DNA (ssDNA)-binding protein (SSB). Previous biochemical studies have identified PriC residues that mediate interactions with ssDNA and SSB. However, the mechanisms by which PriC drives DNA replication restart have remained poorly defined due to the limited structural information available for PriC. Here, we report the NMR structure of full-length PriC from Cronobacter sakazakii PriC forms a compact bundle of α-helices that brings together residues involved in ssDNA and SSB binding at adjacent sites on the protein surface. Disruption of these interaction sites and of other conserved residues leads to decreased DnaB helicase loading onto SSB-bound DNA. We also demonstrate that PriC can directly interact with DnaB and the DnaB·DnaC complex. These data lead to a model in which PriC acts as a scaffold for recruiting DnaB·DnaC to SSB/ssDNA sites present at stalled replication forks. PMID:27382050

  8. Melon Chlorotic Leaf Curl Virus: Characterization and Differential Reassortment with Closest Relatives Reveal Adaptive Virulence in the Squash Leaf Curl Virus Clade and Host Shifting by the Host-Restricted Bean Calico Mosaic Virus▿

    OpenAIRE

    Idris, A. M.; Mills-Lujan, K.; Martin, K; Brown, J K

    2007-01-01

    The genome components of the Melon chlorotic leaf curl virus (MCLCuV) were cloned from symptomatic cantaloupe leaves collected in Guatemala during 2002. The MCLCuV DNA-A and DNA-B components shared their closest nucleotide identities among begomoviruses, at ∼90 and 81%, respectively, with a papaya isolate of MCLCuV from Costa Rica. The closest relatives at the species level were other members of the Squash leaf curl virus (SLCV) clade, which is endemic in the southwestern United States and Me...

  9. The role of template superhelicity in the initiation of bacteriophage lambda DNA replication.

    OpenAIRE

    Alfano, C; McMacken, R

    1988-01-01

    The prepriming steps in the initiation of bacteriophage lambda DNA replication depend on the action of the lambda O and P proteins and on the DnaB helicase, single-stranded DNA binding protein (SSB), and DnaJ and DnaK heat shock proteins of the E. coli host. The binding of multiple copies of the lambda O protein to the phage replication origin (ori lambda) initiates the ordered assembly of a series of nucleoprotein structures that form at ori lambda prior to DNA unwinding, priming and DNA syn...

  10. Host requirements for growth of lambda-P22 hybrid in Escherichia coli.

    OpenAIRE

    Taylor, K.D.; Shizuya, H

    1981-01-01

    The requirements for growth of bacteriophage lambda containing the deoxyribonucleic acid replication region from Salmonella phage P22 were determined in a burst size experiment. The products of genes dnaE, dnaJ, dnaK, dnaY, dnaZ, and seg were required, but not the products of genes dnaA, dnaB, dnaC, and dnaX. This lambda-P22 hybrid phage was also dependent on polA for growth at 32 degrees C.

  11. The 32 kDa subunit of replication protein A (RPA) participates in the DNA replication of Mung bean yellow mosaic India virus (MYMIV) by interacting with the viral Rep protein

    OpenAIRE

    Singh, Dharmendra Kumar; Islam, Mohammad Nurul; Choudhury, Nirupam Roy; Karjee, Sumona; Mukherjee, Sunil Kumar

    2006-01-01

    Mung bean yellow mosaic India virus (MYMIV) is a member of genus begomoviridae and its genome comprises of bipartite (two components, namely DNA-A and DNA-B), single-stranded, circular DNA of about 2.7 kb. During rolling circle replication (RCR) of the DNA, the stability of the genome and maintenance of the stem–loop structure of the replication origin is crucial. Hence the role of host single-stranded DNA-binding protein, Replication protein A (RPA), in the RCR of MYMIV was examined. Two RPA...

  12. RepA and DnaA proteins are required for initiation of R1 plasmid replication in vitro and interact with the oriR sequence.

    OpenAIRE

    Masai, H.; Arai, K.

    1987-01-01

    RepA, an initiation protein of R1 plasmid replication, was purified from an Escherichia coli strain overproducing the protein. The purified RepA protein specifically initiated replication in vitro of plasmid DNA bearing the replication origin of R1 plasmid (oriR). The replication, strictly dependent on added RepA protein, was independent of host RNA polymerase but required other host replication functions (DnaB and DnaC proteins, the single-stranded-DNA-binding protein SSB, and DNA gyrase). T...

  13. Various mechanisms in cyclopeptide production from precursors synthesized independently of non-ribosomal peptide synthetases

    Institute of Scientific and Technical Information of China (English)

    Wenyan Xu; Liling Li; Liangcheng Du; Ninghua Tan

    2011-01-01

    An increasing number of cyclopeptides have been discovered as products of ribosomal synthetic pathway.The biosynthetic study of these cyclopeptides has revealed interesting new mechanisms for cyclization.This review highlighted the recent discoveries in cyclization mechanisms for cyclopeptides synthesized independently of non-ribosomal peptide synthetases,including endopeptidase-catalyzed cyclization,intein-mediated cyclization,and peptide synthetase-catalyzed cyclization.This information may help to design hybrid ribosomal and non-ribosomal biosynthetic systems to produce novel cyclopeptides with various bioactivities.

  14. Molecular characterization of distinct bipartite begomovirus infecting bhendi (Abelmoschus esculentus L.) in India.

    Science.gov (United States)

    Venkataravanappa, V; Lakshminarayana Reddy, C N; Jalali, Salil; Krishna Reddy, M

    2012-06-01

    Yellow vein mosaic disease of okra is a whitefly transmitted begomovirus causing heavy economic loss in different parts of India. The okra isolate (OY131) of this virus from a bhendi plant [(Abelmoschus esculentus L.) Moench] showing yellow vein mosaic, vein twisting, reduced leaves, and a bushy appearance in the Palem region, New Delhi, India, was characterized in the present study. The complete DNA-A and DNA-B sequences have been determined and are comprised of 2,746 and 2,703 nucleotides, respectively. The betasatellite (DNA-β) component was absent in the sample. The genome organization was typically of biparite begomoviruses, which were characterized earlier. Comparison of DNA-A component with other known begomoviruses suggest that this virus, being only distantly related (yellow vein mosaic Delhi virus [BYVDV-IN (India: Delhi: okra)]. DNA-B showed highest sequence identity (87.8% identical) to that of a ToLCNDV (AY158080). The phylogenetic analysis of the present isolate is distinct from all other viruses; however clusters with ToLCNDV group infect different crops. The recombination analysis revealed that this isolate has sequences originated from ToLCNDV. This is the first known bhendi yellow vein mosaic disease associated bipartite begomovirus from India. PMID:22447131

  15. Analysis of watermelon chlorotic stunt virus and tomato leaf curl Palampur virus mixed and pseudo-recombination infections.

    Science.gov (United States)

    Esmaeili, Maryam; Heydarnejad, Jahangir; Massumi, Hossain; Varsani, Arvind

    2015-12-01

    Watermelon chlorotic stunt virus (WmCSV) and tomato leaf curl Palampur virus (ToLCPMV) are limiting factors for cucurbit production in south and southeastern Iran. ToLCPMV infects all cucurbit crops (except watermelons) whereas WmCSV is somewhat limited to watermelon, causing detrimental effects on fruit production. In a survey, we detected WmCSV in all watermelon growing farms in Fars province (southern Iran). Given that WmCSV and ToLCPMV are present in the same geographical location in Iran, we studied the interaction of two viruses. Co-infection using agroinfectious clones of WmCSV and ToLCPMV caused severe symptoms in watermelon and zucchini in comparison to symptoms observed from individual infections. Interestingly, inoculation of zucchini with WmCSV DNA-A and ToLCPMV DNA-B agroinfectious clones or vice versa produced a viable pseudo-recombinant and induced systemic symptoms. This demonstrates that replication-associated protein of DNA-A of each virus is able to bind to cis elements of the DNA-B molecules of another virus. PMID:26433951

  16. Complete nucleotide sequences of a new bipartite begomovirus from Malvastrum sp. plants with bright yellow mosaic symptoms in South Texas.

    Science.gov (United States)

    Alabi, Olufemi J; Villegas, Cecilia; Gregg, Lori; Murray, K Daniel

    2016-06-01

    Two isolates of a novel bipartite begomovirus, tentatively named malvastrum bright yellow mosaic virus (MaBYMV), were molecularly characterized from naturally infected plants of the genus Malvastrum showing bright yellow mosaic disease symptoms in South Texas. Six complete DNA-A and five DNA-B genome sequences of MaBYMV obtained from the isolates ranged in length from 2,608 to 2,609 nucleotides (nt) and 2,578 to 2,605 nt, respectively. Both genome segments shared a 178- to 180-nt common region. In pairwise comparisons, the complete DNA-A and DNA-B sequences of MaBYMV were most similar (87-88 % and 79-81 % identity, respectively) and phylogenetically related to the corresponding sequences of sida mosaic Sinaloa virus-[MX-Gua-06]. Further analysis revealed that MaBYMV is a putative recombinant virus, thus supporting the notion that malvaceous hosts may be influencing the evolution of several begomoviruses. The design of new diagnostic primers enabled the detection of MaBYMV in cohorts of Bemisia tabaci collected from symptomatic Malvastrum sp. plants, thus implicating whiteflies as potential vectors of the virus. PMID:27016928

  17. In vitro inhibition of hyaluronidase by sodium copper chlorophyllin complex and chlorophyllin analogs

    OpenAIRE

    McCook JP; Dorogi PL; Vasily DB; Cefalo DR

    2015-01-01

    John P McCook,1 Peter L Dorogi,2 David B Vasily,3 Dustin R Cefalo4 1Discovery Partners, LLC, Frisco, TX, 2CHL Industries, LLC, Easton, PA, 3Aesthetica Cosmetic and Laser Surgery Center, Bethlehem, PA, 4Frontier Scientific, Inc., Logan, UT, USA Background: Inhibitors of hyaluronidase are potent agents that maintain hyaluronic acid homeostasis and may serve as anti-aging, anti-inflammatory, and anti-microbial agents. Sodium copper chlorophyllin complex is being used therapeutically as a compon...

  18. Palestinian Refugees, Citizenship and the Nation-State

    OpenAIRE

    HANAFI, Sari

    2014-01-01

    In the Bethlehem Fatah communiqué, the authors considered the Palestinian state as a substitute for the right of return. But is there a solution that encompasses the right of return and a Palestinian state? The question is not only one of right or the number of eventual returnees or the technical economic and social capacity for absorption, but is also a question of the nature of both the Palestinian and the Israeli nation-states, the concept of state sovereignty and its inherent violence, an...

  19. Mission as action in hope in the context of white poverty in Pretoria: a case for Betlehem Mission Centre

    OpenAIRE

    Mashau, Thinandavha Derrick

    2012-01-01

    The gap between the rich and the poor is widening daily and the proportion of people living in poverty in South Africa is steadily on the rise. The phenomenon of white poverty has existed since the 1890s and is becoming a more common trend across South Africa. White poverty left a number of whites in South Africa homeless. Consequently, they are forced to live in the streets, in shelters and informal settlements such as Bethlehem Mission Centre in Pretoria. A descriptive study was undertaken ...

  20. Complexities of speech in Palestinian refugee camps Complexités du discours dans les camps de réfugiés palestiniens. Résultats du travail de terrain et réévaluation de la théorie

    Directory of Open Access Journals (Sweden)

    Nancy Hawker

    2011-03-01

    Full Text Available This paper is a report on work in progress. I have recently finished my fieldwork in three refugee camps in the West Bank: Shuafat RC, which is in the Jerusalem Municipal Area, Dheisheh RC, which is in the Bethlehem Governorate, and Tulkarem RC, which is in the north of the West Bank near the Green Line. I have been recording the speech of respondents in these locations to find out whether contact with Hebrew speakers is effecting language variation and change in spoken Palestinian Arabic. So...

  1. Complexities of speech in Palestinian refugee camps Complexités du discours dans les camps de réfugiés palestiniens. Résultats du travail de terrain et réévaluation de la théorie

    OpenAIRE

    Nancy Hawker

    2011-01-01

    This paper is a report on work in progress. I have recently finished my fieldwork in three refugee camps in the West Bank: Shuafat RC, which is in the Jerusalem Municipal Area, Dheisheh RC, which is in the Bethlehem Governorate, and Tulkarem RC, which is in the north of the West Bank near the Green Line. I have been recording the speech of respondents in these locations to find out whether contact with Hebrew speakers is effecting language variation and change in spoken Palestinian Arabic. So...

  2. Complexities of speech in Palestinian refugee camps

    OpenAIRE

    Hawker, Nancy

    2011-01-01

    This paper is a report on work in progress. I have recently finished my fieldwork in three refugee camps in the West Bank: Shuafat RC, which is in the Jerusalem Municipal Area, Dheisheh RC, which is in the Bethlehem Governorate, and Tulkarem RC, which is in the north of the West Bank near the Green Line. I have been recording the speech of respondents in these locations to find out whether contact with Hebrew speakers is effecting language variation and change in spoken Palestinian Arabic. So...

  3. Manual of Decolonization

    DEFF Research Database (Denmark)

    Gigone, Fabio

    2010-01-01

    During a residency in Bethlehem, Venice-based design and research studio Salottobuono formulated a 'strategy of subversion' for Israeli settlements in the West Bank. From this comes the elegant Manual of Decolonization; a generic but detailed resource for all post-occupation scenarios. Assessing...... how evacuated and imposed structures can be transformed in collaboration with local stakeholders, the book's plans, models and projections are divided into themes like 'De-parcelling', 'Re-combining' and 'Un-roofing'; landscape, nature and materials are considered at length. The outcome is an open...

  4. Clean Coal III Project: Blast Furnace Granular Coal Injection Project Trial 1 Report - Blast Furnace Granular Coal Injection - Results with Low Volatile Coal

    Energy Technology Data Exchange (ETDEWEB)

    None, None

    1997-11-01

    This report describes the first coal trial test conducted with the Blast Furnace Granular Coal Injection System at Bethlehem Steel Corporation's Burns Harbor Plant. This demonstration project is divided into three phases: Phase I - Design Phase II - Construction Phase III - Operation The design phase was conducted in 1991-1993. Construction of the facility began in August 1993 and was completed in late 1994. The coal injection facility began operating in January 1995 and Phase III began in November 1995. The Trial 1 base test orI C furnace was carried out in October 1996 as a comparison period for the analysis of the operation during subsequent coal trials.

  5. Clean Coal III Project: Blast Furnace Granular Coal Injection Project Trail 1 Report - Blast Furnace Granular Coal Injection - Results with Low Volatile Coal

    Energy Technology Data Exchange (ETDEWEB)

    None, None

    1997-11-01

    This report describes the first coal trial test conducted with the Blast Furnace Granular Coal Injection System at Bethlehem Steel Corporation's Burns Harbor Plant. This demonstration project is divided into three phases: Phase I - Design Phase II - Construction Phase III - Operation The design phase was conducted in 1991-1993, Construction of the facility began in August 1993 and was completed in late 1994. The coal injection facility began operating in January 1995 and Phase III began in November 1995. The Trial 1 base test on C furnace was carried out in October 1996 as a comparison period for the analysis of the operation during subsequent coal trials.

  6. The early Latin Church Fathers on Herod and the Infanticide

    Directory of Open Access Journals (Sweden)

    M. J. Mans

    1997-01-01

    Full Text Available Since the views of the early Latin Church Fathers on Herod and the carnage at Bethlehem have been neglected by modem scholars, this study is an attempt to discover and interpret  their opinions on the notorious king and this tragic event. Apparently, the main aim of the Latin Church Fathers was to present Herod's heinous deed in the worst possible light, and to exalt the Innocents to the ranks of the martyrs.

  7. Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester

    Directory of Open Access Journals (Sweden)

    Raats Jos MH

    2009-07-01

    Full Text Available Abstract Background Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL. However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging. Results Targeting to the periplasm of E. coli allowed efficient production of correctly-folded single-domain antibody (sdAb-intein fusions proteins. On column purification and 2-mercapthoethanesulfonic acid (MESNA-induced cleavage yielded single-domain antibodies with a reactive C-terminal MESNA thioester in good yields. These thioester-functionalized single-domain antibodies allowed synthesis of immunomicelles via native chemical ligation in a single step. Conclusion A novel procedure was developed to obtain soluble, well-folded single-domain antibodies with reactive C-terminal thioesters in good yields. These proteins are promising building blocks for the chemoselective functionalization via NCL of a broad range of nanoparticle scaffolds, including micelles, liposomes and dendrimers.

  8. Spider silk-like proteins derived from transgenic Nicotiana tabacum.

    Science.gov (United States)

    Peng, Congyue Annie; Russo, Julia; Gravgaard, Charlene; McCartney, Heather; Gaines, William; Marcotte, William R

    2016-08-01

    The high tensile strength and biocompatibility of spider dragline silk makes it a desirable material in many engineering and tissue regeneration applications. Here, we present the feasibility to produce recombinant proteins in transgenic tobacco Nicotiana tabacum with sequences representing spider silk protein building blocks . Recombinant mini-spidroins contain native N- and C-terminal domains of major ampullate spidroin 1 (rMaSp1) or rMaSp2 flanking an abbreviated number (8, 16 or 32) of consensus repeat domains. Two different expression plasmid vectors were tested and a downstream chitin binding domain and self-cleavable intein were included to facilitate protein purification. We confirmed gene insertion and RNA transcription by PCR and reverse-transcriptase PCR, respectively. Mini-spidroin production was detected by N-terminus specific antibodies. Purification of mini-spidroins was performed through chitin affinity chromatography and subsequent intein activation with reducing reagent. Mini-spidroins, when dialyzed and freeze-dried, formed viscous gelatin-like fluids. PMID:27026165

  9. Biosynthesis of the cyclotide Kalata B1 using protein splicing tools

    Energy Technology Data Exchange (ETDEWEB)

    Kimura, R; Krishnan, K; Camarero, J A

    2005-07-13

    Cyclotides are a new emerging family of large cyclic polypeptides ({approx}30 residues long) that share a disulfide-stabilized core (3 disulfide bonds) with an unusual knotted structure (Fig. 1A) [1]. Cyclotides contrast with other circular polypeptides in that they have a highly defined three-dimensional structure, and despite their small size, can be considered as miniproteins. Their unique circular backbone topology and knotted arrangement of 3 disulfide bonds makes them exceptionally stable to thermal and enzymatic degradation. Furthermore, their well defined structures have been also associated with a range of biological activities, including uterotonic activity, inhibition of neurotension binding, hemolytic, anti-HIV, insecticidal as well as trypsin inhibitory activity. Altogether, these characteristics make cyclotides ideal candidates to be used as molecular scaffolds for the development of stable peptide drugs [2]. Access to biosynthetic cyclotides using recombinant DNA expression techniques would offer the exciting possibility of producing large combinatorial libraries of highly stable miniproteins using the tools of molecular biology. This would allow the generation of cell-based combinatorial libraries that could be screened inside living cells for their ability to regulate cellular processes. In the present work we describe for the first time the biosynthesis of the cyclotide Kalata B1 in E. coli. Our approach is based on the use of an intramolecular version of the native chemical ligation combined with the use of a modified protein splicing unit [3]. In order to accomplish the cyclization of Kalata B1, the different linear precursors tested in this work (Fig. 1B) were fused at the N-terminus with a Met residue, and at the C-terminus with an VMA engineered intein (available in the pTXB expression vectors family from New England Biolabs). The Met residue was efficiently removed in vivo in E. coli by an endogenous Met amino peptidase. This in vivo

  10. One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester

    Directory of Open Access Journals (Sweden)

    Merkx Maarten

    2008-10-01

    Full Text Available Abstract Background Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed. Results A novel redox buffer consisting of MESNA and diMESNA showed a refolding efficiency comparable to that of GSH/GSSG and prevented loss of the protein's thioester functionality. Moreover, introduction of the MESNA/diMESNA redox couple in the cleavage buffer allowed simultaneous on-column refolding of Ribonuclease A and intein-mediated cleavage to yield Ribonuclease A with a C-terminal MESNA-thioester. The C-terminal thioester was shown to be active in native chemical ligation. Conclusion An efficient method was developed for the production of disulfide bond containing proteins with C-terminal thioesters. Introduction of a MESNA/diMESNA redox couple resulted in simultaneous on-column refolding, purification and thioester generation of the model protein Ribonuclease A.

  11. TMD2前断裂CFTR翻译后的连接及氯离子通道功能%Post-translational ligation of split CFTR severed before TMD2 and its chloride channel function

    Institute of Scientific and Technical Information of China (English)

    朱甫祥; 宫贤弟; 刘泽隆; 杨树德; 屈慧鸽; 迟晓艳

    2010-01-01

    CFTR基因突变导致一种常染色体隐性遗传疾病--囊性纤维化(CF).利用split Ssp DnaB intein的蛋白质反式剪接技术的真核细胞双载体转CFTR基因,旨在研究翻译后水平CFTR的连接,以及由其建立的氯离子通道功能.于CFTR膜内第2个跨膜结构域(TMD2)前的Glu838密码子后将其cDNA断裂为N端和C端两部分,与具有蛋白质反式剪接作用的split Ssp DnaB intein编码序列融合,分别插入到载体pEGFP-N1和pEYFP-N1,构建一对真核表达载体pEGFP-Nint和pEYFP-IntC.用脂质体将这对载体共转染至幼年仓鼠肾细胞(BHK),瞬时表达实验用Western blotting观察CFTR蛋白质的连接,并用膜片钳技术记录Cl-通道电流.结果显示,基因共转染细胞呈现完整的CFTR蛋白条带,膜片钳记录到全细胞Cl-电流和单个Cl-通道开放活性.结果表明split Ssp DnaB intein的蛋白质反式剪接技术可用于双载体共转移CFTR基因,为CF基因治疗应用双腺相关病毒载体(AAV)转运CFTR基因,克服AAV的容量限制提供了依据.

  12. An HIV1/2 point of care test on sputum for screening TB/HIV co-infection in central India - Will it work?

    Institute of Scientific and Technical Information of China (English)

    Prabha Desikan; Sajal De; Nitika Pant Pai; Pradyumna K Mishra; Kaushal Kumar; Nikita Panwalkar; Mayanka Verma; Zia Ul Hasan; Kewal K Maudar

    2013-01-01

    Objective:To determine whether theOraQuick®HIV-1/2Assay(OraSureTechnologies, Inc.,Bethlehem,PA,USA) in sputum is a valid tool forHIV surveillance amongTB patients. Methods:A cross sectional study was carried out on sputa of patients diagnosed with tuberculosis.Sputa were tested for antibodies toHIV usingOraQuick®HIV-1/2Assay(OraSure Technologies,Inc.,Bethlehem,PA,USA).The results were compared with results of serum ELISA.Results:Compared to serumELISA, theOraQuick®HIV-1/2Assay in sputum specimens reported90% sensitivity(9/10) and100% specificity(307/307), with a positive predictive value of 100%(95%CI:66.37%-100.00%) and a negative predictive value of99.68%(95%CI:98.20%-99.99%). Conclusions:This testing method may provide a useful strategy for conductingHIV surveillance in possible co-infectedTB patients at peripheral centres.Since there is no investment on infrastructure, it may be possible for paramedical health professionals to carry out the test, particularly in areas with lowHIV endemicity.

  13. Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility

    International Nuclear Information System (INIS)

    A common obstacle to NMR studies of proteins is sample preparation. In many cases, proteins targeted for NMR studies are poorly expressed and/or expressed in insoluble forms. Here, we describe a novel approach to overcome these problems. In the protein S tag-intein (PSTI) technology, two tandem 92-residue N-terminal domains of protein S (PrS2) from Myxococcus xanthus is fused at the N-terminal end of a protein to enhance its expression and solubility. Using intein technology, the isotope-labeled PrS2-tag is replaced with non-isotope labeled PrS2-tag, silencing the NMR signals from PrS2-tag in isotope-filtered 1H-detected NMR experiments. This method was applied to the E. coli ribosome binding factor A (RbfA), which aggregates and precipitates in the absence of a solubilization tag unless the C-terminal 25-residue segment is deleted (RbfAΔ25). Using the PrS2-tag, full-length well-behaved RbfA samples could be successfully prepared for NMR studies. PrS2 (non-labeled)-tagged RbfA (isotope-labeled) was produced with the use of the intein approach. The well-resolved TROSY-HSQC spectrum of full-length PrS2-tagged RbfA superimposes with the TROSY-HSQC spectrum of RbfAΔ25, indicating that PrS2-tag does not affect the structure of the protein to which it is fused. Using a smaller PrS-tag, consisting of a single N-terminal domain of protein S, triple resonance experiments were performed, and most of the backbone 1H, 15N and 13C resonance assignments for full-length E. coli RbfA were determined. Analysis of these chemical shift data with the Chemical Shift Index and heteronuclear 1H–15N NOE measurements reveal the dynamic nature of the C-terminal segment of the full-length RbfA protein, which could not be inferred using the truncated RbfAΔ25 construct. CS-Rosetta calculations also demonstrate that the core structure of full-length RbfA is similar to that of the RbfAΔ25 construct.

  14. Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility.

    Science.gov (United States)

    Kobayashi, Hiroshi; Swapna, G V T; Wu, Kuen-Phon; Afinogenova, Yuliya; Conover, Kenith; Mao, Binchen; Montelione, Gaetano T; Inouye, Masayori

    2012-04-01

    A common obstacle to NMR studies of proteins is sample preparation. In many cases, proteins targeted for NMR studies are poorly expressed and/or expressed in insoluble forms. Here, we describe a novel approach to overcome these problems. In the protein S tag-intein (PSTI) technology, two tandem 92-residue N-terminal domains of protein S (PrS(2)) from Myxococcus xanthus is fused at the N-terminal end of a protein to enhance its expression and solubility. Using intein technology, the isotope-labeled PrS(2)-tag is replaced with non-isotope labeled PrS(2)-tag, silencing the NMR signals from PrS(2)-tag in isotope-filtered (1)H-detected NMR experiments. This method was applied to the E. coli ribosome binding factor A (RbfA), which aggregates and precipitates in the absence of a solubilization tag unless the C-terminal 25-residue segment is deleted (RbfAΔ25). Using the PrS(2)-tag, full-length well-behaved RbfA samples could be successfully prepared for NMR studies. PrS(2) (non-labeled)-tagged RbfA (isotope-labeled) was produced with the use of the intein approach. The well-resolved TROSY-HSQC spectrum of full-length PrS(2)-tagged RbfA superimposes with the TROSY-HSQC spectrum of RbfAΔ25, indicating that PrS(2)-tag does not affect the structure of the protein to which it is fused. Using a smaller PrS-tag, consisting of a single N-terminal domain of protein S, triple resonance experiments were performed, and most of the backbone (1)H, (15)N and (13)C resonance assignments for full-length E. coli RbfA were determined. Analysis of these chemical shift data with the Chemical Shift Index and heteronuclear (1)H-(15)N NOE measurements reveal the dynamic nature of the C-terminal segment of the full-length RbfA protein, which could not be inferred using the truncated RbfAΔ25 construct. CS-Rosetta calculations also demonstrate that the core structure of full-length RbfA is similar to that of the RbfAΔ25 construct. PMID:22389115

  15. Mechanism of conjugation and recombination in bacteria 18. Polarity of donor DNA strands transferred to the recipient as determined by DNA-RNA hybridization

    International Nuclear Information System (INIS)

    Polarity of donor DNA strands transferred into recipient during conjugation in Escherichia coli K-12 was determined by DNA-3H-RNA hybridization. Lambda prophage was used as a marker. The defective lysogen Hfr H(lambda t11) as a donor and thermosensitive F-CR34dnaB strain as recipient were used. Two sets of hybridization experiments, with 1-strand specific lambda mRNA and lambda mRNA specific for both phage strands but with large excess of r-strand specific mRNA, were carried out. Strand 1 od lambda DNA was detected preferentially in recipient cells mated at restrictive temperature, when Hfr transferred its genophore in the order gal-lambda-bio. Thus the genophore is transferred with 5'OH at its origin. (author)

  16. Trashing of single-stranded DNA generated during processing of arrested replication fork in E. coli.

    Science.gov (United States)

    Kohiyama, Masamichi; Contremoulins, Vincent; Baudin, Xavier

    2013-11-29

    We analyzed formation of single-stranded DNA (ssDNA) related to SOS induction in nalidixilate (Nal)-treated Escherichia coli, using immunofluorescence microscopy accompanied by computer analysis. We found enhancement of both ssDNA concentrations and cells having ssDNA foci that often localized around cellpoles. Analyzing several mutants deficient in DNA repair or replication, we found, after Nal treatment, that recN, recA, uvrD and dnaB failed to increase ssDNA concentration and that recG and particularly ruvA only partially enhanced it. In Nal-treated recB mutant, despite its failure in SOS induction, ssDNA foci positive cells increased with a slight enhancement of its concentration. These observations suggest the existence of a cellular process that sequesters genotoxic ssDNA as inert form, offering a new concept for SOS suppressor genes action. PMID:23810902

  17. Probe data: 1422 [RED

    Lifescience Database Archive (English)

    Full Text Available R05-049390-1AR RED AU068280 AU068281 C 12919 C B2919 >C ELR148_1(AF025467|pid:g2429509) C aenorhabdi ... tis elegans c osmid R148; c ontains similarity to drosophila DNA-b ... inding protein K10 (NID:g8148); c oded for by C . elegans c DNA yk118e11.5; c oded for b ... y C . elegans c DNA yk64h11.5; c oded for by C . elegans c ... DNA yk172f2.5; c oded for by C . elegans c DNA yk79b6.5; c oded for by ...

  18. Phaeodactylum tricornutum photosynthesis and Thalassiosira pseudonana bio-silica formation genes nucleotide fluctuations

    Science.gov (United States)

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

    2008-08-01

    Diatom bioactivity has been reported to be responsible for about 20% of carbon fixation globally and together with other photosynthetic organisms, the bioactivity can be monitored via satellite ocean imaging. The bioinformatics embedded in the nucleotide fluctuations of photosynthesis and bio-silicate genes in diatoms were studied. The recently reported phosphoenolpyruvate carboxylase PEPC1 and PEPC2 C4-like photosynthesis genes in Phaeodactylum tricornutum were found to have similar fractal dimensions of about 2.01. In comparison, the green alga Chlamydomonas reinhardtii PEPC1 and PEPC2 genes have fractal dimensions of about 2.05. The PEPC CpG dinucleotide content is 8% in P. tricornutum and 10% in C. reinhardtii. Further comparison of the cell wall protein gene showed that the VSP1 gene sequence in C. reinhardtii has a fractal dimension of 2.03 and the bio-silica formation silaffin gene in Thalassiosira pseudonana has a fractal dimension of 2.01. The phosphoenolpyruvate carboxylase PPC1 and PPC2 in T. pseudonana were found to have fractal dimensions and CpG dinucleotide content similar to that of P. tricornutum. The fractal dimension of the dnaB replication helicase gene is about 1.98 for both diatoms as well as for the alga Heterosigma akashiwo. In comparison, the E. coli dnaB gene has a fractal dimension of about 2.03. Given that high fractal dimension and CpG dinucleotide content sequences have been associated with the presence of selective pressures, the relatively low fractal dimension gene sequences of the two unique properties of Earth-bound diatoms (photosynthesis and bio-silica cell wall) suggests the potential for the development of high fractal dimension sequences for adaptation in harsh environments.

  19. Lambda gpP-DnaB Helicase Sequestration and gpP-RpoB Associated Effects: On Screens for Auxotrophs, Selection for Rif(R), Toxicity, Mutagenicity, Plasmid Curing.

    Science.gov (United States)

    Hayes, Sidney; Wang, Wen; Rajamanickam, Karthic; Chu, Audrey; Banerjee, Anirban; Hayes, Connie

    2016-01-01

    The bacteriophage lambda replication initiation protein P exhibits a toxic effect on its Escherichia coli (E. coli) host, likely due to the formation of a dead-end P-DnaB complex, sequestering the replicative DnaB helicase from further activity. Intracellular expression of P triggers SOS-independent cellular filamentation and rapidly cures resident ColE1 plasmids. The toxicity of P is suppressed by alleles of P or dnaB. We asked whether P buildup within a cell can influence E. coli replication fidelity. The influence of P expression from a defective prophage, or when cloned and expressed from a plasmid was examined by screening for auxotrophic mutants, or by selection for rifampicin resistant (Rif(R)) cells acquiring mutations within the rpoB gene encoding the β-subunit of RNA polymerase (RNAP), nine of which proved unique. Using fluctuation assays, we show that the intracellular expression of P evokes a mutator effect. Most of the Rif(R) mutants remained P(S) and localized to the Rif binding pocket in RNAP, but a subset acquired a P(R) phenotype, lost sensitivity to ColE1 plasmid curing, and localized outside of the pocket. One P(R) mutation was identical to rpo*Q148P, which alleviates the UV-sensitivity of ruv strains defective in the migration and resolution of Holliday junctions and destabilizes stalled RNAP elongation complexes. The results suggest that P-DnaB sequestration is mutagenic and supports an earlier observation that P can interact with RNAP. PMID:27338450

  20. Repair and cell-cycle response in cells exposed to environmental biohazards. Comprehensive project report, June 1, 1979-May 31, 1982

    International Nuclear Information System (INIS)

    Agents which cause damage to DNA leading to inhibition of DNA synthesis or faulty DNA replication or repair may cause cell death or mutation. Many organisms possess the ability to circumvent some or all of this DNA damage. Many DNA mutants of E. coli and B. subtilis provide a genetic approach to measuring the role of individual components of the DNA repair and replicative system. The information obtained with prokaryotes provides leads to assess the details of DNA repair and replication in mammalian systems including man. Escherichia coli cells treated with a low concentration of toluene become permeable to a variety of compounds, including the precursors and cofactors necessary for DNA synthesis. By their manipulation various aspects of DNA replication and repair can be selectively emphasized. Observations made by use of this system include: (1) Repair synthesis induced by x irradiation or exposure to alkylating chemicals of toluene-treated cells is more extensive if polynucleotide ligase is inhibited. (2) DNA replication in E. coli is carried out by DNA polymerase III. The replication of DNA is strongly inhibited by methylmethansulfonate, N-methyl-N-nitrosourea, and N-methyl-N'-nitro-N-nitrosoguanidine. (3) Using a po1A1 po1B100 dnaB (po1I-, po1II-, po1III+) mutant of E. coli, it was demonstrated that the dnaB gene product is not necessary for Po1III directed repair synthesis. (4) The physiological stage of cells and tissues affects their response to environmental hazards. (5) Procedures for permeabilizing mammalian cells have been developed or further refined; and (6) In earlier studies involving both alkylating agents and x rays, it was observed that the number of DNA single-strand breaks increased with dose along with repair synthesis. It appears that non-repaired sites do not serve as primer ends for Po1I-dependent repair synthesis in toluene-treated cells

  1. Phylogenetic analysis of Melon chlorotic leaf curl virus from Guatemala: Another emergent species in the Squash leaf curl virus clade

    KAUST Repository

    Brown, J.K.

    2011-06-01

    The genome of a new bipartite begomovirus Melon chlorotic leaf curl virus from Guatemala (MCLCuV-GT) was cloned and the genome sequence was determined. The virus causes distinct symptoms on melons that were not previously observed in melon crops in Guatemala or elsewhere. Phylogenetic analysis of MCLCuV-GT and begomoviruses infecting cucurbits and other host plant species indicated that its closest relative was MCLCuV from Costa Rica (MCLCuV-CR). The DNA-A components of two isolates shared 88.8% nucleotide identity, making them strains of the same species. Further, both MCLCuV-GT and MCLCuV-CR grouped with other Western Hemisphere cucurbit-infecting species in the SLCV-clade making them the most southerly cucurbit-infecting members of the clade to date. Although the common region of the cognate components of MCLCuV-GT and MCLCuV-CR, shared similar to 96.3% nucleotide identity. While DNA-A and DNA-B components of MCLCuV-GT were less than 86% nucleotide identity with the respective DNAA and DNA-B common regions of MCLCuV-CR. The late viral genes of the two strains shared the least nt identity (<88%) while their early genes shared the highest nt identity (>90%). The collective evidence suggests that these two strains of MCLCuV are evolutionarily divergent owing in part to recombination, but also due to the accumulation of a substantial number of mutations. In addition they are differentially host-adapted, as has been documented for other cucurbit-infecting, bean-adapted, species in the SLCV clade. (C) 2011 Elsevier B.V. All rights reserved.

  2. Biosynthesis of a Fully Functional Cyclotide inside Living Bacterial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A; Kimura, R H; Woo, Y; Cantor, J; Shekhtman, A

    2007-04-05

    The cyclotide MCoTI-II is a powerful trypsin inhibitor recently isolated from the seeds of Momordica cochinchinensis, a plant member of cucurbitaceae family. We report for the first time the in vivo biosynthesis of natively-folded MCoTI-II inside live E. coli cells. Our biomimetic approach involves the intracellular backbone cyclization of a linear cyclotide-intein fusion precursor mediated by a modified protein splicing domain. The cyclized peptide then spontaneously folds into its native conformation. The use of genetically engineered E. coli cells containing mutations in the glutathione and thioredoxin reductase genes considerably improves the production of folded MCoTI-II in vivo. Biochemical and structural characterization of the recombinant MCoTI-II confirmed its identity. Biosynthetic access to correctly-folded cyclotides allows the possibility of generating cell-based combinatorial libraries that can be screened inside living cells for their ability to modulate or inhibit cellular processes.

  3. Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering.

    Science.gov (United States)

    Nuñez, James K; Harrington, Lucas B; Doudna, Jennifer A

    2016-03-18

    The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems. PMID:26857072

  4. Recombinant broad-range phospholipase C from Listeria monocytogenes exhibits optimal activity at acidic pH.

    Science.gov (United States)

    Huang, Qiongying; Gershenson, Anne; Roberts, Mary F

    2016-06-01

    The broad-range phospholipase C (PLC) from Listeria monocytogenes has been expressed using an intein expression system and characterized. This zinc metalloenzyme, similar to the homologous enzyme from Bacillus cereus, targets a wide range of lipid substrates. With monomeric substrates, the length of the hydrophobic acyl chain has significant impact on enzyme efficiency by affecting substrate affinity (Km). Based on a homology model of the enzyme to the B. cereus protein, several active site residue mutations were generated. While this PLC shares many of the mechanistic characteristics of the B. cereus PLC, a major difference is that the L. monocytogenes enzyme displays an acidic pH optimum regardless of substrate status (monomer, micelle, or vesicle). This unusual behavior might be advantageous for its role in the pathogenicity of L. monocytogenes. PMID:26976751

  5. Homing endonuclease structure and function.

    Science.gov (United States)

    Stoddard, Barry L

    2005-02-01

    Homing endonucleases are encoded by open reading frames that are embedded within group I, group II and archael introns, as well as inteins (intervening sequences that are spliced and excised post-translationally). These enzymes initiate transfer of those elements (and themselves) by generating strand breaks in cognate alleles that lack the intervening sequence, as well as in additional ectopic sites that broaden the range of intron and intein mobility. Homing endonucleases can be divided into several unique families that are remarkable in several respects: they display extremely high DNA-binding specificities which arise from long DNA target sites (14-40 bp), they are tolerant of a variety of sequence variations in these sites, and they display disparate DNA cleavage mechanisms. A significant number of homing endonucleases also act as maturases (highly specific cofactors for the RNA splicing reactions of their cognate introns). Of the known homing group I endonuclease families, two (HNH and His-Cys box enzymes) appear to be diverged from a common ancestral nuclease. While crystal structures of several representatives of the LAGLIDADG endonuclease family have been determined, only structures of single members of the HNH (I-HmuI), His-Cys box (I-PpoI) and GIY-YIG (I-TevI) families have been elucidated. These studies provide an important source of information for structure-function relationships in those families, and are the centerpiece of this review. Finally, homing endonucleases are significant targets for redesign and selection experiments, in hopes of generating novel DNA binding and cutting reagents for a variety of genomic applications. PMID:16336743

  6. Cloning Rosa hybrid phenylacetaldehyde synthase for the production of 2-phenylethanol in a whole cell Escherichia coli system.

    Science.gov (United States)

    Achmon, Yigal; Ben-Barak Zelas, Zohar; Fishman, Ayelet

    2014-04-01

    2-Phenylethanol (2-PE) is a desirable compound in the food and perfumery industries with a characteristic rose fragrance. Until now, most of the studied biotechnological processes to produce 2-PE were conducted using natural 2-PE-producing yeasts. Only several researches were conducted in other genetically engineered microorganisms that simulated the Ehrlich pathway for the conversion of amino acids to fusel alcohols. Here, a novel metabolic pathway has been designed in Escherichia coli to produce 2-PE, using the Rosa hybrid phenylacetaldehyde synthase (PAAS), a pyridoxal 5'-phosphate (PLP)-dependent enzyme capable of transforming L-phenylalanine (L-phe) into phenylacetaldehyde by decarboxylation and oxidation. To overcome the enzyme insolubility in E. coli, several plasmids and host strains were tested for their expression ability. The desired results were obtained by using the pTYB21 plasmid containing the intein tag from the Saccharomyces cerevisiae VMA1. It was discovered that the intein PAAS activity is temperature-dependent, working well in the range of 25 to 30 °C but losing most of its activity at 37 °C. When external PLP cofactor was added, the cells produced 0.39 g l⁻¹ 2-PE directly from L-phe. In addition, a biotransformation that was based only on internal de novo PLP synthesis produced 0.34 g l⁻¹ 2-PE, thus creating for the first time an E. coli strain that can produce 2-PE from L-phe without the need for exterior cofactor additions. PMID:24081322

  7. Synthetic Fuels Program

    International Nuclear Information System (INIS)

    Studies on waste water derived from synthetic fuel processes included acute toxicity of coal conversion effluent components and acute toxicity of aqueous waste and wastewater components of the ORNL Bench-Scale hydrocarbonization process. The potential hazards of synthetic fuels technology development to terrestrial ecosystems were studied with regard to toxicity of PAH and azaarenes. Aquatic transport studies included the following: aquatic transport of PAH and rates of PAH photolysis at the Bethlehem, Pennsylvania, coking plant site; microbial transformations of polycyclic aromatic hydrocarbons in water and sediment samples; persistence of azaarenes in aquatic systems; bioaccumulation of pathways of azaarenes; sorption of PAH to suspended particles in natural waters; and transport and fate of anthracene in pond microcosms. Studies were also conducted on environmental characterization of solid waste from a Lurgi facility

  8. Hope through Steadfastness: The Journey of "Holy Land Trust"

    Directory of Open Access Journals (Sweden)

    Erin Dyer

    2013-07-01

    Full Text Available Established in 1998, ‘Holy Land Trust’ (HLT serves to empower the Palestinian community in Bethlehem to discover its strengths and resources to confront the present and future challenges of life under occupation. The staff, through a commitment to the principles of nonviolence, seeks to mobilize the local community, regardless of religion, gender, or political affiliation, to resist oppression in all forms and build a model for the future based on justice, equality, and respect. This article places the work of HLT in the literature of nonviolent action and amid the nonviolent movement set by predecessors in the tumultuous history of Palestinian-Israeli relations. HLT programs and projects are presented to demonstrate the progression of nonviolent resistance from lofty goals to strategic empowerment. In a region so often defined by extremes, HLT embodies the Palestinian nonviolent resistance movement.

  9. A cultura e a educação amazônica na arte dos brinquedos de miriti

    Directory of Open Access Journals (Sweden)

    Claudete do Socorro Quaresma da Silva

    2012-01-01

    Full Text Available This paper is about the miriti toy, a secular craft, typically by Amazon contains in their forms elements representing everyday river. Display interfaces of this intangible heritage and culture of the State of Pará, with the Amazonian culture and education. Emphasizes the miriti toys such as the face of the Amazonian culture that bring in their process of making knowledge and practices that perpetuates itself for generations in the region through oral and observation. The city of Abaetetuba, State of Pará, is the main center of manufacture of this toy. This cultural property has a strong presence since the early days in the largest religious festival in Brazil, the Candle of Our Lady of Nazareth, Bethlehem, capital of Pará. Finally, the artisans who pointed toy miriti with his artistic ability and his unique way build education to teach in the Amazon region.

  10. La Seconda Intifada nella stampa italiana. La crisi della Basilica della Natività a Betlemme / The second Intifada in the italian Press: the Church of the Nativity crisis

    Directory of Open Access Journals (Sweden)

    Marzano, Arturo

    2011-02-01

    Full Text Available This paper intends to analyze the way the Italian press reported one of the most sensitive events of the Second Intifada, the siege to the Nativity Church in Bethlehem of April-May 2002.In particular, it aims at investigating whether and to what extend the identified press used a “nexus” between anti-Zionism and anti-Semitism. In some of the articles and of the cartoons published on the Italian newspapers and journals, it is possible to identify stereotypes coming from an anti-Jewish “archive”, that has been combining different discourses, from the Catholic anti-Judaism, to the racial and the political anti-Semitism, both right-wing and Marxist, up to the current anti-Israeli and anti-Zionist positions.

  11. Desulfurization of natural gases and industrial gases using water solutions of alkanolamines - a literature review

    Energy Technology Data Exchange (ETDEWEB)

    Bogdal, S.; Kowalska, T.; Kowalski, P.; Rutkowski, K.; Gwiner, H.

    1984-10-01

    Twenty-eight articles are reviewed on desulfurization of natural gas and industrial gases, among others coal gas, using water solutions of alkanolamines. Processes for production of alkanolamines as well as their chemical and physical properties are evaluated. The Sulfiban Process developed in the USA and used on a commercial scale by the Bethlehem Steel company is characterized: 13 to 18% water solution of monoethanoloamine is used; hydrogen cyanide, oxygen, oxysulfides etc. do not have a negative influence on sorption ability of monoethanolamine. The method is characterized by high desulfurization efficiency, low consumption rates of monoethanolamine solution, uncomplicated design of desulfurization system, low investment and limited waste water discharge. The Thylox Process and the absorption-carbonate methods are used in Poland for coal gas desulfurization. The methods use imported materials. Due to shortage of foreign currency Polish research institutes are investigating the possibility of replacing the Thylox Process with desulfurization methods using alkanolamines. 28 references.

  12. Modeling and Optimization : Theory and Applications Conference

    CERN Document Server

    Terlaky, Tamás

    2015-01-01

    This volume contains a selection of contributions that were presented at the Modeling and Optimization: Theory and Applications Conference (MOPTA) held at Lehigh University in Bethlehem, Pennsylvania, USA on August 13-15, 2014. The conference brought together a diverse group of researchers and practitioners, working on both theoretical and practical aspects of continuous or discrete optimization. Topics presented included algorithms for solving convex, network, mixed-integer, nonlinear, and global optimization problems, and addressed the application of deterministic and stochastic optimization techniques in energy, finance, logistics, analytics, healthcare, and other important fields. The contributions contained in this volume represent a sample of these topics and applications and illustrate the broad diversity of ideas discussed at the meeting.

  13. HIV and HCV prevalence and incarceration-related risks among injecting drug users in three West Bank governorates.

    Science.gov (United States)

    Štulhofer, Aleksandar; Jwehan, Isam; AbuRabie, Randa

    2016-09-01

    In the Middle East, the HIV epidemic among injecting drug users (IDUs) seems to be in an early phase, which increases the importance of prevention and systematic risk surveillance. To gain information about HIV and HCV infection rates among IDUs in the West Bank, a biobehavioral survey was conducted using time-location sampling in the Ramallah, Hebron, and Bethlehem governorates in 2013. The researchers recruited 288 Palestinian IDUs ages 16-64 (Mage = 39.2, SD = 11.11). While no HIV cases were found in the sample, 41% of participants tested positive for HCV. Imprisonment was common among participants (83%), so we explored the association of incarceration experience with HCV infection and HIV testing. In multivariate assessments, incarceration was shown to increase the odds of being infected with HCV and ever tested for HIV. HIV prevention should be strengthened in West Bank prisons and correctional facilities, and imprisonment for drug use re-examined. PMID:26936370

  14. Exploring Ancient Skies An Encyclopedic Survey of Archaeoastronomy

    CERN Document Server

    Kelley, David H

    2005-01-01

    Exploring Ancient Skies brings together the methods of archaeology and the insights of modern astronomy to explore the science of astronomy as it was practiced in various cultures prior to the invention of the telescope. The book reviews an enormous and growing body of literature on the cultures of the ancient Mediterranean, the Far East, and the New World (particularly Mesoamerica), putting the ancient astronomical materials into their archaeological and cultural contexts. The authors begin with an overview of the field and proceed to essential aspects of naked-eye astronomy, followed by an examination of specific cultures. The book concludes by taking into account the purposes of ancient astronomy: astrology, navigation, calendar regulation, and (not least) the understanding of our place and role in the universe. Skies are recreated to display critical events as they would have appeared to ancient observers - events such as the supernova of 1054, the 'lion horoscope' or the 'Star of Bethlehem.' Exploring An...

  15. Exploring Ancient Skies A Survey of Ancient and Cultural Astronomy

    CERN Document Server

    Kelley, David H

    2011-01-01

    Exploring Ancient Skies brings together the methods of archaeology and the insights of modern astronomy to explore the science of astronomy as it was practiced in various cultures prior to the invention of the telescope. The book reviews an enormous and growing body of literature on the cultures of the ancient Mediterranean, the Far East, and the New World (particularly Mesoamerica), putting the ancient astronomical materials into their archaeological and cultural contexts. The authors begin with an overview of the field and proceed to essential aspects of naked-eye astronomy, followed by an examination of specific cultures. The book concludes by taking into account the purposes of ancient astronomy: astrology, navigation, calendar regulation, and (not least) the understanding of our place and role in the universe. Skies are recreated to display critical events as they would have appeared to ancient observers—events such as the supernova of 1054 A.D., the "lion horoscope," and the Star of Bethlehem. Explori...

  16. Not-for-profit hospitals fight tax-exempt challenges.

    Science.gov (United States)

    Hudson, T

    1990-10-20

    The message being sent by local tax boards, state agencies, and the Internal Revenue Service is clear: Not-for-profit hospitals will have to justify their tax-exempt status. But complying with this demand can be a costly administrative burden. Just ask the executives who have been through the experience. CEO Richard Anderson, of St. Luke's Hospital, Bethlehem, PA, is luckier than some executives who have faced tax-exempt challenges. He won his hospital's case. But he still faces a yearly battle: The hospital must prove its compliance annually to the county board of assessors. Other executives report similar experiences. Our cover story takes an in-depth look at how administrators faced challenges to their hospital's tax status and what they learned about their relationship with their communities, as well as a complete state and federal legislative outlook for future developments. PMID:2227856

  17. “A good deal about California does not, on its own preferred terms, add up”: Joan Didion between Dawning Apocalypse and Retrogressive Utopia

    Directory of Open Access Journals (Sweden)

    Eva-Sabine Zehelein

    2011-09-01

    Full Text Available Joan Didion’s depiction of the American West and California is colored by an idiosyncratic sensitivity to her surroundings, intertwined with a sentimental-retrogressive image of the nature, history, character, and meaning of the West as a cultural topos. Her New Journalism-like observations of California are always closely linked to her own self and psyche, are seismographs of her own confusions, of moments of disorientation, insecurity, and loss. Didion is therefore one of those exceptional writers, who, in her fictionalized reportages, comments on American reality, ideas, and her own predicament. Identity and landscape fuse into an auto-psychoanalysis, which at the same time reveals a great deal about the American condition, its constantly strained relationship between rhetoric or auto-mythology and lived reality. This article draws primarily on Slouching Towards Bethlehem (1968 and Where I Was From (2003, but also on After Henry (1992 and The White Album (1979, to illustrate these points.

  18. Plant Line Trial Evaluation of Viable Non-Chromium Passivation Systems for Electrolytin Tinplate, ETP (TRP 9911)

    Energy Technology Data Exchange (ETDEWEB)

    John A. Sinsel

    2003-06-30

    Plant trial evaluations have been completed for two zirconium-based, non-chromium passivation systems previously identified as possible alternatives to cathodic dichromate (CDC) passivation for electrolytic tinplate (ETP). These trials were done on a commercial electrolytic tin plating line at Weirton Steel and extensive evaluations of the materials resulting from these trials have been completed. All this was accomplished as a collaborative effort under the AISI Technology Roadmap Program and was executed by seven North American Tin Mill Products producers [Bethlehem Steel (now acquired by International Steel Group (ISG)), Dofasco Inc., National Steel (now acquired by U.S. Steel), U.S. Steel, USS-Posco, Weirton Steel, and Wheeling-Pittsburgh Steel] with funding partially from the Department of Energy (DOE) and partially on an equal cost sharing basis among project participants. The initial phases of this project involved optimization of application procedures for the non-chromium systems in the laboratories at Bethlehem Steel and Betz Dearborn followed by extensive testing with various lacquer formulations and food simulants in the laboratories at Valspar and PPG. Work was also completed at Dofasco and Weirton Steel to develop methods to prevent precipitation of insoluble solids as a function of time from the zirconate system. The results of this testing indicated that sulfide staining characteristics for the non-chromium passivation systems could be minimized but not totally eliminated and neither system was found to perform quite as good, in this respect, as the standard CDC system. As for the stability of zirconate treatment, a method was developed to stabilize this system for a sufficient period of time to conduct plant trial evaluations but, working with a major supplier of zirconium orthosulfate, a method for long term stabilization is still under development.

  19. Detecção de Begomovirus em maracujazeiro (Passiflora edulis Sims no Estado de Alagoas Begomovirus detection in passion fruit (Passiflora edulis Sims in the State of Alagoas, Brazil

    Directory of Open Access Journals (Sweden)

    Sarah Cavalcanti da Silva

    2006-12-01

    Full Text Available O maracujazeiro é uma cultura de grande importância econômica nos países tropicais, destacando-se o Brasil como o maior produtor. No presente trabalho, é relatada a infecção de plantas de maracujazeiro por um vírus do gênero Begomovirus, família Geminiviridae, no município de Anadia, Estado de Alagoas. As plantas infectadas apresentavam sintomas de mosaico amarelo, redução drástica de crescimento e da área foliar. DNA extraído de plantas sintomáticas foi empregado como molde em PCRs, contendo os pares de primers PAL1v1978 e par1C496 ou PBL1v2040 e PCRc1 que direcionam, respectivamente, a amplificação de regiões de aproximadamente 1,2 kb do DNA-A e 0,6 kb do DNA-B, do genoma dos begomovírus. Fragmentos de tamanho esperado foram amplificados a partir de amostras de plantas sintomáticas, confirmando a infecção por Begomovirus. O seqüenciamento desses fragmentos está em andamento para uma definição precisa da espécie viral.Passion fruit is a crop of great economic importance in Tropical countries, distinguishing Brazil as the greatest producer. The present work reports the infection of passion fruit by a virus belonging to the genus Begomovirus, family Geminiviridae in the state of Alagoas, Brazil. Infected plants showed symptoms of yellow mosaic, drastic reduction of growth and leaf area. The DNA extracted from symptomatic plants was used as template in PCR's, containing the pairs of primers PAL1v1978 and par1C496 or PBL1v2040 and PCRc1, which direct the amplification of regions of approximately 1.2 kb DNA-A and 0.6 kb DNA-B, from begomovírus genome, respectively. Fragments of the components A and B with expected size were amplified from samples of symptomatic plants, confirming the infection by a begomovírus.

  20. Characterization of Tomato yellow spot virus, a novel tomato-infecting begomovirus in Brazil Caracterização do Tomato yellow spot virus, um novo begomovírus isolado de tomateiro no Brasil

    Directory of Open Access Journals (Sweden)

    Renata Faier Calegario

    2007-09-01

    Full Text Available The objective of this work was the biological and molecular characterization of a begomovirus detected in São Joaquim de Bicas, Minas Gerais, Brazil, named TGV-[Bi2], by determining its host range, complete nucleotide sequence and phylogenetic relationships with other begomoviruses. Biological characterization consisted of a host range study using either sap inoculation or particle bombardment as inoculation methods. The yellow spot virus can infect plants in Solanaceae and Amaranthaceae, including economically importat crops as sweet pepper, and weeds as Datura stramonium and Nicotiana silvestris. For the molecular characterization, the full-length genome (DNA-A and DNA-B was amplified, cloned and completely sequenced. Sequence comparisons and phylogenetic analyses indicated that TGV-[Bi2] constitutes a novel begomovirus species named Tomato yellow spot virus (ToYSV, closely related to Sida mottle virus (SiMoV.O objetivo deste trabalho foi a caracterização biológica e molecular de um begomovírus detectado em tomateiros em São Joaquim de Bicas, Minas Gerais, denominado TGV-[Bi2]. A caracterização biológica consistiu em teste de gama de hospedeiros, realizado por meio de inoculação via extrato foliar tamponado ou bombardeamento de partículas. O isolado TGV-[Bi2] infecta plantas das famílias Solanaceae e Amaranthaceae, inclusive espécies economicamente importantes como o pimentão, e algumas plantas daninhas como Datura stramonium e Nicotiana silvestris. A caracterização molecular consistiu na clonagem e seqüenciamento de seu genoma completo (DNA-A e DNA-B. A comparação de seqüências e análise filogenética indicaram que o TGV-[Bi2] constitui uma nova espécie de begomovírus, denominada Tomato yellow spot virus (ToYSV, filogeneticamente relacionado ao Sida mottle virus (SiMoV.

  1. Engineering protein scaffolds for protein separation, biocatalysis and nanotechnology applications

    Science.gov (United States)

    Liu, Fang

    Globally, there is growing appreciation for developing a sustainable economy that uses eco-efficient bio-processes. Biotechnology provides an increasing range of tools for industry to help reduce cost and improve environmental performance. Inspired by the naturally evolved machineries of protein scaffolds and their binding ligands, synthetic protein scaffolds were engineered based on cohesin-dockerin interactions and metal chelating peptides to tackle the challenges and make improvements in three specific areas: (1) protein purification, (2) biofuel cells, and (3) nanomaterial synthesis. The first objective was to develop efficient and cost-effective non-chromatographic purification processes to purify recombinant proteins in an effort to meet the dramatically growing market of protein drugs. In our design, the target protein was genetically fused with a dockerin domain from Clostridium thermocellum and direct purification and recovery was achieved using thermo-responsive elastin-like polypeptide (ELP) scaffold containing the cohesin domain from the same species. By exploiting the highly specific interaction between the dockerin and cohesin domain and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as endoglucanase CelA, chloramphenicol acetyl transferase (CAT) and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single purification step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins by another cycle of thermal precipitation. The purification cost can be further reduced by regenerating and recycling the ELP-cohesin capturing scaffolds. However, due to the high binding affinity between cohesin and dockerin domains, the bound dockerin-intein tag cannot be completely disassociated from ELP-cohesin scaffold after binding. Therefore, a truncated dockerin with the calcium

  2. Suppressors of RNA silencing encoded by tomato leaf curl betasatellites

    Indian Academy of Sciences (India)

    Richa Shukla; Sunita Dalal; V G Malathi

    2013-03-01

    Virus encoded RNA-silencing suppressors (RSSs) are the key components evolved by the viruses to counter RNA-silencing defense of plants. Whitefly-transmitted begomoviruses infecting tomato crop code for five different proteins, ORF AC4, ORF AC2 and ORF AV2 in DNA-A component, ORF BV1 in DNA-B and ORF C1 in satellite DNA which are predicted to function as silencing suppressors. In the present study suppressor function of ORF C1 of three betasatellites Tomato leaf curl Bangalore betasatellite ToLCBB-[IN:Hess:08], Cotton leaf curl Multan betasatellite CLCuMB–[IN:Sri:02] and Luffa leaf distortion betasatellite LuLDB-[IN:Lu:04] were examined. Agroinfiltration of GFP-silenced Nicotiana tabaccum cv. Xanthi with the cells expressing C1 protein resulted in reversal of silenced GFP expression. GFP-siRNA level was more than 50-fold lower compared to silenced plants in plants infiltrated with C1 gene from ToLCBB. However, in the case of 35S-C1 CLCuMB and 35S-C1 LuLDB construct, although GFP was expressed, siRNA level was not reduced, indicating that the step at which C1 interfere in RNA-silencing pathway is different.

  3. Diversity and distribution of begomoviruses infecting tomato in India.

    Science.gov (United States)

    Reddy, R V Chowda; Colvin, J; Muniyappa, V; Seal, S

    2005-05-01

    Leaf curl begomoviruses cause serious yield losses to Indian tomato crops. Total DNAs were extracted from leaves of 69 tomato plants and 34 weeds or neighbouring crops collected from all the major tomato producing areas of India. Eighty-one of the 103 samples were positive by PCRs using begomovirus genus-specific primers. Coat protein (CP) genes from 29 samples were PCR amplified, cloned and sequenced. Phylogenetic analyses of the CP sequences revealed five different tomato leaf curl begomovirus (TLCB) clusters each yellow vein mosaic virus.Sixty-five begomovirus positive samples were characterised further by PCR with DNA-beta, DNA-B, four Indian TLCB species, PALIc1960/PARIv722 (universal begomovirus primers), and by sequencing. The majority of samples represented monopartite TLCBs associated with DNA-beta components. All four known TLCBs appeared to be present throughout India. TLCBs were also present in chilli, cowpea, okra and tobacco crops, as well as in some common weeds. Papaya leaf curl virus and Pepper leaf curl Bangladesh virus sequences were detected in tomato. Mixed begomovirus infections, a prerequisite for recombination, were evident in 13 samples. PMID:15703846

  4. Diversity of Dicotyledenous-Infecting Geminiviruses and Their Associated DNA Molecules in Southern Africa, Including the South-West Indian Ocean Islands

    Directory of Open Access Journals (Sweden)

    Lindy L. Esterhuizen

    2012-09-01

    Full Text Available The family Geminiviridae comprises a group of plant-infecting circular ssDNA viruses that severely constrain agricultural production throughout the temperate regions of the world, and are a particularly serious threat to food security in sub-Saharan Africa. While geminiviruses exhibit considerable diversity in terms of their nucleotide sequences, genome structures, host ranges and insect vectors, the best characterised and economically most important of these viruses are those in the genus Begomovirus. Whereas begomoviruses are generally considered to be either monopartite (one ssDNA component or bipartite (two circular ssDNA components called DNA-A and DNA-B, many apparently monopartite begomoviruses are associated with additional subviral ssDNA satellite components, called alpha- (DNA-as or betasatellites (DNA-βs. Additionally, subgenomic molecules, also known as defective interfering (DIs DNAs that are usually derived from the parent helper virus through deletions of parts of its genome, are also associated with bipartite and monopartite begomoviruses. The past three decades have witnessed the emergence and diversification of various new begomoviral species and associated DI DNAs, in southern Africa, East Africa, and proximal Indian Ocean islands, which today threaten important vegetable and commercial crops such as, tobacco, cassava, tomato, sweet potato, and beans. This review aims to describe what is known about these viruses and their impacts on sustainable production in this sensitive region of the world.

  5. Synthetic peptide inhibitors of DNA replication in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Løbner-Olesen, Anders; Kjelstrup, Susanne

    of clinically important pathogens and is essential for bacterial proliferation. The bacterial replication apparatus fulfill the requirements for a good drug target. The replisome of S. aureus consists of 5 different subunits (2, PolC2, 4, δ and δ`) who’s organization depends on multiple protein......-protein interactions. Centrally in the replisome is the -clamp where to multiple proteins binds through a conserved motif. We have identified the protein-protein interactions in the replisome of S. aureus by use of a bacterial two-hybrid system. A reverse bacterial two-hybrid system (R-BTH) based on Pyr......N (), DnaB and DnaX (). Three peptides identified as inhibitors of DnaN have been purified. Two of these peptides inhibited growth as well as DNA replication in S. aureus. The minimal inhibitory concentration (MIC) of the peptides was approximately 50 g/ml. Overexpression of DnaN reduced the inhibitory...

  6. The effects of polyols on the thermal stability of calf thymus DNA.

    Science.gov (United States)

    Del Vecchio, P; Esposito, D; Ricchi, L; Barone, G

    1999-05-01

    The effects on thermal denaturation of calf thymus DNA (ct-DNA) and its conformational changes induced by the presence in solution of different polyols, namely glycerol, i-erytritol, L( -- ) and D( + ) arabitol, D-mannitol, D-sorbitol and myo-inositol, have been investigated by means of differential scanning calorimetry (DSC) and circular dichroism (CD). By increasing the concentration of these additives a decrease in both the denaturation enthalpy (deltadH) and temperature of the maximum of the denaturation peak (Tmax) of DNA is observed. The values of these thermodynamic parameters depend on both the nature and concentration of the solute. The overall destabilization of DNA molecule has been related to the different capability of polyhydric alcohols to interact with the polynucleotide solvation sites replacing water and to the modification of the electrostatic interactions between the polynucleotide and its surrounding atmosphere of counterions. The particular behaviour of L( -- ) arabitol, which showed a much greater destabilizing ability compared to the other polyols, was further investigated and attributed to a direct more effective interaction with the double helix of DNA. CD spectra showed only a slight alteration of DNA-B structure in the presence of all the molecules here studied, except for L( -- ) arabitol where the DNA molecule seems to undergo a meaningful conformational change. The salt concentration dependence of DNA thermal stability in the presence of L( -- ) arabitol indicates a conformational change of polynucleotide towards a more extended conformation. PMID:10408643

  7. Inhibition of helicase activity by a small molecule impairs Werner syndrome helicase (WRN) function in the cellular response to DNA damage or replication stress.

    Science.gov (United States)

    Aggarwal, Monika; Sommers, Joshua A; Shoemaker, Robert H; Brosh, Robert M

    2011-01-25

    Modulation of DNA repair proteins by small molecules has attracted great interest. An in vitro helicase activity screen was used to identify molecules that modulate DNA unwinding by Werner syndrome helicase (WRN), mutated in the premature aging disorder Werner syndrome. A small molecule from the National Cancer Institute Diversity Set designated NSC 19630 [1-(propoxymethyl)-maleimide] was identified that inhibited WRN helicase activity but did not affect other DNA helicases [Bloom syndrome (BLM), Fanconi anemia group J (FANCJ), RECQ1, RecQ, UvrD, or DnaB). Exposure of human cells to NSC 19630 dramatically impaired growth and proliferation, induced apoptosis in a WRN-dependent manner, and resulted in elevated γ-H2AX and proliferating cell nuclear antigen (PCNA) foci. NSC 19630 exposure led to delayed S-phase progression, consistent with the accumulation of stalled replication forks, and to DNA damage in a WRN-dependent manner. Exposure to NSC 19630 sensitized cancer cells to the G-quadruplex-binding compound telomestatin or a poly(ADP ribose) polymerase (PARP) inhibitor. Sublethal dosage of NSC 19630 and the chemotherapy drug topotecan acted synergistically to inhibit cell proliferation and induce DNA damage. The use of this WRN helicase inhibitor molecule may provide insight into the importance of WRN-mediated pathway(s) important for DNA repair and the replicational stress response. PMID:21220316

  8. The 32 kDa subunit of replication protein A (RPA) participates in the DNA replication of Mung bean yellow mosaic India virus (MYMIV) by interacting with the viral Rep protein.

    Science.gov (United States)

    Singh, Dharmendra Kumar; Islam, Mohammad Nurul; Choudhury, Nirupam Roy; Karjee, Sumona; Mukherjee, Sunil Kumar

    2007-01-01

    Mung bean yellow mosaic India virus (MYMIV) is a member of genus begomoviridae and its genome comprises of bipartite (two components, namely DNA-A and DNA-B), single-stranded, circular DNA of about 2.7 kb. During rolling circle replication (RCR) of the DNA, the stability of the genome and maintenance of the stem-loop structure of the replication origin is crucial. Hence the role of host single-stranded DNA-binding protein, Replication protein A (RPA), in the RCR of MYMIV was examined. Two RPA subunits, namely the RPA70 kDa and RPA32 kDa, were isolated from pea and their roles were validated in a yeast system in which MYMIV DNA replication has been modelled. Here, we present evidences that only the RPA32 kDa subunit directly interacted with the carboxy terminus of MYMIV-Rep both in vitro as well as in yeast two-hybrid system. RPA32 modulated the functions of Rep by enhancing its ATPase and down regulating its nicking and closing activities. The possible role of these modulations in the context of viral DNA replication has been discussed. Finally, we showed the positive involvement of RPA32 in transient replication of the plasmid DNA bearing MYMIV replication origin using an in planta based assay. PMID:17182628

  9. Large-scale Evaluation of Nickel Aluminide Rolls in a Heat-Treat Furnace at Bethelehem Steel's (Now ISG) Burns Harbor Plate Mill

    Energy Technology Data Exchange (ETDEWEB)

    Mengel, J.

    2003-12-16

    At Bethlehem Steel Burns Harbor Plate Division (now ISG Burns Harbor Plate Inc.)'s annealing furnace, new nickel aluminide intermetallic alloy rolls provide greater high-temperature strength and wear resistance compared to the conventional H series cast austenitic alloys currently used in the industry. Oak Ridge National Laboratory and Bethlehem (ISG) partnered under a U.S. Department of Energy, Office of Industrial Technology's Emerging Technology Deployment Program to demonstrate and evaluate the nickel aluminide intermetallic alloy rolls as part of an updated energy efficient large commercial annealing furnace system. Many challenges were involved in this project, including developing welding procedures for joining nickel aluminide intermetallic alloys with H-series austenitic alloys, developing commercial cast roll manufacturing specifications, working with several commercial suppliers to produce a quantity of high quality, reproducible nickel aluminide rolls for a large steel industrial annealing furnace, installing and demonstrating the capability of the rolls in this furnace, performing processing trials to evaluate the benefits of new equipment and processes, and documenting the findings. Updated furnace equipment including twenty-five new automated furnace control dampers have been installed replacing older design, less effective units. These dampers, along with upgraded flame-safety control equipment and new AC motors and roll-speed control equipment, are providing improved furnace control and additional energy efficiency. Energy data shows up to a 34% energy reduction from baseline after the installation of upgraded furnace damper controls along with up to a 34% reduction in greenhouse gases, potential for an additional 3 to 6% energy reduction per campaign of light-up and shutdown, and a 46% energy reduction from baseline for limited trials of a combination of improved damper control and straight-through plate processing. The straight

  10. Correlation of chemical shifts predicted by molecular dynamics simulations for partially disordered proteins

    International Nuclear Information System (INIS)

    There has been a longstanding interest in being able to accurately predict NMR chemical shifts from structural data. Recent studies have focused on using molecular dynamics (MD) simulation data as input for improved prediction. Here we examine the accuracy of chemical shift prediction for intein systems, which have regions of intrinsic disorder. We find that using MD simulation data as input for chemical shift prediction does not consistently improve prediction accuracy over use of a static X-ray crystal structure. This appears to result from the complex conformational ensemble of the disordered protein segments. We show that using accelerated molecular dynamics (aMD) simulations improves chemical shift prediction, suggesting that methods which better sample the conformational ensemble like aMD are more appropriate tools for use in chemical shift prediction for proteins with disordered regions. Moreover, our study suggests that data accurately reflecting protein dynamics must be used as input for chemical shift prediction in order to correctly predict chemical shifts in systems with disorder

  11. Self-Assembly of Synthetic Metabolons through Synthetic Protein Scaffolds: One-Step Purification, Co-immobilization, and Substrate Channeling

    Energy Technology Data Exchange (ETDEWEB)

    You, C; Zhang, YHP

    2013-02-01

    One-step purification of a multi-enzyme complex was developed based on a mixture of cell extracts containing three dockerin-containing enzymes and one family 3 cellulose-binding module (CBM3)-containing scaffoldin through high-affinity adsorption on low-cost solid regenerated amorphous cellulose (RAC). The three-enzyme complex, called synthetic metabolon, was self-assembled through the high-affinity interaction between the dockerin in each enzyme and three cohesins in the synthetic scaffoldin. The metabolons were either immobilized on the external surface of RAC or free when the scaffoldin contained an intein between the CBM3 and three cohesins. The immobilized and free metabolons containing triosephosphate isomerase, aldolase, and fructose 1,6-biphosphatase exhibited initial reaction rates 48 and 38 times, respectively, that of the non-complexed three-enzyme mixture at the same enzyme loading. Such reaction rate enhancements indicated strong substrate channeling among synthetic metabolons due to the close spatial organization among cascade enzymes. These results suggested that the construction of synthetic metabolons by using cohesins, dockerins, and cellulose-binding modules from cellulosomes not only decreased protein purification labor and cost for in vitro synthetic biology projects but also accelerated reaction rates by 1 order of magnitude compared to non-complexed enzymes. Synthetic metabolons would be an important biocatalytic module for in vitro and in vivo synthetic biology projects.

  12. Engineering and optimization of an allosteric biosensor protein for peroxisome proliferator-activated receptor γ ligands.

    Science.gov (United States)

    Li, Jingjing; Gierach, Izabela; Gillies, Alison R; Warden, Charles D; Wood, David W

    2011-11-15

    The peroxisome proliferator-activated receptor gamma (PPARγ or PPARG) belongs to the nuclear receptor superfamily, and is a potential drug target for a variety of diseases. In this work, we constructed a series of bacterial biosensors for the identification of functional PPARγ ligands. These sensors entail modified Escherichia coli cells carrying a four-domain fusion protein, comprised of the PPARγ ligand binding domain (LBD), an engineered mini-intein domain, the E. coli maltose binding protein (MBD), and a thymidylate synthase (TS) reporter enzyme. E. coli cells expressing this protein exhibit hormone ligand-dependent growth phenotypes. Unlike our published estrogen (ER) and thyroid receptor (TR) biosensors, the canonical PPARγ biosensor cells displayed pronounced growth in the absence of ligand. They were able to distinguish agonists and antagonists, however, even in the absence of agonist. To improve ligand sensitivity of this sensor, we attempted to engineer and optimize linker peptides flanking the PPARγ LBD insertion point. Truncation of the original linkers led to decreased basal growth and significantly enhanced ligand sensitivity of the PPARγ sensor, while substitution of the native linkers with optimized G(4)S (Gly-Gly-Gly-Gly-Ser) linkers further increased the sensitivity. Our studies demonstrate that the properties of linkers, especially the C-terminal linker, greatly influence the efficiency and fidelity of the allosteric signal induced by ligand binding. Our work also suggests an approach to increase allosteric behavior in this multidomain sensor protein, without modification of the functional LBD. PMID:21893405

  13. Thermal stabilization of an endoglucanase by cyclization.

    Science.gov (United States)

    van Lieshout, Johan F T; Pérez Gutiérrez, Odette N; Vroom, Wietse; Planas, Antoni; de Vos, Willem M; van der Oost, John; Koutsopoulos, Sotirios

    2012-08-01

    An intein-driven protein splicing approach allowed for the covalent linkage between the N- and C-termini of a polypeptide chain to create circular variants of the endo-β-1,3-1,4-glucanase, LicA, from Bacillus licheniformis. Two circular variants, LicA-C1 and LicA-C2, which have connecting loops of 20 and 14 amino acids, respectively, showed catalytic activities that are approximately two and three times higher, respectively, compared to that of the linear LicA (LicA-L1). The thermal stability of the circular variants was significantly increased compared to the linear form. Whereas the linear glucanase lost half of its activity after 3 min at 65 °C, the two circular variants have 6-fold (LicA-C1) and 16-fold (LicA-C2) increased half-life time of inactivation. In agreement with this, fluorescence spectroscopy and differential scanning calorimetry studies revealed that circular enzymes undergo structural changes at higher temperatures compared to that of the linear form. The effect of calcium on the conformational stability and function of the circular LicAs was also investigated, and we observed that the presence of calcium ions results in increased thermal stability. The impact of the length of the designed loops on thermal stability of the circular proteins is discussed, and it is suggested that cyclization may be an efficient strategy for the increased stability of proteins. PMID:22653681

  14. Antibacterial Peptide CecropinB2 Production via Various Host and Construct Systems

    Directory of Open Access Journals (Sweden)

    Wei-Shiang Lai

    2016-01-01

    Full Text Available Cecropin is a cationic antibacterial peptide composed of 35–39 residues. This peptide has been identified as possessing strong antibacterial activity and low toxicity against eukaryotic cells, and it has been claimed that some types of the cecropin family of peptides are capable of killing cancer cells. In this study, the host effect of cloning antibacterial peptide cecropinB2 was investigated. Three different host expression systems were chosen, i.e., Escherichia coli, Bacillus subtilis and Pichia pastoris. Two gene constructs, cecropinB2 (cecB2 and intein-cecropinB2 (INT-cecB2, were applied. Signal peptide and propeptide from Armigeres subalbatus were also attached to the gene construct. The results showed that the best host for cloning cecropinB2 was P. pastoris SMD1168 harboring the gene of pGAPzαC-prepro-cecB2 via Western blot confirmation. The cecropinB2 that was purified using immobilized-metal affinity chromatography resin showed strong antibacterial activity against the Gram-negative strains, including the multi-drug-resistant bacteria Acinetobacter baumannii.

  15. A genetically encoded bioluminescent indicator for illuminating proinflammatory cytokines.

    Science.gov (United States)

    Kim, Sung Bae; Ozawa, Takeaki; Umezawa, Yoshio

    2016-01-01

    We introduce a method to evaluate the activities of cytokines based on the nuclear transport of NF-κB. A pair of bioluminescent indicators was made for conferring cytokine sensitivity to cervical carcinoma-derived HeLa cells. The principle is based on reconstitution of split fragments of Renilla reniformis luciferase (RLuc) by protein splicing with a DnaE intein from Synechocystis sp. PCC6803. The bioluminescence intensity of thus reconstituted RLuc in the HeLa cells was used as a measure of the activities for cytokines. With the present method, we evaluated the activities of various cytokines based on the nuclear transport of NF-κB in human cervical carcinoma-derived HeLa cells carrying the indicators. The present approach to evaluating the activities of cytokines may provide a potential clinical value in monitoring drug activity and directing treatment for various diseases related with NF-κB. The method highlights the experimental procedure from our original publications, Anal. Biochem. 2006, 359, 147-149 and Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 11542. The summary of the method is: •Cytokine activities are determined within 2 h after stimulation.•Temporarily inactivated split-luciferase fragments are reconstituted by protein splicing.•Nucleartrafficking of NF-κB was illuminated for gauging the ligand-driven activity. PMID:27489781

  16. Genome and Proteome of Campylobacter jejuni Bacteriophage NCTC 12673▿†

    Science.gov (United States)

    Kropinski, Andrew M.; Arutyunov, Denis; Foss, Mary; Cunningham, Anna; Ding, Wen; Singh, Amit; Pavlov, Andrey R.; Henry, Matthew; Evoy, Stephane; Kelly, John; Szymanski, Christine M.

    2011-01-01

    Campylobacter jejuni continues to be the leading cause of bacterial food-borne illness worldwide, so improvements to current methods used for bacterial detection and disease prevention are needed. We describe here the genome and proteome of C. jejuni bacteriophage NCTC 12673 and the exploitation of its receptor-binding protein for specific bacterial detection. Remarkably, the 135-kb Myoviridae genome of NCTC 12673 differs greatly from any other proteobacterial phage genome described (including C. jejuni phages CP220 and CPt10) and instead shows closest homology to the cyanobacterial T4-related myophages. The phage genome contains 172 putative open reading frames, including 12 homing endonucleases, no visible means of packaging, and a putative trans-splicing intein. The phage DNA appears to be strongly associated with a protein that interfered with PCR amplification and estimation of the phage genome mass by pulsed-field gel electrophoresis. Identification and analyses of the receptor-binding protein (Gp48) revealed features common to the Salmonella enterica P22 phage tailspike protein, including the ability to specifically recognize a host organism. Bacteriophage receptor-binding proteins may offer promising alternatives for use in pathogen detection platforms. PMID:21965409

  17. The Lehigh Steel Collection: a new open dataset for document recognition research

    Science.gov (United States)

    Bruno, Barri; Lopresti, Daniel

    2013-12-01

    Document image analysis is a data-driven discipline. For a number of years, research was focused on small, homogeneous datasets such as the University of Washington corpus of scanned journal pages. More recently, library digitization efforts have raised many interesting problems with respect to historical documents and their recognition. In this paper, we present the Lehigh Steel Collection (LSC), a new open dataset we are currently assembling which will be, in many ways, unique to the field. LSC is an extremely large, heterogeneous set of documents dating from the 1960's through the 1990's relating to the wide-ranging research activities of Bethlehem Steel, a now-bankrupt company that was once the second-largest steel producer and the largest shipbuilder in the United States. As a result of the bankruptcy process and the disposition of the company's assets, an enormous quantity of documents (we estimate hundreds of thousands of pages) were left abandoned in buildings recently acquired by Lehigh University. Rather than see this history destroyed, we stepped in to preserve a portion of the collection via digitization. Here we provide an overview of LSC, including our efforts to collect and scan the documents, a preliminary characterization of what the collection contains, and our plans to make this data available to the research community for non-commercial purposes.

  18. A molecular epidemiology survey of respiratory adenoviruses circulating in children residing in Southern Palestine.

    Directory of Open Access Journals (Sweden)

    Lina Qurei

    Full Text Available A molecular epidemiology survey was performed in order to establish and document the respiratory adenovirus pathogen profiles among children in Southern Palestine. Three hundred and thirty-eight hospitalized pediatric cases with adenovirus-associated respiratory tract infections were analyzed. Forty four cases out of the 338 were evaluated in more detail for the adenoviruses types present. All of the children resided in Southern Palestine, that is, in city, village and refugee camp environments within the districts of Hebron and Bethlehem. Human adenoviruses circulated throughout 2005-2010, with major outbreaks occurring in the spring months. A larger percent of the children diagnosed with adenoviral infections were male infants. DNA sequence analysis of the hexon genes from 44 samples revealed that several distinct adenovirus types circulated in the region; these were HAdV-C1, HAdV-C2, HAdV-B3 and HAdV-C5. However, not all of these types were detected within each year. This is the first study ever conducted in Palestine of the genetic epidemiology of respiratory adenovirus infections.

  19. Spatial risk modelling for water shortage and nitrate pollution in the lower Jordan valley

    International Nuclear Information System (INIS)

    This report summarizes the results of the spatial risk modeling activities (work package WP-4.4, 'GIS Risk Modeling') of the INCO-DC project 'Developing Sustainable Water Management in the Jordan Valley'. The project was funded by European Commission's INCO-DC research program. The main objective of the project was to develop the scientific basis for an integral management plan of water resources and their use in the Lower Jordan Valley. The outputs of the project were expected to allow a better understanding of the water management situation, and to provide a sound basis for a better future water management - not only separately in the three countries, but in the overall valley region. The risk modeling was done by the ARCS Seibersdorf research (ARCS), based on information and data provided by the regional partners from Israel (Hebrew University, Jerusalem, HUJ), Palestine (Applied Research Institute, Jerusalem, Bethlehem, ARIJ) and Jordan (EnviroConsult Office, Amman, ECO). The land use classification has been established through a cooperation between ARCS and the Yale University Center for Earth Observation (YUCEO). As a result of the work, the spatial patterns of agricultural and domestic water demand in the Lower Jordan Valley were established, and the spatial dimension of driving forces for water usage and water supply was analyzed. Furthermore, a conceptual model for nitrate leakage (established by HUJ) was translated into a GIS system, and the risks for nitrate pollution of groundwater were quantified. (author)

  20. Preparation of hospitals for handling victims of radiation accidents

    International Nuclear Information System (INIS)

    This chapter is devoted to a generalized discussion of the inter-and intraorganizational structure of hospitals for handling radiation emergencies of the kind suggested above as well as the isolated remote minor accident involving radiation. The general elements of hospital planning for radiation accidents have been discussed and a detailed protocol for handling the radioactive patient is presented. Minor additions and emphasis to parts of these earlier works are summarized, reflecting experiences gained in receiving simulated radioactively contaminated victims in drills at St. Luke's Hospital of Bethlehem, Pennsylvania. Two accidents were simulated involving mock radioactive materials over a two year period. One such ''accident'' was staged at A-B-E Airport, Lehigh County, in 1981 and the other in the Saucon Valley in 1983. It should be mentioned that in neither case was the release of radioactive material possible, in reality, as portrayed. In planning mock radiation accident drills for emergency care units and support staff, one is best-advised not to pay too much attention to the logic of how the release occurred but rather that there are victims who must be treated, decontaminated and evaluated for the necessity of continued medical care

  1. Blast furnace granular coal injection project. Annual report, January--December 1995

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-05-01

    This annual report describes the Blast Furnace Granular Coal Injection project being implemented at Bethlehem Steel Corporation`s (BSC) Burns Harbor Plant. The project is receiving cost-sharing from the U.S. Department of Energy (DOE), and is being administrated by the Morgantown Energy Technology Center in accordance with the DOE Cooperative Agreement No. DE-FC21-91MC27362. This installation is the first in the United States to employ British Steel technology that uses granular coal to provide part of the fuel requirement of blast furnaces. The project will demonstrate/assess a broad range of technical/economic issues associated with the use of coal for this purpose. To achieve the program objectives, the demonstration project is divided into the following three Phases: (1) Phase I - Design. (2) Phase II - Construction. (3) Phase III - Operation. Preliminary Design (Phase I) began in 1991 with detailed design commencing in 1993. Construction at Burns Harbor (Phase II) began in August 1993 and was completed at the end of 1994. The demonstration test program (Phase III) started in the fourth quarter of 1995.

  2. An update on blast furnace granular coal injection

    Energy Technology Data Exchange (ETDEWEB)

    Hill, D.G. [Bethlehem Steel Corp., Burns Harbor, IN (United States); Strayer, T.J.; Bouman, R.W. [Bethlehem Steel Corp., PA (United States)

    1997-12-31

    A blast furnace coal injection system has been constructed and is being used on the furnace at the Burns Harbor Division of Bethlehem Steel. The injection system was designed to deliver both granular (coarse) and pulverized (fine) coal. Construction was completed on schedule in early 1995. Coal injection rates on the two Burns Harbor furnaces were increased throughout 1995 and was over 200 lbs/ton on C furnace in September. The injection rate on C furnace reached 270 lbs/ton by mid-1996. A comparison of high volatile and low volatile coals as injectants shows that low volatile coal replaces more coke and results in a better blast furnace operation. The replacement ratio with low volatile coal is 0.96 lbs coke per pound of coal. A major conclusion of the work to date is that granular coal injection performs very well in large blast furnaces. Future testing will include a processed sub-bituminous coal, a high ash coal and a direct comparison of granular versus pulverized coal injection.

  3. Factors Affecting D-7-Stigmastenol in Palestinian Olive Oil

    Directory of Open Access Journals (Sweden)

    K. Abu-Alruz

    2011-01-01

    Full Text Available The level of delta-7-stigmastenol (D-7-stigmastenol contained in olive oil is a new criterion for oil quality, particularly its purity from adulteration with other seed oils. In this study, 79 olive samples were collected and analyzed from different areas of Palestine to study the factors affecting D-7-stigmastenol levels in the oil. These areas included the provinces of Jericho, Hebron, Bethlehem, Ramallah, Salfeet, Nablus, Jenin, Tulkarem and Qalqilyah. The study began in October 2007 and ended in July 2008. The following 11 factors were taken into consideration during sample collection: olive fly infection, topography, olive storage before pressing, geographical area, effect of olive seeds during oil extraction, effect of pressing temperature, presence of olive leaves during oil extraction, soil type, maturity index of the olive fruit, olive variety and oil preservation and storage in terms of storage container types. The results show that soil type, region, maturity index and olive fly infection are the main factors affecting D-7-stigmastenol. Pressing temperature, olive storage before pressing, olive variety and oil storage showed a moderate effect. Olive seeds, topography and presence of olive leaves had a negligible effect on D-7-stigmastenol levels in the oil.

  4. Hot cracking studies on CrMoV and NiCrMoV turbine rotor steels during welding

    International Nuclear Information System (INIS)

    Four different rotor materials, three CrMoV steels and a NiCrMoV steel, were investigated both with respect to solidification and HAZ liquation cracking. It involved the Varestraint testing using autogeneous gas tungsten arc welding at two different heat inputs, and metallographic examinations using optical, scanning and/or transmission electron microscopy. An increase in heat input/unit length (from 1.2 to 2.7 KJ/mm) or an increase in travel speed at the same low heat input (1.2 KJ/mm) tends to produce more solidification cracking. In the case of CrMoV steels, 1950s air melted rotors (Buck and the Gallatin rotors) showed worse solidification cracking susceptibility than the modern Bethlehem forging vacuum poured, 2A. The modern NiCrMoV forging, also vacuum-poured, 3A, however, showed worse solidification cracking susceptibility than the old air-melted Gallatin rotor. Therefore, the harmful effects of Ni on solidification cracking was confirmed. Its role is understood as having favored the austenite formation, thus resulting in more primary austenite solidification. This gives rise to heavy segregation of impurities such as S and P at the austenite grain boundaries. Detrimental effects of sulfur and phosphorus both on solidification and the HAZ hot cracking were confirmed. Addition of Ce or Ti to the Buck rotor with the greatest solidification cracking susceptibility helped improve cracking resistance

  5. Hot cracking studies on CrMoV and NiCrMoV turbine rotor steels during welding

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, S.Y.

    1986-01-01

    Four different rotor materials, three CrMoV steels and a NiCrMoV steel, were investigated both with respect to solidification and HAZ liquation cracking. It involved the Varestraint testing using autogeneous gas tungsten arc welding at two different heat inputs, and metallographic examinations using optical, scanning and/or transmission electron microscopy. An increase in heat input/unit length (from 1.2 to 2.7 KJ/mm) or an increase in travel speed at the same low heat input (1.2 KJ/mm) tends to produce more solidification cracking. In the case of CrMoV steels, 1950s air melted rotors (Buck and the Gallatin rotors) showed worse solidification cracking susceptibility than the modern Bethlehem forging vacuum poured, 2A. The modern NiCrMoV forging, also vacuum-poured, 3A, however, showed worse solidification cracking susceptibility than the old air-melted Gallatin rotor. Therefore, the harmful effects of Ni on solidification cracking was confirmed. Its role is understood as having favored the austenite formation, thus resulting in more primary austenite solidification. This gives rise to heavy segregation of impurities such as S and P at the austenite grain boundaries. Detrimental effects of sulfur and phosphorus both on solidification and the HAZ hot cracking were confirmed. Addition of Ce or Ti to the Buck rotor with the greatest solidification cracking susceptibility helped improve cracking resistance.

  6. Software Developed for Analyzing High- Speed Rolling-Element Bearings

    Science.gov (United States)

    Fleming, David P.

    2005-01-01

    COBRA-AHS (Computer Optimized Ball & Roller Bearing Analysis--Advanced High Speed, J.V. Poplawski & Associates, Bethlehem, PA) is used for the design and analysis of rolling element bearings operating at high speeds under complex mechanical and thermal loading. The code estimates bearing fatigue life by calculating three-dimensional subsurface stress fields developed within the bearing raceways. It provides a state-of-the-art interactive design environment for bearing engineers within a single easy-to-use design-analysis package. The code analyzes flexible or rigid shaft systems containing up to five bearings acted upon by radial, thrust, and moment loads in 5 degrees of freedom. Bearing types include high-speed ball, cylindrical roller, and tapered roller bearings. COBRA-AHS is the first major upgrade in 30 years of such commercially available bearing software. The upgrade was developed under a Small Business Innovation Research contract from the NASA Glenn Research Center, and incorporates the results of 30 years of NASA and industry bearing research and technology.

  7. Twee Bybelse Verhale van Abraham H. De Vries

    Directory of Open Access Journals (Sweden)

    P. van der Merwe

    1986-05-01

    Full Text Available Abraham de Vries is not so much known as the author of Biblical stories, but two stories can be pointed out very clearly as belonging to this category. These stories are "Skoenmaker diepe water" from Volmoed se gasie and "Die verdeling van die kind” from Vliegoog.The first story, "Skoenmaker diepe water”, refers to Matthew 14:22 - 32 in which Jesus walks on water. In this story the fairy tale given appears at the first level, and the religious motif on the second level. The main character, Vel Binneman, is clearly depicted in the story as being the true believer. “Die verdeling van die kind” also has a Biblical background, and then Herod's infanticide in Bethlehem of which we read in Matthew 2:16 as well as the crucifixion of Christ. In the story It is not only the Biblical motif that is dealt with, but there is a strong Palestinian and New Testament aura. "Die verdeling van die kind” can, with the data at our disposal, be interpreted as a new crucifixion, and the story in fact does become an illustration of God’s redeeming grace.

  8. 人细小病毒B19感染与小儿川崎病的关系%Relationship between human parvovirus B19 infection and Kawasaki disease in children

    Institute of Scientific and Technical Information of China (English)

    曹玉红; 张光运; 张国成; 丁翠玲; 曹艳华; 杨欣伟; 沈青

    2005-01-01

    目的: 探讨人细小病毒B19感染与小儿川崎病的关系.方法: 用巢式PCR法对46例川崎病患儿进行B19-DNA检测,用ELISA法对其中的30例患儿进行B19-VP2-IgM检测.结果: 病例组46例,B19-DNA阳性14例(30%),对照组50例B19-DNA阳性2例,两组间差异显著(P<0.01).病例组B19-VP2-IgM阳性7/30例,对照组50例均阴性.两组间差异极显著(P<0.01).30例川崎病患者中B19-DNA,B19-VP2-IgM均阳性6例;1例仅B19-VP2-IgM阳性;4例仅B19-DNA阳性;B19-DNA和B19-VP2-IgM 同时阴性19例,B19-DNA和B19-VP2-IgM 一致率为83.3%,有一致性(P<0.01).B19阳性与阴性川崎病患儿在性别、年龄、常见临床表现、预后等方面无差别.结论: 我国川崎病患儿B19病毒感染率较高可能是导致川崎病的主要病原体之一.

  9. Tomato 26S Proteasome subunit RPT4a regulates ToLCNDV transcription and activates hypersensitive response in tomato.

    Science.gov (United States)

    Sahu, Pranav Pankaj; Sharma, Namisha; Puranik, Swati; Chakraborty, Supriya; Prasad, Manoj

    2016-01-01

    Involvement of 26S proteasomal subunits in plant pathogen-interactions, and the roles of each subunit in independently modulating the activity of many intra- and inter-cellular regulators controlling physiological and defense responses of a plant were well reported. In this regard, we aimed to functionally characterize a Solanum lycopersicum 26S proteasomal subunit RPT4a (SlRPT4) gene, which was differentially expressed after Tomato leaf curl New Delhi virus (ToLCNDV) infection in tolerant cultivar H-88-78-1. Molecular analysis revealed that SlRPT4 protein has an active ATPase activity. SlRPT4 could specifically bind to the stem-loop structure of intergenic region (IR), present in both DNA-A and DNA-B molecule of the bipartite viral genome. Lack of secondary structure in replication-associated gene fragment prevented formation of DNA-protein complex suggesting that binding of SlRPT4 with DNA is secondary structure specific. Interestingly, binding of SlRPT4 to IR inhibited the function of RNA Pol-II and subsequently reduced the bi-directional transcription of ToLCNDV genome. Virus-induced gene silencing of SlRPT4 gene incited conversion of tolerant attributes of cultivar H-88-78-1 into susceptibility. Furthermore, transient overexpression of SlRPT4 resulted in activation of programmed cell death and antioxidant enzymes system. Overall, present study highlights non-proteolytic function of SlRPT4 and their participation in defense pathway against virus infection in tomato. PMID:27252084

  10. FANCJ helicase uniquely senses oxidative base damage in either strand of duplex DNA and is stimulated by replication protein A to unwind the damaged DNA substrate in a strand-specific manner.

    Science.gov (United States)

    Suhasini, Avvaru N; Sommers, Joshua A; Mason, Aaron C; Voloshin, Oleg N; Camerini-Otero, R Daniel; Wold, Marc S; Brosh, Robert M

    2009-07-01

    FANCJ mutations are genetically linked to the Fanconi anemia complementation group J and predispose individuals to breast cancer. Understanding the role of FANCJ in DNA metabolism and how FANCJ dysfunction leads to tumorigenesis requires mechanistic studies of FANCJ helicase and its protein partners. In this work, we have examined the ability of FANCJ to unwind DNA molecules with specific base damage that can be mutagenic or lethal. FANCJ was inhibited by a single thymine glycol, but not 8-oxoguanine, in either the translocating or nontranslocating strands of the helicase substrate. In contrast, the human RecQ helicases (BLM, RECQ1, and WRN) display strand-specific inhibition of unwinding by the thymine glycol damage, whereas other DNA helicases (DinG, DnaB, and UvrD) are not significantly inhibited by thymine glycol in either strand. In the presence of replication protein A (RPA), but not Escherichia coli single-stranded DNA-binding protein, FANCJ efficiently unwound the DNA substrate harboring the thymine glycol damage in the nontranslocating strand; however, inhibition of FANCJ helicase activity by the translocating strand thymine glycol was not relieved. Strand-specific stimulation of human RECQ1 helicase activity was also observed, and RPA bound with high affinity to single-stranded DNA containing a single thymine glycol. Based on the biochemical studies, we propose a model for the specific functional interaction between RPA and FANCJ on the thymine glycol substrates. These studies are relevant to the roles of RPA, FANCJ, and other DNA helicases in the metabolism of damaged DNA that can interfere with basic cellular processes of DNA metabolism. PMID:19419957

  11. DNA repair and replication fork helicases are differentially affected by alkyl phosphotriester lesion.

    Science.gov (United States)

    Suhasini, Avvaru N; Sommers, Joshua A; Yu, Stephen; Wu, Yuliang; Xu, Ting; Kelman, Zvi; Kaplan, Daniel L; Brosh, Robert M

    2012-06-01

    DNA helicases are directly responsible for catalytically unwinding duplex DNA in an ATP-dependent and directionally specific manner and play essential roles in cellular nucleic acid metabolism. It has been conventionally thought that DNA helicases are inhibited by bulky covalent DNA adducts in a strand-specific manner. However, the effects of highly stable alkyl phosphotriester (PTE) lesions that are induced by chemical mutagens and refractory to DNA repair have not been previously studied for their effects on helicases. In this study, DNA repair and replication helicases were examined for unwinding a forked duplex DNA substrate harboring a single isopropyl PTE specifically positioned in the helicase-translocating or -nontranslocating strand within the double-stranded region. A comparison of SF2 helicases (RecQ, RECQ1, WRN, BLM, FANCJ, and ChlR1) with a SF1 DNA repair helicase (UvrD) and two replicative helicases (MCM and DnaB) demonstrates unique differences in the effect of the PTE on the DNA unwinding reactions catalyzed by these enzymes. All of the SF2 helicases tested were inhibited by the PTE lesion, whereas UvrD and the replication fork helicases were fully tolerant of the isopropyl backbone modification, irrespective of strand. Sequestration studies demonstrated that RECQ1 helicase was trapped by the PTE lesion only when it resided in the helicase-translocating strand. Our results are discussed in light of the current models for DNA unwinding by helicases that are likely to encounter sugar phosphate backbone damage during biological DNA transactions. PMID:22500020

  12. Characterization of Rhynchosia yellow mosaic Yucatan virus, a new recombinant begomovirus associated with two fabaceous weeds in Yucatan, Mexico.

    Science.gov (United States)

    Hernández-Zepeda, C; Brown, J K; Moreno-Valenzuela, O A; Argüello-Astorga, G; Idris, A M; Carnevali, G; Rivera-Bustamante, R F

    2010-10-01

    Rhynchosia minima (L.) DC. (Fabaceae) plants exhibiting bright golden mosaic symptoms were previously associated with begomovirus infection in Yucatan, México [1]. To characterize the begomovirus infecting these plants, the complete bipartite genome was cloned and sequenced. Sequence comparisons indicated that the virus was distinct from all other begomoviruses known to date, including those previously identified from symptomatic R. minima, and the name Rhynchosia yellow mosaic Yucatan virus (RhYMYuV) is proposed. Pairwise comparisons indicated that RhYMYuV DNA-A [2,597 nt, (EU021216)] and DNA-B [2,542 nt, (FJ792608)] components shared the highest nt sequence identity with Cabbage leaf curl virus (CaLCuV), 87% for component A and 71% for component B. Phylogenetic analysis indicated that both components of RhYMYuV are most closely related to other New World begomoviruses, having as closest relatives immediate outliers to the major Squash leaf curl virus (SLCV) clade. Recombination analysis of the RhYMYuV genome indicated that the DNA-A component has arisen through intermolecular recombination. R. minima plants inoculated with the monomeric clones developed a bright yellow mosaic similar to symptoms observed in naturally infected plants, confirming that the clones were infectious. Nicotiana benthamiana plants biolistically inoculated with monomeric clones developed curling and chlorosis in the newly emerging leaves. RhYMYuV was also detected in symptomatic Desmodium sect. Scorpiurus Benth. (Fabaceae) that were collected near the RhYMYuV-infected plants. PMID:20574644

  13. Coincidence of cleavage sites of intron endonuclease I-TevI and critical sequences of the host thymidylate synthase gene.

    Science.gov (United States)

    Edgell, David R; Stanger, Matthew J; Belfort, Marlene

    2004-11-01

    To maximize spread of their host intron or intein, many homing endonucleases recognize nucleotides that code for important and conserved amino acid residues of the target gene. Here, we examine the cleavage requirements for I-TevI, which binds a stretch of thymidylate synthase (TS) DNA that codes for functionally critical residues in the TS active site. Using an in vitro selection scheme, we identified two base-pairs in the I-TevI cleavage site region as important for cleavage efficiency. These were confirmed by comparison of I-TevI cleavage efficiencies on mutant and on wild-type substrates. We also showed that nicking of the bottom strand by I-TevI is not affected by mutation of residues surrounding the bottom-strand cleavage site, unlike other homing endonucleases. One of these two base-pairs is universally conserved in all TS sequences, and is identical with a previously identified cleavage determinant of I-BmoI, a related GIY-YIG endonuclease that binds a homologous stretch of TS-encoding DNA. The other base-pair is conserved only in a subset of TS genes that includes the I-TevI, but not the I-BmoI, target sequence. Both the I-TevI and I-BmoI cleavage site requirements correspond to functionally critical residues involved in an extensive hydrogen bond network within the TS active site. Remarkably, these cleavage requirements correlate with TS phylogeny in bacteria, suggesting that each endonuclease has individually adapted to efficiently cleave distinct TS substrates. PMID:15491609

  14. The genome and proteome of a Campylobacter coli bacteriophage vB_CcoM-IBB_35 reveal unusual features

    Directory of Open Access Journals (Sweden)

    Carvalho Carla M

    2012-01-01

    Full Text Available Abstract Background Campylobacter is the leading cause of foodborne diseases worldwide. Bacteriophages (phages are naturally occurring predators of bacteria, ubiquitous in the environment, with high host specificity and thus considered an appealing option to control bacterial pathogens. Nevertheless for an effective use of phages as antimicrobial agents, it is important to understand phage biology which renders crucial the analysis of phage genomes and proteomes. The lack of sequence data from Campylobacter phages adds further importance to these studies. Methods vB_CcoM-IBB_35 is a broad lytic spectrum Myoviridae Campylobacter phage with high potential for therapeutic use. The genome of this phage was obtained by pyrosequencing and the sequence data was further analyzed. The proteomic analysis was performed by SDS-PAGE and Mass spectrometry. Results and conclusions The DNA sequence data of vB_CcoM-IBB_35 consists of five contigs for a total of 172,065 bp with an average GC content of 27%. Attempts to close the gaps between contigs were unsuccessful since the DNA preparations appear to contain substances that inhibited Taq and ϕ29 polymerases. From the 210 identified ORFs, around 60% represent proteins that were not functionally assigned. Homology exists with members of the Teequatrovirinae namely for T4 proteins involved in morphogenesis, nucleotide metabolism, transcription, DNA replication and recombination. Tandem mass spectrometric analysis revealed 38 structural proteins as part of the mature phage particle. Conclusions Genes encoding proteins involved in the carbohydrate metabolism along with several incidences of gene duplications, split genes with inteins and introns have been rarely found in other phage genomes yet are found in this phage. We identified the genes encoding for tail fibres and for the lytic cassette, this later, expressing enzymes for bacterial capsular polysaccharides (CPS degradation, which has not been reported

  15. Recombinant nematode anticoagulant protein c2 inhibits cell invasion by decreasing uPA expression in NSCLC cells.

    Science.gov (United States)

    Tong, Yu; Yue, Jun; Mao, Meng; Liu, Qingqing; Zhou, Jing; Yang, Jiyun

    2015-04-01

    Nematode anticoagulant protein c2 (NAPc2) is an 85-residue polypeptide originally isolated from the hematophagous hookworm, Ancylostoma caninum. Several studies have shown that rNAPc2 inhibits the growth of primary and metastatic tumors in mice independently of its ability to initiate coagulation. We obtained bioactive recombinant rNAPc2 by splicing of the rNAPc2-intein-CBD fusion proteins expressed in E. coli ER2566. In the in vitro assay, rNAPc2 obviously inhibited the invasive ability of non-small cell lung cancer (NSCLC) cells in a dose-dependent manner. Furthermore, rNAPc2 suppressed tumor growth in vivo by daily intraperitoneal injection of rNAPc2 in an NSCLC cell xenograft model of nude mice. Respectively, rNAPc2 downregulated the production of urokinase plasminogen activator (uPA) (P<0.05) and suppressed nuclear factor-κB (NF-κB) activity. We also identified that inhibition of NF-κB activity impaired cell invasion and reduced the uPA production in NSCLC cells. Meanwhile, NF-κB was found to directly bind to the uPA promoter in vitro. These results demonstrated that rNAPc2 inhibits cell invasion at least in part through the downregulation of the NF-κB-dependent metastasis-related gene expression in NSCLC. Our results also suggest that uPA, a known metastasis-promoting gene, is indirectly regulated by rNAPc2 through NF-κB activation. These results indicate that rNAPc2 may be a potent agent for the prevention of NSCLC progression. PMID:25672417

  16. A Cartografia Medieval. O mundo dos homens e o mundo de Deus

    Directory of Open Access Journals (Sweden)

    Maria Eurydice Barros Ribeiro

    2010-01-01

    Full Text Available The article suggests the study of medieval cartography, the language integrated to the communication system promoted and controlled by the Church. Two world maps located in the manuscripts were taken as examples: the Beatus of Liébana’s The Apocalypse (the British Library, Ms 11695 and the Honorius of Autun’s Imago Mundi (Cambridge, Ms 66 and two mural world maps: the Ebstorf map, originally found in the Ebstorf monastery cloister (Germany and the Hereford map that can be seen on the altar of the Hereford Cathedral (England. It was attempted to demonstrate in this article how the world maps put into practice the art of memory, the rhetoric resource, by repeating the information from one charter to the other. The maps faced east, maintaining the Homeric tradition of land surrounded by ocean and the division of the world in three continents. The sacred took hold of the physical geography by locating the biblical places equally, on the same positions: On top - on Asia -, Heaven; in the center, celestial Jerusalem. The other vetero and neo-testamentary references such as Noah’s ark and the cities of Bethlehem and Babel are located correctly in the east part (on the top. It is in the monastery context - particularly the Benedictine Rule principle in which, fighting inactivity, determined besides handwork, the monks take some time of the day to read – that it is possible to understand the purpose of the world maps inside the medieval manuscripts, which could function as educational instrument to the aristocracy and nobility in the Court world. Similarly, it is through liturgy that mural maps become not cloister or the cathedral altar ornaments, but images capable of sending the greatest message of Christianity, the resurrection and salvation of the soul, announcing the Last Judgment.

  17. Performing the renaissance body and mind: somaesthetic style and devotional practice at the Sacro Monte di Varallo

    Directory of Open Access Journals (Sweden)

    Allie Terry-Fritsch

    2015-02-01

    Full Text Available This essay examines the somaesthetic experience of renaissance pilgrims to the Sacro Monte di Varallo, a late fifteenthcentury simulation of the Holy Land located in northern Italy. It reconstructs how pilgrims once cultivated their bodies and minds to enhance aesthetic and devotional experience to offer a re-evaluation of artistic style at the site. Built by a team of architects, painters and sculptors at the behest of Franciscan friars, the Sacro Monte di Varallo transformed the mountainous terrain of the Val Sesia into a ‘true representation’ of Bethlehem and Jerusalem. The Holy Land was presented to the pilgrim in a series of interactive spaces housed in independent architectural units, each containing life-sized wooden or terracotta sculptures of Biblical figures adorned with real hair, clothes and shoes, and situated in frescoed narratival environments. Pilgrims were led to each architectural site along a fixed path and encountered the Biblical scenes in a strict sequence that was narrated by a Franciscan friar. If the pilgrim engaged in proper performances of body-mindfulness, the site served as a pilgrimage destination that was equally enriching as ‘the real thing’. The essay questions how the somaesthetics of experience at Varallo served to enfold pilgrims into multi-sensory, immersive scenarios and thereby allowed pilgrims to activate the art and architecture of the Franciscan campus in personalised ways. Through their physical and mental participation in the works, pilgrims actively constructed the means for the art and architecture of the holy mountain to fulfil and even surpass the power of the real Holy Land.

  18. Real time detection of exhaled human breath using quantum cascade laser based sensor technology

    Science.gov (United States)

    Tittel, Frank K.; Lewicki, Rafal; Dong, Lei; Liu, Kun; Risby, Terence H.; Solga, Steven; Schwartz, Tim

    2012-02-01

    The development and performance of a cw, TE-cooled DFB quantum cascade laser based sensor for quantitative measurements of ammonia (NH3) and nitric oxide (NO) concentrations present in exhaled breath will be reported. Human breath contains ~ 500 different chemical species, usually at ultra low concentration levels, which can serve as biomarkers for the identification and monitoring of human diseases or wellness states. By monitoring NH3 concentration levels in exhaled breath a fast, non-invasive diagnostic method for treatment of patients with liver and kidney disorders, is feasible. The NH3 concentration measurements were performed with a 2f wavelength modulation quartz enhanced photoacoustic spectroscopy (QEPAS) technique, which is suitable for real time breath measurements, due to the fast gas exchange inside a compact QEPAS gas cell. A Hamamatsu air-cooled high heat load (HHL) packaged CW DFB-QCL is operated at 17.5°C, targeting the optimum interference free NH3 absorption line at 967.35 cm-1 (λ~10.34 μm), with ~ 20 mW of optical power. The sensor architecture includes a reference cell, filled with a 2000 ppmv NH3 :N2 mixture at 130 Torr, which is used for absorption line-locking. A minimum detection limit (1σ) for the line locked NH3 sensor is ~ 6 ppbv (with a 1σ 1 sec time resolution of the control electronics). This NH3 sensor was installed in late 2010 and is being clinically tested at St. Luke's Hospital in Bethlehem, PA.

  19. Genetic variability of begomoviruses associated with cotton leaf curl disease originating from India.

    Science.gov (United States)

    Kirthi, N; Priyadarshini, C G P; Sharma, P; Maiya, S P; Hemalatha, V; Sivaraman, P; Dhawan, P; Rishi, N; Savithri, H S

    2004-10-01

    Cotton leaf curl disease (CLCuD) causing viruses belong to the Begomovirus genus of the family Geminiviridae. Most begomoviruses are bipartite with two molecules of circular single stranded DNA (A and B) encapsidated in icosahedral geminate particles. However, the begomoviruses associated with CLCuD have DNA-beta instead of DNA-B. In this communication we report the complete genomic sequence of DNA-A component of two CLCuD-causing begomoviruses, cotton leaf curl Kokhran virus-Dabawali (CLCuKV-Dab), tomato leaf curl Bangalore virus-Cotton [Fatehabad] (ToLCBV-Cotton [Fat]) and partial sequences of two other isolates cotton leaf curl Rajasthan virus-Bangalore (CLCuRV-Ban) and cotton leaf curl Kokhran virus-Ganganagar (CLCuKV-Gang). A phylogenetic analysis of these isolates along with other related begomoviruses showed that ToLCBV-Cotton [Fat] isolate was closest to the tomato leaf curl Bangalore virus-5 (ToLCBV-Ban5) where as CLCuKV-Dab isolate was close to the cotton leaf curl Kokhran virus-Faisalabad1 (CLCuKV-Fai1), cotton leaf curl Kokhran virus-72b (CLCuKV-72b) and cotton leaf curl Kokhran virus-806b (CLCuKV-806b) isolates from Pakistan. The phylogenetic analysis further showed that the ToLCBV-Cotton [Fat] and CLCuKV-Dab isolates along with CLCuKV-Fai1, CLCuKV-72b and CLCuKV-806b are closer to the ToLCBV, tomato leaf curl Gujarat virus (ToLCGV), tomato leaf curl Gujarat virus-Varanasi (ToLCGV-Var) and tomato leaf curl Sri Lanka virus (ToLCSLV) isolates, where as cotton leaf curl Alabad virus-804a (CLCuAV-804a), cotton leaf curl Multhan virus (CLCuMV) cluster with the isolates from cotton leaf curl Rajasthan virus (CLCuRV) and okra yellow vein mosaic virus (OYVMV). These results demonstrate the extensive variability observed in this group of viruses. The AC4 ORF is the least conserved among these viruses. In order to further asses the variability in the CLCuD-causing begomoviruses, the region showing minimum similarity in the DNA-A sequence was first determined by

  20. 双载体断裂CFTR基因转移及翻译后的连接和功能%Post-translational ligation and function of dual-vector transferred split CFTR gene

    Institute of Scientific and Technical Information of China (English)

    朱甫祥; 刘泽隆; 屈慧鸽; 迟晓艳

    2010-01-01

    囊性纤维化跨膜电导调节体(CFTR)基因突变导致一种常染色体隐性遗传病囊性纤维化(CF),利用腺辅助病毒(AAV)载体转运CFTR基因的基因疗法受到AAV载体容量的限制.本文采用intein的蛋白质反式剪接技术,以双载体转运CFTR基因,研究了于其调节结构域(R)断裂成两部分的CFTR基因翻译后的连接及其产生的Cl~-通道功能.将人CFTR cDNA于R结构域的Ser~(713)密码子前断裂,构建一对融合Ssp DnaB intein编码序列的真核表达载体.将这对载体共转染培养的幼年仓鼠肾(BHK)细胞,通过瞬时表达,全细胞和单通道膜片钳记录Cl~-电流,并用Western blotting观察CFTR蛋白的剪接.结果表明,共转染细胞显示较高的全细胞Cl~-电流和单个Cl~-通道开放活性,说明CFTR的Cl~-通道功能的恢复,用CFTR特异性抗体进行的细胞总蛋白Western blotting显示有完整的CFTR蛋白条带形成,表明intcin可有效连接翻译后的两部分CFTR蛋白.结果提示,蛋白质剪接技术可有效用于双载体系统转运CFTR基因,为进一步应用双AAV载体转运CFTR基因的CF基因治疗研究提供了实验依据.

  1. Impact of age-associated cyclopurine lesions on DNA repair helicases.

    Science.gov (United States)

    Khan, Irfan; Suhasini, Avvaru N; Banerjee, Taraswi; Sommers, Joshua A; Kaplan, Daniel L; Kuper, Jochen; Kisker, Caroline; Brosh, Robert M

    2014-01-01

    8,5' cyclopurine deoxynucleosides (cPu) are locally distorting DNA base lesions corrected by nucleotide excision repair (NER) and proposed to play a role in neurodegeneration prevalent in genetically defined Xeroderma pigmentosum (XP) patients. In the current study, purified recombinant helicases from different classifications based on sequence homology were examined for their ability to unwind partial duplex DNA substrates harboring a single site-specific cPu adduct. Superfamily (SF) 2 RecQ helicases (RECQ1, BLM, WRN, RecQ) were inhibited by cPu in the helicase translocating strand, whereas helicases from SF1 (UvrD) and SF4 (DnaB) tolerated cPu in either strand. SF2 Fe-S helicases (FANCJ, DDX11 (ChlR1), DinG, XPD) displayed marked differences in their ability to unwind the cPu DNA substrates. Archaeal Thermoplasma acidophilum XPD (taXPD), homologue to the human XPD helicase involved in NER DNA damage verification, was impeded by cPu in the non-translocating strand, while FANCJ was uniquely inhibited by the cPu in the translocating strand. Sequestration experiments demonstrated that FANCJ became trapped by the translocating strand cPu whereas RECQ1 was not, suggesting the two SF2 helicases interact with the cPu lesion by distinct mechanisms despite strand-specific inhibition for both. Using a protein trap to simulate single-turnover conditions, the rate of FANCJ or RECQ1 helicase activity was reduced 10-fold and 4.5-fold, respectively, by cPu in the translocating strand. In contrast, single-turnover rates of DNA unwinding by DDX11 and UvrD helicases were only modestly affected by the cPu lesion in the translocating strand. The marked difference in effect of the translocating strand cPu on rate of DNA unwinding between DDX11 and FANCJ helicase suggests the two Fe-S cluster helicases unwind damaged DNA by distinct mechanisms. The apparent complexity of helicase encounters with an unusual form of oxidative damage is likely to have important consequences in the

  2. Impact of age-associated cyclopurine lesions on DNA repair helicases.

    Directory of Open Access Journals (Sweden)

    Irfan Khan

    Full Text Available 8,5' cyclopurine deoxynucleosides (cPu are locally distorting DNA base lesions corrected by nucleotide excision repair (NER and proposed to play a role in neurodegeneration prevalent in genetically defined Xeroderma pigmentosum (XP patients. In the current study, purified recombinant helicases from different classifications based on sequence homology were examined for their ability to unwind partial duplex DNA substrates harboring a single site-specific cPu adduct. Superfamily (SF 2 RecQ helicases (RECQ1, BLM, WRN, RecQ were inhibited by cPu in the helicase translocating strand, whereas helicases from SF1 (UvrD and SF4 (DnaB tolerated cPu in either strand. SF2 Fe-S helicases (FANCJ, DDX11 (ChlR1, DinG, XPD displayed marked differences in their ability to unwind the cPu DNA substrates. Archaeal Thermoplasma acidophilum XPD (taXPD, homologue to the human XPD helicase involved in NER DNA damage verification, was impeded by cPu in the non-translocating strand, while FANCJ was uniquely inhibited by the cPu in the translocating strand. Sequestration experiments demonstrated that FANCJ became trapped by the translocating strand cPu whereas RECQ1 was not, suggesting the two SF2 helicases interact with the cPu lesion by distinct mechanisms despite strand-specific inhibition for both. Using a protein trap to simulate single-turnover conditions, the rate of FANCJ or RECQ1 helicase activity was reduced 10-fold and 4.5-fold, respectively, by cPu in the translocating strand. In contrast, single-turnover rates of DNA unwinding by DDX11 and UvrD helicases were only modestly affected by the cPu lesion in the translocating strand. The marked difference in effect of the translocating strand cPu on rate of DNA unwinding between DDX11 and FANCJ helicase suggests the two Fe-S cluster helicases unwind damaged DNA by distinct mechanisms. The apparent complexity of helicase encounters with an unusual form of oxidative damage is likely to have important consequences in

  3. The Escherichia coli Cryptic Prophage Protein YfdR Binds to DnaA and Initiation of Chromosomal Replication Is Inhibited by Overexpression of the Gene Cluster yfdQ-yfdR-yfdS-yfdT

    Science.gov (United States)

    Noguchi, Yasunori; Katayama, Tsutomu

    2016-01-01

    The initiation of bacterial chromosomal replication is regulated by multiple pathways. To explore novel regulators, we isolated multicopy suppressors for the cold-sensitive hda-185 ΔsfiA(sulA) mutant. Hda is crucial for the negative regulation of the initiator DnaA and the hda-185 mutation causes severe replication overinitiation at the replication origin oriC. The SOS-associated division inhibitor SfiA inhibits FtsZ ring formation, an essential step for cell division regulation during the SOS response, and ΔsfiA enhances the cold sensitivity of hda-185 cells in colony formation. One of the suppressors comprised the yfdQ-yfdR-yfdS-yfdT gene cluster carried on a cryptic prophage. Increased copy numbers of yfdQRT or yfdQRS inhibited not only hda-185-dependent overinitiation, but also replication overinitiation in a hyperactive dnaA mutant, and in a mutant lacking an oriC-binding initiation-inhibitor SeqA. In addition, increasing the copy number of the gene set inhibited the growth of cells bearing specific, initiation-impairing dnaA mutations. In wild-type cells, multicopy supply of yfdQRT or yfdQRS also inhibited replication initiation and increased hydroxyurea (HU)-resistance, as seen in cells lacking DiaA, a stimulator of DnaA assembly on oriC. Deletion of the yfdQ-yfdR-yfdS-yfdT genes did not affect either HU resistance or initiation regulation. Furthermore, we found that DnaA bound specifically to YfdR in soluble protein extracts oversupplied with YfdQRST. Purified YfdR also bound to DnaA, and DnaA Phe46, an amino acid residue crucial for DnaA interactions with DiaA and DnaB replicative helicase was important for this interaction. Consistently, YfdR moderately inhibited DiaA-DnaA and DnaB-DnaA interactions. In addition, protein extracts oversupplied with YfdQRST inhibited replication initiation in vitro. Given the roles of yfdQ and yfdS in cell tolerance to specific environmental stresses, the yfdQ-yfdR-yfdS-yfdT genes might downregulate the initiator Dna

  4. Evolution of hedgehog and hedgehog-related genes, their origin from Hog proteins in ancestral eukaryotes and discovery of a novel Hint motif

    Directory of Open Access Journals (Sweden)

    Bürglin Thomas R

    2008-03-01

    gene family must have arisen in very early eukaryotic evolution, and eventually gave rise to hh and hh-related genes in animals. The results indicate a hitherto unsuspected ability of Hog domain encoding genes to evolve new N-termini. In one instance in Cnidaria, the Hh N-terminal signaling domain is associated with a VWA domain and lacks a Hog domain, suggesting a modular mode of evolution also for the N-terminal domain. The Hog domain proteins, the inteins and VWA-Vint proteins represent three different families of Hint domain proteins that evolved in parallel in eukaryotes.

  5. AISI/DOE Advanced Process Control Program Vol. 1 of 6: Optical Sensors and Controls for Improved Basic Oxygen Furnace Operations

    Energy Technology Data Exchange (ETDEWEB)

    Sarah Allendorf; David Ottesen; Donald Hardesty

    2002-01-31

    The development of an optical sensor for basic oxygen furnace (BOF) off-gas composition and temperature in this Advanced Process Control project has been a laboratory spectroscopic method evolve into a pre-commercialization prototype sensor system. The sensor simultaneously detects an infrared tunable diode laser ITDL beam transmitted through the process off-gas directly above the furnace mouth, and the infrared greybody emission from the particulate-laden off-gas stream. Following developmental laboratory and field-testing, the sensor prototype was successfully tested in four long-term field trials at Bethlehem Steel's Sparrows Point plant in Baltimore, MD> The resulting optical data were analyzed and reveal correlations with four important process variables: (1) bath turndown temperature; (2) carbon monoxide post-combustion control; (2) bath carbon concentration; and (4) furnace slopping behavior. The optical sensor measurement of the off-gas temperature is modestly correlated with bath turndown temperature. A detailed regression analysis of over 200 heats suggests that a dynamic control level of +25 Degree F can be attained with a stand-alone laser-based optical sensor. The ability to track off-gas temperatures to control post-combustion lance practice is also demonstrated, and may be of great use in optimizing post-combustion efficiency in electric furnace steelmaking operations. In addition to the laser-based absorption spectroscopy data collected by this sensor, a concurrent signal generated by greybody emission from the particle-laden off-gas was collected and analyzed. A detailed regression analysis shows an excellent correlation of a single variable with final bath turndown carbon concentration. Extended field trials in 1998 and early 1999 show a response range from below 0.03% to a least 0.15% carbon concentration with a precision of +0.0007%. Finally, a strong correlation between prolonged drops in the off-gas emission signal and furnace slopping

  6. THE BIBLICAL CHRONOTOPE IN THE “TRAVEL POEMS” BY IVAN A. BUNIN “THE BIRD’S SHADOW”

    Directory of Open Access Journals (Sweden)

    Tat’yana N. Kovalyova

    2015-11-01

    Full Text Available Ivan Bunin created his “Travel Poems” — “The Bird’s Shadow” — based on the impressions of his wanderings, between 1903 and 1909, across the Middle East countries including Turkey, Judaea, Palestine, Syria, Egypt, Algeria, Tunisia and Greece. Modern researchers present the East of Bunin’s “Travel Poems” as a certain generalized image of a “culturological aspect”which comprises the historical and cultures features of diff erent countries of the Levant. Meanwhile, Bunin emphasized that he took his journeys to Judaea and Palestine as a pilgrimage to the Holy Land, not as mere leisure travels. The importance of the Bible East in the “Travel Poems” is also determined by the fact that the largest part of his routes and most of his essays (7 essays of 11 are related to the Holy Land. Th is article examines the artistic space-time of the Palestinian Essays by Bunin and reveals and characterizes the biblical chronotope and the role of the key topos of the Old Testament and the New Testament. However, the author emphasizes a special meaning of some biblical topos. These are the places associated with the key biblical events and with intentionality of author’s consciousness, which generate a broad range of the keynotes of the cycle of stories: the Valley of Josaphat as the place of the upcoming Last Judgement; the Dead Sea as the symbol of visitation of God for the people’s sins; the Judean Desert where Jesus was tempted by devil; Jerusalem, Bethlehem, Nazareth, Gennisaret as the cities of ancient Palestine related to the events of the Terrestrial Life of Jesus Christ. The hero’s perception of the holy places, domination of the biblical space-time in the “Travel Poems” devoted to the pilgrimage to the Holy Land, aspiration for being projected to the biblical times, unity of the hero’s chronotope with the biblical chronotope — all this indicates the extreme importance of the biblical events for the author

  7. Patient satisfaction at haematology and oncology clinics in the Free State & Northern Cape

    Directory of Open Access Journals (Sweden)

    W.R. Davies

    2002-09-01

    Full Text Available The Free State and Northern Cape make up some 40% of the land area of South Africa, while being home to only 10% of the total population. Haematology and Oncotherapy outreach clinics were established in Kimberley, Bethlehem and Welkom to provide a more accessible service to the thinly spread population. A previous study showed these clinics to be cost-effective, but we had no idea how the patients experience them. Our aim was to obtain information about the demographics of the patients, the logistical support of the clinics, the medical needs of the patients and how they experience the clinics. This can help us to improve the service. A questionnaire was tested in a pilot study. The demographic questions covered age, sex and ethnicity. The logistical questions dealt with distance travelled to the clinic, mode of transport, length of time as a patient and cost. The medical need questions dealt with type of disease, treatment received, type of doctor seen and origin of referral. The questions about experience covered satisfaction with the service, staff, waiting times and involvement of non-governmental organizations. Of the 95 patients interviewed 42% were from the haematology clinics. The mean age was 59.5 and the male: female ratio was 0.6:1. Forty-six percent of the patients spoke Afrikaans and 31 % spoke South Sotho. The black:white ratio was 1:1. Twenty-eight percent used the government ambulances (of whom 80% were satisfied and 56% used their own cars. The median payment at a clinic was R20 (R0 to R200. Only 19% of patients were paying privately. Ninety-five percent of the patients were follow-ups, with the median length of follow-up being 24 months (1 to 468. The patients were mainly referred by local hospitals. Twentytwo percent of the patients had chronic haematological malignancies, while 68% had solid tumours. Thirty-seven percent of the patients received drugs to take home and only 6% got intravenous chemotherapy. Consultants saw 44

  8. Communication is the Key Skill for Reference Librarians. A review of: Taylor, Robert S. “Question‐Negotiation and Information Seeking in Libraries.” College & Research Libraries 29.3 (1968: 178‐94.

    Directory of Open Access Journals (Sweden)

    Christina K. Pikas

    2007-12-01

    Full Text Available Objective – To better understand the question negotiation process in libraries both in intermediated and in self‐helpsituations. To achieve a richer understanding of the relationship between library users and library systems in order to establish a research agenda and inform librarian education.Design – The first part consisted of qualitative research involving interviews. The second part consisted of a diary study.Setting – Special engineering libraries in the United States and a university campus (Lehigh in Bethlehem, Pennsylvania.Subjects – The participants in the interviews were special librarians. Special librarians were selected because they have more specialized knowledge and respond to more substantive questions in greater depth than do public and academic librarians who emphasize instruction and who encounter staffing restrictions that prevent them from spending too much time on each inquiry. Detailed information on the selection of the individual participants is not provided. The participants in the diary study were twenty undergraduate students who were enrolled in an information science course.Methods – The interviews were open‐ended and unstructured. The interviews lasted sixty to ninety minutes and were taped. No information is provided on transcription or analysis methods or paradigms. In the second part, the students were given areading assignment on information seeking. They then had to select a search topic and document the steps they took, decisions they made, and resources they used to answer the question. The participants were asked to analyze their original question, the type of answer required, and decisions they made in the process. No details are provided on the analysis of the diaries.Main results – Taylor found five filters required for search definition: 1. Determination of subject; 2. Objective and motivation; 3. Personal characteristics of the inquirer; 4. Relationship of inquiry description to file

  9. In silico ordinary differential equation/partial differential equation hemodialysis model estimates methadone removal during dialysis

    Directory of Open Access Journals (Sweden)

    Linares OA

    2015-07-01

    Full Text Available Oscar A Linares,1 William E Schiesser,2 Jeffrey Fudin,3–6 Thien C Pham,6 Jeffrey J Bettinger,6 Roy O Mathew,6 Annemarie L Daly7 1Translational Genomic Medicine Lab, Plymouth Pharmacokinetic Modeling Study Group, Plymouth, MI, 2Department of Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, PA, 3University of Connecticut School of Pharmacy, Storrs, CT, 4Western New England College of Pharmacy, Springfield, MA, 5Albany College of Pharmacy and Health Sciences, Albany, NY, 6Stratton VA Medical Center, Albany, NY, 7Grace Hospice of Ann Arbor, Ann Arbor, MI, USA Background: There is a need to have a model to study methadone’s losses during hemodialysis to provide informed methadone dose recommendations for the practitioner. Aim: To build a one-dimensional (1-D, hollow-fiber geometry, ordinary differential equation (ODE and partial differential equation (PDE countercurrent hemodialyzer model (ODE/PDE model. Methodology: We conducted a cross-sectional study in silico that evaluated eleven hemodialysis patients. Patients received a ceiling dose of methadone hydrochloride 30 mg/day. Outcome measures included: the total amount of methadone removed during dialysis; methadone’s overall intradialytic mass transfer rate coefficient, km; and, methadone’s removal rate, jME. Each metric was measured at dialysate flow rates of 250 mL/min and 800 mL/min. Results: The ODE/PDE model revealed a significant increase in the change of methadone’s mass transfer with increased dialysate flow rate, %Δ km=18.56, P=0.02, N=11. The total amount of methadone mass transferred across the dialyzer membrane with high dialysate flow rate significantly increased (0.042±0.016 versus 0.052±0.019 mg/kg, P=0.02, N=11. This was accompanied by a small significant increase in methadone’s mass transfer rate (0.113±0.002 versus 0.014±0.002 mg/kg/h, P=0.02, N=11. The ODE/PDE model accurately predicted methadone’s removal during dialysis. The absolute value

  10. UMA ANÁLISE DAS REPERCUSSÕES DO PROUNI NA VISÂO DOS EGRESSOS DA UNAMA NO PERÍODO DE 2009 A 2014

    Directory of Open Access Journals (Sweden)

    Sonia Andrea Pimentel Rodrigues Ferreira

    2015-09-01

    Uni access through the vision of the UNAMA graduates, the intention in researching the University for All Program as a public access policy in Bethlehem, has scientific relevance, policy and social. From the point, scientific, we seek answers to research in our research, since the topic in question acts dialectically in reality as well, so controversial in higher education literature, since it refers to constant discussion and reflection of the right direction the program takes, the national scene. Occasionally the program has been the subject of several studies that discuss the issue of inclusion / exclusion proposed by the policy. Methodologically adopt the literature search that allowed us to know the opinions for and against the program, the Field Research at the locus of research, desk research by collecting information from the Ministry of Education and the institution itself, to entrdermos the Logistics of ProUni, the materialization of their information not sharp in official documents. Preliminary findings point to the importance of the program in the evaluation of the graduates, however, it is denounced the immense difficulties to stay on course. The development of the research focused on the ongoing high tuition, (Law, Social Communication, Nursing, Physiotherapy, Psiclogia, Engineering as they are the most social demand for courses in the research university, which leveraged our investigation in relation to access and retention of graduates scholars of politics. The time frame of the survey was 2006-20014, covering 500 graduates of which was obtained in 26 of those surveyed answers through questionnaires.PALABRAS CLAVE: acceso, ProUni y graduadosRecebido em: 22/05/2015  – Aceito em 27/07/2015

  11. Obituary: Ben Hawkins Moore, 1921-2003

    Science.gov (United States)

    Moore, James F.

    2004-12-01

    at the public schools and the general public. These regularly scheduled programs provided a way for the university and for the science programs to achieve a level of prominence in the community and they opened vistas of wonder for budding scientists in the schools. After his experiences at the Adler Planetarium, he developed a particular presentation on the Star of Bethlehem that he gave not only in St. Cloud but also in Texas where he spent winters in the last few years. His program was designed to highlight the scientific questions that arise when one thinks about the possible explanations of such an event. On the other hand, the popular knowledge of, and interest in, this story became a vehicle for Ben to draw an even greater appreciation for the sciences from public audiences. Ben married Alice Winifred Bassett in 1943 in Kansas City, Missouri; she died in 1971. A year later he married Marjorie Rotnem who survives him. He is also survived by three sons (John, James and Robert Moore), and one daughter (Donna Habermeyer) from his first marriage as well as Richard and Diane Rotnem from his second marriage; there are seven grandchildren. His devotion to his family was perhaps even more central to his life than his love for teaching and science. He is also survived by a host of friends, colleagues and students who hold him in the highest regard. In the last few years of his life, Ben took on a project in thinking about the relation between science and religion, partly at my urging. His written comments on this topic are more than two hundred pages. Throughout his career he had fought for ways to be Christian and to be an authentic scientist. This meant, for him, a level of humility for both disciplines as well as clear and reasonable thinking. Among the many other things that Ben's life models for us is this life long passion to be both religious and rigorously scientific at the same time, finding no ultimate conflict in doing that. In my view, his influence on these