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Sample records for bethlehem dnab intein

  1. The Arthrobacter species FB24 Arth_1007 (DnaB intein is a pseudogene.

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    Kazuo Tori

    Full Text Available An Arthrobacter species FB24 gene (locus tag Arth_1007 was previously annotated as a putative intein-containing DnaB helicase of phage origin (Arsp-FB24 DnaB intein. However, it is not a helicase gene because the sequence similarity is limited to inteins. In fact, the flanking exteins total only 66 amino acids. Therefore, the intein should be referred to as the Arsp-FB24 Arth_1007 intein. The Arsp-FB24 Arth_1007 intein failed to splice in its native precursor and in a model precursor. We previously noted that the Arsp-FB24 Arth_1007 intein is the only putative Class 3 intein that is missing the catalytically essential Cys at position 4 of intein Motif F, which is one of the three defining signature residues of this class. Additionally, a catalytically essential His in position 10 of intein Motif B is also absent; this His is the most conserved residue amongst all inteins. Splicing activity was not rescued when these two catalytically important positions were 'reverted' back to their consensus residues. This study restores the unity of the Class 3 intein signature sequence in active inteins by demonstrating that the Arsp-FB24 Arth_1007 intein is an inactive pseudogene.

  2. [Ssp DnaB intein-mediated ligation of heavy and light chains of coagulation factor VIII in Escherichia coli].

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    Zhu, Fuxiang; Liu, Zelong; Qu, Huige; Xin, Xiaolin; Dong, Hongxin; Liu, Xiangqin

    2009-07-01

    We studied the ligation of coagulation factor VIII heavy and light chains in Escherichia coli by utilizing the intein-mediated protein trans-splicing. A B-domain deleted factor VIII (BDD-FVIII) gene was broken into two halves of heavy and light chains before Ser1657 which meets the splicing required conserved residue and then fused to 106 and 48 amino acid-containing N-part termed Int-N and C-part termed Int-C coding sequences of split mini Ssp DnaB intein respectively. These two fusion genes were constructed into a prokaryotic expression vector pBV220. Through induction for expression of recombinant protein it displayed an obvious protein band as predicted size of BDD-FVIII protein on SDS-PAGE gel. Western blotting using factor VIII specific antibodies confirmed that this protein band is BDD-FVIII produced by protein trans-splicing. It demonstrated that the heavy and light chains of BDD-FVIII can be efficiently ligated with the Ssp DnaB intein-mediated protein trans-splicing. These results provided evidence for encouraging our ongoing investigation with intein as a means in dual AAV vectors carrying the factor VIII gene to overcome the packaging size limitation of a single AAV vector in hemophilia A gene therapy.

  3. Split-Inteins for Simultaneous, site-specific conjugation of Quantum Dots to multiple protein targets In vivo

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    Christodoulou Neophytos

    2011-09-01

    Full Text Available Abstract Background Proteins labelled with Quantum Dots (QDs can be imaged over long periods of time with ultrahigh spatial and temporal resolution, yielding important information on the spatiotemporal dynamics of proteins within live cells or in vivo. However one of the major problems regarding the use of QDs for biological imaging is the difficulty of targeting QDs onto proteins. We have recently developed a DnaE split intein-based method to conjugate Quantum Dots (QDs to the C-terminus of target proteins in vivo. In this study, we expand this approach to achieve site-specific conjugation of QDs to two or more proteins simultaneously with spectrally distinguishable QDs for multiparameter imaging of cellular functions. Results Using the DnaE split intein we target QDs to the C-terminus of paxillin and show that paxillin-QD conjugates become localized at focal adhesions allowing imaging of the formation and dissolution of these complexes. We go on to utilize a different split intein, namely Ssp DnaB mini-intein, to demonstrate N-terminal protein tagging with QDs. Combination of these two intein systems allowed us to simultaneously target two distinct proteins with spectrally distinguishable QDs, in vivo, without any cross talk between the two intein systems. Conclusions Multiple target labeling is a unique feature of the intein based methodology which sets it apart from existing tagging methodologies in that, given the large number of characterized split inteins, the number of individual targets that can be simultaneously tagged is only limited by the number of QDs that can be spectrally distinguished within the cell. Therefore, the intein-mediated approach for simultaneous, in vivo, site-specific (N- and C-terminus conjugation of Quantum Dots to multiple protein targets opens up new possibilities for bioimaging applications and offers an effective system to target QDs and other nanostructures to intracellular compartments as well as specific

  4. Inteins in Microbial Genomes: Distribution, Mechanism, and Function

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    Maurice W. Southworth

    2002-01-01

    Full Text Available Inteins are self-splicing protein elements (134 to 608 amino acids. Over 125 inteins have been cataloged in InBase, the on-line intein database (http://www.neb.com/neb/inteins.html, which includes the Intein Registry[1]. Inteins naturally present in pathogenic microbes represent novel, yet unexploited drug targets. Understanding the chemistry of the splicing reaction has allowed the manipulation of inteins, which are now used in many protein engineering applications[2].

  5. Properties of DnaB helicase in [lambda] DNA replication

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    Stephens, K.M.

    1991-01-01

    A tailed nicked-circle DNA substrate was used to measure the rapid replication fork (RF) movement catalyzed by E. Coli DnaB helicase and DNA polymerase III holoenzyme (pol III HE) (DnaB-RFs) (30 DnaB hexamers/substrate). The DnaB RFs can efficiently utilize the DNA substrate (60% in 5 min at 30C), and the forks move at a rapid rate (550-780 bp/sec at 30C). The DnaB-RFs have an average maximal processivity of 40,000 nt, and addition of either SSB or primase increase the processivity (150,000 nt + SSB, 70,000-140,000 nt + primase). However, SSB and primase do not affect the rate of fork movement or the amount of substrate utilized in the assay. The [lambda] SS proteins are effective at transferring DnaB onto the DNA substrate (8 DnaB hexamers/substrate). The [lambda] SS proteins do not change the rate of RF movement or the amount of substrate utilized. However, the amount of synthesis measured in the assay is [approximately]2-fold higher in the presence of the [lambda] SS proteins. Therefore, the [lambda] SS proteins increase the processivity of DnaB at the RF (100,000 nt). The [lambda] SS proteins do not appear to play a role in elongation because the processivity of the RF in the presence of SSB and primase is equivalent to the processivity of the [lambda] SS-RFs. [lambda] P protein blocks DnaB helicase activity if added to the RF assay prior to initiation or during elongation. DnaB helicase is more resistant to P inhibition, if the helicase is allowed to bind to the substrate prior to addition of [lambda] P or if primase and rNTPs are included in the assay. These results suggest that the conformation of the RF complex (DNA or nucleoprotein structure) blocks the attack of P on DnaB helicase. The heat shock proteins may play an auxiliary role in mediating the effects of [lambda] P if the concentration of P protein in the cells are high.

  6. Protein trans-splicing of multiple atypical split inteins engineered from natural inteins.

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    Ying Lin

    Full Text Available Protein trans-splicing by split inteins has many uses in protein production and research. Splicing proteins with synthetic peptides, which employs atypical split inteins, is particularly useful for site-specific protein modifications and labeling, because the synthetic peptide can be made to contain a variety of unnatural amino acids and chemical modifications. For this purpose, atypical split inteins need to be engineered to have a small N-intein or C-intein fragment that can be more easily included in a synthetic peptide that also contains a small extein to be trans-spliced onto target proteins. Here we have successfully engineered multiple atypical split inteins capable of protein trans-splicing, by modifying and testing more than a dozen natural inteins. These included both S1 split inteins having a very small (11-12 aa N-intein fragment and S11 split inteins having a very small (6 aa C-intein fragment. Four of the new S1 and S11 split inteins showed high efficiencies (85-100% of protein trans-splicing both in E. coli cells and in vitro. Under in vitro conditions, they exhibited reaction rate constants ranging from ~1.7 × 10(-4 s(-1 to ~3.8 × 10(-4 s(-1, which are comparable to or higher than those of previously reported atypical split inteins. These findings should facilitate a more general use of trans-splicing between proteins and synthetic peptides, by expanding the availability of different atypical split inteins. They also have implications on understanding the structure-function relationship of atypical split inteins, particularly in terms of intein fragment complementation.

  7. Mira Ceti and the Star of Bethlehem

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    Sigismondi, Costantino

    2014-05-01

    We consider the probability that the Gospel of Matthew could report the earliest observation of Mira Ceti. Some biblical remarks have to be considered in order to distinguish a scientific text in the modern acceptation and the content of Gospels regarding some astronomical arguments. Mira fulfills the basic requirements to be the Star of Bethlehem as described in the Gospel according to Matthew (Mt 2:1-12). In fact it was visible at least two times with a time interval (not specified in Mt text) in which it disappeared. Moreover Mira was close to the position were the triple conjunction of Jupiter and Saturn occurred in the years 7-6 b. C. and it could be observed during that period by ancient astronomers. The discovery of Mira in 1596 and its second observation 12.5 years later, made by David Fabricius, occurred when Jupiter approached it. Because of those reasons we study the maxima of Mira in order to evaluate both the frequency of one and of two consecutive bright apparitions eventually as observed by the Magi. We did an evaluation of the correlation between two following maxima in order to verify the probability of occurrence of two consecutive bright maxima, because that condition would have been indeed the most favorable for the candidature of Mira as the Bethlehem Star. Analyzing the maxima of Mira we found a probability of seeing it brighter than alpha Ceti once every 21 years. In this case, as in February 1997, Mira can be detected at the first sight as a new component near the most significant asterism in its zone, composed by alpha, gamma and delta Ceti. This condition could have happened in the case of the Bethlehem Star. We found also a correlation between the magnitude of two consecutive maxima: if a bright maximum occurs it is more probable that the following is a faint one.

  8. Inteins as Indicators of Gene Flow in the Halobacteria

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    Shannon Margaret Soucy

    2014-06-01

    Full Text Available This research uses inteins, a type of mobile genetic element, to infer patterns of gene transfer within the Halobacteria. We surveyed one hundred and eighteen genomes representing twenty-six genera of Halobacteria for intein sequences. We then used the presence-absence profile, sequence similarity and phylogenies from the inteins recovered to explore how intein distribution can provide insight on the dynamics of gene flow between closely related and divergent organisms. We identified twenty-four proteins in the Halobacteria that have been invaded by inteins at some point in their evolutionary history, including two proteins not previously reported to contain an intein. Furthermore, the size of an intein is used as a heuristic for the phase of the intein’s life cycle. Larger size inteins are assumed to be the canonical two domain inteins, consisting of self-splicing and homing endonuclease domains (HEN; smaller sizes are assumed to have lost the HEN domain. For many halobacterial groups the consensus phylogenetic signal derived from intein sequences is compatible with vertical inheritance or with a strong gene transfer bias creating these clusters. Regardless, the coexistence of intein-free and intein-containing alleles reveal ongoing transfer and loss of inteins within these groups. Inteins were frequently shared with other Euryarchaeota and among the Bacteria, with members of the Cyanobacteria (Cyanothece, Anabaena, Bacteriodetes (Salinibacter, Betaproteobacteria (Delftia, Acidovorax, Firmicutes (Halanaerobium, Actinobacteria (Longispora, and Deinococcus-Thermus-group.

  9. [Intein-mediated F309SfVIII ligation with enhanced secretion of its heavy chain.].

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    Zhu, Fu-Xiang; Liu, Ze-Long; Qu, Hui-Ge; Chi, Xiao-Yan

    2009-12-25

    Coagulation factor VIII (fVIII) is a secretion protein and plays a crucial role in the coagulation cascade. Hemophilia A resulted from deficiency of fVIII is the most common X-linked recessive bleeding disorder. Gene therapy is recognized as an attractive strategy for the eventual cure of this disease. However, the gene therapy is hampered by the big size of fVIII gene when using the most promising gene vectors, adeno-associated virus (AAV) vectors. In this study we explored the intein-mediated protein trans-splicing to deliver a Phe(309)-->Ser mutant full-length fVIII (F309SfVIII) gene by using a dual-vector system. An intein is a protein sequence embedded within a precursor protein and can excise itself through protein splicing. The F309SfVIII is proven to be beneficial to its secretion. The F309SfVIII gene was broken into heavy and light chains before Ser(1239) in B domain and fused with the coding sequences of Ssp DnaB intein respectively to construct a pair of plasmid vectors by inserting them into the pcDNA3.1 vectors. Forty-eight hours after co- or separate transfection of 293 cells, the co-transfected cell lysate showed an obvious ligated F309SfVIII protein band by Western blot with a polyclonal antibody against fVIII. The amounts of secreted F309SfVIII protein in culture supernatants and their bioactivities were (71+/-9) ng/mL and (0.38+/-0.09) IU/mL determined by ELISA and Coatest assay respectively. The supernatant from combined cells with separate transfections also displayed lower levels of F309SfVIII antigen and fVIII activity [(25+/-6) ng/mL and (0.12+/-0.05) IU/mL], indicating the F309SfVIII could be formed by splicing both before and after secretion. The content of F309SfVIII heavy chain protein from co-transfected cell supernatant was higher than that of intein-fused heavy chain transfection alone [(135+/-10) ng/mL vs (37+/-7) ng/mL, Pintein could be used as a technical strategy in a dual-vector system delivering F309SfVIII gene with improved

  10. Mycobacteriophages as Incubators for Intein Dissemination and Evolution

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    Kelley, Danielle S.; Lennon, Christopher W.; Novikova, Olga

    2016-01-01

    ABSTRACT Inteins are self-splicing protein elements that are mobile at the DNA level and are sporadically distributed across microbial genomes. Inteins appear to be horizontally transferred, and it has been speculated that phages may play a role in intein distribution. Our attention turns to mycobacteriophages, which infect mycobacteria, where both phage and host harbor inteins. Using bioinformatics, mycobacteriophage genomes were mined for inteins. This study reveals that these mobile elements are present across multiple mycobacteriophage clusters and are pervasive in certain genes, like the large terminase subunit TerL and a RecB-like nuclease, with the majority of intein-containing genes being phage specific. Strikingly, despite this phage specificity, inteins localize to functional motifs shared with bacteria, such that intein-containing genes have similar roles, like hydrolase activity and nucleic acid binding, indicating a global commonality among intein-hosting proteins. Additionally, there are multiple insertion points within active centers, implying independent invasion events, with regulatory implications. Several phage inteins were shown to be splicing competent and to encode functional homing endonucleases, important for mobility. Further, bioinformatic analysis supports the potential for phages as facilitators of intein movement among mycobacteria and related genera. Analysis of catalytic intein residues finds the highly conserved penultimate histidine inconsistently maintained among mycobacteriophages. Biochemical characterization of a noncanonical phage intein shows that this residue influences precursor accumulation, suggesting that splicing has been tuned in phages to modulate generation of important proteins. Together, this work expands our understanding of phage-based intein dissemination and evolution and implies that phages provide a context for evolution of splicing-based regulation. PMID:27703073

  11. Mycobacteriophages as Incubators for Intein Dissemination and Evolution

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    Danielle S. Kelley

    2016-10-01

    Full Text Available Inteins are self-splicing protein elements that are mobile at the DNA level and are sporadically distributed across microbial genomes. Inteins appear to be horizontally transferred, and it has been speculated that phages may play a role in intein distribution. Our attention turns to mycobacteriophages, which infect mycobacteria, where both phage and host harbor inteins. Using bioinformatics, mycobacteriophage genomes were mined for inteins. This study reveals that these mobile elements are present across multiple mycobacteriophage clusters and are pervasive in certain genes, like the large terminase subunit TerL and a RecB-like nuclease, with the majority of intein-containing genes being phage specific. Strikingly, despite this phage specificity, inteins localize to functional motifs shared with bacteria, such that intein-containing genes have similar roles, like hydrolase activity and nucleic acid binding, indicating a global commonality among intein-hosting proteins. Additionally, there are multiple insertion points within active centers, implying independent invasion events, with regulatory implications. Several phage inteins were shown to be splicing competent and to encode functional homing endonucleases, important for mobility. Further, bioinformatic analysis supports the potential for phages as facilitators of intein movement among mycobacteria and related genera. Analysis of catalytic intein residues finds the highly conserved penultimate histidine inconsistently maintained among mycobacteriophages. Biochemical characterization of a noncanonical phage intein shows that this residue influences precursor accumulation, suggesting that splicing has been tuned in phages to modulate generation of important proteins. Together, this work expands our understanding of phage-based intein dissemination and evolution and implies that phages provide a context for evolution of splicing-based regulation.

  12. The distribution and evolutionary history of the PRP8 intein

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    Goodwin Timothy JD

    2006-05-01

    Full Text Available Abstract Background We recently described a mini-intein in the PRP8 gene of a strain of the basidiomycete Cryptococcus neoformans, an important fungal pathogen of humans. This was the second described intein in the nuclear genome of any eukaryote; the first nuclear encoded intein was found in the VMA gene of several saccharomycete yeasts. The evolution of eukaryote inteins is not well understood. In this report we describe additional PRP8 inteins (bringing the total of these to over 20. We compare and contrast the phylogenetic distribution and evolutionary history of the PRP8 intein and the saccharomycete VMA intein, in order to derive a broader understanding of eukaryote intein evolution. It has been suggested that eukaryote inteins undergo horizontal transfer and the present analysis explores this proposal. Results In total, 22 PRP8 inteins have been detected in species from three different orders of euascomycetes, including Aspergillus nidulans and Aspergillus fumigatus (Eurotiales, Paracoccidiodes brasiliensis, Uncinocarpus reesii and Histoplasma capsulatum (Onygales and Botrytis cinerea (Helotiales. These inteins are all at the same site in the PRP8 sequence as the original Cryptococcus neoformans intein. Some of the PRP8 inteins contain apparently intact homing endonuclease domains and are thus potentially mobile, while some lack the region corresponding to the homing endonuclease and are thus mini-inteins. In contrast, no mini-inteins have been reported in the VMA gene of yeast. There are several examples of pairs of closely related species where one species carries the PRP8 intein while the intein is absent from the other species. Bio-informatic and phylogenetic analyses suggest that many of the ascomycete PRP8 homing endonucleases are active. This contrasts with the VMA homing endonucleases, most of which are inactive. Conclusion PRP8 inteins are widespread in the euascomycetes (Pezizomycota and apparently their homing endonucleases are

  13. Intein-mediated purification system: mechanism and applications

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    Sarra setrerrahmane; Shuhua Tan

    2013-01-01

    The incorporation of self-cleaving protein elements into a variety of fusion-based purification systems; has been an important development in the area of recombinant protein purification. The self-cleaving capability of these tags has recently been combined with additional purification tags to generate novel and convenient protein purification methods. This review elucidates the properties of intein, the mechanism of the intein-based protein splicing and the progress of intein-based protein purification procedures, and recent advances in the applications of intein.

  14. Streamlined expressed protein ligation using split inteins.

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    Vila-Perelló, Miquel; Liu, Zhihua; Shah, Neel H; Willis, John A; Idoyaga, Juliana; Muir, Tom W

    2013-01-09

    Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.

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

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    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

  16. Intein-Promoted Cyclization of Aspartic Acid Flanking the Intein Leads to Atypical N-Terminal Cleavage.

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    Minteer, Christopher J; Siegart, Nicolle M; Colelli, Kathryn M; Liu, Xinyue; Linhardt, Robert J; Wang, Chunyu; Gomez, Alvin V; Reitter, Julie N; Mills, Kenneth V

    2017-02-28

    Protein splicing is a post-translational reaction facilitated by an intein, or intervening protein, which involves the removal of the intein and the ligation of the flanking polypeptides, or exteins. A DNA polymerase II intein from Pyrococcus abyssi (Pab PolII intein) can promote protein splicing in vitro on incubation at high temperature. Mutation of active site residues Cys1, Gln185, and Cys+1 to Ala results in an inactive intein precursor, which cannot promote the steps of splicing, including cleavage of the peptide bond linking the N-extein and intein (N-terminal cleavage). Surprisingly, coupling the inactivating mutations to a change of the residue at the C-terminus of the N-extein (N-1 residue) from the native Asn to Asp reactivates N-terminal cleavage at pH 5. Similar "aspartic acid effects" have been observed in other proteins and peptides but usually only occur at lower pH values. In this case, however, the unusual N-terminal cleavage is abolished by mutations to catalytic active site residues and unfolding of the intein, indicating that this cleavage effect is mediated by the intein active site and the intein fold. We show via mass spectrometry that the reaction proceeds through cyclization of Asp resulting in anhydride formation coupled to peptide bond cleavage. Our results add to the richness of the understanding of the mechanism of protein splicing and provide insight into the stability of proteins at moderately low pH. The results also explain, and may help practitioners avoid, a side reaction that may complicate intein applications in biotechnology.

  17. A new example of viral intein in Mimivirus

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    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.

  18. Intein-mediated backbone cyclization of VP1 protein enhanced protection of CVB3-induced viral myocarditis

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    Qi, Xingmei; Xiong, Sidong

    2017-01-01

    CVB3 is a common human pathogen to be highly lethal to newborns and causes viral myocarditis and pancreatitis in adults. However, there is no vaccine available for clinical use. CVB3 capsid protein VP1 is an immunodominant structural protein, containing several B- and T-cell epitopes. However, immunization of mice with VP1 protein is ineffective. Cyclization of peptide is commonly used to improve their in vivo stability and biological activity. Here, we designed and synthesizd cyclic VP1 protein by using engineered split Rma DnaB intein and the cyclization efficiency was 100% in E. coli. As a result, the cyclic VP1 was significantly more stable against irreversible aggregation upon heating and against carboxypeptidase in vitro and the degradation rate was more slowly in vivo. Compared with linear VP1, immunization mice with circular VP1 significantly increased CVB3-specific serum IgG level and augmented CVB3-specific cellular immune responses, consequently afforded better protection against CVB3-induced viral myocarditis. The cyclic VP1 may be a novel candidate protein vaccine for preventing CVB3 infection and similar approaches could be employed to a variety of protein vaccines to enhance their protection effect. PMID:28148910

  19. PRP8 intein in cryptic species of Histoplasma capsulatum: evolution and phylogeny

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    The PRP8 intein is the most widespread intein among the Kingdom Fungi. This genetic element occurs within the prp8 gene, and is transcribed and translated simultaneously with the gene. After translation, the intein excises itself from the Prp8 protein by an autocatalytic splicing reaction, subsequen...

  20. An evolved Mxe GyrA intein for enhanced production of fusion proteins.

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    Marshall, Carrie J; Grosskopf, Vanessa A; Moehling, Taylor J; Tillotson, Benjamin J; Wiepz, Gregory J; Abbott, Nicholas L; Raines, Ronald T; Shusta, Eric V

    2015-02-20

    Expressing antibodies as fusions to the non-self-cleaving Mxe GyrA intein enables site-specific, carboxy-terminal chemical modification of the antibodies by expressed protein ligation (EPL). Bacterial antibody-intein fusion protein expression platforms typically yield insoluble inclusion bodies that require refolding to obtain active antibody-intein fusion proteins. Previously, we demonstrated that it was possible to employ yeast surface display to express properly folded single-chain antibody (scFv)-intein fusions, therefore permitting the direct small-scale chemical functionalization of scFvs. Here, directed evolution of the Mxe GyrA intein was performed to improve both the display and secretion levels of scFv-intein fusion proteins from yeast. The engineered intein was shown to increase the yeast display levels of eight different scFvs by up to 3-fold. Additionally, scFv- and green fluorescent protein (GFP)-intein fusion proteins can be secreted from yeast, and while fusion of the scFvs to the wild-type intein resulted in low expression levels, the engineered intein increased scFv-intein production levels by up to 30-fold. The secreted scFv- and GFP-intein fusion proteins retained their respective binding and fluorescent activities, and upon intein release, EPL resulted in carboxy-terminal azide functionalization of the target proteins. The azide-functionalized scFvs and GFP were subsequently employed in a copper-free, strain-promoted click reaction to site-specifically immobilize the proteins on surfaces, and it was demonstrated that the functionalized, immobilized scFvs retained their antigen binding specificity. Taken together, the evolved yeast intein platform provides a robust alternative to bacterial intein expression systems.

  1. Evolution and Application of Inteins in Candida species: a Review

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    José Alex Lourenço Fernandes

    2016-10-01

    Full Text Available Inteins are invasive intervening sequences that perform an autocatalytic splicing from their host proteins. Among eukaryotes, these elements are present in many fungal species, including those considered opportunistic or primary pathogens, such as Candida spp. Here we reviewed and updated the list of Candida species containing inteins in the genes VMA, THRRS and GLT1 and pointed out the importance of these elements as molecular markers for molecular epidemiological researches and species-specific diagnosis, since the presence, as well as the size of these inteins, is polymorphic among the different species. Although absent in Candida albicans, these elements are present in different sizes, in some environmental Candida spp. and also in most of the non-albicans Candida spp. considered emergent opportunistic pathogens. Besides, the possible role of these inteins in yeast physiology was also discussed in the light of the recent findings on the importance of these elements as post-translational modulators of gene expression, reinforcing their relevance as alternative therapeutic targets for the treatment of non-albicans Candida infections, because, once the splicing of an intein is inhibited, its host protein, which is usually a housekeeping protein, becomes nonfunctional.

  2. Construction of a Bacterial Assay for Estrogen Detection Based on an Estrogen-Sensitive Intein ▿ †

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    Liang, Rubing; Zhou, Jing; Liu, Jianhua

    2011-01-01

    Escherichia coli strain DIER was constructed for estrogen detection by inserting an estrogen-sensitive intein (VMAER intein) into the specific site of the constitutively expressed chromosomal lacZ gene. This VMAER intein was generated by replacing the endonuclease region of the Saccharomyces cerevisiae VMA intein with the estrogen binding region of the human estrogen receptor α (hERα). When there were estrogens or analogs, the splicing of the VMAER intein was induced to produce the mature Lac...

  3. Intein-modified enzymes, their production and industrial applications

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    Apgar, James; Lessard, Philip; Raab, Michael R.; Shen, Binzhang; Lazar, Gabor; de la Vega, Humberto

    2016-10-11

    A method of predicting an intein insertion site in a protein that will lead to a switching phenotype is provided. The method includes identifying a plurality of C/T/S sites within the protein; selecting from the plurality of C/T/S/ sites those that are ranked 0.75 or higher by a support vector machine, within ten angstroms of the active site of the protein, and at or near a loop-.beta.-sheet junction or a loop-.alpha.-helix junction. A method of controlling protein activity and hosts including proteins with controlled activity are also provided. Also, intein modified proteins and plants containing intein modified proteins are provided.

  4. Cloning and Expression of Ontak Immunotoxin Using Intein Tag

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    SA Moosavizadeh

    2016-06-01

    Full Text Available Introduction: Inteins (INT are internal parts of a number of proteins in yeast and some other unicellular eukaryotes, which can be separated from the immature protein during protein splicing process. After identifying the mechanism of intein action, applications of these sequences are be considered in the single- step purification of recombinant proteins and different intein tags were developed. The most important advantage of using intein tags in purification of recombinant proteins than other affinity tags is no requirement of expensive protease enzymes and following additional steps to remove protease that make intein tags economically are considered more important. In the present study, denileukin diftitox immunotoxin (brand name Ontak, be fused with an intein tag and it was inserted in pTXB1 plasmid. Methods: In this study, with respect to multiple cloning sites (MCS of pTXB1, specific primers were designed. Polymerase Chain Reaction (PCR was performed and encoding sequence of ONTAK was cloned using restriction sites of NdeI and SapI. Recombinant vector (PTX-IDZ was transformed into E. coli strain ER2566 and expression of gene was studied. Results: The accuracy of recombinant construct was confirmed by PCR and enzymatic digestion. The produced recombinant proteins were confirmed by SDS-PAGE and Western blotting. Conclusion: Restriction site of SapI guarantees no additional residues incorporate in primary protein sequence. Also, the expression of this construct was analyzed in compare with fused protein to poly-His tag. According to the appropriate expression of fused protein in both constructs it was expected that one step- purification of considered drug protein will be success in the following steps.

  5. 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

    2016-08-09

    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.

  6. 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; Gophna, Uri

    2016-01-01

    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

  7. Analysis of the roles of NrdR and DnaB from Streptococcus pyogenes in response to host defense.

    Science.gov (United States)

    Zhang, Yan; Okada, Ryo; Isaka, Masanori; Tatsuno, Ichiro; Isobe, Ken-Ichi; Hasegawa, Tadao

    2015-03-01

    Toxic shock syndrome caused by Streptococcus pyogenes (S. pyogenes) is a re-emerging infectious disease. Many virulence-associated proteins play important roles in its pathogenesis and the production of these proteins is controlled by many regulatory factors. CovS is one of the most important two-component sensor proteins in S. pyogenes, and it has been analyzed extensively. Our recent analyses revealed the existence of a transposon between covS and nrdR in several strains, and we speculated that this insertion has some importance. Hence, we examined the significances of the NrdR stand-alone regulator and DnaB, which is encoded by the gene located immediately downstream of nrdR in S. pyogenes infection. We established an nrdR-only knockout strain, and both nrdR and partial dnaB knockout strain. These established knockout strains exhibited a deteriorated response to H2 O2 exposure. nrdR and partial dnaB knockout strain was more easily killed by human polynuclear blood cells, but the nrdR-only knockout strain had no significant difference compared to wild type in contrast to the combined knockout strain. In addition, the mouse infection model experiment illustrated that nrdR and partial dnaB knockout strain, but not the nrdR-only knockout strain, was less virulent compared with the parental strain. These results suggest that DnaB is involved in response to host defense.

  8. Behind the Scene Role of Conserved Threonine in Intein Splicing

    Science.gov (United States)

    Dearden, Albert; Callahan, Brian; Belfort, Marlene; Nayak, Saroj

    2012-02-01

    Protein splicing is an autocatalytic process where an ``intein'' self-cleaves from a precursor protein and catalyzes ligation of the flanking fragments. Inteins occur in all domains of life and have myriad uses in biotechnology. While reaction steps of intein splicing are known, mechanistic details remain incomplete. Here, we investigate the possible role of a highly conserved active-site Threonine residue in bringing about the initial step of splicing: peptide bond rearrangement at a conserved Glycine-Cysteine motif. We report that although not part of the active transition state in this reaction, Threonine plays an important role in reducing the energy barrier through charge screening of active residues in the transition state. Interestingly, Threonine-Glycine hydrogen bonding makes sulfur of the attacking Cysteine less nucleophilic, thereby minimizing Coulomb repulsion in the transition state. These non-intuitive results are obtained through a combination of crystal structure, quantum mechanical simulations, and mutagenesis data. Our results further predict that the sluggish reaction rates observed with intein mutants harboring Threonine-Alanine substitutions can be accelerated in the presence of non-aqueous solvents.

  9. Recombinant production of peptide C-terminal α-amides using an engineered intein

    DEFF Research Database (Denmark)

    Albertsen, Louise; Shaw, Allan C; Norrild, Jens Chr.;

    2013-01-01

    is that they contain a C-terminal that is α-amidated, and this amidation is crucial for biological function. A challenge is to generate such peptides by recombinant means and particularly in a production scale. Here, we have examined an intein-mediated approach to generate a PYY derivative in a larger scale. Initially...... of the 198 amino acid intein with an eight amino acid linker. The optimized intein construct was used to produce the PYY derivative under high cell density cultivation conditions, generating the peptide thioester precursor in good yields and subsequent amidation provided the target peptide......., we experienced challenges with hydrolysis of the intein fusion protein, which was reduced by a T3C mutation in the intein. Subsequently, we further engineered the intein to decrease the absolute size and improve the relative yield of the PYY derivative, which was achieved by substituting 54 residues...

  10. Protein trans-splicing of an atypical split intein showing structural flexibility and cross-reactivity.

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    Huiling Song

    Full Text Available Inteins catalyze a protein splicing reaction to excise the intein from a precursor protein and join the flanking sequences (exteins with a peptide bond. In a split intein, the intein fragments (I(N and I(C can reassemble non-covalently to catalyze a trans-splicing reaction that joins the exteins from separate polypeptides. An atypical split intein having a very small I(N and a large I(C is particularly useful for joining synthetic peptides with recombinant proteins, which can be a generally useful method of introducing site-specific chemical labeling or modifications into proteins. However, a large I(C derived from an Ssp DnaX intein was found recently to undergo spontaneous C-cleavage, which raised questions regarding its structure-function and ability to trans-splice. Here, we show that this I(C could undergo trans-splicing in the presence of I(N, and the trans-splicing activity completely suppressed the C-cleavage activity. We also found that this I(C could trans-splice with small I(N sequences derived from two other inteins, showing a cross-reactivity of this atypical split intein. Furthermore, we found that this I(C could trans-splice even when the I(N sequence was embedded in a nearly complete intein sequence, suggesting that the small I(N could project out of the central pocket of the intein to become accessible to the I(C. Overall, these findings uncovered a new atypical split intein that can be valuable for peptide-protein trans-splicing, and they also revealed an interesting structural flexibility and cross-reactivity at the active site of this intein.

  11. Salt-Dependent Conditional Protein Splicing of an Intein from Halobacterium salinarum.

    Science.gov (United States)

    Reitter, Julie N; Cousin, Christopher E; Nicastri, Michael C; Jaramillo, Mario V; Mills, Kenneth V

    2016-03-01

    An intein from Halobacterium salinarum can be isolated as an unspliced precursor protein with exogenous exteins after Escherichia coli overexpression. The intein promotes protein splicing and uncoupled N-terminal cleavage in vitro, conditional on incubation with NaCl or KCl at concentrations of >1.5 M. The protein splicing reaction also is conditional on reduction of a disulfide bond between two active site cysteines. Conditional protein splicing under these relatively mild conditions may lead to advances in intein-based biotechnology applications and hints at the possibility that this H. salinarum intein could serve as a switch to control extein activity under physiologically relevant conditions.

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

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    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

  13. Semisynthesis of Ribonuclease A using Intein-Mediated Protein Ligation

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    Ulrich Arnold

    2002-01-01

    Full Text Available The introduction of non-natural amino acid residues or modules into proteins provides a new means to explore the basis for conformational stability, folding/unfolding behavior, or biological function. We exploited intein-mediated protein ligation to produce a semisynthetic ribonuclease A. Of the 124 residues of RNase A, residues 1–94 were linked to an intein. After expression of the fusion protein and thiol-induced cleavage, the RNase A(1–94 fragment possessed a C-terminal thioester. A peptide identical to the C-terminal residues 95–124 of RNase A (with residue 95 being cysteine was successfully ligated to that thioester thereby reconstituting full-length wild-type RNase A. In mass spectrometry, this semisynthetic RNase A proved to be undistinguishable from the control protein, namely recombinant wild-type RNase A. Recombinant wild-type RNase A was obtained by expression of RNase A(1–124–intein fusion protein followed by thiol-induced cleavage and hydrolysis of the thioester. Both proteins showed thermal stabilities (Tm and catalytic activities comparable to the wild-type enzyme, indicating that both proteins folded properly. These results might serve as basis for the semisynthesis of RNase A variants containing non-natural modules in the aforementioned peptide.

  14. An Engineered Split Intein for Photoactivated Protein Trans-Splicing.

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    Stanley Wong

    Full Text Available Protein splicing is mediated by inteins that auto-catalytically join two separated protein fragments with a peptide bond. Here we engineered a genetically encoded synthetic photoactivatable intein (named LOVInC, by using the light-sensitive LOV2 domain from Avena sativa as a switch to modulate the splicing activity of the split DnaE intein from Nostoc punctiforme. Periodic blue light illumination of LOVInC induced protein splicing activity in mammalian cells. To demonstrate the broad applicability of LOVInC, synthetic protein systems were engineered for the light-induced reassembly of several target proteins such as fluorescent protein markers, a dominant positive mutant of RhoA, caspase-7, and the genetically encoded Ca2+ indicator GCaMP2. Spatial precision of LOVInC was demonstrated by targeting activity to specific mammalian cells. Thus, LOVInC can serve as a general platform for engineering light-based control for modulating the activity of many different proteins.

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

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    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. Analysis of inteins in the Candida parapsilosis complex for simple and accurate species identification.

    Science.gov (United States)

    Prandini, Tâmara Heloísa Rocha; Theodoro, Raquel Cordeiro; Bruder-Nascimento, Ariane C M O; Scheel, Christina M; Bagagli, Eduardo

    2013-09-01

    Inteins are coding sequences that are transcribed and translated with flanking sequences and then are excised by an autocatalytic process. There are two types of inteins in fungi, mini-inteins and full-length inteins, both of which present a splicing domain containing well-conserved amino acid sequences. Full-length inteins also present a homing endonuclease domain that makes the intein a mobile genetic element. These parasitic genetic elements are located in highly conserved genes and may allow for the differentiation of closely related species of the Candida parapsilosis (psilosis) complex. The correct identification of the three psilosis complex species C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis is very important in the clinical setting for improving antifungal therapy and patient care. In this work, we analyzed inteins that are present in the vacuolar ATPase gene VMA and in the threonyl-tRNA synthetase gene ThrRS in 85 strains of the Candida psilosis complex (46 C. parapsilosis, 17 C. metapsilosis, and 22 C. orthopsilosis). Here, we describe an accessible and accurate technique based on a single PCR that is able to differentiate the psilosis complex based on the VMA intein. Although the ThrRS intein does not distinguish the three species of the psilosis complex by PCR product size, it can differentiate them by sequencing and phylogenetic analysis. Furthermore, this intein is unusually present as both mini- and full-length forms in C. orthopsilosis. Additional population studies should be performed to address whether this represents a common intraspecific variability or the presence of subspecies within C. orthopsilosis.

  17. A predictive model of intein insertion site for use in the engineering of molecular switches.

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    James Apgar

    Full Text Available Inteins are intervening protein domains with self-splicing ability that can be used as molecular switches to control activity of their host protein. Successfully engineering an intein into a host protein requires identifying an insertion site that permits intein insertion and splicing while allowing for proper folding of the mature protein post-splicing. By analyzing sequence and structure based properties of native intein insertion sites we have identified four features that showed significant correlation with the location of the intein insertion sites, and therefore may be useful in predicting insertion sites in other proteins that provide native-like intein function. Three of these properties, the distance to the active site and dimer interface site, the SVM score of the splice site cassette, and the sequence conservation of the site showed statistically significant correlation and strong predictive power, with area under the curve (AUC values of 0.79, 0.76, and 0.73 respectively, while the distance to secondary structure/loop junction showed significance but with less predictive power (AUC of 0.54. In a case study of 20 insertion sites in the XynB xylanase, two features of native insertion sites showed correlation with the splice sites and demonstrated predictive value in selecting non-native splice sites. Structural modeling of intein insertions at two sites highlighted the role that the insertion site location could play on the ability of the intein to modulate activity of the host protein. These findings can be used to enrich the selection of insertion sites capable of supporting intein splicing and hosting an intein switch.

  18. The intein of the Thermoplasma A-ATPase A subunit: Structure, evolution and expression in E. coli

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    Gogarten J Peter

    2001-11-01

    Full Text Available Abstract Background Inteins are selfish genetic elements that excise themselves from the host protein during post translational processing, and religate the host protein with a peptide bond. In addition to this splicing activity, most reported inteins also contain an endonuclease domain that is important in intein propagation. Results The gene encoding the Thermoplasma acidophilum A-ATPase catalytic subunit A is the only one in the entire T. acidophilum genome that has been identified to contain an intein. This intein is inserted in the same position as the inteins found in the ATPase A-subunits encoding gene in Pyrococcus abyssi, P. furiosus and P. horikoshii and is found 20 amino acids upstream of the intein in the homologous vma-1 gene in Saccharomyces cerevisiae. In contrast to the other inteins in catalytic ATPase subunits, the T. acidophilum intein does not contain an endonuclease domain. T. acidophilum has different codon usage frequencies as compared to Escherichia coli. Initially, the low abundance of rare tRNAs prevented expression of the T. acidophilum A-ATPase A subunit in E. coli. Using a strain of E. coli that expresses additional tRNAs for rare codons, the T. acidophilum A-ATPase A subunit was successfully expressed in E. coli. Conclusions Despite differences in pH and temperature between the E. coli and the T. acidophilum cytoplasms, the T. acidophilum intein retains efficient self-splicing activity when expressed in E. coli. The small intein in the Thermoplasma A-ATPase is closely related to the endonuclease containing intein in the Pyrococcus A-ATPase. Phylogenetic analyses suggest that this intein was horizontally transferred between Pyrococcus and Thermoplasma, and that the small intein has persisted in Thermoplasma apparently without homing.

  19. Fractured genes: a novel genomic arrangement involving new split inteins and a new homing endonuclease family.

    Science.gov (United States)

    Dassa, Bareket; London, Nir; Stoddard, Barry L; Schueler-Furman, Ora; Pietrokovski, Shmuel

    2009-05-01

    Inteins are genetic elements, inserted in-frame into protein-coding genes, whose products catalyze their removal from the protein precursor via a protein-splicing reaction. Intein domains can be split into two fragments and still ligate their flanks by a trans-protein-splicing reaction. A bioinformatic analysis of environmental metagenomic data revealed 26 different loci with a novel genomic arrangement. In each locus, a conserved enzyme coding region is broken in two by a split intein, with a free-standing endonuclease gene inserted in between. Eight types of DNA synthesis and repair enzymes have this 'fractured' organization. The new types of naturally split-inteins were analyzed in comparison to known split-inteins. Some loci include apparent gene control elements brought in with the endonuclease gene. A newly predicted homing endonuclease family, related to very-short patch repair (Vsr) endonucleases, was found in half of the loci. These putative homing endonucleases also appear in group-I introns, and as stand-alone inserts in the absence of surrounding intervening sequences. The new fractured genes organization appears to be present mainly in phage, shows how endonucleases can integrate into inteins, and may represent a missing link in the evolution of gene breaking in general, and in the creation of split-inteins in particular.

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

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    Raquel Cordeiro Theodoro

    2009-05-01

    Full Text Available 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 widespread inteins, occurring in important pathogens such as Cryptococcus neoformans (varieties grubii and neoformans, Cryptococcus gattii, Histoplasma capsulatum and Paracoccidioides brasiliensis. The knowledge of conserved and non-conserved domains in inteins have opened up new opportunities for the study of population variability in pathogenic fungi, including their phylogenetic relationships and recognition or diagnoses of species. Furthermore, inteins in pathogenic fungi should also be considered a promising therapeutic drug target, since once the autocatalytic splicing is inhibited, the host protein, which is typically vital, will not be able to perform its normal function and the fungal cell will not survive or reproduce.

  1. An Unprecedented Combination of Serine and Cysteine Nucleophiles in a Split Intein with an Atypical Split Site.

    Science.gov (United States)

    Bachmann, Anne-Lena; Mootz, Henning D

    2015-11-27

    Protein splicing mediated by inteins is a self-processive reaction leading to the excision of the internal intein domain from a precursor protein and the concomitant ligation of the flanking sequences, the extein-N and extein-C parts, thereby reconstituting the host protein. Most inteins employ a splicing pathway in which the upstream scissile peptide bond is consecutively rearranged into two thioester or oxoester intermediates before intein excision and rearrangement into the new peptide bond occurs. The catalytically critical amino acids involved at the two splice junctions are cysteine, serine, or threonine. Notably, the only potential combination not observed so far in any of the known or engineered inteins corresponds to the transesterification from an oxoester to a thioester, which suggested that this formal uphill reaction with regard to the thermodynamic stability might be incompatible with intein-mediated catalysis. We show that corresponding mutations also led to inactive gp41-1 and AceL-TerL inteins. We report the novel GOS-TerL split intein identified from metagenomic databases as the first intein harboring the combination of Ser1 and Cys+1 residues. Mutational analysis showed that its efficient splicing reaction indeed follows the shift from oxoester to thioester and thus represents a rare diversion from the canonical pathway. Furthermore, the GOS-TerL intein has an atypical split site close to the N terminus. The Int(N) fragment could be shortened from 37 to 28 amino acids and exchanged with the 25-amino acid Int(N) fragment from the AceL-TerL intein, indicating a high degree of promiscuity of the Int(C) fragment of the GOS-TerL intein.

  2. The effect of modified medium on the growth of Begonia imperialis and Begonia ‘Bethlehem Star’

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    DYAN MEININGSASI SISWOYO PUTRI

    2006-04-01

    Full Text Available The aim of the research was to know the effect of modified medium on the growth of Begonia imperialis and Begonia ‘Bethlehem Star’. The design of the research used was Experimental by Factorial Cluster Random Sampling, by using types of media (I=soil (control; II=soil:sand:compost; III=soil:sand:’kompenit’; IV=soil:sand:humus; V=soil:sand:manure and the material (B. imperialis and B. ‘Bethlehem Star’. Sampling time (three replicated was chosen in-groups. Observed parameters were plants tall, root, the numbers of shoots and the environmental factors (temperature and humidity. Analysist of variant showed that ‘kompenit’ is the best medium to support on the propagation of B. imperialis and B. ‘Bethlehem Star’.

  3. Circular permutation prediction reveals a viable backbone disconnection for split proteins: an approach in identifying a new functional split intein.

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    Yun-Tzai Lee

    Full Text Available Split-protein systems have emerged as a powerful tool for detecting biomolecular interactions and reporting biological reactions. However, reliable methods for identifying viable split sites are still unavailable. In this study, we demonstrated the feasibility that valid circular permutation (CP sites in proteins have the potential to act as split sites and that CP prediction can be used to search for internal permissive sites for creating new split proteins. Using a protein ligase, intein, as a model, CP predictor facilitated the creation of circular permutants in which backbone opening imposes the least detrimental effects on intein folding. We screened a series of predicted intein CPs and identified stable and native-fold CPs. When the valid CP sites were introduced as split sites, there was a reduction in folding enthalpy caused by the new backbone opening; however, the coincident loss in entropy was sufficient to be compensated, yielding a favorable free energy for self-association. Since split intein is exploited in protein semi-synthesis, we tested the related protein trans-splicing (PTS activities of the corresponding split inteins. Notably, a novel functional split intein composed of the N-terminal 36 residues combined with the remaining C-terminal fragment was identified. Its PTS activity was shown to be better than current reported two-piece intein with a short N-terminal segment. Thus, the incorporation of in silico CP prediction facilitated the design of split intein as well as circular permutants.

  4. Cloning and expression of aequorin photoprotein using intein tag

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    Elah sadat Seyed Hosseini

    2015-01-01

    Full Text Available Background: Intein (INT, is the internal parts of the protein which can be separated from the immature protein during protein splicing process. This sequence requires no specific enzyme or cofactor for separation. This protein sequence and their characteristic of self-cleavage by thiol induction, temperature and pH changes is used for protein purification. The advantage of this method compared to the other protein purification methods is that it doesn’t require any protease enzyme and protease removal steps that make this method important economically. In this study, aequorin photoprotein was hybridized with INT in molecular form and its expression was evaluated. Materials and Methods: In this study, aequorin coding gene that was cloned in pET21-a in the previous studies, was cloned in pTYB21 vector containing INT tag by specific primers and restriction enzymes. Then the resulting pTY-aequarin was transformed to the ER2566 expression strain and cloning accuracy was confirmed by electrophoresis, western blotting and sequencing. Results: The photoprotein aequorin was cloned into SapI/PstI restriction site of pTYB21 plasmid accurately and successfully. Aequorin- INT hybrid protein expression confirmed using traditional methods. Conclusion: The photoprotein aequorin constract in fused with INT confirmed by molecular methods. Also rate of Aequorin- INT expression determined about %25 of cell total protein.

  5. Conservation of intron and intein insertion sites: implications for life histories of parasitic genetic elements

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    Senejani Alireza G

    2009-12-01

    Full Text Available Abstract Background Inteins and introns are genetic elements that are removed from proteins and RNA after translation or transcription, respectively. Previous studies have suggested that these genetic elements are found in conserved parts of the host protein. To our knowledge this type of analysis has not been done for group II introns residing within a gene. Here we provide quantitative statistical support from an analyses of proteins that host inteins, group I introns, group II introns and spliceosomal introns across all three domains of life. Results To determine whether or not inteins, group I, group II, and spliceosomal introns are found preferentially in conserved regions of their respective host protein, conservation profiles were generated and intein and intron positions were mapped to the profiles. Fisher's combined probability test was used to determine the significance of the distribution of insertion sites across the conservation profile for each protein. For a subset of studied proteins, the conservation profile and insertion positions were mapped to protein structures to determine if the insertion sites correlate to regions of functional activity. All inteins and most group I introns were found to be preferentially located within conserved regions; in contrast, a bacterial intein-like protein, group II and spliceosomal introns did not show a preference for conserved sites. Conclusions These findings demonstrate that inteins and group I introns are found preferentially in conserved regions of their respective host proteins. Homing endonucleases are often located within inteins and group I introns and these may facilitate mobility to conserved regions. Insertion at these conserved positions decreases the chance of elimination, and slows deletion of the elements, since removal of the elements has to be precise as not to disrupt the function of the protein. Furthermore, functional constrains on the targeted site make it more difficult

  6. In vivo and in vitro protein ligation by naturally occurring and engineered split DnaE inteins.

    Directory of Open Access Journals (Sweden)

    A Sesilja Aranko

    Full Text Available BACKGROUND: Protein trans-splicing by naturally occurring split DnaE inteins is used for protein ligation of foreign peptide fragments. In order to widen biotechnological applications of protein trans-splicing, it is highly desirable to have split inteins with shorter C-terminal fragments, which can be chemically synthesized. PRINCIPAL FINDINGS: We report the identification of new functional split sites in DnaE inteins from Synechocystis sp. PCC6803 and from Nostoc punctiforme. One of the newly engineered split intein bearing C-terminal 15 residues showed more robust protein trans-splicing activity than naturally occurring split DnaE inteins in a foreign context. During the course of our experiments, we found that protein ligation by protein trans-splicing depended not only on the splicing junction sequences, but also on the foreign extein sequences. Furthermore, we could classify the protein trans-splicing reactions in foreign contexts with a simple kinetic model into three groups according to their kinetic parameters in the presence of various reducing agents. CONCLUSION: The shorter C-intein of the newly engineered split intein could be a useful tool for biotechnological applications including protein modification, incorporation of chemical probes, and segmental isotopic labelling. Based on kinetic analysis of the protein splicing reactions, we propose a general strategy to improve ligation yields by protein trans-splicing, which could significantly enhance the applications of protein ligation by protein trans-splicing.

  7. Blast furnace granular coal injection at Bethlehem Steel's Burns Harbor Plant

    Energy Technology Data Exchange (ETDEWEB)

    D. Gregory Hill; Leo I.E. Makovsky; Thomas A. Sarkus; Howard G. McIlvried [Bethlehem Steel Corporation, Chesterton, IN (USA)

    2004-03-01

    The paper discusses the demonstration of the British Steel/CPC-Macawber Blast Furnace Granular Coal Injection (BFGCI) technology that was installed on the blast furnaces at Bethlehem Steel's Burns Harbor Plant in Indiana as a highly successful Clean Coal Technology project, cofunded by the U.S. Department of Energy. In the BFGCI process, granular coal (10%-30% through a 200-mesh screen) is injected into a blast furnace as a fuel supplement to decrease coke requirements, thus reducing costs. Tests run to determine the effect of process variables on furnace operations showed that granular coal works as well as pulverized coal and is easier to handle and cheaper to produce because of reduced grinding costs.

  8. Production of a polar fish antimicrobial peptide in Escherichia coli using an ELP-intein tag.

    Science.gov (United States)

    Sousa, Daniel A; Mulder, Kelly C L; Nobre, Kethly S; Parachin, Nádia S; Franco, Octávio L

    2016-09-20

    An important aspect related to infectious pathogens is their exceptional adaptability in developing resistance, which leads to a perpetual challenge in the discovery of antimicrobial drugs with novel mechanisms of action. Among them, antimicrobial peptides (AMPs) stand out as promising anti-infective molecules. In order to overcome the high costs associated with isolation from natural sources or chemical synthesis of AMPs we propose the expression of Pa-MAP 2, a polyalanine AMP. Pa-MAP 2 was fused to an ELP-intein tag where the ELP (Elastin-like polypeptide) was used to promote aggregation and fast and cost-effective isolation after expression, and the intein was used to stimulate a controlled AMP release. For these, the vector pET21a was used to produce Pa-MAP 2 fused to the N-termini region of a modified Mxe GyrA intein followed by 60 repetitions of ELP. Purified Pa-MAP 2 showed a MIC of 25μM against E. coli ATCC 8739. Batch fermentation demonstrated that Pa-MAP-2 can be produced in both rich and defined media at yields 50-fold higher than reported for other AMPs produced by the ELP-intein system, and in comparable yields to expression systems with protease or chemical cleavage.

  9. 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.

  10. Intein-Fused Lucine Zippers Increase Plasma Coagulation Activity by Improving Protein Trans-Splicing in Dual-Vector Factor Ⅷ Gene Delivered Mice%亮氨酸拉链提高小鼠血浆中剪接的凝血因子Ⅷ凝血活性

    Institute of Scientific and Technical Information of China (English)

    朱甫祥; 刘泽隆; 缪静; 屈慧鸽; 迟晓艳

    2012-01-01

    培养细胞实验表明,亮氨酸拉链通过改善内含肽(intein)的蛋白质剪接效率,提高双载体转B区缺失型凝血因子Ⅷ(BDD-FⅧ)基因细胞剪接FⅧ蛋白的分泌量和活性.本文从C57BL/6小鼠门静脉注射含亮氨酸拉链和Ssp DnaB内含肽融合的BDD-FⅧ的重链和轻链基因双表达载体,48 h后,检测到血浆的重链分泌量和FⅧ活性分别为(298±67) μg/L和(1.15±0.29) U/mL,明显高于不含亮氨酸拉链的双载体转BDD-FⅧ基因对照小鼠((179±59) μg/L和(0.58±0.19) U/mL).结果表明,亮氨酸拉链通过改善蛋白质反式剪接,提高基于蛋白质剪接的双载体转BDD-FⅧ基因小鼠血浆的凝血活性,为进一步双腺相关病毒(AAV)载体转BDD-FⅧ基因的甲型血友病基因治疗研究提供了依据.%We previously demonstrated that leucine zippers fused to intein could increase secretion of spliced B-domain-deleted coagulation factor Ⅷ (BDD-FⅧ) protein and activity by dual-vector based BDD-Ⅷ gene transfected cell in vitro through improving protein trans -splicing. In this study, a pair of plasmid vectors expressing human BDD-FⅧ heavy and light chain fused with lucine zipper and split Ssp DnaB intein was co-injected into C57BL/6 mice via the portal vein. Fourty-eight hours post-injection, the level of heavy chain and FⅧ coagulation activity in collected plasma were determined and shown as (298±67)μg/L and (1.15±0.29) U/ml respectively, greater than that of control mice injected with both vectors without leucine zippers ((179±59) μg/L and (0.58±0.19) U/ml). It demonstrated that leucine zippers fused intein could increase FⅧ coagulation activity in plasma of mice with intein-based dual-vector BDD-FⅧ gene delivery through improved protein trans -splicing. It provided evidence for ongoing hemophilia A gene therapy using dual-AAV vecors.

  11. 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.

  12. Establishment of intein-mediated protein ligation under denaturing conditions: C-terminal labeling of a single-chain antibody for biochip screening.

    Science.gov (United States)

    Sydor, Jens R; Mariano, Maria; Sideris, Steve; Nock, Steffen

    2002-01-01

    Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins. This technique requires a correctly folded intein mutant that is fused to the C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine (1, 2). Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins. A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions. The intein showed activity after refolding in nondenaturing and slightly denaturing conditions. A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form. The intein-mediated ligation was established for this single chain antibody-intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step. Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding. This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format.

  13. Generation of an affinity column for antibody purification by intein-mediated protein ligation.

    Science.gov (United States)

    Sun, Luo; Ghosh, Inca; Xu, Ming-Qun

    2003-11-01

    Coupling an antigenic peptide to a solid support is a crucial step in the affinity purification of a peptide-specific antibody. Conventional methods for generating reactive agarose, cellulose or other matrices for peptide conjugation are laborious and can result in a significant amount of chemical waste. In this report, we present a novel method for the facile production of a peptide affinity column by employing intein-mediated protein ligation (IPL) in conjunction with chitin affinity chromatography. A reactive thioester was generated at the C-terminal of the chitin binding domain (CBD) from the chitinase A1 of Bacillus circulans WL-2 by thiol-induced cleavage of the peptide bond between the CBD and a modified intein. Peptide epitopes possessing an N-terminal cysteine were ligated to the chitin bound CBD tag. We demonstrate that the resulting peptide columns permit the highly specific and efficient affinity purification of antibodies from animal sera.

  14. Site-specific modification of ED-B-targeting antibody using intein-fusion technology

    Directory of Open Access Journals (Sweden)

    Greven Simone

    2011-07-01

    Full Text Available Abstract Background A promising new approach in cancer therapy is the use of tumor specific antibodies coupled to cytotoxic agents. Currently these immunoconjugates are prepared by rather unspecific coupling chemistries, resulting in heterogeneous products. As the drug load is a key parameter for the antitumor activity, site-specific strategies are desired. Expressed protein ligation (EPL and protein trans-splicing (PTS are methods for the specific C-terminal modification of a target protein. Both include the expression as an intein fusion protein, followed by the exchange of the intein for a functionalized moiety. Results A full-length IgG specific for fibronectin ED-B was expressed as fusion protein with an intein (Mxe GyrA or Npu DnaE attached to each heavy chain. In vitro protocols were established to site-specifically modify the antibodies in high yields by EPL or PTS, respectively. Although reducing conditions had to be employed during the process, the integrity or affinity of the antibody was not affected. The protocols were used to prepare immunoconjugates containing two biotin molecules per antibody, attached to the C-termini of the heavy chains. Conclusion Full-length antibodies can be efficiently and site-specifically modified at the C-termini of their heavy chains by intein-fusion technologies. The described protocols can be used to prepare immunoconjugates of high homogeneity and with a defined drug load of two. The attachment to the C-termini is expected to retain the affinity and effector functions of the antibodies.

  15. 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.

  16. High-Yield Soluble Expression and Simple Purification of the Antimicrobial Peptide OG2 Using the Intein System in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Yong-Gang Xie

    2013-01-01

    Full Text Available OG2 is a modified antimicrobial peptide, that is, derived from the frog peptide Palustrin-OG1. It has high antimicrobial activity and low cytotoxicity, and it is therefore promising as a therapeutic agent. Both prokaryotic (Escherichia coli and eukaryotic (Pichia pastoris production host systems were used to produce OG2 in our previous study; however, it was difficult to achieve high expression yields and efficient purification. In this study, we achieved high-yield OG2 expression using the intein fusion system. The optimized OG2 gene was cloned into the pTWIN1 vector to generate pTWIN-OG2-intein2 (C-terminal fusion vector and pTWIN-intein1-OG2 (N-terminal fusion vector. Nearly 70% of the expressed OG2-intein2 was soluble after the IPTG concentration and induction temperature were decreased, whereas only 42% of the expressed of intein1-OG2 was soluble. Up to 75 mg of OG2-intein2 was obtained from a 1 l culture, and 85% of the protein was cleaved by 100 mM DTT. Intein1-OG2 was less amenable to cleavage due to the inhibition of cleavage by the N-terminal amino acid of OG2. The purified OG2 exhibited strong antimicrobial activity against E. coli K88. The intein system is the best currently available system for the cost-effective production of OG2.

  17. 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.

  18. Measurements of indoor radon concentration levels in dwellings in Bethlehem, Palestine.

    Science.gov (United States)

    Leghrouz, Amin A; Abu-Samreh, Mohammad M; Shehadeh, Ayah K

    2013-02-01

    Indoor radon level measurements were carried out in 42 dwellings in Bethlehem, Palestine, using CR-39 solid state nuclear track detectors. The measurements were performed during winter and spring seasons of the year 2010, for a period ranging from 97-118 d using a total of 100 detectors. The detectors were installed in living rooms, bedrooms, kitchens, and storage areas of 39 houses, as well as in three schools, selected randomly in the surveyed area. The results of indoor radon levels and the annual effective dose in houses were found to vary from 26 - 611 Bq m(-3) and 0.65 - 14.1 m Sv y(-1), with average values of 117.0 Bq m(-3) and 2.95 m Sv y(-1), respectively. The mean values of radon concentration levels in bedrooms, kitchens, living rooms, basements, and storage areas are, respectively, 106.5, 113.1, 101.5, and 164.2 Bq m(-3). The corresponding mean values of annual effective dose for the bedrooms, kitchens, living rooms, basements, and storage areas are 2.66, 2.83, 2.54, 14.1 m Sv y(-1), respectively. In schools, the radon levels are found to vary from 31 - 400 Bq m(-3) with an average value of 125.1 Bq m(-3). The average annual effective dose in schools is found to be 3.12 mSv y(-1). This value is higher than the assigned international value. In general, the results show that radon concentration levels in 83% of the investigated dwellings are lower than the indoor radon action level of 150 Bq m(-3) for the United States.

  19. In vitro splicing of erythropoietin by the Mycobacterium tuberculosis RecA intein without substituting amino acids at the splice junctions.

    Science.gov (United States)

    Gangopadhyay, Jaya Pal; Jiang, Shu-qin; van Berkel, Patrick; Paulus, Henry

    2003-01-20

    Protein splicing is a self-catalyzed process involving the excision of an intervening polypeptide sequence, the intein, and joining of the flanking polypeptide sequences, the extein, by a peptide bond. We have studied the in vitro splicing of erythropoietin (EPO) using a truncated form of the Mycobacterium tuberculosis RecA mini-intein in which the homing endonuclease domain was replaced with a hexahistidine sequence (His-tag). The intein was inserted adjacent to cysteine residues to assure that the spliced product had the natural amino acid sequence. When expressed in Escherichia coli, intein-containing EPO was found entirely as inclusion bodies but could be refolded in soluble form in the presence of 0.5 M arginine. Protein splicing of the refolded protein could be induced with a reducing agent such as DTT or tris(2-carboxyethyl)phosphine and led to the formation of EPO and mini-intein along with some cleavage products. Protein splicing mediated by the RecA intein requires the presence of a cysteine residue adjacent to the intein insertion site. We compared the efficiencies of protein splicing adjacent to three of the four cysteine residues of EPO (Cys29, Cys33 and Cys161) and found that insertion of intein adjacent to Cys29 allowed far more efficient protein splicing than insertion adjacent to Cys33 or Cys161. For ease of purification, our experiments involved a His-tagged EPO fusion protein and a His-tagged intein and the spliced products (25 kDa EPO and 24 kDa mini-intein) were identified by Western blotting using anti-EPO and anti-His-tag antibodies and by mass spectroscopy. The optimal splicing yield at Cys29 (40%) occurred at pH 7.0 after refolding at 4 degrees C and splicing for 18 h at 25 degrees C in the presence of 1 mM DTT.

  20. 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...

  1. Facile chemical functionalization of proteins through intein-linked yeast display.

    Science.gov (United States)

    Marshall, Carrie J; Agarwal, Nitin; Kalia, Jeet; Grosskopf, Vanessa A; McGrath, Nicholas A; Abbott, Nicholas L; Raines, Ronald T; Shusta, Eric V

    2013-09-18

    Intein-mediated expressed protein ligation (EPL) permits the site-specific chemical customization of proteins. While traditional techniques have used purified, soluble proteins, we have extended these methods to release and modify intein fusion proteins expressed on the yeast surface, thereby eliminating the need for soluble protein expression and purification. To this end, we sought to simultaneously release yeast surface-displayed proteins and selectively conjugate with chemical functionalities compatible with EPL and click chemistry. Single-chain antibodies (scFv) and green fluorescent protein (GFP) were displayed on the yeast surface as fusions to the N-terminus of the Mxe GyrA intein. ScFv and GFP were released from the yeast surface with either a sulfur nucleophile (MESNA) or a nitrogen nucleophile (hydrazine) linked to an azido group. The hydrazine azide permitted the simultaneous release and azido functionalization of displayed proteins, but nonspecific reactions with other yeast proteins were detected, and cleavage efficiency was limited. In contrast, MESNA released significantly more protein from the yeast surface while also generating a unique thioester at the carboxy-terminus of the released protein. These protein thioesters were subsequently reacted with a cysteine alkyne in an EPL reaction and then employed in an azide-alkyne cycloaddition to immobilize the scFv and GFP on an azide-decorated surface with >90% site-specificity. Importantly, the immobilized proteins retained their activity. Since yeast surface display is also a protein engineering platform, these approaches provide a particularly powerful tool for the rapid assessment of engineered proteins.

  2. Protein C-terminal labeling and biotinylation using synthetic peptide and split-intein.

    Directory of Open Access Journals (Sweden)

    Gerrit Volkmann

    Full Text Available BACKGROUND: Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling. METHODOLOGY: A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein. CONCLUSIONS: We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups.

  3. The Star of Bethlehem and the Magi : Interdisciplinary Perspectives from Experts on the Ancient Near East, the Greco-Roman World, and Modern Astronomy

    NARCIS (Netherlands)

    van Kooten, George; Barthel, Peter

    2015-01-01

    This book is the fruit of the first ever interdisciplinary international scientific conference on the biblical story of the Magi and the Star of Bethlehem. The conference, held in 2014 at the University of Groningen, was attended by world-leading specialists in astronomy, the history of science, the

  4. 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

  5. Diverse organo-peptide macrocycles via a fast and catalyst-free oxime/intein-mediated dual ligation.

    Science.gov (United States)

    Satyanarayana, Maragani; Vitali, Francesca; Frost, John R; Fasan, Rudi

    2012-02-01

    Macrocyclic Organo-Peptide Hybrids (MOrPHs) can be prepared from genetically encoded polypeptides via a chemoselective and catalyst-free reaction between a trifunctional oxyamino/amino-thiol synthetic precursor and an intein-fusion protein incorporating a bioorthogonal keto group.

  6. Native-sized spider silk proteins synthesized in planta via intein-based multimerization.

    Science.gov (United States)

    Hauptmann, Valeska; Weichert, Nicola; Menzel, Matthias; Knoch, Dominic; Paege, Norman; Scheller, Jürgen; Spohn, Uwe; Conrad, Udo; Gils, Mario

    2013-04-01

    The synthesis of native-sized proteins is a pre-requisite for exploiting the potential of spider silk as a bio-based material. The unique properties of spider silk, such as extraordinary tensile strength and elasticity, result from the highly repetitive nature of spider silk protein motifs. The present report describes the combination of spider silk flagelliform protein (FLAG) production in the endoplasmic reticulum of tobacco plant leaf cells with an intein-based posttranslational protein fusion technology. The repeated ligation of FLAG monomers resulted in the formation of large multimers. This method avoids the need for highly repetitive transgenes, which may result in a higher genetic and transcriptional stability. Here we show, for the first time, the production of synthetic, high molecular weight spider silk proteins larger than 250 kDa based on the assembly of protein monomers via intein-mediated trans-splicing in planta. The resulting multimeric structures form microfibers, thereby demonstrating their great potential as a biomaterial.

  7. Use of intein-mediated phosphoprotein arrays to study substrate specificity of protein phosphatases.

    Science.gov (United States)

    Kochinyan, Samvel; Sun, Luo; Ghosh, Inca; Barshevsky, Tanya; Xu, Jie; Xu, Ming-Qun

    2007-01-01

    Synthetic peptides incorporating various chemical moieties, for example, phosphate groups, are convenient tools for investigating protein modification enzymes, such as protein phosphatases (PPs). However, short peptides are sometimes poor substrates, and their binding to commonly used matrices is unpredictable and variable. In general, protein substrates for PPs are superior for enzymatic assays, binding to various matrices, and Western blot analysis. The preparation and characterization of phosphoproteins, however can be difficult and technically demanding. In this study, the intein-mediated protein ligation (IPL) technique was used to readily generate phosphorylated protein substrates by ligating a synthetic phosphopeptide to an intein-generated carrier protein (CP) possessing a carboxyl-terminal thioester with a one-to-one stoichiometry. The ligated phosphoprotein (LPP) substrate was treated with a PP and subsequently subjected to array or Western blot analysis with a phospho-specific antibody. This approach is highly effective in producing arrays of protein substrates containing phosphorylated amino acid residues and has been applied for screening of PPs with specificity toward phosphorylated tyrosine, serine, or threonine residues, resulting in an approximately 240-fold increase in sensitivity in dot blot analysis compared with the use of synthetic peptides. The IPL technique overcomes the disadvantages of current methods and is a versatile system for the facile production of protein substrates containing well-defined structural motifs for the study of protein modification enzymes.

  8. Multiplex Detection of Protease Activity with Quantum Dot Nanosensors Prepared by Intein-Mediated Specific Bioconjugation

    Science.gov (United States)

    Xia, Zuyong; Xing, Yun; So, Min-Kyung; Koh, Ai Leen; Sinclair, Robert; Rao, Jianghong

    2009-01-01

    We report here a protease sensing nanoplatform based on semiconductor nanocrystals or quantum dots (QDs) and bioluminescence resonance energy transfer (QD-BRET) to detect the protease activity in complex biological samples. These nanosensors consist of bioluminescent proteins as the BRET donor, quantum dots as the BRET acceptor, and protease substrates sandwiched between the two as a sensing group. An intein-mediated conjugation strategy was developed for site-specific conjugation of proteins to QDs in preparing these QD nanosensors. In this traceless ligation, the intein itself is spliced out and excluded from the final conjugation product. With this method, we have synthesized a series of QD nanosensors for highly sensitive detection of an important class of protease matrix metalloproteinase (MMP) activity. We demonstrated that these nanosensors can detect the MMP activity in buffers and in mouse serum with the sensitivity to a few ng/ml, and secreted proteases by tumor cells. The suitability of these nanosensors for a multiplex protease assay has also been shown. PMID:18922019

  9. A cost-effective ELP-intein coupling system for recombinant protein purification from plant production platform.

    Directory of Open Access Journals (Sweden)

    Li Tian

    Full Text Available BACKGROUND: Plant bioreactor offers an efficient and economical system for large-scale production of recombinant proteins. However, high cost and difficulty in scaling-up of downstream purification of the target protein, particularly the common involvement of affinity chromatography and protease in the purification process, has hampered its industrial scale application, therefore a cost-effective and easily scale-up purification method is highly desirable for further development of plant bioreactor. METHODOLOGY/PRINCIPAL FINDINGS: To tackle this problem, we investigated the ELP-intein coupling system for purification of recombinant proteins expressed in transgenic plants using a plant lectin (PAL with anti-tumor bioactivity as example target protein and rice seeds as production platform. Results showed that ELP-intein-PAL (EiP fusion protein formed novel irregular ER-derived protein bodies in endosperm cells by retention of endogenous prolamins. The fusion protein was partially self-cleaved in vivo, but only self-cleaved PAL protein was detected in total seed protein sample and deposited in protein storage vacuoles (PSV. The in vivo uncleaved EiP protein was accumulated up to 2-4.2% of the total seed protein. The target PAL protein could be purified by the ELP-intein system efficiently without using complicated instruments and expensive chemicals, and the yield of pure PAL protein by the current method was up to 1.1 mg/g total seed protein. CONCLUSION/SIGNIFICANCE: This study successfully demonstrated the purification of an example recombinant protein from rice seeds by the ELP-intein system. The whole purification procedure can be easily scaled up for industrial production, providing the first evidence on applying the ELP-intein coupling system to achieve cost-effective purification of recombinant proteins expressed in plant bioreactors and its possible application in industry.

  10. Intein-mediated peptide arrays for epitope mapping and kinase/phosphatase assays.

    Science.gov (United States)

    Xu, Ming-Qun; Ghosh, Inca; Kochinyan, Samvel; Sun, Luo

    2007-01-01

    Synthetic peptides are widely used for production and analysis of antibodies as well as in the study of protein modification enzymes. To circumvent the technical challenges of the existing techniques regarding peptide quantization and normalization, a new method of producing peptide arrays has been developed. This approach utilizes intein-mediated protein ligation that involves linkage of a carrier protein possessing a reactive carboxyl-terminal thioester to a peptide with an amino-terminal cysteine through a native peptide bond. Ligated protein substrates or enzyme-treated samples are arrayed on nitrocellulose membranes with a standard dot-blot apparatus and analyzed by immunoassay. This technique has improved sensitivity and reproducibility, and is suitable for various peptide-based applications. In this report, several experimental procedures including epitope mapping and the study of protein modifications were described.

  11. 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

  12. 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

  13. Producing peptide arrays for epitope mapping by intein-mediated protein ligation.

    Science.gov (United States)

    Sun, Luo; Rush, John; Ghosh, Inca; Maunus, Jeremy R; Xu, Ming-Qun

    2004-09-01

    Peptide arrays are increasingly used to define antibody epitopes and substrate specificities of protein kinases. Their use is hampered, however, by ineffective and variable binding efficiency of peptides, which often results in low sensitivity and inconsistent results. To overcome these limitations, we have developed a novel method for making arrays of synthetic peptides on various membranes after ligating the peptide substrates to an intein-generated carrier protein. We have conducted screening for optimal carrier proteins by immunoreactivity and direct assessment of binding using a peptide derivatized at a lysine sidechain with fluorescein, CDPEK(fluorescein)DS. Ligation of a synthetic peptide antigen to a carrier protein, HhaI methylase, resulted in an improved retention of peptides and an increased sensitivity of up to 10(4)-fold in immunoassay- and epitope-scanning experiments. Denaturing the ligation products with 2% sodium dodecyl sulfate (SDS) or an organic solvent (20% methanol) prior to arraying did not significantly affect the immunoreactivity of the HhaI methylase-peptide product. Because the carrier protein dominates the binding of ligation products and contains one peptide reactive site, the amount of peptide arrayed onto the membranes can be effectively normalized. This technique was utilized in the alanine scanning of hemagglutinin (HA) antigen using two monoclonal antibodies, resulting in distinguishing the different antigen epitope profiles. Furthermore, we show that this method can be used to characterize the antibodies that recognize phosphorylated peptides. This novel approach allows for synthetic peptides to be uniformly arrayed onto membranes, compatible with a variety of applications.

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    Wood David W

    2010-10-01

    Full Text Available 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 ELP-tagged targets in a supplemented minimal medium at a 2.5 liter fermentation scale, with increased yields and purity compared to traditional shake flask cultures. This demonstration of ELP expression in supplemented minimal media is juxtaposed to previous expression of ELP tags in extract-based rich media. We also describe several sets of fed-batch conditions and their impact on ELP expression and growth medium cost. Conclusions By using fed batch E. coli fermentation at high cell density, ELP-intein-tagged proteins can be expressed and purified at high yield with low cost. Further, the impact of media components and fermentation design can significantly impact the overall process cost, particularly at large scale. This work thus demonstrates an important advances in the scale up of self-cleaving ELP tag-mediated processes.

  16. 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

  17. DnaG interacts with a linker region that joins the N- and C-domains of DnaB and induces the formation of 3-fold symmetric rings

    Science.gov (United States)

    Thirlway, Jenny; Turner, Ian J.; Gibson, Christopher T.; Gardiner, Laurence; Brady, Kevin; Allen, Stephanie; Roberts, Clive J.; Soultanas, Panos

    2004-01-01

    Loading of the replicative ring helicase onto the origin of replication (oriC) is the final outcome of a well coordinated series of events that collectively constitute a primosomal cascade. Once the ring helicase is loaded, it recruits the primase and signals the switch to the polymerization mode. The transient nature of the helicase–primase (DnaB–DnaG) interaction in the Escherichia coli system has hindered our efforts to elucidate its structure and function. Taking advantage of the stable DnaB–DnaG complex in Bacillus stearothermophilus, we have reviewed conflicting mutagenic data from other bacterial systems and shown that DnaG interacts with the flexible linker that connects the N- and C-terminal domains of DnaB. Furthermore, atomic force microscopy (AFM) imaging experiments show that binding of the primase to the helicase induces predominantly a 3-fold symmetric morphology to the hexameric ring. Overall, three DnaG molecules appear to interact with the hexameric ring helicase but a small number of complexes with two and even one DnaG molecule bound to DnaB were also detected. The structural/functional significance of these data is discussed and a speculative structural model for this complex is suggested. PMID:15173380

  18. Change in diet of the Eurasian eagle owl (Bubo bubo suggests decline in biodiversity in Wadi Al Makhrour, Bethlehem Governorate, Palestinian Territories

    Directory of Open Access Journals (Sweden)

    Amr Zuhair S.

    2016-12-01

    Full Text Available The diet of the Eurasian eagle owl (Bubo bubo was studied in Wadi Al Makhrour, Bethlehem, Palestinian Territories in 2015 with fresh and several year old pellets. Three species of arthropods, one reptile species, at least four bird species, and six species of mammals were recovered from the studied pellets. Black rat (Rattus rattus was the most common prey (37.0%, followed by the southern white-breasted hedgehog (Erinaceus concolor (29.4% and birds (21.8%. Comparison of recent and older pellets showed change in diet composition. Recent pellets contained more Rattus rattus compared to older ones. Older pellets included more naturally-occurring species such as Meriones tristrami, Microtus guentheri, and Rousettus aegyptiacus, which were absent in newer pellets.

  19. Reconstruction of enzymatic activity from split genes encoding glyphosate-tolerant EPSPS protein of Psedomonas fluorescens G2 strain by intein mediated protein complementation

    Institute of Scientific and Technical Information of China (English)

    DUN Baoqing; ZHAO Zhonglin; LIANG Aimin; HOU Songna; XU Ming-Qun; LIN Min; LU Wei; ZHANG Wei; PING Shuzhen; WANG Xujing; CHEN Ming; XU Yuquan; JIN Dan; WANG Jin

    2006-01-01

    A mutagenesis library was constructed using GPS-LS system to insert a random 5 aa into the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by aroA gene. Active EPSPS proteins were identified by the ability to rescue growth of aroA-deleted mutant ER2799 on M9 minimal media.12 unique sites, which can tolerate a 5-aa insertion,were identified. In all of the 12 sites, only F295/T296site was found to split the G2-EPSPS properly by co-transformation of plasmids into E. coli ER2799.The G2-EPSPS gene was then divided into N-terminal and C-terminal from F295/T296 site which were fused to the N-terminal and C-terminal of Ssp. DnaE intein, respectively, creating two plasmids pMEPSN295IN and pKEPSc296Ic. Co-transformation of plasmids, pMEPSN295IN and pKEPSc296Ic, rescued growth of ER2799 in M9 minimal media, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. Reconsituted activity of splitted G2-EPSPS enzyme was 4.48 U/mg.

  20. Spatially addressable chemoselective C-terminal ligation of an intein fusion protein from a complex mixture to a hydrazine-terminated surface.

    Science.gov (United States)

    Yang, Peng; Marinakos, Stella M; Chilkoti, Ashutosh

    2011-02-15

    Protein immobilization on surfaces is useful in many areas of research, including biological characterization, antibody purification, and clinical diagnostics. A critical limitation in the development of protein microarrays and heterogeneous protein-based assays is the enormous amount of work and associated costs in the purification of proteins prior to their immobilization onto a surface. Methods to address this problem would simplify the development of interfacial diagnostics that use a protein as the recognition element. Herein, we describe an approach for the facile, site-specific immobilization of proteins on a surface without any preprocessing or sample purification steps that ligates an intein fusion protein at its C-terminus by reaction with a hydrazine group presented by a surface. Furthermore, we demonstrate that this methodology can directly immobilize a protein directly from cell lysate onto a protein-resistant surface. This methodology is also compatible with soft lithography and inkjet printing so that one or more proteins can be patterned on a surface without the need for purification.

  1. 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.

  2. 重组鹅IFN-α蛋白在内含肽-冷激诱导表达系统中的表达%Expression of recombinant GoIFN-α in the intein-cold shock induced expression system

    Institute of Scientific and Technical Information of China (English)

    王艺萌; 崔子寅; 高明春; 马波; 王君伟

    2011-01-01

    To achieve high-level expression of soluble recombinant GoIFN-α in Escherichia coli, the Ssp Dnabmini-intein(SDI) coding sequence was merged at the upstream of the GoIFN-α cDNA,and then cloned into vector pET43. 1a(+). Finally, the recombinant pET-43. 1a (+)-SDI-GoIFN-α plasmid was digested with the restriction enzymes and the small fragment was ligated into the expression vector pColdⅢ predigested with the same enzymes to construct the intein-cold shock expression plasmid, named as pCold Ⅲ-SDI- GoIFN-α. The recombinant expression plasmids were transformed to E. coli Rosetta and induced by IPTG. The result showed that the fusion protein was expressed in the form of solubility and then the protein was purified through Ni+ column. The purified protein could be detected by CGBQ-GPMV,indicating that the fusion protein had the antiviral activity. Overall,the successful expression is of significance for further purification of GoIFN-α.%为了在大肠杆菌系统中高效表达具有抗病毒活性的重组鹅IFN-α(GoIFN-α)蛋白,在GoIFN-α基因上游融合内含肽Ssp Dnabmini-intein(SDI)序列,然后克隆至pET-43.1a(+)载体中,再将获得的pET-43.h(+)-SDI-GoIFN-α重组载体和pColdⅢ载体分别进行双酶切,构建内含肽-冷激表达载体pColdⅢ-NusASDI-GoIFN-α,将重组载体转化至大肠杆菌Rosetta感受态细胞,IPTG低温诱导表达.结果表明,融合基因获得高效表达,表达的融合蛋白主要以可溶形式存在.表达产物经Ni柱纯化后可得到纯度较高的目的蛋白.经CGBQ-GPMV系统检测,证实表达的融合蛋白具有生物学活性,为后续的纯化工作和研究具有天然活性的重组鹅IFN-α奠定了基础.

  3. Effect of location of the His-tag on the production of soluble and functional Buthus martensii Karsch insect toxin.

    Science.gov (United States)

    Xu, Cheng-Gang; Fan, Xiao-Jun; Fu, Yue-Jun; Liang, Ai-Hua

    2008-05-01

    The low yield and poor folding efficiency in vivo of soluble and active recombinant cysteine-rich proteins expressed in Escherichia coli are a major challenge for large-scale protein production and purification. Expression vectors containing Buthus martensii Karsch insect toxin (BmK IT) fused to the C terminus of the intein Ssp DnaB were constructed in an attempt to overcome this problem. Following purification and intein self-cleavage, the fusion protein His(6)-intein-IT produced insoluble BmK IT, while intein-IT-His(6) generated soluble and properly folded BmK IT. This result indicated that the positioning of the His(6) tag has a key role in the production of soluble and functional BmK IT.

  4. 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.

  5. 大肠杆菌BL21(DE3)中DnaE intein介导ABCA1蛋白的连接%Separate expression of ABCA1 and ligation mediated by Ssp DnaE intein in Escherichia coli BL21 (DE3)

    Institute of Scientific and Technical Information of China (English)

    朱甫祥; 缪静; 屈慧鸽; 迟晓艳

    2009-01-01

    [Objective]By exploring Ssp DnaE intein-catalyzed protein trans-splicing we investigated the ligation of expression product of ATP-binding cassette transporter Al(ABCAl) gene in E. coli.[Methods]The ABCA1 cDNA was broken into two halves of N-part and C-part before Cys~(978) codon which meets the splicing required conserved residue, and then fused to 123 and 36 amino acid-containing N terminal and C terminal coding sequences of Ssp DnaE intein naturally occurring trans-splicing intein respectively. These two fusion genes were constructed into a prokaryotic expression vector pET-28a(+). After transformation into E. coli BL21(DE3) cells followed by induction the expression of recombinant proteins and the ligation of ABCA1 were observed.[Results]Through IPTG induction for expression of recombinant protein it displayed an obvious protein band as predicted size of ABCA1 on SDS-PAGE gel. Western blotting using His-Tag specific antibody confirmed that this protein band is trans-spliced ABCA1.[Conclusion]The data demonstrated that Ssp DnaE inlein can efficiently catalyze the ligation of ABCA1 providing an evidence for our ongoing study on ABCA1 gene transfer by a dual AAV vector system to circumvent AAV volume limitation in gene therapy of Tangier disease which resulted from ABCA1 gene mutations.%[目的]利用Ssp DnaE intein的蛋白质反式剪接技术研究在大肠杆菌中对ABCAl基因表达产物的连接作用.[方法]将ABC:A1的cDNA于满足剪接所需的保守性氨基酸Cys~(978)密码子前断裂为N端和C端两部分,分别与天然存在的反式作用Ssp DnaE intein的123个氨基酸的N端和36个氨基酸的C端编码序列融合,构建到原核表达载体pET-28a(+).转化感受态大肠杆菌BL21(DE3)细胞,诱导表达后观察重组蛋白的表达和ABCA1的连接.[结果]转化菌经IPTG诱导表达,SDS-PAGE分析显示预期大小的ABCAl剪接蛋白条带,并进一步为His-Tag抗体进行的Westem blotting证实.[

  6. [Post-translational ligation of split CFTR severed before TMD2 and its chloride channel function].

    Science.gov (United States)

    Zhu, Fuxiang; Gong, Xiandi; Liu, Zelong; Yang, Shude; Qu, Huige; Chi, Xiaoyan

    2010-12-01

    Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to cystic fibrosis, an autosomal recessive genetic disorder affecting a number of organs including the lung airways, pancreas and sweat glands. In order to investigate the post-translational ligation of CFTR with reconstructed functional chloride ion channel and the split Ssp DnaB intein-mediated protein trans-splicing was explored to co-deliver CFTR gene into eukaryotic cells with two vectors. The human CFTR cDNA was split after Glu838 codon before the second transmembrane dome (TMD2) into two halves of N- and C-parts and fused with the coding sequences of split Ssp DnaB intein. Pair of eukaryotic expression vectors pEGFP-NInt and pEYFP-IntC were constructed by inserting them into the vectors pEGFP-N1 and pEYFP-N1 respectively. The transient expression was carried out for observing the ligation of CFTR by Western blotting and recording the chloride current by patch clamps when cotransfection of the pair of vectors into baby hamster kidney (BHK) cells. The results showed that an obvious protein band proven to be ligated intact CFTR can be seen and a higher chloride current and activity of chloride channel were recorded after cotransfection. These data demonstrated that split Ssp DnaB intein could be used as a strategy in delivering CFTR gene by two vectors providing evidence for application of dual adeno-associated virus (AAV) vectors to overcome the limitation of packaging size in cystic fibrosis gene therapy.

  7. 类弹性蛋白和Mxe内含肽介导的非洲猪瘟病毒重组蛋白K205R的表达与纯化%Mxe intein and elastin-like polypeptide-mediated expression and purification of African swine fever virus recombinant protein K205R

    Institute of Scientific and Technical Information of China (English)

    刘文俊; 张鑫宇; 夏晓莉; 孙怀昌

    2013-01-01

    为了构建Mxe内含肽(mxe intein,MI)和类弹性蛋白(elastin-like peptide,ELP)介导的重组蛋白表达与纯化系统,将PCR扩增的MI序列和限制性内切酶切取的ELP序列插入原核表达载体pET-30a,获得融合表达载体pET-MIE;将PCR扩增的非洲猪瘟病毒(ASFV) K205R基因插入pET-MIE载体,获得重组载体pET-K205R-MIE;分别将pET-MIE和pET-K205R-MIE转化大肠杆菌,对其重组蛋白表达、逆向相变循环沉淀及MI自体裂解条件进行优化.结果显示,在20℃条件下K205R-MIE融合蛋白为可溶性表达,26℃条件下逆向相变循环沉淀融合蛋白的回收率达73.2%,26℃孵育4h的裂解效率达91.4%,K205R蛋白的回收率为51.3%,纯度高达92.2%,能被ASFV免疫血清识别.结果提示,成功构建了简单、经济的重组蛋白表达与纯化系统,制备的K205R抗原可用于ASFV诊断.

  8. 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.

  9. Microbial polyhydroxyalkanote synthesis repression protein PhaR as an affinity tag for recombinant protein purification

    Directory of Open Access Journals (Sweden)

    Chen Guo

    2010-05-01

    Full Text Available Abstract Background PhaR which is a repressor protein for microbial polyhydroxyalkanoates (PHA biosynthesis, is able to attach to bacterial PHA granules in vivo, was developed as an affinity tag for in vitro protein purification. Fusion of PhaR-tagged self-cleavable Ssp DnaB intein to the N-terminus of a target protein allowed protein purification with a pH and temperature shift. During the process, the target protein was released to the supernatant while PhaR-tagged intein was still immobilized on the PHA nanoparticles which were then separated by centrifugation. Results Fusion protein PhaR-intein-target protein was expressed in recombinant Escherichia coli. The cell lysates after sonication and centrifugation were collected and then incubated with PHA nanoparticles to allow sufficient absorption onto the PHA nanoparticles. After several washing processes, self-cleavage of intein was triggered by pH and temperature shift. As a result, the target protein was released from the particles and purified after centrifugation. As target proteins, enhanced green fluorescent protein (EGFP, maltose binding protein (MBP and β-galactosidase (lacZ, were successfully purified using the PhaR based protein purification method. Conclusion The successful purification of EGFP, MBP and LacZ indicated the feasibility of this PhaR based in vitro purification system. Moreover, the elements used in this system can be easily obtained and prepared by users themselves, so they can set up a simple protein purification strategy by themselves according to the PhaR method, which provides another choice instead of expensive commercial protein purification systems.

  10. [Post-translational ligation and function of dual-vector transferred split CFTR gene].

    Science.gov (United States)

    Zhu, Fu-Xiang; Liu, Ze-Long; Qu, Hui-Ge; Chi, Xiao-Yan

    2010-01-01

    The mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to an autosomal recessive genetic disorder cystic fibrosis (CF). The gene therapy for CF using adeno-associated virus (AAV) vectors delivering CFTR gene is restricted by the contents limitation of AAV vectors. In this study the split CFTR genes severed at its regulatory domain were delivered by a dual-vector system with an intein-mediated protein trans-splicing as a technique to investigate the post-translational ligation of CFTR half proteins and its function as a chloride ion channel. A pair of eukaryotic expression vectors was constructed by breaking the human CFTR cDNA before Ser712 codon and fusing with Ssp DnaB intein coding sequences. After co-transfection into baby hamster kidney (BHK) cells followed by transient expression, patch clamps were carried out to record the chloride current of whole-cell and the activity of a single channel, and the ligation of two halves of CFTR was observed by Western blotting. The results showed that the intein-fused half genes co-transfected cells displayed a high whole cell chloride current and activity of a single channel indicating the functional recovery of chloride channel, and an intact CFTR protein band was figured out by CFTR-specific antibodies indicating that intein can efficiently ligate the separately expressed half CFTR proteins. The data demonstrated that protein splicing strategy could be used as a strategy in delivering CFTR gene by two vectors, encouraging our ongoing research program on dual AAV vector system based gene transfer in gene therapy for cystic fibrosis.

  11. Automated control for coal handling operations at Bethlehem Steel, Burns Harbor Division

    Energy Technology Data Exchange (ETDEWEB)

    Zendzian, T.N. [Bethlehem Steel Corp., Chesterton, IN (United States). Burns Harbor Div.

    1997-12-31

    The Burns Harbor coal handling operation processes 7,200 tons of coal per day to supply two 82 oven, six meter batteries. The operations in coal handling are subdivided into three separate sections: the coal field and stacker reclaimer operation, the crushing and storage of coal, and the coal blending operation. In 1996 a supervisory system was developed and installed to fully automate all the operations and equipment in the coal handling unit, add additional instrumentation and logic controls to prevent coal contamination, and improve data collection and logging. The supervisory system is operated from a computer based workstation and is based on a distributed control philosophy utilizing programmable logic controllers, set point controllers, and man-machine interface displays. The previous control system for the coal handling operation consisted of a switchboard from which an operator controller the set up and running of the conveyor systems and equipment to stack, reclaim, and blend coal. The new supervisory system was installed in parallel with the original control system to safeguard continued operation during the system installation and commissioning. The original system still exists and can be operated in even of failure of the supervisory system.

  12. SwissProt search result: AK110500 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110500 002-167-D06 (O33845) DNA polymerase (EC 2.7.7.7) (Pol Tfu) [Contains: Tag pol-1 intein... (Tsp-TY pol-1) (Intein I); Tag pol-2 intein (Tsp-TY pol-2) (Intein II); Tag pol-3 intein (Tsp-TY pol-3) (Intein III)] DPOL_THEAG 3e-16 ...

  13. Science at Harvard University: Historical Perspectives, edited by Clark A. Elliott and Margaret W. Rossiter. Lehigh University Press, Bethlehem, 1992

    Directory of Open Access Journals (Sweden)

    Andrew L. Christenson

    1992-05-01

    Full Text Available This volume contains historical studies of several sciences as practiced at Harvard University. Two of these studies have relevance to the history of archaeology. A chapter by Toby Appel focuses upon the scientific career of Jeffries Wyman, first curator of Harvard's Peabody Museum. She contrasts Wyman's unassuming character with the dominating personality of his mentor and contemporary Louis Agassiz. Trained as a medical doctor, Wyman's main love was zoology, particularly comparative anatomy. In his mid-40s, he encountered his first shell midden and was bitten by the archaeology bug. Soon he was doing pioneering excavation in both New England and Florida. In 1866, he was selected to be the curator of the Peabody Museum, primarily upon his strong museum background but also because of the high regard with which he was held by certain influential people. His selection to this position may have made him America's first professional archaeologist. His principal responsibilities were to collect and display archaeological and ethnological specimens and he made great steps in this direction prior to his death in 1874. Wyman's scientific work was poorly known or studied (he is best noted for having made the first scientific description of the gorilla, in part, Appel argues, because he did not seek acclaim or controversy. His greatest influence was locally through personal interactions with students and colleagues. His archaeological work is only briefly discussed in this and the following article, and there is still much to be written about this man of high character.

  14. Final environmental information volume for the coke oven gas cleaning project at the Bethlehem Steel Corporation Sparrows Point Plant

    Energy Technology Data Exchange (ETDEWEB)

    1990-04-24

    Bethelehem Steel Corporation (BSC) is planning to conduct a demonstration project involving an integrated system that can be retrofitted into coke oven gas handling systems to address a variety of environmental and operational factors in a more cost-effective manner. Successful application of this technology to existing US coke plants could: (1) reduce emissions of sulfur dioxide, cyanide, and volatile organic compounds (including benzene) (2) reduce the cost and handling of processing feed chemicals, (3) disposal costs of nuisance by-products and (4) increase reliability and reduce operation/maintenance requirements for coke oven gas desulfurization systems. The proposed system will remove sulfur from the coke oven gas in the form of hydrogen sulfide using the ammonia indigenous to the gas as the primary reactive chemical. Ammonia and hydrogen cyanide are also removed in this process. The hydrogen sulfide removed from the coke oven gas in routed to a modified Claus plant for conversion to a saleable sulfur by-product. Ammonia and hydrogen cyanide will be catalytically converted to hydrogen, nitrogen, carbon dioxide, and carbon monoxide. The tail gas from the sulfur recovery unit is recycled to the coke oven gas stream, upstream of the new gas cleaning system. The proposed demonstration project will be installed at the existing coke oven facilities at BSC's Sparrows Point Plant. This volume describes the proposed actions and the resulting environmental impacts. 21 refs., 19 figs., 9 tabs.

  15. Comparison of Phytoconstituents, Total Phenol Contents and Free Radical Scavenging Capacities between Four Arum Species from Jerusalem and Bethlehem

    Directory of Open Access Journals (Sweden)

    Nidal Jaradat, Murad Abualhasan

    2016-06-01

    Full Text Available Background: Palestine is a rich land with wild edible plants which used from the ancient times as food and medicine. Arum is a valuable genus of medicinal plants which is used in the ethnic medicine for treatment of cancer or consumed as integrated food. This work aimed to evaluate and compare the phytoconstituents, total phenols contents and free radical scavenging potential for Arum dioscoridis, Arum elongatum, Arum hygrophilum and Arum palaestinum a members of Palestinian flora. Methods: Phytoconstituents screened by using standard analytical methods, total phenols determined by using Folin Ciocalteu's method and antioxidant activities were assessed by DPPH assay. Results: The crude extracts of Arum plant studied species revealed the presence of several biologically active phytochemicals with the highest quantity of saponin, alkaloid, phenols and flavonoids. For A. dioscoridis, A. elongatum, A. hygrophilum and A. palaestinum free radical scavenging activities were 6.7±0.75µg/ml, 19.9±0.63µg/ml, 9.9±0.49µg/ml, and 6.9±0.62µg/ml respectively, while the IC50 for Trolox was 4.8±0.39µg/ml as well as the total phenols contents for these species were 60.07 ±0.12, 27.49 ±0.32, 41.75±0.12 and 53.17±0.22 (mg GAE/g extract, ±SD respectively. Conclusion: The antioxidant activities in the studied Arum plant species showed a marked correlation with their total phenols contents. A. dioscoridis and A. palaestinum had the highest antioxidant activities with high contents of total phenols and they can be used as perfect choices for manufacturing of pharmaceutical, cosmeticeuticals and nutraceutical formulations.

  16. International Conference on Defects in Semiconductors (16th) Held in Bethlehem, Pennsylvania on 22-26 July 1991

    Science.gov (United States)

    1992-04-30

    found to have a different magnitude (sometimes even changing sign ). Some RTSs show complex behaviour due to defect-defect interactions and...configurations. [1] M. Baj, P. Dreszer and A. Babinski , Phys. Rev. B 412070 (1991) [2 M. BaJ and P. Dreszer Mat. Sci. Forum vols. 84101 (1988) [3 J. Dabrowski and...defect. As in the case of the Pt’ defect, the values of B -B and Bz for each defect have the opposite sign to those values for V. The electronic

  17. 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.

  18. Inteins and affinity resin substitutes for protein purification and scale up

    Directory of Open Access Journals (Sweden)

    Wood David W

    2005-11-01

    Full Text Available Abstract The development of self-cleaving fusion-tag technology has greatly simplified the purification of recombinant proteins at laboratory scale. The self-cleaving capability of these tags has recently been combined with additional purification tags to generate novel and convenient protein purification methods at a variety of scales. In this review, we describe some of these methods, and provide a rudimentary economic analysis of hypothetical large-scale applications. This work is expected to provide a rough outline for the evaluation of these methods for large-scale bioprocessing of a variety of products.

  19. Defects in Semiconductors 16: Proceedings of the International Conference (16th) Held in Bethlehem, Pennsylvania on 22-26 July 1991. Part 3

    Science.gov (United States)

    1992-01-01

    defect to an interstitial Si e tom 35 was dismissed. Further studies in p-Si 2 revealed that as-delivered ( unpolished ) Si wafers, implanted with 350 keV...have described the depths of RIE induced defect penetration from 200A to 7gm. Transmission electron spectroscopy ( gEM ) has shown a modified silicon

  20. 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.

  1. 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

  2. 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

  3. 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

  4. Methylphenidate, Add, and Classroom Performance

    OpenAIRE

    1993-01-01

    The degree to which methylphenidate (MPH) normalized the classroom behavior and academic functioning of 31 children with attention deficit disorder (ADD) was evaluated at Lehigh University, Bethlehem, PA.

  5. 78 FR 54816 - Approval and Promulgation of Implementation Plans; Oklahoma; Revisions to Excess Emissions...

    Science.gov (United States)

    2013-09-06

    ... submittal draw upon the concepts of ``separability'' as expressed in Bethlehem Steel Corp. v. Gorsuch, 742 F... Corp. v. Gorsuch, 742 F.2d 1028, 1036-37 (7th Cir. 1984); see also 1992 Calcagni Memo. Furthermore....\\8\\ \\8\\ See Bethlehem Steel Corp. v. Gorsuch, 742 F.2d 1028, 1036-37 (7th Cir. 1984); see also...

  6. 76 FR 5576 - Combined Notice of Filings #1

    Science.gov (United States)

    2011-02-01

    ...., Calpine Mid-Atlantic Marketing, LLC, Calpine Bethlehem, LLC, Calpine Mid-Atlantic Generation, LLC, Calpine... filing per 35.13(a)(2)(iii): WMPA No. 2715, Queue No. V4-077, Sustainable Energy Holding, LLC &...

  7. Quantification of African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV-UG) in single and mixed infected Cassava (Manihot esculenta Crantz) using quantitative PCR.

    Science.gov (United States)

    Naseem, Saadia; Winter, Stephan

    2016-01-01

    The quantity of genomic DNA-A and DNA-B of African cassava mosaic virus (ACMV) and East African cassava mosaic virus Uganda (Uganda variant, EACMV-UG) was analysed using quantitative PCR to assess virus concentrations in plants from susceptible and tolerant cultivars. The concentrations of genome components in absolute and relative quantification experiments in single and mixed viral infections were determined. Virus concentration was much higher in symptomatic leaf tissues compared to non-symptomatic leaves and corresponded with the severity of disease symptoms. In general, higher titres were recorded for EACMV-UG Ca055 compared to ACMV DRC6. The quantitative assessment also showed that the distribution of both viruses in the moderately resistant cassava cv. TMS 30572 was not different from the highly susceptible cv. TME 117. Natural mixed infections with both viruses gave severe disease symptoms. Relative quantification of virus genomes in mixed infections showed higher concentrations of EACMV-UG DNA-A compared to ACMV DNA-A, but a marked reduction of EACMV-UG DNA-B. The higher concentrations of EACMV-UG DNA-B compared to EACMV DNA-A accumulation in single infections were consistent. Since DNA-B is implicated in virus cell-to-cell spread and systemic movement, the abundance of the EACMV-UG DNA-B may be an important factor driving cassava mosaic disease epidemic.

  8. 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.

  9. 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.

  10. Pre-replication assembly of E. coli replisome components.

    Science.gov (United States)

    den Blaauwen, Tanneke; Aarsman, Mirjam E G; Wheeler, Linda J; Nanninga, Nanne

    2006-11-01

    The localization of SeqA, thymidylate synthase, DnaB (helicase) and the DNA polymerase components alpha and tau, has been studied by immunofluorescence microscopy. The origin has been labelled through GFP-LacI bound near oriC. SeqA was located in the cell centre for one replication factory (RF) and at 1/4 and 3/4 positions in pre-divisional cells harbouring two RFs. The transition of central to 1/4 and 3/4 positions of SeqA appeared abrupt. Labelled thymidylate synthetase was found all over the cell, thus not supporting the notion of a dNTP-synthesizing complex exclusively localized near the RF. More DnaB, alpha and tau foci were found than expected. We have hypothesized that extra foci arise at pre-replication assembly sites, where the number of sites equals the number of origins, i.e. the number of future RFs. A reasonable agreement was found between predicted and found foci. In the case of multifork replication the number of foci appeared consistent with the assumption that three RFs are grouped into a higher-order structure. The RF is probably separate from the foci containing SeqA and the hemi-methylated SeqA binding sites because these foci did not coincide significantly with DnaB as marker of the RF. Co-labelling of DnaB and oriC revealed limited colocalization, indicating that DnaB did not yet become associated with oriC at a pre-replication assembly site. DnaB and tau co-labelled in the cell centre, though not at presumed pre-replication assembly sites. By contrast, alpha and tau co-labelled consistently suggesting that they are already associated before replication starts.

  11. Characterization of a New World Monopartite Begomovirus Causing Leaf Curl Disease of Tomato in Ecuador and Peru Reveals a New Direction in Geminivirus Evolution

    OpenAIRE

    Melgarejo, Tomas A.; Kon, Tatsuya; Rojas, Maria R.; Paz-Carrasco, Lenin; Zerbini, F. Murilo; Gilbertson, Robert L.

    2013-01-01

    All characterized whitefly-transmitted geminiviruses (begomoviruses) with origins in the New World (NW) have bipartite genomes composed of a DNA-A and DNA-B component. Recently, an NW begomovirus lacking a DNA-B component was associated with tomato leaf curl disease (ToLCD) in Peru, and it was named Tomato leaf deformation virus (ToLDeV). Here, we show that isolates of ToLDeV associated with ToLCD in Ecuador and Peru have a single, genetically diverse genomic DNA that is most closely related ...

  12. 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.

  13. 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.

  14. SPLICEFINDER - a fast and easy screening method for active protein trans-splicing positions.

    Directory of Open Access Journals (Sweden)

    Joachim Zettler

    Full Text Available Split intein enabled protein trans-splicing (PTS is a powerful method for the ligation of two protein fragments, thereby paving the way for various protein modification or protein function control applications. PTS activity is strongly influenced by the amino acids directly flanking the splice junctions. However, to date no reliable prediction can be made whether or not a split intein is active in a particular foreign extein context. Here we describe SPLICEFINDER, a PCR-based method, allowing fast and easy screening for active split intein insertions in any target protein. Furthermore we demonstrate the applicability of SPLICEFINDER for segmental isotopic labeling as well as for the generation of multi-domain and enzymatically active proteins.

  15. Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR.

    OpenAIRE

    1997-01-01

    The DNA polymerase gene from the archaeon Pyrococcus sp. strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671). Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conse...

  16. 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

  17. Educational and Nonprofit Institutions Receiving Prime Contract Awards for RDT&E, Fiscal Year 1989

    Science.gov (United States)

    1989-01-01

    Nida Oklahoma 90 UN:VERSITY OF VIRGINIA 3,473 Norman Ok Iahoma 683 Austin Texas 09 Charlotteevill Virginia 3,264 UNIVERSITY OF OREGON 9158 Eugene ...ARIZONA STATE UNIVERSITY 1,655 * Tulsa Oklahoma 77 Tempe Arizona 1,605 Eugene Oregon 96 Tucson Arizona 50 Bethlehem Penn 46 Philadelphia Penn 34 ASSOC

  18. Sociology, Writing, and Reading and the Community College Learning Community: The Skills/Content Tango Principles of Sociology and Freshman English and Critical Reading and Principles of Sociology.

    Science.gov (United States)

    Trautmann, Nancy; Boes, Chris

    This document details the planning process and briefly discusses the experiences of faculty and students in two distinct paired-course learning communities at Northampton Community College in Bethlehem (Pennsylvania). One learning community paired a critical reading course with principles of sociology, while the other paired a freshmen composition…

  19. 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…

  20. Microtubule Control of Metabolism in Prostate Cancer

    Science.gov (United States)

    2013-11-01

    reduced risk of cancer development, compared to diabetics taking other types of medication (7). Use of metformin to treat cancers is currently under...9. Kisfalvi K, Moro A, Sinnett-Smith J, Eibl G, Rozengurt E. 2013. Metformin inhibits the growth of human pancreatic cancer xenografts. Pancreas 42...Prostate Cancer PRINCIPAL INVESTIGATOR: Lynne Cassimeris CONTRACTING ORGANIZATION: Lehigh University Bethlehem, PA 18015-3008 REPORT

  1. 75 FR 51960 - Proposed Rule To Implement the 1997 8-Hour Ozone National Ambient Air Quality Standard: New...

    Science.gov (United States)

    2010-08-24

    ... FURTHER INFORMATION CONTACT: For information on 1-hour major NSR, contact: Mr. David Painter, Office of....david@epa.gov . To request a public hearing, contact Mrs. Pamela Long, Office of Air Quality Planning.... Island, NY-NJ-CT. PA Allentown-Bethlehem- Subpart 1 Allentown- Marginal. Easton, PA*....

  2. 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.

  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...

  4. 40 CFR 52.1670 - Identification of plans.

    Science.gov (United States)

    2010-07-01

    ... orders for Bethlehem Steel Corporation, referenced by this document, are not being incorporated as part... Environmental Conservation which includes three listings of permanent projects, demonstration projects and... projects relating to transportation control measures which are part of the SIP; —An improved program...

  5. Versuch, über Grenzen zu sprechen

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    Ein Briefwechsel zwischen Rana und liana Der folgende Briefwechsel ist auf Anregung einer Lehrerin entstanden. Rana aus dem palāstinensischen Bethlehem schrieb diesen Brief an eine imaginǎre israelische Adressatin. Der Brief gelangte durch Vermittlung der Lehrerin an die israelische Studentin liana, die in Jerusalem wohnt und sofort darauf antwortete.

  6. The Joseph Bellamy House: Great Awakening in Puritan New England. Teaching with Historic Places.

    Science.gov (United States)

    Pape, Barbara Bradbury

    The small rural town of Bethlehem, Connecticut, contains pristine examples of modest 18th-century houses that surround a charming village green. Opposite the village green, the Reverend Joseph Bellamy's immense white clapboard house rises from a hilltop, an imposing presence that makes the village appear diminutive. The house stands today as a…

  7. Reconstrucción virtual de la Estancia Jesuítica de Nuestra Señora de Belén (Calera de las Huérfanas

    Directory of Open Access Journals (Sweden)

    Marcelo Payssé Álvarez

    2010-04-01

    Full Text Available The present project consists of the three-dimensional recreation of the Jesuitical Farm of Bethlehem, as it was approximately in 1790, including the topography, architecture, notable buildings, territory, equipment, etc., that allow tours across animations, stereoscopic images, layouts and engravings of the epoch, by means of immersive applications.

  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.

  9. Complete genome sequence of a new bipartite begomovirus infecting fluted pumpkin (Telfairia occidentalis) plants in Cameroon.

    Science.gov (United States)

    Leke, Walter N; Khatabi, Behnam; Fondong, Vincent N; Brown, Judith K

    2016-08-01

    The complete genome sequence was determined and characterized for a previously unreported bipartite begomovirus from fluted pumpkin (Telfairia occidentalis, family Cucurbitaceae) plants displaying mosaic symptoms in Cameroon. The DNA-A and DNA-B components were ~2.7 kb and ~2.6 kb in size, and the arrangement of viral coding regions on the genomic components was like those characteristic of other known bipartite begomoviruses originating in the Old World. While the DNA-A component was more closely related to that of chayote yellow mosaic virus (ChaYMV), at 78 %, the DNA-B component was more closely related to that of soybean chlorotic blotch virus (SbCBV), at 64 %. This newly discovered bipartite Old World virus is herein named telfairia mosaic virus (TelMV).

  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. Yellow mosaic symptom caused by the nuclear shuttle protein gene of mungbean yellow mosaic virus is associated with single-stranded DNA accumulation and mesophyll spread of the virus.

    Science.gov (United States)

    Kuruba, B L; Buvani, A P; Veluthambi, K

    Mungbean yellow mosaic virus-[India:Vigna] (MYMV-[IN:Vig]), a blackgram isolate of MYMV, causes yellow mosaic disease in blackgram and mungbean. Two variable DNA-B components, KA22 and KA27, cause distinct symptoms in blackgram [V. mungo (L.) Hepper] with the same DNA-A component. KA22 + DNA-A-agroinoculated blackgram plants displayed yellow mosaic symptom and accumulated high levels of viral single-stranded (ss) DNA. KA27 + DNA-A-agroinoculated blackgram plants displayed severe stunting symptom and accumulated very low levels of viral ssDNA. However, in mungbean [V. radiata (L.) Wilczek], KA27 + DNA-A caused yellow mosaic symptom and a high level of viral ssDNA accumulated. Swapping of KA27 DNA-B with the nuclear shuttle protein gene (NSP) of KA22 DNA-B (KA27xKA22 NSP) caused yellow mosaic symptom in blackgram, suggesting that KA22 NSP is the determinant of yellow mosaic symptom. Interestingly, KA27xKA22 NSP-infected blackgram plants accumulated high levels of viral ssDNA, comparable to that of KA22 DNA-B infection, suggesting that the KA22 NSP is responsible for accumulation of high levels of viral ssDNA. MYMV distribution was studied in blackgram and mungbean plants by leaf tissue hybridization, which showed mesophyll spread of the virus in KA22-infected blackgram leaflets and in KA27-infected mungbean leaflets, both of which displayed yellow mosaic symptom. However, the virus did not accumulate in the mesophyll in the case of KA27-infected blackgram leaflets. Interestingly, the swapped KA27xKA22 NSP-infected blackgram leaflets showed mesophyll accumulation of the virus, suggesting that KA22 NSP determines its mesophyll spread.

  12. Development of Cold-Formed Steel Seismic Design Recommendations

    Science.gov (United States)

    2015-08-01

    strap strength was equal to the maxi - mum strength in accordance with the Bethlehem Steel study. For example, the measured yield strength of the A3...intermediate studs, which carried a small amount of lateral load in weak -axis bending, to screwed connections to the track. The intermediate stud...is a reasonable upper bound, having a value of on- ly 0.32 kips, which is an increase to 7.75 kips. The average of the maxi - mum positive and negative

  13. Seventh International Symposium on Nondestructive Characterization of Materials

    Science.gov (United States)

    1995-01-01

    Hall Social Function 3400 N. Charles Street Monday, June 19 Baltimore, MD 21218-2689, U.S.A. Opening Reception (410) 516-5397 Bethlehem Chapel FAX (410...Photothermal Investigation of Silicon Wafers with Diamond-like coating- 10:10 BREAK -J. Bodzenta, J. Mazur, & R. Bukowski , Silesian Technical University... Bukowski , J. Bodzenta, J. Mazur, & Z. Kleszczewski, Nonlinear Ultrasonics for Materials Silesian Technical University, Poland Characterization-M

  14. 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...

  15. Studies of Elastic Properties on Stretch Films of Polycarbonate by Brillouin Scattering.

    Science.gov (United States)

    1982-02-22

    78u472-608 TECHNICAL REPORT DISTRZBUTON LIST, 356A NO . No. Copies Copies Mr. Robert W. Jones Dr. T. J. Reinhart, Jr., Chief Advanced Projects Manager...Engineering Lehigh University University of Cincinnati Behlh Unsy Cincinnati, Ohio 45221 Bethlehem, Pennsylvania 18015 Dr. Robert E. Cohen Dr. R. F...25. D. B. Cavanaugh and C. H. Wang, J. Appl. Phys. (in press) 26. R. Vacher and L. Boyer, Phys. Rev. 13, 6, 693 (1972) 27. G. D. Enright and B. P

  16. Welding Research for Shipbuilding: SP-7 Panel Program From 1972 to 1992

    Science.gov (United States)

    1992-11-01

    and which consistently results in cracks to HY 80 steel castings. Toughness properties are equal to HY80 castings. The next step in this development...Bethlehem Steel Shipyard at Sparrows Point, Maryland. In 1980 the 01041 contract was awarded to Sun Shipbuilding in Chester, Pennsylvania. The contract...for shipbuilding applications. Some projects have contributed simultaneously to quality and producibility, as well as to the metallurgy of steel

  17. Intracellular Production of Cyclic Peptide Libraries with SICLOPPS.

    Science.gov (United States)

    Osher, Eliot L; Tavassoli, Ali

    2017-01-01

    Cyclic peptides are an important class of molecules that are increasingly viewed as an ideal scaffold for inhibition of protein-protein interactions (PPI). Here we detail an approach that enables the intracellular synthesis of cyclic peptide libraries of around 10(8) members. The method utilizes split intein mediated circular ligation of peptides and proteins (SICLOPPS), taking advantage of split intein splicing to cyclize a library of peptide sequences. SICLOPPS allows the ring size, set residues and number of random residues within a library to be predetermined by the user. SICLOPPS libraries have been combined with a variety of cell-based screens to identify cyclic peptide inhibitors of a variety of enzymes and protein-protein interactions.

  18. Gene-splitting technology: a novel approach for the containment of transgene flow in Nicotiana tabacum.

    Directory of Open Access Journals (Sweden)

    Xu-Jing Wang

    Full Text Available The potential impact of transgene escape on the environment and food safety is a major concern to the scientists and public. This work aimed to assess the effect of intein-mediated gene splitting on containment of transgene flow. Two fusion genes, EPSPSn-In and Ic-EPSPSc, were constructed and integrated into N. tabacum, using Agrobacterium tumefaciens-mediated transformation. EPSPSn-In encodes the first 295 aa of the herbicide resistance gene 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS fused with the first 123 aa of the Ssp DnaE intein (In, whereas Ic-EPSPSc encodes the 36 C-terminal aa of the Ssp DnaE intein (Ic fused to the rest of EPSPS C terminus peptide sequences. Both EPSPSn-In and Ic-EPSPSc constructs were introduced into the same N. tabacum genome by genetic crossing. Hybrids displayed resistance to the herbicide N-(phosphonomethyl-glycine (glyphosate. Western blot analysis of protein extracts from hybrid plants identified full-length EPSPS. Furthermore, all hybrid seeds germinated and grew normally on glyphosate selective medium. The 6-8 leaf hybrid plants showed tolerance of 2000 ppm glyphosate in field spraying. These results indicated that functional EPSPS protein was reassembled in vivo by intein-mediated trans-splicing in 100% of plants. In order to evaluate the effect of the gene splitting technique for containment of transgene flow, backcrossing experiments were carried out between hybrids, in which the foreign genes EPSPSn-In and Ic-EPSPSc were inserted into different chromosomes, and non-transgenic plants NC89. Among the 2812 backcrossing progeny, about 25% (664 plantlets displayed glyphosate resistance. These data indicated that transgene flow could be reduced by 75%. Overall, our findings provide a new and highly effective approach for biological containment of transgene flow.

  19. Synthesis and application of a N-1' fluorescent biotinyl derivative inducing the specific carboxy-terminal dual labeling of a novel RhoB-selective scFv.

    Science.gov (United States)

    Chaisemartin, L; Chinestra, P; Favre, G; Blonski, C; Faye, J C

    2009-05-20

    The fluorescent site-specific labeling of protein would provide a new, easy-to-use alternative to biochemical and immunochemical methods. We used an intein-mediated strategy for covalent labeling of the carboxy-terminal amino acid of a RhoB-selective scFv previously isolated from a phage display library (a human synthetic V(H) + V(L) scFv phage library). The scFv fused to the Mxe intein was produced in E. coli and purified and was then labeled with a newly synthesized fluorescent biotinyl cysteine derivative capable of inducing scFv-Mxe intein splicing. In this study, we investigated the splicing and labeling properties of various amino acids in the hinge domain between scFv and Mxe under thiol activation. In this dual labeling system, the fluorescein is used for antibody detection and biotin is used for purification, resulting in a high specific activity for fluorescence. We then checked that the purified biotinylated fluorescent scFv retained its selectivity for RhoB without modification of its affinity.

  20. Watermelon chlorotic stunt virus (WmCSV): a serious disease threatening watermelon production in Jordan.

    Science.gov (United States)

    Al-Musa, A; Anfoka, G; Al-Abdulat, A; Misbeh, S; Haj Ahmed, F; Otri, I

    2011-08-01

    The incidence of watermelon chlorotic stunt disease and the molecular characterization of the Jordanian isolate of Watermelon chlorotic stunt virus (WmCSV-[JO]) are described in this study. Symptomatic leaf samples obtained from watermelon (Citrullus lanatus Thunb.), melon (Cucumis melo L.), squash (Cucurbita pepo), cucumber (Cucumis sativus L.), and bottle gourd (Lagenaria siceraria) plants were tested for WmCSV-[JO] infection by PCR. The virus could be detected in 8 melon and 87 watermelon samples obtained from Ghor Assafi (southern part of Jordan Valley). Three samples collected from Mafraq (eastern part of Jordan) were found mixed infected with WmCSV-[JO] and Squash leaf curl virus. The full-length DNA-A and DNA-B genomes of WmCSV-[JO] were amplified, and sequences were deposited in the GenBank under accession numbers EU561237 and EU561236, respectively. Sequence analysis reveals that WmCSV-[JO] is closely related to other virus isolates from Israel (WmCSV-[IL]), Yemen (WmCSV-[YE]), Iran (WmCSV-[IR]), Lebanon (WmCSV-[LB]), and Sudan (WmCSV-[SD]). DNA-A of WmCSV-[JO] showed highest nucleotide identity (99.42%) with WmCSV-[IL], while DNA-B had highest nucleotide identity (95.52%) with WmCSV-[YE]. Data of this study demonstrate that digestion of DNA-B genome of WmCSV isolates with ApaI enzyme can discriminate between these isolates at the molecular level. Infectious clones of WmCSV-[JO] were constructed and agroinoculated to Nicotiana benthamiana plants. Inoculated plants developed mild disease symptoms 4 weeks post inoculation, while watermelon plants biolistically inoculated with WmCSV-[JO] developed characteristic mottling, yellowing and severe leaf curling symptoms 3 weeks post inoculation.

  1. Multiple genetic pathways for restarting DNA replication forks in Escherichia coli K-12.

    OpenAIRE

    Sandler, S J

    2000-01-01

    In Escherichia coli, the primosome assembly proteins, PriA, PriB, PriC, DnaT, DnaC, DnaB, and DnaG, are thought to help to restart DNA replication forks at recombinational intermediates. Redundant functions between priB and priC and synthetic lethality between priA2::kan and rep3 mutations raise the possibility that there may be multiple pathways for restarting replication forks in vivo. Herein, it is shown that priA2::kan causes synthetic lethality when placed in combination with either Delt...

  2. Mitochondrial genome of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa): A linear DNA molecule encoding a putative DNA-dependent DNA polymerase.

    Science.gov (United States)

    Shao, Zhiyong; Graf, Shannon; Chaga, Oleg Y; Lavrov, Dennis V

    2006-10-15

    The 16,937-nuceotide sequence of the linear mitochondrial DNA (mt-DNA) molecule of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa) - the first mtDNA sequence from the class Scypozoa and the first sequence of a linear mtDNA from Metazoa - has been determined. This sequence contains genes for 13 energy pathway proteins, small and large subunit rRNAs, and methionine and tryptophan tRNAs. In addition, two open reading frames of 324 and 969 base pairs in length have been found. The deduced amino-acid sequence of one of them, ORF969, displays extensive sequence similarity with the polymerase [but not the exonuclease] domain of family B DNA polymerases, and this ORF has been tentatively identified as dnab. This is the first report of dnab in animal mtDNA. The genes in A. aurita mtDNA are arranged in two clusters with opposite transcriptional polarities; transcription proceeding toward the ends of the molecule. The determined sequences at the ends of the molecule are nearly identical but inverted and lack any obvious potential secondary structures or telomere-like repeat elements. The acquisition of mitochondrial genomic data for the second class of Cnidaria allows us to reconstruct characteristic features of mitochondrial evolution in this animal phylum.

  3. Complete nucleotide sequence and host range of South African cassava mosaic virus: further evidence for recombination amongst begomoviruses.

    Science.gov (United States)

    Berrie, L C; Rybicki, E P; Rey, M E

    2001-01-01

    Complete nucleotide sequences of the DNA-A (2800 nt) and DNA-B (2760 nt) components of a novel cassava-infecting begomovirus, South African cassava mosaic virus (SACMV), were determined and compared with various New World and Old World begomoviruses. SACMV is most closely related to East African cassava mosaic virus (EACMV) in both its DNA-A (85% with EACMV-MH and -MK) and -B (90% with EACMV-UG2-Mld and EACMV-UG3-Svr) components; however, percentage sequence similarities of less than 90% in the DNA-A component allowed SACMV to be considered a distinct virus. One significant recombination event spanning the entire AC4 open reading frame was identified; however, there was no evidence of recombination in the DNA-B component. Infectivity of the cloned SACMV genome was demonstrated by successful agroinoculation of cassava and three other plant species (Phaseolus vulgaris, Malva parviflora and Nicotiana benthamiana). This is the first description of successful infection of cassava with a geminivirus using Agrobacterium tumefaciens.

  4. Sulfuric Acid Regeneration Waste Disposal Technology.

    Science.gov (United States)

    1986-11-01

    tons calcium sulfate (gypsum) per ton of titanium oxide (TiO2 ) produced. Because of the shear magnitude of the calcium sulfate disposal problem, one... pickling liquors that used as high as a 40:1 seed recycle ratio (we did not talk directly with Bethlehem Steel on their process). The Dorr Oliver...I I I 4-14 / Arthur D. Little, Inc. SECTION 5 BIBLIOGRAPHY 1. Aarons, R. and Taylor, R.A. (1967), The DuPont Waste Pickle Liquor Process, 22 Ind

  5. Workshop and Conference on Clay Microstructure: The Microstructure of Fine-Grained Terrigenous Marine Sediments - From Muds to Shale Held in Stennis, Mississippi on October 4-7, 1988

    Science.gov (United States)

    1988-10-07

    IN 47907 (371 494-5028 ANDERSON, DR. A. DEPT. OF OCNY TAMU COLLEGE STATION, TX 77843 [409] 845-7211 ASPER DR VERNON USM/CMS BLDG 1105 STENNIS SPACE... SOMERSET TAl 4XW UK KODAMA, DR KENNETH P. DEPT GEOLOGICAL SCIENCES LEHIGH UNIV BETHLEHEM, PA 18015 [215] 758-3663 KNAUER DR GEORGE A m USM/CMS BLDG...CONCENTRATION SUSPENSIONS R. Kirby Ravensrodd Consultants Ltd 6 Queens Drive l n Taunton, Somerset TAl 4XW UK Latest findings on the behaviour of fine sediment

  6. 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...

  7. 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.

  8. A novel recombinant, VPAC2-selective agonist enhancing insulin release and glucose disposal

    Institute of Scientific and Technical Information of China (English)

    Rong-jie YU; Ngai-lik TAM; Yuan GAO; Zhi-hong ZENG; Tian-hong ZHOU; An HONG

    2007-01-01

    Aim: To obtain the recombinant, VPAC2 -selective ( VPAC2: type 3 receptor of pituitary adenylate cyclase activating polypeptide which shared by vasoactive intestinal peptide) agonist with effects on glucose disposal by intein-mediated,single column purification. Methods: A gene encoding 32-amino acid peptide named rMBAY was designed and synthesized and cloned into Escherichia coli expression vector, pKYB (NEB, USA). The recombinant vector was transferred into E coli ER2566 strain and the target protein was overexpressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the fusion protein was puri-fied by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by β-mercaptoethanol and the target peptide, rMBAY, was released from the chitin-bound intein tag. Results: Approximately 53 mg rMBAY with the purity over 95% was obtained by single column purification from 1 L induced culture fermented in 5 L fermenter. The results of the competitive binding assay and cAMP accumulation assay indicated that the recombinant rMBAY had special binding selectivity and potency for VPAC2. The recombinant peptide, rMBAY,enhanced insulin release and decreased the plasma glucose level after intraperito-neal injection (50 ng/kg) with a high concentration of glucose (1.8 mmol/kg) in the NIH mice. Conclusion: An efficient production procedure of a recombinant VPAC2-selective agonist with corresponding effects on glucose disposal was established.

  9. Streamlined protein expression and purification using cleavable self-aggregating tags

    Directory of Open Access Journals (Sweden)

    Zhou Bihong

    2011-06-01

    Full Text Available Abstract Background Recombinant protein expression and purification remains a fundamental issue for biotechnology. Recently we found that two short self-assembling amphipathic peptides 18A (EWLKAFYEKVLEKLKELF and ELK16 (LELELKLKLELELKLK can induce the formation of active protein aggregates in Escherichia coli (E. coli, in which the target proteins retain high enzymatic activities. Here we further explore this finding to develop a novel, facile, matrix-free protein expression and purification approach. Results In this paper, we describe a streamlined protein expression and purification approach by using cleavable self-aggregating tags comprising of one amphipathic peptide (18A or ELK16 and an intein molecule. In such a scheme, a target protein is first expressed as active protein aggregate, separated by simple centrifugation, and then released into solution by intein-mediated cleavage. Three target proteins including lipase A, amadoriase II and β-xylosidase were used to demonstrate the feasibility of this approach. All the target proteins released after cleavage were highly active and pure (over 90% in the case of intein-ELK16 fusions. The yields were in the range of 1.6-10.4 μg/mg wet cell pellet at small laboratory scale, which is comparable with the typical yields from the classical his-tag purification, the IMPACT-CN system (New England Biolabs, Beverly, MA, and the ELP tag purification scheme. Conclusions This tested single step purification is capable of producing proteins with high quantity and purity. It can greatly reduce the cost and time, and thus provides application potentials for both industrial scale up and laboratorial usage.

  10. Identifying and Prioritizing Genes involved in Bovine Mastitis

    DEFF Research Database (Denmark)

    Jiang, Li

    In the "omics" era, identification of biological entities underlying complex traits or common diseases is characterized by the integration of high-throughput experiments and knowledge that have benn published or refined in biomedical repositories. Studies in this thesis generate, collect and inte......In the "omics" era, identification of biological entities underlying complex traits or common diseases is characterized by the integration of high-throughput experiments and knowledge that have benn published or refined in biomedical repositories. Studies in this thesis generate, collect...

  11. Cyclic peptide inhibitors of the β-sliding clamp in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Kjelstrup, Susanne; Hansen, Paula Melo Paulon; Thomsen, Line Elnif;

    2013-01-01

    Interaction between pairs of Staphylococcus aureus replication proteins was detected in an Escherichia coli based two-hybrid analysis. A reverse two-hybrid system was constructed for selection of compounds that hindered interaction between interacting protein pairs. A number of cyclic peptides......, from a library generated by the split intein-mediated circular ligation of peptides and proteins technology, were found to interfere with dimerization of the β-sliding clamp of the replisome. Two 8-mer peptides were analyzed in more detail. Both inhibited DNA replication, led to SOS induction, altered...

  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. 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.

  14. An improved method for utilization of peptide substrates for antibody characterization and enzymatic assays.

    Science.gov (United States)

    Ghosh, Inca; Sun, Luo; Evans, Thomas C; Xu, Ming-Qun

    2004-10-01

    Synthetic peptides have become an important tool in antibody production and enzyme characterization. The small size of peptides, however, has hindered their use in assays systems, such as Western blots, and as immunogens. Here, we present a facile method to improve the properties of peptides for multiple applications by ligating the peptides to intein-generated carrier proteins. The stoichiometric ligation of peptide and carrier achieved by intein-mediated protein ligation (IPL) results in the ligation product migrating as a single band on a SDS-PAGE gel. The carrier proteins, HhaI methylase (M.HhaI) and maltose-binding protein (MBP), were ligated to various peptides; the ligated carrier-peptide products gave sharp, reproducible bands when used as positive controls for antibodies raised against the same peptides during Western blot analysis. We further show that ligation of the peptide antigens to a different thioester-tagged carrier protein, paramyosin, produced immunogens for the production of antisera in rabbits or mice. Furthermore, we demonstrate the generation of a substrate for enzymatic assays by ligating a peptide containing the phosphorylation site for Abl protein tyrosine kinase to a carrier protein. This carrier-peptide protein was used as a kinase substrate that could easily be tested for phosphorylation using a phosphotyrosine antibody in Western blot analysis. These techniques do not require sophisticated equipment, reagents, or skills thereby providing a simple method for research and development.

  15. 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.

  16. 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.

  17. 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

  18. Two- and three-input TALE-based AND logic computation in embryonic stem cells.

    Science.gov (United States)

    Lienert, Florian; Torella, Joseph P; Chen, Jan-Hung; Norsworthy, Michael; Richardson, Ryan R; Silver, Pamela A

    2013-11-01

    Biological computing circuits can enhance our ability to control cellular functions and have potential applications in tissue engineering and medical treatments. Transcriptional activator-like effectors (TALEs) represent attractive components of synthetic gene regulatory circuits, as they can be designed de novo to target a given DNA sequence. We here demonstrate that TALEs can perform Boolean logic computation in mammalian cells. Using a split-intein protein-splicing strategy, we show that a functional TALE can be reconstituted from two inactive parts, thus generating two-input AND logic computation. We further demonstrate three-piece intein splicing in mammalian cells and use it to perform three-input AND computation. Using methods for random as well as targeted insertion of these relatively large genetic circuits, we show that TALE-based logic circuits are functional when integrated into the genome of mouse embryonic stem cells. Comparing construct variants in the same genomic context, we modulated the strength of the TALE-responsive promoter to improve the output of these circuits. Our work establishes split TALEs as a tool for building logic computation with the potential of controlling expression of endogenous genes or transgenes in response to a combination of cellular signals.

  19. The molecular characterisation of a Sida-infecting begomovirus from Jamaica.

    Science.gov (United States)

    Stewart, Cheryl; Kon, Tatsuya; Rojas, Maria; Graham, André; Martin, Darren; Gilbertson, Robert; Roye, Marcia

    2014-02-01

    The complete DNA sequence of both genome components of a new begomovirus (Sida golden mosaic Buckup virus-[Jamaica:St. Elizabeth:2004]; SiGMBuV-[JM:SE:04]) was determined from a field-infected Sida sp. sample from Buckup, St. Elizabeth, Jamaica. Phylogenetically, both genome components of SiGMBuV-[JM:SE:04] are most closely related to malvaceous weed-infecting Floridian and Mexican begomoviruses. Its DNA-B is a recombinant molecule, the majority of which was derived from a virus resembling Sida yellow mosaic Yucatan virus-[Mexico:Yucatan:2005] (SiYMYuV-[MX:Yuc:05]), while nucleotides 43-342 were derived from a virus resembling Sida golden mosaic virus-[United States of America:Florida] (SiGMV-[US:Flo]). Symptomatic infectivity of our cloned SiGMBuV-[JM:SE:04] components was confirmed in Nicotiana benthamiana.

  20. Three distinct begomoviruses associated with soybean in central Brazil.

    Science.gov (United States)

    Fernandes, Fernanda R; Cruz, A R R; Faria, J C; Zerbini, F M; Aragão, Francisco J L

    2009-01-01

    We report the complete nucleotide sequences of geminiviruses of the genus Begomovirus infecting soybean (Glycine max) in central Brazil. Samples obtained from soybean plants collected at Santo Antonio de Goiás, Goiás State, showing typical symptoms of viral infection, were analyzed. Infection was confirmed by PCR-based amplification of a DNA-A fragment with universal begomovirus primers. Total DNA from infected plants was then subjected to rolling-circle amplification (RCA), and 2.6-kb molecules were cloned into plasmid vectors. Sequencing of the three DNA-A and two DNA-B clones thus obtained confirmed infection by three distinct begomoviruses: bean golden mosaic virus, Sida micrantha mosaic virus and okra mottle virus, the last of which was reported recently to be a novel virus infecting okra plants in Brazil. Begomovirus infection of soybean plants has been reported sporadically in Brazil and has generally not been considered to be of economic relevance.

  1. Complete genome sequence of bean leaf crumple virus, a novel begomovirus infecting common bean in Colombia.

    Science.gov (United States)

    Carvajal-Yepes, Monica; Zambrano, Leidy; Bueno, Juan M; Raatz, Bodo; Cuellar, Wilmer J

    2017-02-10

    A copy of the complete genome of a novel bipartite begomovirus infecting common bean (Phaseolus vulgaris L.) in Colombia was obtained by rolling-circle amplification (RCA), cloned, and sequenced. The virus is associated with leaf crumple symptoms and significant yield losses in Andean and Mesoamerican beans. Such symptoms have been reported increasingly in Colombia since at least 2002, and we detected the virus in leaf material collected since 2008. Sequence analysis showed that the virus is a member of a distinct species, sharing 81% and 76% nucleotide (nt) sequence identity (in DNA-A and DNA-B, respectively) to other begomoviruses infecting common bean in the Americas. The data obtained support the taxonomic status of this virus (putatively named 'bean leaf crumple virus', BLCrV) as a member of a novel species in the genus Begomovirus.

  2. 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.

  3. PriC-mediated DNA replication restart requires PriC complex formation with the single-stranded DNA-binding protein.

    Science.gov (United States)

    Wessel, Sarah R; Marceau, Aimee H; Massoni, Shawn C; Zhou, Ruobo; Ha, Taekjip; Sandler, Steven J; Keck, James L

    2013-06-14

    Frequent collisions between cellular DNA replication complexes (replisomes) and obstacles such as damaged DNA or frozen protein complexes make DNA replication fork progression surprisingly sporadic. These collisions can lead to the ejection of replisomes prior to completion of replication, which, if left unrepaired, results in bacterial cell death. As such, bacteria have evolved DNA replication restart mechanisms that function to reload replisomes onto abandoned DNA replication forks. Here, we define a direct interaction between PriC, a key Escherichia coli DNA replication restart protein, and the single-stranded DNA-binding protein (SSB), a protein that is ubiquitously associated with DNA replication forks. PriC/SSB complex formation requires evolutionarily conserved residues from both proteins, including a pair of Arg residues from PriC and the C terminus of SSB. In vitro, disruption of the PriC/SSB interface by sequence changes in either protein blocks the first step of DNA replication restart, reloading of the replicative DnaB helicase onto an abandoned replication fork. Consistent with the critical role of PriC/SSB complex formation in DNA replication restart, PriC variants that cannot bind SSB are non-functional in vivo. Single-molecule experiments demonstrate that PriC binding to SSB alters SSB/DNA complexes, exposing single-stranded DNA and creating a platform for other proteins to bind. These data lead to a model in which PriC interaction with SSB remodels SSB/DNA structures at abandoned DNA replication forks to create a DNA structure that is competent for DnaB loading.

  4. Tomato yellow spot virus, a tomato-infecting begomovirus from Brazil with a closer relationship to viruses from Sida sp., forms pseudorecombinants with begomoviruses from tomato but not from Sida.

    Science.gov (United States)

    Andrade, E C; Manhani, G G; Alfenas, P F; Calegario, R F; Fontes, E P B; Zerbini, F M

    2006-12-01

    Geminiviruses are characterized by a circular, single-stranded DNA genome and twinned icosahedral particles. Begomoviruses (whitefly-transmitted geminiviruses) are a major constraint to crop production worldwide. In Brazil, tomato-infecting begomoviruses emerged as serious pathogens over the last 10 years, due to the introduction of a new biotype of the insect vector. Tomato yellow spot virus (ToYSV) is a newly described begomovirus originally isolated from tomato, but phylogenetically closer to viruses from Sida sp. A study was performed to determine the viability of pseudorecombinants formed between the DNA components of ToYSV and other weed- and tomato-infecting begomoviruses from Brazil. Despite its closer relationship to weed-infecting viruses, ToYSV was only capable of forming viable pseudorecombinants with tomato viruses. An infectious pseudorecombinant formed between ToYSV DNA-A and tomato crinkle leaf yellows virus (TCrLYV) DNA-B induced severe symptoms in Nicotiana benthamiana. This was attributed, at least in part, to the fact that the origins of replication of both components had identical Rep-binding sequences. However, this was not the case for another infectious pseudorecombinant formed between tomato golden mosaic virus (TGMV) DNA-A and ToYSV DNA-B, which have different Rep-binding sequences. These results reinforce the notion that pseudorecombinant formation cannot be explained solely on the basis of phylogenetic relationships and conserved iteron sequences, and suggest that the TGMV Rep protein may be more versatile in terms of recognizing heterologous DNA components than that of ToYSV.

  5. Complete genome sequencing of two causative viruses of cassava mosaic disease in Ghana.

    Science.gov (United States)

    Oteng-Frimpong, R; Levy, Y; Torkpo, S K; Danquah, E Y; Offei, S K; Gafni, Y

    2012-01-01

    Cassava mosaic disease (CMV), caused by one or a combination of cassava mosaic geminiviruses, is ranked among the most important constraints to profitable and efficient production of cassava. Effective control measures require in-depth knowledge of the viral causative agent. Using rolling-circle amplification and unique enzymes, the full genome of two species of cassava mosaic geminivirus isolated from infected cassava plants in Ghana were cloned into pCambia 1300 and pET-28b. The sequences of the genome were determined on an ABI sequencer and a pairwise comparison was performed with other cassava-infecting geminiviruses from different countries. It was revealed that cassava grown in Ghana is attacked by two species of geminivirus in either single or mixed infections. These are the African cassava mosaic virus (ACMV) and the East African cassava mosaic virus (EACMV)-like, with high sequence similarity of 94% and 80%, respectively, between the DNA-A and DNA-B components of each virus, and 66% and 41% similarity of the common region (CR) (for A and B accordingly). The DNA-A of ACMV and EACMV-like contained 2781 and 2800 nucleotides, respectively, while their DNA-B components had 2725 and 2734 nucleotides, respectively. ACMV DNA-A was over 97% similar to those of other ACMVs from the continent. In contrast, EACMV-like DNA-A was over 98% similar to the isolates from Cameroon and other West African countries, and less than 88% similar to other EACMV species. Thus ACMV and EACMV-like were named African cassava mosaic virus-Ghana and East African cassava mosaic Cameroon virus-Ghana. Computer analysis revealed that their genome arrangement follows the typical old world bipartite begomovirus genome. The association of these two species and their interaction might account for the severe symptoms observed on infected plants in the field and in the greenhouse.

  6. Squash leaf curl virus (SLCV): a serious disease threatening cucurbits production in Palestine.

    Science.gov (United States)

    Ali-Shtayeh, M S; Jamous, R M; Hussein, E Y; Mallah, O B; Abu-Zeitoun, S Y

    2014-04-01

    The incidence of squash leaf curl disease and molecular characterization of the Palestinian isolate of Squash leaf curl virus [SLCV-(PAL)] are described in this study. Symptomatic leaf samples obtained from squash (Cucurbita pepo), watermelon [Citrullus lanatus (Thunb.)], and cucumber (Cucumis sativus L.) plants were tested for SLCV-[PAL] infection by PCR and RCA. SLCV was also found to occur naturally in Chenopodium murale, Convolvulus sp, and Prosporis farcta which showed yellowing. The disease incidence was 85 % in samples collected from Nablus in summer season, while it was 98 % in samples collected from Qalqilia in autumn. On the other hand, SLCV incidence did not exceed 25 % in winter season. The full-length DNA-A and DNA-B genomes of SLCV-[PAL] were amplified and sequenced, and the sequences were deposited in the GenBank. Sequence analysis reveals that SLCV-[PAL] is closely related to other isolates from Lebanon (SLCV-LB2), Jordan (SLCV-JO), Israel (SLCV-IL), and Egypt (SLCV-EG). DNA-A of SLCV-[PAL] showed the highest nucleotide identity (99.4 %) with SLCV-JO, and SLCV-LB2, while DNA-B had the highest nucleotide identity (99.3 %) with SLCV-IL. However, following genome sequencing, it was found that due to two separate point mutations, two viral open reading frames (ORF) were altered in some SLCV Palestinian isolates. The AC2 ORF was extended by 141 nucleotides, while the AC4 ORF was extended by 36 nucleotides.

  7. 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.

  8. Le massacre de la mariée

    Directory of Open Access Journals (Sweden)

    Bastiaan van der Velden

    2016-06-01

    Full Text Available Le Massacre des innocents a été pour Marcel Duchamp l’une de ses sources en composant Le Grand Verre. Ce ‘massacre’ est dans ce cas plus précisément une attraction foraine. D’autre part l’infanticide à Bethlehem constitue un sujet maintes fois représenté par différents peintres. Pour Alfred Jarry, auteur littéraire et de théâtre qui a plus particulièrement inspiré Duchamp, le tableau Le Massacre des innocents de Breughel l’Ancien était une pièce clé qui lui permettait de démontrer pourquoi il rejetait l’art historisant en peinture et dans le théâtre.

  9. 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...

  10. A woman praised by women is better than a woman praised by seven men

    Directory of Open Access Journals (Sweden)

    James A. Loader

    2004-11-01

    Full Text Available The title, a parody on Ruth 4:15bb and Proverbs 31:28, counterposes the motif of praise in the final scene of what is probably the opus classicum for the foregrounding of women in the Old Testament with the same motif in a text notorious for praising women into subservience. After a short presentation of the text of Ruth 4:13-17, its main ideas and compositional relationships with the rest of the Book, the focus falls on the praise of the women of Bethlehem, its presuppositions, logic, use of terms and the role of its speakers in the story. It is concluded that a non-feminist, intentional reading highlights the critical perspective of women in the narrative, which means that the gist of mainstream feminist readings of the Ruth story is corroborated even from a perspective independent of feminist hermeneutic.

  11. Iatrogenic migration of VenaTech LP IVC filter to superior vena cava secondary to guidewire entrapment: case report and review of literature.

    Science.gov (United States)

    Almestady, Rajaa; Spain, James; Bayona-Molano, Maria Del Pilar; Wang, Weiping

    2013-01-01

    Modern inferior vena cava (IVC) filters are generally safe devices for preventing pulmonary embolus, with fewer complications compared to earlier techniques of caval interruption. Despite continuing improvement in filter designs and insertion methods, complications still occur. The IVC filter complications resulting from iatrogenic causes are rare and include but are not limited to misplacement, filter tilting, incomplete deployment, and filter migration. We recently experienced a problem in which the Vena Tech LP filter (B. Braun, Bethlehem, Pennsylvania) migrated to the superior vena cava (SVC) immediately after successful deployment of the filter in the infrarenal venacava. The root cause analysis of this case revealed that the complication was related to blind pullout of the J-tipped guidewire following deployment of the filter in the IVC. This report highlights the potential risks of using a wire while an IVC filter is in place.

  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. 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...

  14. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kobayashi, Hiroshi [Center for Advanced Biotechnology and Medicine, Department of Biochemistry, Robert Wood Johnson Medical School (United States); Swapna, G. V. T. [State University of New Jersey, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers (United States); Wu, Kuen-Phon; Afinogenova, Yuliya [Center for Advanced Biotechnology and Medicine, Department of Biochemistry, Robert Wood Johnson Medical School (United States); Conover, Kenith; Mao, Binchen [State University of New Jersey, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers (United States); Montelione, Gaetano T.; Inouye, Masayori, E-mail: inouye@cabm.rutgers.edu [Center for Advanced Biotechnology and Medicine, Department of Biochemistry, Robert Wood Johnson Medical School (United States)

    2012-04-15

    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{sub 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{sub 2}-tag is replaced with non-isotope labeled PrS{sub 2}-tag, silencing the NMR signals from PrS{sub 2}-tag in isotope-filtered {sup 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 (RbfA{Delta}25). Using the PrS{sub 2}-tag, full-length well-behaved RbfA samples could be successfully prepared for NMR studies. PrS{sub 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{sub 2}-tagged RbfA superimposes with the TROSY-HSQC spectrum of RbfA{Delta}25, indicating that PrS{sub 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 {sup 1}H, {sup 15}N and {sup 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 {sup 1}H-{sup 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 RbfA{Delta}25 construct. CS-Rosetta calculations also demonstrate that the core structure of full-length RbfA is similar to that of the RbfA{Delta}25

  16. The genome of Hyperthermus butylicus: a sulfur-reducing, peptide fermenting, neutrophilic Crenarchaeote growing up to 108 degrees C

    DEFF Research Database (Denmark)

    Brügger, Kim; Chen, Lanming; Stark, Markus;

    2007-01-01

    Hyperthermus butylicus, a hyperthermophilic neutrophile and anaerobe, is a member of the archaeal kingdom Crenarchaeota. Its genome consists of a single circular chromosome of 1,667,163 bp with a 53.7% G+C content. A total of 1672 genes were annotated, of which 1602 are protein-coding, and up to ...... elements, no inteins, and introns are exclusive to tRNA genes. This suggests that the genome structure is quite stable, possibly reflecting a constant, and relatively uncompetitive, natural environment....... clusters of regularly interspaced repeats (CRISPRs) are present, one of which is associated with a crenarchaeal-type cas gene superoperon; none of the spacer sequences yielded good sequence matches with known archaeal chromosomal elements. The genome carries no detectable transposable or integrated...

  17. Split-gene system for hybrid wheat seed production.

    Science.gov (United States)

    Kempe, Katja; Rubtsova, Myroslava; Gils, Mario

    2014-06-24

    Hybrid wheat plants are superior in yield and growth characteristics compared with their homozygous parents. The commercial production of wheat hybrids is difficult because of the inbreeding nature of wheat and the lack of a practical fertility control that enforces outcrossing. We describe a hybrid wheat system that relies on the expression of a phytotoxic barnase and provides for male sterility. The barnase coding information is divided and distributed at two loci that are located on allelic positions of the host chromosome and are therefore "linked in repulsion." Functional complementation of the loci is achieved through coexpression of the barnase fragments and intein-mediated ligation of the barnase protein fragments. This system allows for growth and maintenance of male-sterile female crossing partners, whereas the hybrids are fertile. The technology does not require fertility restorers and is based solely on the genetic modification of the female crossing partner.

  18. 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.

  19. 通过染色体整合表达古菌乙酰化酶合成N-末端乙酰化胸腺肽β4%Biosynthesis of Nα-acetylated Thymosin β4 by Co-expressing an Archaeal Acetylase Integrated in Chromosome of Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    曹赛; 谢达平; 周长林; 戴红梅; 孙旭; 颛孙丹丹; 和金周; 司信喜; 李树龙; 方宏清; 陈惠鹏

    2011-01-01

    Thymosin β4 (T(34), a 43-amino acid Na-acetylated peptide, has multiple significant biological functions. There are two bottlenecks to its biosynthesis: the difficulties in obtaining Na-acetylation and expressing small peptides. In this study we found that ssArdl, an archaeal acetylase from Sulfolobus solfataricus can acetylate the N-terminal residue Ser of T|34. A modified E. Coli BL21(DE3) with ssArdl-expressing cassette integrated in the locus of lpxM of chromosome by Red recombination was constructed and named as E. Coli BDA for Na-acetylation of proteins expressed in it. To obtain Na-acetylated T|34 efficiently, a fusion protein, Tβ4-Intein, was constructed, in which T(34 and a His tag were fused respectively to the N-terminus and the C-terminus of a smallest mini-intein, 136-amino acid Spl DnaX. After expression in E. Coli BDA and purification by Ni-Sepharose affinity chromatography, the fusion protein was induced by P-mercaptoethanol to release Na-acetylated Tβ4 through intein-mediated N-terminal cleavage. Three mutants of T|34 were prepared in the same way. All of Tβ4 and its three mutants have the ability to bind actin. This study laid a foundation for further investigation of the function and application of Tβ4.%胸腺肽β4(Tβ4)是N-末端乙酰化的43肽,具有多种重要生物学功能.其生物合成存在两大难点,即乙酰化修饰和小分子肽的表达.本研究发现来自古菌Sulfolobus solfataricus 的乙酰化酶ssArd1可以催化Tβ4的N-末端乙酰化修饰.利用Red同源重组技术将ssArd1 基因表达盒整合至E coli BL21(DE3)染色体的lpxM位点上,构建了可以实现Tβ4 N-末端乙酰化修饰的新型宿主E coli BDA.将Tβ4编码基因融合在改造的微型Spl DnaX Intein的N 端,并在Intein的C端添加His标签,构建了表达载体pET-Tβ4-Intein.在E.coli BDA中表达的融合蛋白,经镍亲和层析纯化后用β-巯基乙醇诱导融合蛋白切割释放小分子多肽,获得了

  20. Substrate Recognition of Histone H2B by DUBm

    Science.gov (United States)

    Henderson, Elizabeth; Berndsen, Christopher; Wolberger, Cynthia

    2011-03-01

    The SAGA complex is a transcriptional coactivator that regulates gene expression in eukaryotes via histone acetylation and deubiquitination, which are crucial for transcription. Our lab is investigating the SAGA-dependent deubiquitination of histone H2B. The deubiquitinating module (DUBm) of SAGA is comprised of a ubiquitin-specific protease, Ubp8, and three other proteins. It is known that Ubp8 cleaves ubiquitin from histone H2B, however, the specific way in which the enzyme binds to the substrate remains elusive. In order to unravel this mechanism, we attempted to determine the crystal structure of the substrate binding complex. We obtained this substrate by exploiting the techniques of intein chemistry to artificially ubiquitinate a histone H2B peptide, which we then co-crystallized with DUBm. Additionally, we synthesized Ub-K63R-linked chains and Ub-K48-linked chains and co-crystallized them with DUBm.

  1. Expression of an insect excitatory toxin, BmK IT, from the scorpion, Buthus martensii Karsch, and its biological activity.

    Science.gov (United States)

    Hao, Chan-juan; Xu, Cheng-gang; Wang, Wei; Chai, Bao-feng; Liang, Ai-hua

    2005-12-01

    An insect excitatory toxin from Buthus martensii Karsch (BmK IT) was cloned into the expression vector, pTWIN1, and expressed into Escherichia coli BL21 (DE3) host cells. The soluble fusion expression of CBD-intein-BmK IT was obtained. The recombinant BmK IT was purified by two anion-exchange chromatography columns and one gel chromatography column. Bioassays were carried out to verify the toxicity of this recombinant toxin. At the end of a 96 h experimental period, 83% of cotton bollworm larvae were killed with an LT(50) value of 58-62 h. Furthermore, the average weight of larvae fed on BmK IT-containing media was approx 4% of that of the control groups. The results indicate that the expressed and purified recombinant BmK IT has biological activity.

  2. Access to site-specific Fc-cRGD peptide conjugates through streamlined expressed protein ligation.

    Science.gov (United States)

    Frutos, S; Jordan, J B; Bio, M M; Muir, T W; Thiel, O R; Vila-Perelló, M

    2016-10-12

    An ideal drug should be highly effective, non-toxic and be delivered by a convenient and painless single dose. We are still far from such optimal treatment but peptides, with their high target selectivity and low toxicity profiles, provide a very attractive platform from which to strive towards it. One of the major limitations of peptide drugs is their high clearance rates, which limit dosage regimen options. Conjugation to antibody Fc domains is a viable strategy to improve peptide stability by increasing their hydrodynamic radius and hijacking the Fc recycling pathway. We report the use of a split-intein based semi-synthetic approach to site-specifically conjugate a synthetic integrin binding peptide to an Fc domain. The strategy described here allows conjugating synthetic peptides to Fc domains, which is not possible via genetic methods, fully maintaining the ability of both the Fc domain and the bioactive peptide to interact with their binding partners.

  3. Recombinant Expression and Phenotypic Screening of a Bioactive Cyclotide Against α-Synuclein-Induced Cytotoxicity in Baker's Yeast.

    Science.gov (United States)

    Jagadish, Krishnappa; Gould, Andrew; Borra, Radhika; Majumder, Subhabrata; Mushtaq, Zahid; Shekhtman, Alexander; Camarero, Julio A

    2015-07-13

    We report for the first time the recombinant expression of fully folded bioactive cyclotides inside live yeast cells by using intracellular protein trans-splicing in combination with a highly efficient split-intein. This approach was successfully used to produce the naturally occurring cyclotide MCoTI-I and the engineered bioactive cyclotide MCoCP4. Cyclotide MCoCP4 was shown to reduce the toxicity of human α-synuclein in live yeast cells. Cyclotide MCoCP4 was selected by phenotypic screening from cells transformed with a mixture of plasmids encoding MCoCP4 and inactive cyclotide MCoTI-I in a ratio of 1:5×10(4). This demonstrates the potential for using yeast to perform phenotypic screening of genetically encoded cyclotide-based libraries in eukaryotic cells.

  4. 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.

  5. The fingerprint of antimitochondrial antibodies and the etiology of primary biliary cholangitis.

    Science.gov (United States)

    Shuai, Zongwen; Wang, Jinjun; Badamagunta, Madhu; Choi, Jinjung; Yang, Guoxiang; Zhang, Weici; Kenny, Thomas P; Guggenheim, Kathryn; Kurth, Mark J; Ansari, Aftab A; Voss, John; Coppel, Ross L; Invernizzi, Pietro; Leung, Patrick S C; Gershwin, M Eric

    2017-01-18

    The identification of environmental factors that lead to loss of tolerance has been coined the holy grail of autoimmunity. Our work has focused on the reactivity of antimitochondrial autoantibodies (AMA) to chemical xenobiotics and has hypothesized that a modified peptide within PDC-E2, the major mitochondrial autoantigen, will have been immunologically recognized at the time of loss of tolerance. Herein, we successfully applied intein technology to construct a PDC-E2 protein fragment containing amino acid residues 177-314 of PDC-E2 by joining a recombinant peptide spanning residues 177-252 (PDC-228) with a 62-residue synthetic peptide from 253 to 314 (PP), which encompasses PDC-E2 inner lipoyl domain (ILD). We named this intein-constructed fragment PPL. Importantly, PPL, as well as lipoic acid conjugated PPL (LA-PPL) and xenobiotic 2-octynoic acid conjugated PPL (2OA-PPL), are recognized by AMA. Of great importance, AMA has specificity for the 2OA-modified PDC-E2 ILD peptide backbone distinct from antibodies that react with native lipoylated PDC-E2 peptide. Interestingly, this unique AMA subfraction is of the immunoglobulin M isotype and more dominant in early-stage primary biliary cholangitis (PBC), suggesting that exposure to 2OA-PPL-like compounds occurs early in the generation of AMA. To understand the structural basis of this differential recognition, we analyzed PPL, LA-PPL, and 2OA-PPL using electron paramagnetic resonance spectroscopy, with confirmations by enzyme-linked immunosorbent assay, immunoblotting, and affinity antibody analysis. We demonstrate that the conformation of PDC-E2 ILD is altered when conjugated with 2OA, compared to conjugation with lipoic acid.

  6. Design, preparation and use of ligated phosphoproteins: a novel approach to study protein phosphatases by dot blot array, ELISA and Western blot assays.

    Science.gov (United States)

    Sun, Luo; Ghosh, Inca; Barshevsky, Tanya; Kochinyan, Samvel; Xu, Ming-Qun

    2007-07-01

    The study of substrate specificity of protein phosphatases (PPs) is very challenging since it is difficult to prepare a suitable phosphorylated substrate. Phosphoproteins, phosphorylated by a protein kinase, or chemically synthesized phosphopeptides are commonly used substrates for PPs. Both types of these substrates have their advantages and limitations. Phosphoproteins mimic more closely the physiologically relevant PP substrates, but their preparation is technically demanding. Synthetic phosphopeptides present advantages over proteins because they can be easily produced in large quantity and their amino acid sequence can be designed to contain potential determinants of substrate specificity. However, short peptides are less optimal compared to in vivo PP substrates and often display poor and variable binding to different matrices, resulting in low sensitivity in analysis of PP activity on solid support. In this work we utilize the intein-mediated protein ligation (IPL) technique to generate substrates for PPs, combining the advantages of proteins and synthetic peptides in one molecule. The ligation of a synthetic phosphopeptide to an intein-generated carrier protein (CP) with a one-to-one stoichiometry results in the formation of a ligated phosphoprotein (LPP). Three widely used assays, dot blot array, Western blot and ELISA were employed to study the PP activity on LPP substrates. Dephosphorylation was measured by detection of the remaining phosphorylation, or lack of it, with a phospho-specific antibody. The data show the advantage of LPPs over free peptides in assays on solid supports. LPPs exhibited enhanced binding to the matrices used in the study, which significantly improved sensitivity and consistency of the assays. In addition, saturation of the signal was circumvented by serial dilution of the assay samples. This report describes detailed experimental procedures for preparation of LPP substrates and their use in PP assays based on immobilization on

  7. Semisynthesis and characterization of the first analogues of pro-neuropeptide y.

    Science.gov (United States)

    von Eggelkraut-Gottanka, Regula; Machova, Zuzana; Grouzmann, Eric; Beck-Sickinger, Annette G

    2003-05-09

    Enzymatic cleavage of prohormone neuropeptide Y (proNPY) leads to mature neuropeptide Y (NPY), a widely distributed neuropeptide with multiple functions both peripherally and centrally. A single dibasic pair of amino acids, Lys38-Arg39, represents the recognition motif for a class of hormone-processing enzymes known as prohormone convertases (PCs). Two members of this PC family, PC1/3 and PC2, are involved in proNPY cleavage. The aim of this work was to establish an effective method for the generation of full-length 69-amino acid proNPY analogues for further studies of prohormone convertase interaction. We have chosen two ligation sites in order to perform the semisynthesis of proNPY analogues by expressed protein ligation (EPL). By using the intein-mediated purification system (IMPACT) with improved conditions for intein splicing, we were able to isolate proNPY 1-40 and proNPY 1-54 fragments as Cterminal thioesters. Peptides bearing Nterminal cysteine instead of the naturally occurring Ser41 and Thr55 residues, respectively, were generated by solid-phase peptide synthesis. Moreover, labels (carboxyfluorescein and biotin) were inserted into the peptide sequences. The synthesis of the [C41]proNPY 41-69 fragment, which proved to be a difficult peptide sequence, could be achieved by the incorporation of two pseudo-proline derivatives. Western blot analysis revealed that all five proNPY analogues are recognized by monoclonal antibodies directed against NPY as well as against the Cflanking peptide of NPY (CPON).

  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.

    Science.gov (United States)

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

    2008-02-01

    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 approximately 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 Mexico. Biolistic inoculation of cantaloupe seedlings with the MCLCuV DNA-A and -B components resulted in the development of characteristic disease symptoms, providing definitive evidence of causality. MCLCuV experimentally infected species within the Cucurbitaceae, Fabaceae, and Solanaceae. The potential for interspecific reassortment was examined for MCLCuV and its closest relatives, including the bean-restricted Bean calico mosaic virus (BCaMV), and three other cucurbit-infecting species, Cucurbit leaf crumple virus (CuLCrV), SLCV, and SMLCV. The cucurbit viruses have distinct but overlapping host ranges. All possible reassortants were established using heterologous combinations of the DNA-A or DNA-B components. Surprisingly, only certain reassortants arising from MCLCuV and BCaMV, or MCLCuV and CuLCrV, were viable in bean, even though it is a host of all of the "wild-type" (parent) viruses. The bean-restricted BCaMV was differentially assisted in systemically infecting the cucurbit test species by the components of the four cucurbit-adapted begomoviruses. In certain heterologous combinations, the BCaMV DNA-A or -B component was able to infect one or more cucurbit species. Generally, the reassortants were less virulent in the test hosts than the respective wild-type (parent) viruses, strongly implicating adaptive modulation of virulence. This is the first illustration of reassortment resulting in the host range expansion of a host-restricted begomovirus.

  9. 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.

  10. Near-atomic structural model for bacterial DNA replication initiation complex and its functional insights.

    Science.gov (United States)

    Shimizu, Masahiro; Noguchi, Yasunori; Sakiyama, Yukari; Kawakami, Hironori; Katayama, Tsutomu; Takada, Shoji

    2016-12-13

    Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.

  11. Promoters, Transcripts, and Regulatory Proteins of Mungbean Yellow Mosaic Geminivirus†

    Science.gov (United States)

    Shivaprasad, P. V.; Akbergenov, Rashid; Trinks, Daniela; Rajeswaran, R.; Veluthambi, K.; Hohn, Thomas; Pooggin, Mikhail M.

    2005-01-01

    Geminiviruses package circular single-stranded DNA and replicate in the nucleus via a double-stranded intermediate. This intermediate also serves as a template for bidirectional transcription by polymerase II. Here, we map promoters and transcripts and characterize regulatory proteins of Mungbean yellow mosaic virus-Vigna (MYMV), a bipartite geminivirus in the genus Begomovirus. The following new features, which might also apply to other begomoviruses, were revealed in MYMV. The leftward and rightward promoters on DNA-B share the transcription activator AC2-responsive region, which does not overlap the common region that is nearly identical in the two DNA components. The transcription unit for BC1 (movement protein) includes a conserved, leader-based intron. Besides negative-feedback regulation of its own leftward promoter on DNA-A, the replication protein AC1, in cooperation with AC2, synergistically transactivates the rightward promoter, which drives a dicistronic transcription unit for the coat protein AV1. AC2 and the replication enhancer AC3 are expressed from one dicistronic transcript driven by a strong promoter mapped within the upstream AC1 gene. Early and constitutive expression of AC2 is consistent with its essential dual function as an activator of viral transcription and a suppressor of silencing. PMID:15956560

  12. Promoters, transcripts, and regulatory proteins of Mungbean yellow mosaic geminivirus.

    Science.gov (United States)

    Shivaprasad, P V; Akbergenov, Rashid; Trinks, Daniela; Rajeswaran, R; Veluthambi, K; Hohn, Thomas; Pooggin, Mikhail M

    2005-07-01

    Geminiviruses package circular single-stranded DNA and replicate in the nucleus via a double-stranded intermediate. This intermediate also serves as a template for bidirectional transcription by polymerase II. Here, we map promoters and transcripts and characterize regulatory proteins of Mungbean yellow mosaic virus-Vigna (MYMV), a bipartite geminivirus in the genus Begomovirus. The following new features, which might also apply to other begomoviruses, were revealed in MYMV. The leftward and rightward promoters on DNA-B share the transcription activator AC2-responsive region, which does not overlap the common region that is nearly identical in the two DNA components. The transcription unit for BC1 (movement protein) includes a conserved, leader-based intron. Besides negative-feedback regulation of its own leftward promoter on DNA-A, the replication protein AC1, in cooperation with AC2, synergistically transactivates the rightward promoter, which drives a dicistronic transcription unit for the coat protein AV1. AC2 and the replication enhancer AC3 are expressed from one dicistronic transcript driven by a strong promoter mapped within the upstream AC1 gene. Early and constitutive expression of AC2 is consistent with its essential dual function as an activator of viral transcription and a suppressor of silencing.

  13. 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.

  14. Hasse-Schmidt derivations and the Hopf algebra of noncommutative symmetric functions

    CERN Document Server

    Hazewinkel, Michiel

    2011-01-01

    Let A be an associative algebra (or any other kind of algebra for that matter). A derivation on A is an endomorphism \\del of the underlying Abelian group of A such that \\del(ab)=a(\\del b)+(\\del a)b for all a,b\\in A (1.1) A Hasse-Schmidt derivation is a sequence (d_0=id,d_1,d_2,...,d_n,...) of endomorphisms of the underlying Abelian group such that for all n \\ge 1 d_n(ab)= \\sum_{i=0}^n (d_ia)(d_{n-i}b) (1.2) Note that d_1 is a derivation as defined by (1.1). The individual d_n that occur in a Hasse-Schmidt derivation are also sometimes called higher derivations. A question of some importance is whether Hasse-Schmidt derivations can be written down in terms of polynomials in ordinary derivations. For instance in connection with automatic continuity for Hasse-Schmidt derivations on Banach algebras. Such formulas have been written down by, for instance, Heerema and Mirzavaziri in [5] and [6]. They also will be explicitly given below. It is the purpose of this short note to show that such formulas follow directly ...

  15. Begomoviruses infecting weeds in Cuba: increased host range and a novel virus infecting Sida rhombifolia.

    Science.gov (United States)

    Fiallo-Olivé, Elvira; Navas-Castillo, Jesús; Moriones, Enrique; Martínez-Zubiaur, Yamila

    2012-01-01

    As a result of surveys conducted during the last few years to search for wild reservoirs of begomoviruses in Cuba, we detected a novel bipartite begomovirus, sida yellow mottle virus (SiYMoV), infecting Sida rhombifolia plants. The complete genome sequence was obtained, showing that DNA-A was 2622 nucleotides (nt) in length and that it was most closely related (87.6% nucleotide identity) to DNA-A of an isolate of sida golden mosaic virus (SiGMV) that infects snap beans (Phaseolus vulgaris) in Florida. The DNA-B sequence was 2600 nt in length and shared the highest nucleotide identity (75.1%) with corchorus yellow spot virus (CoYSV). Phylogenetic relationship analysis showed that both DNA components of SiYMoV were grouped in the Abutilon clade, along with begomoviruses from Florida and the Caribbean islands. We also present here the complete nucleotide sequence of a novel strain of sida yellow vein virus found infecting Malvastrum coromandelianum and an isolate of euphorbia mosaic virus that was found for the first time infecting Euphorbia heterophylla in Cuba.

  16. First report of the complete sequence of Sida golden yellow vein virus from Jamaica.

    Science.gov (United States)

    Stewart, Cheryl S; Kon, Tatsuya; Gilbertson, Robert L; Roye, Marcia E

    2011-08-01

    Begomoviruses are phytopathogens that threaten food security [18]. Sida spp. are ubiquitous weed species found in Jamaica. Sida samples were collected island-wide, DNA was extracted via a modified Dellaporta method, and the viral genome was amplified using degenerate and sequence-specific primers [2, 11]. The amplicons were cloned and sequenced. Sequence analysis revealed that a DNA-A molecule isolated from a plant in Liguanea, St. Andrew, was 90.9% similar to Sida golden yellow vein virus-[United States of America:Homestead:A11], making it a strain of SiGYVV. It was named Sida golden yellow vein virus-[Jamaica:Liguanea 2:2008] (SiGYVV-[JM:Lig2:08]). The cognate DNA-B, previously unreported, was successfully cloned and was most similar to that of Malvastrum yellow mosaic Jamaica virus (MaYMJV). Phylogenetic analysis suggested that this virus was most closely related to begomoviruses that infect malvaceous hosts in Jamaica, Cuba and Florida in the United States.

  17. Host growth temperature and a conservative amino acid substitution in the replication protein of pPS10 influence plasmid host range.

    Science.gov (United States)

    Fernández-Tresguerres, M E; Martín, M; García de Viedma, D; Giraldo, R; Díaz-Orejas, R

    1995-01-01

    pPS10 is a replicon isolated from Pseudomonas syringe pv. savastanoi that can be established at 37 degrees C efficiently in Pseudomonas aeruginosa but very inefficiently in Escherichia coli. The establishment of the wild-type pPS10 replicon in E. coli is favored at low temperatures (30 degrees C or below). RepA protein of pPS10 promotes in vitro plasmid replication in extracts from E. coli, and this replication depends on host proteins DnaA, DnaB, DnaG, and SSB. Mutant plasmids able to efficiently replicate in E. coli at 37 degrees C were obtained. Three of four mutants whose mutations were mapped show a conservative Ala-->Val change in the amino-terminal region of the replication protein RepA. Plasmids carrying this mutation maintain the capacity to replicate in P. aeruginosa and have a fourfold increase in copy number in this host. The mutation does not substantially alter the autoregulation mediated by RepA. These results show that the physiological conditions of the host as well as subtle changes in the plasmid replication protein can modulate the host range of the pPS10 replicon. PMID:7635822

  18. Molecular identification of a new begomovirus infecting yellow passion fruit (Passiflora edulis) in Colombia.

    Science.gov (United States)

    Vaca-Vaca, Juan Carlos; Carrasco-Lozano, Emerson Clovis; López-López, Karina

    2017-02-01

    The complete genome sequence of a bipartite begomovirus (genus Begomovirus, family Geminiviridae) infecting yellow passion fruit (Passiflora edulis) in the state of Valle del Cauca (Colombia) has been determined. The complete DNA-A and DNA-B components were determined to be 2600 and 2572 nt in length, respectively. The DNA-A showed the highest nucleotide sequence identity (87.2 %) to bean dwarf mosaic virus (M88179), a begomovirus found in common bean crops in Colombia, and only 77.4 % identity to passion fruit severe leaf distortion virus (FJ972767), a begomovirus identified infecting passion fruit in Brazil. Based on its sequence identity to all other begomoviruses known to date and in accordance with the ICTV species demarcation criterion for the genus Begomovirus (≥91 % sequence identity for the complete DNA-A), the name passion fruit leaf distortion virus is proposed for this new begomovirus. To our knowledge, this is the first report of a bipartite begomovirus affecting passion fruit in Colombia and the second report of a geminivirus affecting this crop worldwide.

  19. 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.

  20. Diversity of dicotyledenous-infecting geminiviruses and their associated DNA molecules in southern Africa, including the South-west Indian ocean islands.

    Science.gov (United States)

    Rey, Marie E C; Ndunguru, Joseph; Berrie, Leigh C; Paximadis, Maria; Berry, Shaun; Cossa, Nurbibi; Nuaila, Valter N; Mabasa, Ken G; Abraham, Natasha; Rybicki, Edward P; Martin, Darren; Pietersen, Gerhard; Esterhuizen, Lindy L

    2012-09-01

    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-αs) 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.

  1. 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

  2. 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

  3. Hydrogeologic data for the lower Housatonic River basin, Connecticut

    Science.gov (United States)

    Grossman, I.G.; Wilson, William E.

    1970-01-01

    This report contains hydrologic and geologic data collected for an investigation of the lower Housatonic River basin by the U.S. Geological Survey in financial cooperation with the Connecticut Water Resources Commission. The report also summarizes data that are available in other publications. The towns within the 557 square mile area of the basin in western Connecticut include all of Beacon Falls, Middlebury, Naugatuck, Oxford, Seymour, Thomaston, Waterbury, Watertown, and Woodbury; and parts of Ansonia, Bethany, Bethlehem, Bristol, Burlington, Cheshire, Derby, Easton, Goshen, Narwinton, Litchfield, Milford, Monroe, Morris, New Hartford, Newtown, Norfolk, Orange, Plymouth, Prospect, Roxbury, Shelton, Southbury, Stratford, Torrington, Trumbull, Washington, Winchester, Wolcott, and Woodbridge. The factual information on the following pages was the basis for a companion interpretive report, Connecticut Water Resources Bulletin No. 19 (Wilson, W. E., and others, in preparation, 1970). The basic-data report can be used alone for detailed information needed in planning water resources development at specific sites or it can be used to supplement the interpretive report.

  4. Die een wat sal kom: 'Messiaanse tekste' in die Ou Testament en ander Joodse geskrifte

    Directory of Open Access Journals (Sweden)

    Alphonso Groenewald

    2010-03-01

    Full Text Available The one who is to come: �Messianic texts� in the Old Testament and other Jewish writingsAccording to the New-Testament authors, the life of Jesus, as Christ, should be seen in light of the Old-Testament texts. It seems that all the messianic texts in the Old Testament had been fulfilled in Jesus. The Messiah, who had been expected for a long time, was born in Bethlehem. This interpretation by the New-Testament authors has caused the church and Christians throughout the centuries to read the Old Testament as a prophecy, which is fulfilled in Jesus Christ. This interpretation has caused impatience with Jews, who did not accept Jesus of Nazareth as the Messiah. This article addresses the question: How did ancient Israel understand the concept �messiah�? It seems that the term is much more complex than a single meaning would allow the reader to believe. This article thus focuses on the theological functioning of the term within the Hebrew Bible as well as in other Jewish writings.

  5. 从布鲁盖尔画中的典故再析奥登的《美术馆》%A Re - analysis of Auden's Musee des Beaux from the Allusions in Breughel's Paintings

    Institute of Scientific and Technical Information of China (English)

    王唯一

    2012-01-01

    Auden' s poem Musee des Beaux Arts is inspired by Breughel's painting -Fall of Icarus and by the Legend of Icarus. Two other Breughel's paintings -The Numbering at Bethlehem and The Mas- sacre of the Innocents are also alluded to in the poem. The knowledge of allusions is the first essential to the understanding of the poem. This paper focuses on the allusions of the poem, including Bible stories and Greek myth, combining Mr. Zha Liangzheng's translation of the poem, reanalyzes Auden's poem Musee des Beaux Arts, to make its meaning clearer.%奥登的诗歌《美术馆》是因布鲁盖尔的名画《伊卡洛斯的坠落》以及伊卡洛斯的传说而创作的,诗中还间接提到了布鲁盖尔的另两幅画——《伯利恒的户口调查》和《屠杀无辜者》。典故知识是理解这首诗的第一要素.该诗典故包括圣经故事和希腊神话,在此基础上再析《美术馆》,诗歌的意义将更为明了。

  6. The Effectiveness of a Geospatial Technologies-Integrated Curriculum to Promote Climate Literacy

    Science.gov (United States)

    Anastasio, D. J.; Bodzin, A. M.; Peffer, T.; Sahagian, D. L.; Cirucci, L.

    2011-12-01

    This study examined the effectiveness of a geospatial technologies - integrated climate change curriculum (http://www.ei.lehigh.edu/eli/cc/) to promote climate literacy in an urban school district. Five 8th grade Earth and Space Science classes in an urban middle school (Bethlehem, Pennsylvania) consisting of three different ability level tracks participated in the study. Data gathering methods included pre/posttest assessments, daily classroom observations, daily teacher meetings, and examination of student produced artifacts. Data was gathered using a climate change literacy assessment instrument designed to measure students' climate change content knowledge. The items included distractors that address misunderstandings and knowledge deficits about climate change from the existing literature. Paired-sample t-test analyses were conducted to compare the pre- and post-test assessment results. The results of these analyses were used to compare overall gains as well as ability level track groups. Overall results regarding the use of the climate change curriculum showed significant improvement in urban middle school students' understanding of climate change concepts. Effect sizes were large (ES>0.8) and significant (p<0.001) for the entire assessment and for each ability level subgroup. Findings from classroom observations, assessments embedded in the curriculum, and the examination of all student artifacts revealed that the use of geospatial technologies enable middle school students to improve their knowledge of climate change and improve their spatial thinking and reasoning skills.

  7. A journey to the bible lands: a call from the past to the present in astonishing ways.

    Science.gov (United States)

    Brown, Geraldine

    2013-01-01

    I traveled to the Middle East six times between 1978 and 200. These trips will always remain among my most precious memories. These trips included visiting Tele Aviv, Jerusalem (Old and New), Bethlehem, Haifa, Galilee, Golan Heights, Tiberius, Temple Mount, Ein Gedi Beach (Dead Sea), the Masada, Sinai, Caesarea, and Megiddo (Armageddon). The Church of Our Lord Jesus Christ, Inc. (New York City) was the Host Church, with Dr. Robert Spellman (a Senior Professor at Essex County College (New Jersey), Bishop/Pastor of the Macedonia Church of Our Lord Jesus Christ (Newark, NJ), and Bible Seminar Teacher, was the Tour Leader. During these travels, many airlines were used, including British Airways, El Al, Olympia, Lufthansa, and Alitalia. Side trips included Egypt, Jordan (Petra), and Europe (Greece, London, Paris, Rome). This tour in my own words, hopefully will entice and enlighten others to plan a trip to the most sacred ground on earth. I trust there will be as much enjoyment as I have had and am still having in sharing the biblical knowledge as it relates to the Bible lessons I learned over the years in Sunday School and actually comparing them today with the Holy Land itself.

  8. [Jonathan Swift's asylum in Dublin--Ireland's introduction to institutional psychiatry 250 years ago].

    Science.gov (United States)

    Reuber, M

    1995-09-01

    250 years ago, the satirical writer and clergyman Jonathan Swift from Dublin (1667-1745) founded the first Irish lunatic asylum. Rejecting the theories put forward by the English philosopher Thomas Hobbes and the doctor Thomas Willis, he was influenced by the ideas of the Scottish doctor and the "enlightened" thinker John Locke. Swift's St. Patrick's Hospital did not, however, realise a new philosophical concept: architecture and therapeutic approach of the new institution were clearly modelled on the much older Hospital of St. Mary of Bethlehem ( = Bedlam). Despite its conservative conceptual basis, the first institution dedicated to the mentally ill and intellectually subnormal in Ireland became a starting point for the apparantly unstoppable expansion of the, at one time, most comprehensive asylum system in the world. After Swift's Hospital had been enlarged twice at the tax-payers' expense (1778, 1793), the administration decided to relieve the institution by erecting the Richmond Asylum (1810), the first public asylum in Ireland. When this establishment also became overcrowded, in 1817, legislation was passed which led to the establishment of the oldest system of public asylums in Europe.

  9. 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.

  10. Another step towards zero waste, using pollution control residuals to make steel

    Energy Technology Data Exchange (ETDEWEB)

    Easterly, T.W.; Berquist, W.G. [Bethlehem Steel Corp., Chesterton, IN (United States); Lynn, J.D. [Bethlehem Steel Corp., PA (United States)

    1997-12-31

    Environmental legislation and regulations plus the economies of disposal are directing the steel industry to look for ways of minimizing the generation of waste and to maximize the recycling of collected materials. Further, the increasing use and efficiency of end of pipe pollution controls capture ever increasing amounts of materials that were previously discharged to the environment. These newly captured pollution control dusts and sludges often have chemical or physical properties that may prevent their direct recycle into the iron and steelmaking process. This paper describes how Bethlehem Steel`s Burns Harbor Division is using a variety of material handling and recycling technologies in an integrated pollution control dust and sludge management program to recycle its daily generation of pollution control dusts and sludges. This program has been designed to be consistent with the operating requirements of the iron and steelmaking processes while insuring conformance with all environmental requirements. When fully operational, this program will reuse over 90% of the plant`s pollution control dusts and sludges to make the product steel.

  11. New Numismatic Evidence about the Comets of Mithradates the Great of Pontus (134 and 119 BC).

    Science.gov (United States)

    Molnar, M. R.

    1997-12-01

    The historian, Justinus, tells us that the life of Mithradates the Great of Pontus (ca. 134 - 63 BC) was marked by two unusually large comets: one at his birth in ca. 134 BC and another at his coronation ca. 119 BC. Often these comets are cited as proof that sometimes comets heralded great, good events (such as the Star of Bethlehem.) We now have evidence that counters that notion. Mithradates struck some bronze coins that depict a foreboding hippeus (horse) comet. Pliny, the Roman naturalist, tells us that this kind of comet had plumes much like horses manes in very rapid motion and moving in a circle. The evidence is that the horses mains are synchronic bands. The visibility of these bands indicates that the hippeus comet is a class of comets that had a close encounter with the earth, perhaps on the order of a million kilometers. Hephaistion of Thebes tells us that the hippeus comet foretold the quick fall of kings and tyrants and rapid changes in the affairs of these countries. It is likely that the comet was interpreted as an omen of violent revolution, but Mithradates apparently altered the focus of the portent, namely that the comet signified his struggle to evict the Romans from Asia Minor.

  12. Blast Furnace Granulated Coal Injection System Demonstration Project public design report. Topical report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-03-01

    The public design report describes the Blast Furnace Granulated Coal Injection (BFGCI) project under construction at Bethlehem Steel Corporation`s (BSC) Burns Harbor, Indiana, 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. The project is the first installation in the United States for the British Steel technology using granular coal in blast furnaces. The objective is to demonstrate that granular coal is an economic and reliable fuel which can successfully be applied to large North American blast furnaces. These include: coal grind size, coal injection rate, coal source (type) and blast furnace conversion method. To achieve the program objectives, the demonstration project is divided into the following three Phases: Phase I-Design; Phase II-Procurement & Construction; and Phase III-Operation. Preliminary design (Phase I) began in 1991 with detailed design commencing in April 1993. Construction at Burns Harbor (Phase II) began August 1993. Construction is expected to be complete in the first quarter of 1995 which will be followed by a demonstration test program (Phase III).

  13. Aux frontières poreuses des cartes palestiniennes et de l'art contemporain

    Directory of Open Access Journals (Sweden)

    Anaïs Farine

    2013-12-01

    Full Text Available Un certain nombre d'artistes contemporains palestiniens se saisissent de la carte comme motif (simple présence de carte traditionnelle dans l'espace des œuvres ou comme modèle conceptuel structurant leurs travaux. A travers l'analyse des vidéos M* of Bethlehem [Ayreen Anastas, 2003], Red, Dead and Mediterranean [Akram Al-Ashqar, 2006], et We Began by Measuring Distance [Basma Al-sharif, 2009], cet article entend étudier le déplacement des frontières artistiques ainsi que des représentations de l'espace géopolitique dans les images en mouvements palestiniennes contemporaines. En interrogeant les différents dispositifs mis en place par les trois artistes pour se réapproprier le territoire en lui donnant un sens et une unité, cet article indique que ces œuvres, parce qu'elles répondent avec une grande subtilité aux préoccupations spatiales d'une certaine mouvance de l'art contemporain, sont devenues incontournables.

  14. 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.

  15. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Karp, Jerome M.; Erylimaz, Ertan; Cowburn, David, E-mail: cowburn@cowburnlab.org, E-mail: David.cowburn@einstein.yu.edu [Albert Einstein College of Medicine of Yeshiva University, Department of Biochemistry (United States)

    2015-01-15

    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.

  17. Evolution of cyclic peptide protease inhibitors.

    Science.gov (United States)

    Young, Travis S; Young, Douglas D; Ahmad, Insha; Louis, John M; Benkovic, Stephen J; Schultz, Peter G

    2011-07-05

    We report a bacterial system for the evolution of cyclic peptides that makes use of an expanded set of amino acid building blocks. Orthogonal aminoacyl-tRNA synthetase/tRNA(CUA) pairs, together with a split intein system were used to biosynthesize a library of ribosomal peptides containing amino acids with unique structures and reactivities. This peptide library was subsequently used to evolve an inhibitor of HIV protease using a selection based on cellular viability. Two of three cyclic peptides isolated after two rounds of selection contained the keto amino acid p-benzoylphenylalanine (pBzF). The most potent peptide (G12: GIXVSL; X=pBzF) inhibited HIV protease through the formation of a covalent Schiff base adduct of the pBzF residue with the ε-amino group of Lys 14 on the protease. This result suggests that an expanded genetic code can confer an evolutionary advantage in response to selective pressure. Moreover, the combination of natural evolutionary processes with chemically biased building blocks provides another strategy for the generation of biologically active peptides using microbial systems.

  18. Spider wrapping silk fibre architecture arising from its modular soluble protein precursor

    Science.gov (United States)

    Tremblay, Marie-Laurence; Xu, Lingling; Lefèvre, Thierry; Sarker, Muzaddid; Orrell, Kathleen E.; Leclerc, Jérémie; Meng, Qing; Pézolet, Michel; Auger, Michèle; Liu, Xiang-Qin; Rainey, Jan K.

    2015-06-01

    Spiders store spidroins in their silk glands as high concentration aqueous solutions, spinning these dopes into fibres with outstanding mechanical properties. Aciniform (or wrapping) silk is the toughest spider silk and is devoid of the short amino acid sequence motifs characteristic of the other spidroins. Using solution-state NMR spectroscopy, we demonstrate that the 200 amino acid Argiope trifasciata AcSp1 repeat unit contrasts with previously characterized spidroins, adopting a globular 5-helix bundle flanked by intrinsically disordered N- and C-terminal tails. Split-intein-mediated segmental NMR-active isotope-enrichment allowed unambiguous demonstration of modular and malleable “beads-on-a-string” concatemeric behaviour. Concatemers form fibres upon manual drawing with silk-like morphology and mechanical properties, alongside secondary structuring and orientation consistent with native AcSp1 fibres. AcSp1 structural stability varies locally, with the fifth helix denaturing most readily. The structural transition of aciniform spidroin from a mostly α-helical dope to a mixed α-helix/β-sheet-containing fibre can be directly related to spidroin architecture and stability.

  19. Bacterial superglue enables easy development of efficient virus-like particle based vaccines

    DEFF Research Database (Denmark)

    Thrane, Susan; Janitzek, Christoph M; Matondo, Sungwa;

    2016-01-01

    for VLP-based antigen display require labor-intensive trial-and-error optimization, and often fail to generate dense antigen display. Here we utilize the split-intein (SpyTag/SpyCatcher) conjugation system to generate stable isopeptide bound antigen-VLP complexes by simply mixing of the antigen and VLP...... components. RESULTS: Genetic fusion of SpyTag or SpyCatcher to the N-terminus and/or C-terminus of the Acinetobacter phage AP205 capsid protein resulted in formation of stable, nonaggregated VLPs expressing one SpyCatcher, one SpyTag or two SpyTags per capsid protein. Mixing of spy-VLPs with eleven different...... vaccine antigens fused to SpyCatcher or SpyTag resulted in formation of antigen-VLP complexes with coupling efficiencies (% occupancy of total VLP binding sites) ranging from 22-88 %. In mice, spy-VLP vaccines presenting the malaria proteins Pfs25 or VAR2CSA markedly increased antibody titer, affinity...

  20. Mobile elements in a single-filament orange Guaymas Basin Beggiatoa ("Candidatus Maribeggiatoa") sp. draft genome: evidence for genetic exchange with cyanobacteria.

    Science.gov (United States)

    MacGregor, Barbara J; Biddle, Jennifer F; Teske, Andreas

    2013-07-01

    The draft genome sequence of a single orange Beggiatoa ("Candidatus Maribeggiatoa") filament collected from a microbial mat at a hydrothermal site in Guaymas Basin (Gulf of California, Mexico) shows evidence of extensive genetic exchange with cyanobacteria, in particular for sensory and signal transduction genes. A putative homing endonuclease gene and group I intron within the 23S rRNA gene; several group II catalytic introns; GyrB and DnaE inteins, also encoding homing endonucleases; multiple copies of sequences similar to the fdxN excision elements XisH and XisI (required for heterocyst differentiation in some cyanobacteria); and multiple sequences related to an open reading frame (ORF) (00024_0693) of unknown function all have close non-Beggiatoaceae matches with cyanobacterial sequences. Sequences similar to the uncharacterized ORF and Xis elements are found in other Beggiatoaceae genomes, a variety of cyanobacteria, and a few phylogenetically dispersed pleiomorphic or filamentous bacteria. We speculate that elements shared among filamentous bacterial species may have been exchanged in microbial mats and that some of them may be involved in cell differentiation.

  1. Romulus and Remus, two phage isolates representing a distinct clade within the Twortlikevirus genus, display suitable properties for phage therapy applications.

    Science.gov (United States)

    Vandersteegen, Katrien; Kropinski, Andrew M; Nash, John H E; Noben, Jean-Paul; Hermans, Katleen; Lavigne, Rob

    2013-03-01

    The renewed interest in controlling Staphylococcus aureus infections using their natural enemies, bacteriophages, has led to the isolation of a limited number of virulent phages so far. These phages are all members of the Twortlikevirus, displaying little variance. We present two novel closely related (95.9% DNA homology) lytic myoviruses, Romulus and Remus, with double-stranded DNA (dsDNA) genomes of 131,333 bp and 134,643 bp, respectively. Despite their relatedness to Staphylococcus phages K, G1, ISP, and Twort and Listeria phages A511 and P100, Romulus and Remus can be proposed as isolates of a new species within the Twortlikevirus genus. A distinguishing feature for these phage genomes is the unique distribution of group I introns compared to that in other staphylococcal myoviruses. In addition, a hedgehog/intein domain was found within their DNA polymerase genes, and an insertion sequence-encoded transposase exhibits splicing behavior and produces a functional portal protein. From a phage therapy application perspective, Romulus and Remus infected approximately 70% of the tested S. aureus isolates and displayed promising lytic activity against these isolates. Furthermore, both phages showed a rapid initial adsorption and demonstrated biofilm-degrading capacity in a proof-of-concept experiment.

  2. Broad nucleotide cofactor specificity of DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus and its evolutionary significance.

    Science.gov (United States)

    Kim, Jun-Hwan; Lee, Kang-Keun; Sun, Younguk; Seo, Gang-Jin; Cho, Sung Suk; Kwon, Suk Hyung; Kwon, Suk-Tae

    2013-05-01

    The nucleotide cofactor specificity of the DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus (Hbu) was studied to investigate the evolutionary relationship of DNA ligases. The Hbu DNA ligase gene was expressed under control of the T7lac promoter of pTARG in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was purified using the IMPACT™-CN system (intein-mediated purification with an affinity chitin-binding tag) and cation-ion (Arg-tag) chromatography. The optimal temperature for Hbu DNA ligase activity was 75 °C, and the optimal pH was 8.0 in Tris-HCl. The activity was highly dependent on MgCl2 or MnCl2 with maximal activity above 5 mM MgCl2 and 2 mM MnCl2. Notably, Hbu DNA ligase can use ADP and GTP in addition to ATP. The broad nucleotide cofactor specificity of Hbu DNA ligase might exemplify an undifferentiated ancestral stage in the evolution of DNA ligases. This study provides new evidence for possible evolutionary relationships among DNA ligases.

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

    Directory of Open Access Journals (Sweden)

    Nicola eWeichert

    2016-01-01

    Full Text Available 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 routinely produced, which is above the native size of the FLAG protein. The storage of seeds for eight weeks and one year at an ambient temperature of 15°C does not influence the accumulation level. Even the extended storage time does not influence the typical pattern of multimerized bands. These results show that seeds are the method of choice for stable accumulation of products of complex transgenes and have the capability for long-term storage at moderate conditions, an important feature for the development of suitable downstream processes.

  4. Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-splicing.

    Science.gov (United States)

    Mehler, Michaela; Eckert, Carl Elias; Busche, Alena; Kulhei, Jennifer; Michaelis, Jonas; Becker-Baldus, Johanna; Wachtveitl, Josef; Dötsch, Volker; Glaubitz, Clemens

    2015-11-13

    Protein trans-splicing using split inteins is well established as a useful tool for protein engineering. Here we show, for the first time, that this method can be applied to a membrane protein under native conditions. We provide compelling evidence that the heptahelical proteorhodopsin can be assembled from two separate fragments consisting of helical bundles A and B and C, D, E, F, and G via a splicing site located in the BC loop. The procedure presented here is on the basis of dual expression and ligation in vivo. Global fold, stability, and photodynamics were analyzed in detergent by CD, stationary, as well as time-resolved optical spectroscopy. The fold within lipid bilayers has been probed by high field and dynamic nuclear polarization-enhanced solid-state NMR utilizing a (13)C-labeled retinal cofactor and extensively (13)C-(15)N-labeled protein. Our data show unambiguously that the ligation product is identical to its non-ligated counterpart. Furthermore, our data highlight the effects of BC loop modifications onto the photocycle kinetics of proteorhodopsin. Our data demonstrate that a correctly folded and functionally intact protein can be produced in this artificial way. Our findings are of high relevance for a general understanding of the assembly of membrane proteins for elucidating intramolecular interactions, and they offer the possibility of developing novel labeling schemes for spectroscopic applications.

  5. High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli.

    Science.gov (United States)

    Ma, Yi; Yu, Jieying; Lin, Jinglian; Wu, Shaomin; Li, Shan; Wang, Jufang

    2016-01-01

    Human epidermal growth factor (hEGF) is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe) at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO) and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10. The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development.

  6. High Efficient Expression, Purification, and Functional Characterization of Native Human Epidermal Growth Factor in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Yi Ma

    2016-01-01

    Full Text Available Human epidermal growth factor (hEGF is a small, mitotic growth polypeptide that promotes the proliferation of various cells and is widely applied in clinical practices. However, high efficient expression of native hEGF in Escherichia coli has not been successful, since three disulfide bonds in monomer hEGF made it unable to fold into correct 3D structure using in vivo system. To tackle this problem, we fused Mxe GyrA intein (Mxe at the C-terminal of hEGF followed by small ubiquitin-related modifier (SUMO and 10x His-tag to construct a chimeric protein hEGF-Mxe-SUMO-H10. The fusion protein was highly expressed at the concentration of 281 mg/L and up to 59.5% of the total cellular soluble proteins. The fusion protein was purified by affinity chromatography and 29.4 mg/L of native hEGF can be released by thiol induced N-terminal cleavage without any proteases. The mitotic activity in Balb/c 3T3 cells is proliferated by commercial and recombinant hEGF measured with methylthiazolyldiphenyl-tetrazolium bromide (MTT assay which indicated that recombinant hEGF protein stimulates the cell proliferation similar to commercial protein. This study significantly improved the yield and reduced the cost of hEGF in the recombinant E. coli system and could be a better strategy to produce native hEGF for pharmaceutical development.

  7. Home and away- the evolutionary dynamics of homing endonucleases

    Directory of Open Access Journals (Sweden)

    Barzel Adi

    2011-11-01

    Full Text Available Abstract Background Homing endonucleases (HEases are a large and diverse group of site-specific DNAases. They reside within self-splicing introns and inteins, and promote their horizontal dissemination. In recent years, HEases have been the focus of extensive research due to their promising potential use in gene targeting procedures for the treatment of genetic diseases and for the genetic engineering of crop, animal models and cell lines. Results Using mathematical analysis and computational modeling, we present here a novel account for the evolution and population dynamics of HEase genes (HEGs. We describe HEGs as paradoxical selfish elements whose long-term persistence in a single population relies on low transmission rates and a positive correlation between transmission efficiency and toxicity. Conclusion Plausible conditions allow HEGs to sustain at high frequency through long evolutionary periods, with the endonuclease frequency being either at equilibrium or periodically oscillating. The predictions of our model may prove important not only for evolutionary theory but also for gene therapy and bio-engineering applications of HEases.

  8. Characterizing Aciniform Silk Repetitive Domain Backbone Dynamics and Hydrodynamic Modularity

    Directory of Open Access Journals (Sweden)

    Marie-Laurence Tremblay

    2016-08-01

    Full Text Available Spider aciniform (wrapping silk is a remarkable fibrillar biomaterial with outstanding mechanical properties. It is a modular protein consisting, in Argiope trifasciata, of a core repetitive domain of 200 amino acid units (W units. In solution, the W units comprise a globular folded core, with five α-helices, and disordered tails that are linked to form a ~63-residue intrinsically disordered linker in concatemers. Herein, we present nuclear magnetic resonance (NMR spectroscopy-based 15N spin relaxation analysis, allowing characterization of backbone dynamics as a function of residue on the ps–ns timescale in the context of the single W unit (W1 and the two unit concatemer (W2. Unambiguous mapping of backbone dynamics throughout W2 was made possible by segmental NMR active isotope-enrichment through split intein-mediated trans-splicing. Spectral density mapping for W1 and W2 reveals a striking disparity in dynamics between the folded core and the disordered linker and tail regions. These data are also consistent with rotational diffusion behaviour where each globular domain tumbles almost independently of its neighbour. At a localized level, helix 5 exhibits elevated high frequency dynamics relative to the proximal helix 4, supporting a model of fibrillogenesis where this helix unfolds as part of the transition to a mixed α-helix/β-sheet fibre.

  9. 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.

  10. The genome of Hyperthermus butylicus: a sulfur-reducing, peptide fermenting, neutrophilic Crenarchaeote growing up to 108 °C

    Directory of Open Access Journals (Sweden)

    Kim Brügger

    2007-01-01

    Full Text Available Hyperthermus butylicus, a hyperthermophilic neutrophile and anaerobe, is a member of the archaeal kingdom Crenarchaeota. Its genome consists of a single circular chromosome of 1,667,163 bp with a 53.7% G+C content. A total of 1672 genes were annotated, of which 1602 are protein-coding, and up to a third are specific to H. butylicus. In contrast to some other crenarchaeal genomes, a high level of GUG and UUG start codons are predicted. Two cdc6 genes are present, but neither could be linked unambiguously to an origin of replication. Many of the predicted metabolic gene products are associated with the fermentation of peptide mixtures including several peptidases with diverse specificities, and there are many encoded transporters. Most of the sulfur-reducing enzymes, hydrogenases and electron-transfer proteins were identified which are associated with energy production by reducing sulfur to H2S. Two large clusters of regularly interspaced repeats (CRISPRs are present, one of which is associated with a crenarchaeal-type cas gene superoperon; none of the spacer sequences yielded good sequence matches with known archaeal chromosomal elements. The genome carries no detectable transposable or integrated elements, no inteins, and introns are exclusive to tRNA genes. This suggests that the genome structure is quite stable, possibly reflecting a constant, and relatively uncompetitive, natural environment.

  11. Cyclic peptide inhibitors of the β-sliding clamp in Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Susanne Kjelstrup

    Full Text Available Interaction between pairs of Staphylococcus aureus replication proteins was detected in an Escherichia coli based two-hybrid analysis. A reverse two-hybrid system was constructed for selection of compounds that hindered interaction between interacting protein pairs. A number of cyclic peptides, from a library generated by the split intein-mediated circular ligation of peptides and proteins technology, were found to interfere with dimerization of the β-sliding clamp of the replisome. Two 8-mer peptides were analyzed in more detail. Both inhibited DNA replication, led to SOS induction, altered cell morphology and cell death. The peptides were active when added to bacterial cultures indicating that they could traverse the bacterial membrane to find their intracellular target. Peptide specificity was confirmed by overproduction of the putative target (DnaN which resulted in resistance. The minimum inhibitory concentration was ∼50 μg/ml for S. aureus cells. These compounds may serve as lead candidates for future development into novel classes of antibiotics as well as provide information on the function of the S. aureus replication process.

  12. Locked and proteolysis-based transcription activator-like effector (TALE) regulation.

    Science.gov (United States)

    Lonzarić, Jan; Lebar, Tina; Majerle, Andreja; Manček-Keber, Mateja; Jerala, Roman

    2016-02-18

    Development of orthogonal, designable and adjustable transcriptional regulators is an important goal of synthetic biology. Their activity has been typically modulated through stimulus-induced oligomerization or interaction between the DNA-binding and activation/repression domain. We exploited a feature of the designable Transcription activator-like effector (TALE) DNA-binding domain that it winds around the DNA which allows to topologically prevent it from binding by intramolecular cyclization. This new approach was investigated through noncovalent ligand-induced cyclization or through a covalent split intein cyclization strategy, where the topological inhibition of DNA binding by cyclization and its restoration by a proteolytic release of the topologic constraint was expected. We show that locked TALEs indeed have diminished DNA binding and regain full transcriptional activity by stimulation with the rapamycin ligand or site-specific proteolysis of the peptide linker, with much higher level of activation than rapamycin-induced heterodimerization. Additionally, we demonstrated reversibility, activation of genomic targets and implemented logic gates based on combinations of protein cyclization, proteolytic cleavage and ligand-induced dimerization, where the strongest fold induction was achieved by the proteolytic cleavage of a repression domain from a linear TALE.

  13. A novel begomovirus isolated from sida contains putative cis- and trans-acting replication specificity determinants that have evolved independently in several geographical lineages.

    Science.gov (United States)

    Mauricio-Castillo, J A; Torres-Herrera, S I; Cárdenas-Conejo, Y; Pastor-Palacios, G; Méndez-Lozano, J; Argüello-Astorga, G R

    2014-09-01

    A novel begomovirus isolated from a Sida rhombifolia plant collected in Sinaloa, Mexico, was characterized. The genomic components of sida mosaic Sinaloa virus (SiMSinV) shared highest sequence identity with DNA-A and DNA-B components of chino del tomate virus (CdTV), suggesting a vertical evolutionary relationship between these viruses. However, recombination analysis indicated that a short segment of SiMSinV DNA-A encompassing the plus-strand replication origin and the 5´-proximal 43 codons of the Rep gene was derived from tomato mottle Taino virus (ToMoTV). Accordingly, the putative cis- and trans-acting replication specificity determinants of SiMSinV were identical to those of ToMoTV but differed from those of CdTV. Modeling of the SiMSinV and CdTV Rep proteins revealed significant differences in the region comprising the small β1/β5 sheet element, where five putative DNA-binding specificity determinants (SPDs) of Rep (i.e., amino acid residues 5, 8, 10, 69 and 71) were previously identified. Computer-assisted searches of public databases led to identification of 33 begomoviruses from three continents encoding proteins with SPDs identical to those of the Rep encoded by SiMSinV. Sequence analysis of the replication origins demonstrated that all 33 begomoviruses harbor potential Rep-binding sites identical to those of SiMSinV. These data support the hypothesis that the Rep β1/β5 sheet region determines specificity of this protein for DNA replication origin sequences.

  14. A melting pot of Old World begomoviruses and their satellites infecting a collection of Gossypium species in Pakistan.

    Science.gov (United States)

    Nawaz-ul-Rehman, Muhammad Shah; Briddon, Rob W; Fauquet, Claude M

    2012-01-01

    CLCuD in southern Asia is caused by a complex of multiple begomoviruses (whitefly transmitted, single-stranded [ss]DNA viruses) in association with a specific ssDNA satellite; Cotton leaf curl Multan betasatellite (CLCuMuB). A further single ssDNA molecule, for which the collective name alphasatellites has been proposed, is also frequently associated with begomovirus-betasatellite complexes. Multan is in the center of the cotton growing area of Pakistan and has seen some of the worst problems caused by CLCuD. An exhaustive analysis of the diversity of begomoviruses and their satellites occurring in 15 Gossypium species (including G. hirsutum, the mainstay of Pakistan's cotton production) that are maintained in an orchard in the vicinity of Multan has been conducted using φ29 DNA polymerase-mediated rolling-circle amplification, cloning and sequence analysis. The non-cultivated Gossypium species, including non-symptomatic plants, were found to harbor a much greater diversity of begomoviruses and satellites than found in the cultivated G. hirsutum. Furthermore an African cassava mosaic virus (a virus previously only identified in Africa) DNA-A component and a Jatropha curcas mosaic virus (a virus occurring only in southern India) DNA-B component were identified. Consistent with earlier studies of cotton in southern Asia, only a single species of betasatellite, CLCuMuB, was identified. The diversity of alphasatellites was much greater, with many previously unknown species, in the non-cultivated cotton species than in G. hirsutum. Inoculation of newly identified components showed them to be competent for symptomatic infection of Nicotiana benthamiana plants. The significance of the findings with respect to our understanding of the role of host selection in virus diversity in crops and the geographical spread of viruses by human activity are discussed.

  15. A melting pot of Old World begomoviruses and their satellites infecting a collection of Gossypium species in Pakistan.

    Directory of Open Access Journals (Sweden)

    Muhammad Shah Nawaz-ul-Rehman

    Full Text Available CLCuD in southern Asia is caused by a complex of multiple begomoviruses (whitefly transmitted, single-stranded [ss]DNA viruses in association with a specific ssDNA satellite; Cotton leaf curl Multan betasatellite (CLCuMuB. A further single ssDNA molecule, for which the collective name alphasatellites has been proposed, is also frequently associated with begomovirus-betasatellite complexes. Multan is in the center of the cotton growing area of Pakistan and has seen some of the worst problems caused by CLCuD. An exhaustive analysis of the diversity of begomoviruses and their satellites occurring in 15 Gossypium species (including G. hirsutum, the mainstay of Pakistan's cotton production that are maintained in an orchard in the vicinity of Multan has been conducted using φ29 DNA polymerase-mediated rolling-circle amplification, cloning and sequence analysis. The non-cultivated Gossypium species, including non-symptomatic plants, were found to harbor a much greater diversity of begomoviruses and satellites than found in the cultivated G. hirsutum. Furthermore an African cassava mosaic virus (a virus previously only identified in Africa DNA-A component and a Jatropha curcas mosaic virus (a virus occurring only in southern India DNA-B component were identified. Consistent with earlier studies of cotton in southern Asia, only a single species of betasatellite, CLCuMuB, was identified. The diversity of alphasatellites was much greater, with many previously unknown species, in the non-cultivated cotton species than in G. hirsutum. Inoculation of newly identified components showed them to be competent for symptomatic infection of Nicotiana benthamiana plants. The significance of the findings with respect to our understanding of the role of host selection in virus diversity in crops and the geographical spread of viruses by human activity are discussed.

  16. The Crimean War: a Clash of Civilizations

    Directory of Open Access Journals (Sweden)

    Tatyana V. Vakulova

    2016-12-01

    Full Text Available The need to use the civilizational approach to the analysis of the causes and outcomes of the Crimean war (campaign 1854-1855 is revealed in this article. The author analyzes the causes of the war related to the religious factor. The Soviet historiography had not considered the religious factor to be relevant for the progressive development of the state, and therefore the provisions of the mentioned approach had not been used in the analysis of historical events. The conflict that arose between France and Russia about the Holy places, is characterized by the fact that the keys of the Bethlehem Church had been taken from the Orthodox community, which they traditionally belonged to, and had been handed over to the Catholic community by the Turkish authorities of Palestine under France’s constraint. The author points to the main cause of the war – violation of the Russian law on the protection of Orthodoxy in Turkey fixed by international treaties. It is argued that it is natural to name this war – The battle for the Manger of the Lord. That is why the main events of inter-civilizational conflict took place in the Crimea and in Sevastopol, which had not only been the military base of Russia on the Black Sea, but also the cradle of Russian Orthodoxy. Analyzing the events, the author comes to the conclusion that the outcome of the war testifies to the victory of the Orthodox state and the Russian diplomacy, because the status quo of the Holy places was maintained in accordance with the state of Affairs which had existed in the Byzantine Empire. The preservation of the integrity and sovereignty of Orthodox state is the confirmation of this victory. It is shown the ability to evaluate the results of the war is based on the positions of a civilizational approach.

  17. Another look at Emergency Department HIV screening in practice: no need to revise expectations

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    Czarnogosrki Maggie

    2010-01-01

    Full Text Available Abstract Background A recent study reported a lower than expected specificity and positive predictive value of the rapid oral HIV test in the setting of routine emergency department (ED screening. These results appeared inconsistent with the findings in another urban Emergency Department during the same time period. Objective To compare the specificity and positive predictive vale (PPV of an oral rapid HIV test used in an ED screening program in Washington DC with that performed in the USHER clinical trial. Design Period cross-sectional analysis of rapid oral HIV testing conducted in an ongoing HIV screening program emergency department patients. Setting The George Washington University Emergency Department (Washington DC from 7 February to 1 October 2007. Patients 1,560 adults seen in the ED for non-HIV-related presenting complaints, who participated in the HIV screening program. Intervention Rapid HIV testing with the OraQuick ADVANCE Rapid HIV-1/2 Antibody Test (OraSure Technologies, Bethlehem, Pennsylvania. Patients with reactive rapid test results were offered Western blot testing for confirmation. Measurements Specificity and positive predictive value for the program were determined. Findings were compared to those found in the USHER trial. Results Of 1,560 patients screened for HIV, 13 [0.8%, 95% CI 0.38% to 1.28%] had a reactive HIV screening test, and all were confirmed to be positive by Western Blot. The specificity was 100% (95% CI 99.6%-100%. Limitation Since non-reactive tests were not confirmed, the test sensitivity cannot be determined. Conclusion Review of our data conflict with findings from the USHER study surrounding false positive OraQuick HIV screening. Our data suggest that rapid HIV screening protocols implemented in EDs outside of the clinical trial paradigm perform effectively without an excess of false positive results. Compared with other screening tests, HIV rapid screening should remain an essential component of ED

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

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    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.

  19. The Good, the Bad, and the Ugly: Tales of Mold-Ripened Cheese.

    Science.gov (United States)

    Marcellino O S B, Sister Noëlla; Benson, David R

    2013-10-01

    The history of cheese manufacture is a "natural history" in which animals, microorganisms, and the environment interact to yield human food. Part of the fascination with cheese, both scientifically and culturally, stems from its ability to assume amazingly diverse flavors as a result of seemingly small details in preparation. In this review, we trace the roots of cheesemaking and its development by a variety of human cultures over centuries. Traditional cheesemakers observed empirically that certain environments and processes produced the best cheeses, unwittingly selecting for microorganisms with the best biochemical properties for developing desirable aromas and textures. The focus of this review is on the role of fungi in cheese ripening, with a particular emphasis on the yeast-like fungus Geotrichum candidum. Conditions that encourage the growth of problematic fungi such as Mucor and Scopulariopsis as well as Arachnida (cheese mites), and how such contaminants might be avoided, are discussed. Bethlehem cheese, a pressed, uncooked, semihard, Saint-Nectaire-type cheese manufactured in the United Sates without commercial strains of bacteria or fungi, was used as a model for the study of stable microbial succession during ripening in a natural environment. The appearance of fungi during a 60-day ripening period was documented using light and scanning electron microscopy, and it was shown to be remarkably reproducible and parallel to the course of ripening of authentic Saint-Nectaire cheese in the Auvergne region of France. Geotrichum candidum, Mucor, and Trichothecium roseum predominate the microbiotas of both cheese types. Geotrichum in particular was shown to have high diversity in different traditional cheese ripening environments, suggesting that traditional manufacturing techniques selected for particular fungi. This and other studies suggest that strain diversity arises in relation to the lore and history of the regions from which these types of cheeses arose.

  20. 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.

  1. Sequence and analysis of the genome of the pathogenic yeast Candida orthopsilosis.

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    Alessandro Riccombeni

    Full Text Available Candida orthopsilosis is closely related to the fungal pathogen Candida parapsilosis. However, whereas C. parapsilosis is a major cause of disease in immunosuppressed individuals and in premature neonates, C. orthopsilosis is more rarely associated with infection. We sequenced the C. orthopsilosis genome to facilitate the identification of genes associated with virulence. Here, we report the de novo assembly and annotation of the genome of a Type 2 isolate of C. orthopsilosis. The sequence was obtained by combining data from next generation sequencing (454 Life Sciences and Illumina with paired-end Sanger reads from a fosmid library. The final assembly contains 12.6 Mb on 8 chromosomes. The genome was annotated using an automated pipeline based on comparative analysis of genomes of Candida species, together with manual identification of introns. We identified 5700 protein-coding genes in C. orthopsilosis, of which 5570 have an ortholog in C. parapsilosis. The time of divergence between C. orthopsilosis and C. parapsilosis is estimated to be twice as great as that between Candida albicans and Candida dubliniensis. There has been an expansion of the Hyr/Iff family of cell wall genes and the JEN family of monocarboxylic transporters in C. parapsilosis relative to C. orthopsilosis. We identified one gene from a Maltose/Galactoside O-acetyltransferase family that originated by horizontal gene transfer from a bacterium to the common ancestor of C. orthopsilosis and C. parapsilosis. We report that TFB3, a component of the general transcription factor TFIIH, undergoes alternative splicing by intron retention in multiple Candida species. We also show that an intein in the vacuolar ATPase gene VMA1 is present in C. orthopsilosis but not C. parapsilosis, and has a patchy distribution in Candida species. Our results suggest that the difference in virulence between C. parapsilosis and C. orthopsilosis may be associated with expansion of gene families.

  2. Biosynthesis of the Cyclotide Kalata B1 using a Protein Splicing Unit

    Energy Technology Data Exchange (ETDEWEB)

    Kimura, R H; Tran, A T; Camarero, J A

    2005-08-13

    Cyclotides are a newly emerging family of large backbone cyclic polypeptides ({approx}30 residues long) characterized by a disulfide-stabilized core (3 disulfide bonds) with an unusual knotted structure. In contrast to other cyclic polypeptides, cyclotides have a well-defined three-dimensional structure. Therefore, despite their small size, they can be considered miniproteins. The unique cyclic-backbone topology and knotted arrangement of 3 disulfide bonds endow cyclotides with exceptional stability and resistance to chemical, enzymatic and thermal degradation. Furthermore, their well-defined structures have been associated with a range of biological functions. Together, these characteristics suggest that cyclotides are ideal molecular scaffolds for the development of stable peptide drugs. Despite the fact that the chemical synthesis of circular peptides has been well explored and a number different approaches involving solid-phase or liquid-phase exist, recent developments in the fields of molecular biology and protein engineering have now made possible the biosynthesis of cyclic peptides. This progress has been made mainly in two areas, non-ribosomal peptide synthesis and Expressed Protein Ligation (EPL)/protein trans-splicing. Access to biosynthetic cyclotides using recombinant DNA expression techniques offers the exciting possibility of producing large combinatorial libraries of highly stable miniproteins. This would allow the generation of cell-based combinatorial libraries that could be screened either in vitro or in vivo for their ability to regulate cellular processes. In the present work, we describe the biosynthesis of the cyclotide Kalata B1 (KB1) in E. coli using an engineered intein. Our approach (Figure 1) is based on an intramolecular version of Native Chemical Ligation (NCL). NCL involves the chemoselective reaction between a N-terminal Cys residue of one peptide and an {alpha}-thioester group of a second peptide. Importantly, incorporation of these

  3. Sequence and analysis of the genome of the pathogenic yeast Candida orthopsilosis.

    Science.gov (United States)

    Riccombeni, Alessandro; Vidanes, Genevieve; Proux-Wéra, Estelle; Wolfe, Kenneth H; Butler, Geraldine

    2012-01-01

    Candida orthopsilosis is closely related to the fungal pathogen Candida parapsilosis. However, whereas C. parapsilosis is a major cause of disease in immunosuppressed individuals and in premature neonates, C. orthopsilosis is more rarely associated with infection. We sequenced the C. orthopsilosis genome to facilitate the identification of genes associated with virulence. Here, we report the de novo assembly and annotation of the genome of a Type 2 isolate of C. orthopsilosis. The sequence was obtained by combining data from next generation sequencing (454 Life Sciences and Illumina) with paired-end Sanger reads from a fosmid library. The final assembly contains 12.6 Mb on 8 chromosomes. The genome was annotated using an automated pipeline based on comparative analysis of genomes of Candida species, together with manual identification of introns. We identified 5700 protein-coding genes in C. orthopsilosis, of which 5570 have an ortholog in C. parapsilosis. The time of divergence between C. orthopsilosis and C. parapsilosis is estimated to be twice as great as that between Candida albicans and Candida dubliniensis. There has been an expansion of the Hyr/Iff family of cell wall genes and the JEN family of monocarboxylic transporters in C. parapsilosis relative to C. orthopsilosis. We identified one gene from a Maltose/Galactoside O-acetyltransferase family that originated by horizontal gene transfer from a bacterium to the common ancestor of C. orthopsilosis and C. parapsilosis. We report that TFB3, a component of the general transcription factor TFIIH, undergoes alternative splicing by intron retention in multiple Candida species. We also show that an intein in the vacuolar ATPase gene VMA1 is present in C. orthopsilosis but not C. parapsilosis, and has a patchy distribution in Candida species. Our results suggest that the difference in virulence between C. parapsilosis and C. orthopsilosis may be associated with expansion of gene families.

  4. Phylogenomic Methods to Guide Paleontological Searches for the Early Cyanobacteria

    Science.gov (United States)

    Blank, C. E.

    2004-12-01

    Phylogenomic methods can help paleontologists target their searches for early microbial microfossils and potentially help them better interpret the early fossil record. In this study, the deep-branching relationships in the cyanobacteria were resolved using whole genome sequences, multiple genes for taxa lacking genomes, and intein presence/absence in the DnaE protein. Once a framework tree was produced, characters were mapped onto the tree. Characters included morphology (unicellular vs. filamentous), habitat (marine vs. freshwater), metabolism (use of sulfide as electron donor, nitrogen fixation), presence/absence of complex morphological traits (akinetes, heterocysts, hormogonia), salt tolerance, and thermal tolerance. It was found that the earliest cyanobacteria were unicellular coccoids, with cell diameters cyanobacteria to freshwater deposits (lakes, streams) and to small diameter coccoids (not mats, not filaments). The earliest "cyanobacterial" microfossils (Eosynechococcus and Eoentophysalis) are large-diameter coccoids found in shallow marine platform carbonates. Because these cells have large diameters, if they were cyanobacteria one would also expect to see their sister taxa in the fossil record (i.e., large-diameter filamentous forms with sheaths, also akinetes). Because these are not found until 2.0 Ga (and akinetes until 1.5 Ga), this suggests that these earliest microfossils are not cyanobacteria. There are several instances in the cyanobacterial tree where ancestors with low salt tolerance gave rise to lineages that grow in brackish, marine, and/or hypersaline environments. This suggests that either the cyanobacteria first originated on continents and later colonized more saline environments, or that the cyanobacteria first originated in shallow "seas" that were not very saline but gradually became more saline by about 2.0 Ga. Because the continents were likely harsh environments (due to lack of an ozone layer and increased chemical and physical

  5. Study on CCR5 analogs and affinity peptides.

    Science.gov (United States)

    Wu, Yingping; Deng, Riqiang; Wu, Wenyan

    2012-03-01

    The G protein-coupled receptor of human chemokine receptor 5 (CCR5) is a key target in the human immunodeficiency virus (HIV) infection process due to its major involvement in binding to the HIV type 1 (HIV-1) envelope glycoprotein gp120 and facilitating virus entry into the cells. The identification of naturally occurring CCR5 mutations (especially CCR5 delta-32) has allowed us to address the CCR5 molecule as a promising target to prevent or resist HIV infection in vivo. To obtain high-affinity peptides that can be used to block CCR5, CCR5 analogs with high conformational similarity are required. In this study, two recombinant proteins named CCR5 N-Linker-E2 and CCR5 mN-E1-E2 containing the fragments of the CCR5 N-terminal, the first extracellular loop or the second extracellular loop are cloned from a full-length human CCR5 cDNA. The recombinant human CCR5 analogs with self-cleavage activity of the intein Mxe or Ssp in the vector pTwinI were then produced with a high-yield expression and purification system in Escherichia coli. Experiments of extracellular epitope-activity identification (such as immunoprecipitation and indirective/competitive enzyme-linked immunosorbent assay) confirmed the close similarity between the epitope activity of the CCR5 analogs and that of the natural CCR5, suggesting the applicability of the recombinant CCR5 analogs as antagonists of the chemokine ligands. Subsequent screening of high-affinity peptides from the phage random-peptides library acquired nine polypeptides, which could be used as CCR5 peptide antagonists. The CCR5 analogs and affinity peptides elucidated in this paper provide us with a basis for further study of the mechanism of inhibition of HIV-1 infection.

  6. Site-specific immobilization of enzymes on magnetic nanoparticles and their use in organic synthesis.

    Science.gov (United States)

    Yu, Ching-Ching; Kuo, Yu-Ying; Liang, Chien-Fu; Chien, Wei-Ting; Wu, Huan-Ting; Chang, Tsung-Che; Jan, Fan-Dan; Lin, Chun-Cheng

    2012-04-18

    Magnetic nanoparticles (MNPs) are attractive materials that serve as a support for enzyme immobilization and facilitate separations by applying an external magnetic field; this could facilitate the recycling of enzymes and broaden their applications in organic synthesis. Herein, we report the methods for the immobilization of water-soluble and membrane-bound enzymes, and the activity difference between free and immobilized enzymes is discussed. Sialyltransferase (PmST1, from Pasteurella multocida ) and cytidine monophosphate (CMP)-sialic acid synthetase (CSS, from Neisseria meningitides ) were chosen as water-soluble enzymes and expressed using an intein expression system. The enzymes were site-specifically and covalently immobilized on PEGylated-N-terminal cysteine MNPs through native chemical ligation (NCL). Increasing the length of the PEG linker between the enzyme and the MNP surface increased the activity of the immobilized enzymes relative to the free parent enzymes. In addition, the use of a fluorescent acceptor tag for PmST1 affected enzyme kinetics. In contrast, sialyltransferase from Neisseria gonorrheae (NgST, a membrane-bound enzyme) was modified with a biotin-labeled cysteine at the C-terminus using NCL, and the enzyme was then assembled on streptavidin-functionalized MNPs. Using a streptavidin-biotin interaction, it was possible to immobilize NgST on a solid support under mild ligation conditions, which prevented the enzyme from high-temperature decomposition and provided an approximately 2-fold increase in activity compared to other immobilization methods on MNPs. Finally, the ganglioside GM3-derivative (sialyl-lactose derivative) was synthesized in a one-pot system by combining the use of immobilized PmST1 and CSS. The enzymes retained 50% activity after being reused ten times. Furthermore, the results obtained using the one-pot two-immobilized-enzyme system demonstrated that it can be applied to large-scale reactions with acceptable yields and

  7. Replication factor C from the hyperthermophilic archaeon Pyrococcus abyssi does not need ATP hydrolysis for clamp-loading and contains a functionally conserved RFC PCNA-binding domain.

    Science.gov (United States)

    Henneke, Ghislaine; Gueguen, Yannick; Flament, Didier; Azam, Philippe; Querellou, Joël; Dietrich, Jacques; Hübscher, Ulrich; Raffin, Jean-Paul

    2002-11-08

    The molecular organization of the replication complex in archaea is similar to that in eukaryotes. Only two proteins homologous to subunits of eukaryotic replication factor C (RFC) have been detected in Pyrococcus abyssi (Pab). The genes encoding these two proteins are arranged in tandem. We cloned these two genes and co-expressed the corresponding recombinant proteins in Escherichia coli. Two inteins present in the gene encoding the small subunit (PabRFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the PabRFC-small subunit could be purified, while the large subunit (PabRFC-large) alone was completely insoluble. The highly purified PabRFC complex possessed an ATPase activity, which was not enhanced by DNA. The Pab proliferating cell nuclear antigen (PCNA) activated the PabRFC complex in a DNA-dependent manner, but the PabRFC-small ATPase activity was neither DNA-dependent nor PCNA-dependent. The PabRFC complex was able to stimulate PabPCNA-dependent DNA synthesis by the Pabfamily D heterodimeric DNA polymerase. Finally, (i) the PabRFC-large fraction cross-reacted with anti-human-RFC PCNA-binding domain antibody, corroborating the conservation of the protein sequence, (ii) the human PCNA stimulated the PabRFC complex ATPase activity in a DNA-dependent way and (iii) the PabRFC complex could load human PCNA onto primed single-stranded circular DNA, suggesting that the PCNA-binding domain of RFC has been functionally conserved during evolution. In addition, ATP hydrolysis was not required either for DNA polymerase stimulation or PCNA-loading in vitro.

  8. Design, synthesis, and characterization of a 39 amino acid peptide mimic of the main immunogenic region of the Torpedo acetylcholine receptor.

    Science.gov (United States)

    Trinh, Vu B; Foster, Alex J; Fairclough, Robert H

    2014-05-01

    We have designed a 39 amino acid peptide mimic of the conformation-dependent main immunogenic region (MIR) of the Torpedo acetylcholine receptor (TAChR) that joins three discontinuous segments of the Torpedo α-subunit, α(1-12), α(65-79), and α(110 - 115) with two GS linkers: This 39MIR-mimic was expressed in E. coli as a fusion protein with an intein-chitin-binding domain (IChBD) to permit affinity collection on chitin beads. Six MIR-directed monoclonal antibodies (mAbs) bind to this complex and five agonist/antagonist site directed mAbs do not. The complex of MIR-directed mAb-132A with 39MIR has a Kd of (2.11±0.11)×10(-10)M, which is smaller than (7.13±1.20)×10(-10)M for the complex of mAb-132A with α(1-161) and about the same as 3.4×10(-10)M for that of mAb-132A with TAChR. Additionally, the 39MIR-IChBD adsorbs all MIR-directed antibodies (Abs) from an experimental autoimmune myasthenia gravis (EAMG) rat serum. Hence, the 39MIR-mimic has the potential to inactivate or remove pathogenic Torpedo MIR-directed Abs from EAMG sera and to direct a magic bullet to the memory B-cells that produce those pathogenic Abs. The hope is to use this as a guide to produce a mimic of the human MIR on the way to an antigen specific therapeutic agent to treat MG.

  9. Genus paracoccidioides: Species recognition and biogeographic aspects.

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    Raquel Cordeiro Theodoro

    Full Text Available BACKGROUND: Paracoccidioidomycosis is a systemic mycosis caused by Paracoccidioides brasiliensis (species S1, PS2, PS3, and Paracoccidioides lutzii. This work aimed to differentiate species within the genus Paracoccidioides, without applying multilocus sequencing, as well as to obtain knowledge of the possible speciation processes. METHODOLOGY/PRINCIPAL FINDINGS: Single nucleotide polymorphism analysis on GP43, ARF and PRP8 intein genes successfully distinguished isolates into four different species. Morphological evaluation indicated that elongated conidia were observed exclusively in P. lutzii isolates, while all other species (S1, PS2 and PS3 were indistinguishable. To evaluate the biogeographic events that led to the current geographic distribution of Paracoccidioides species and their sister species, Nested Clade and Likelihood Analysis of Geographic Range Evolution (LAGRANGE analyses were applied. The radiation of Paracoccidioides started in northwest South America, around 11-32 million years ago, as calculated on the basis of ARF substitution rate, in the BEAST program. Vicariance was responsible for the divergence among S1, PS2 and P. lutzii and a recent dispersal generated the PS3 species, restricted to Colombia. Taking into account the ancestral areas revealed by the LAGRANGE analysis and the major geographic distribution of L. loboi in the Amazon basin, a region strongly affected by the Andes uplift and marine incursions in the Cenozoic era, we also speculate about the effect of these geological events on the vicariance between Paracoccidioides and L. loboi. CONCLUSIONS/SIGNIFICANCE: The use of at least 3 SNPs, but not morphological criteria, as markers allows us to distinguish among the four cryptic species of the genus Paracoccidioides. The work also presents a biogeographic study speculating on how these species might have diverged in South America, thus contributing to elucidating evolutionary aspects of the genus Paracoccidioides.

  10. Changing the topology of protein backbone: the effect of backbone cyclization on the structure and dynamics of a SH3 domain

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    Frank H Schumann

    2015-04-01

    Full Text Available Understanding of the effects of the backbone cyclization on the structure and dynamics of a protein is essential for using protein topology engineering to alter protein stability and function. Here we have determined, for the first time, the structure and dynamics of the linear and various circular constructs of the N-SH3 domain from protein c-Crk. These constructs differ in the length and amino acid composition of the cyclization region. The backbone cyclization was carried out using intein-mediated intramolecular chemical ligation between the juxtaposed N- and the C-termini. The structure and backbone dynamics studies were performed using solution NMR. Our data suggest that the backbone cyclization has little effect on the overall three-dimensional structure of the SH3 domain: besides the termini, only minor structural changes were found in the proximity of the cyclization region. In contrast to the structure, backbone dynamics are significantly affected by the cyclization. On the subnanosecond time scale, the backbone of all circular constructs on average appears more rigid than that of the linear SH3 domain; this effect is observed over the entire backbone and is not limited to the cyclization site. The backbone mobility of the circular constructs becomes less restricted with increasing length of the circularization loop. In addition, significant conformational exchange motions (on the sub-millisecond time scale were found in the N-Src loop and in the adjacent β-strands in all circular constructs studied in this work. These effects of backbone cyclization on protein dynamics have potential implications for the stability of the protein fold and for ligand binding.

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

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    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

  12. pTWIN1-EgAgB8/3自剪切融合蛋白原核表达载体的构建及表达%Construction and expression of protein self-splicing prokaryotic expression vector pTWIN1-EgAgB8/3

    Institute of Scientific and Technical Information of China (English)

    张瑞妮; 吾拉木·马木提; 于春洋; 法蒂玛·木特力甫

    2013-01-01

    The objective was to establish expression vector pTWINl-AgB8/3 of a self-splicing prokaryotic expression system and obtain pure recombinant antigen of Echinococcus granulosus AgB8/3 (rEgAgB8/3). The specific primers were designed according to the published nucleotide sequence of EgAgB8/3 deposited in the GenBank database(AF362442). The nucle-otide sequence corresponding to the secreted form of EgAgB8/3 was amplified by PCR, and the product was directionally liga-ted into Prokaryotic Expression Vector pTWINl. The ligation was transferred into the competent cell ER2566, the fusion protein CBD-inteinl-EgAgB8/3 was expressed by inducing with IPTG, and the target protein rEgAgB8/3 was purified by chitin binding affinity purification column. The expression product and the target protein were analyzed by SDS-PAGE and western blot. Results showed that the EgAgB8/3 gene was successfully cloned and ligated into pTWINl to form protein self-splicing prokaryotic recombimant expression vector pTWINl-EgAgB8/3. The SDS-PAGE and western blot analysis showed that the fusion protein CBD-inteinl-EgAgB8/3 was expressed as soluble form. The chitin binding domain (CBD) and inteinl were removed from target protein rEgAgB8/3 at one step during the chitin column affinity purification. It suggested that the high level of soluble target protein rEgAgB8/3 with few additional amino acid residue was successfully purified by simple treatment after expression, which may allow us to keep the original amino acid sequence and possible activity of the target protein and facilitate the process of production of anti-rEgAgB8/3 monoclonal antibodies.%目的 表达获得较纯的细粒棘球绦虫AgB8/3重组抗原(rEgAgB8/3).方法 根据GeneBank登陆号(AF362442)下载目的 基因核酸序列,利用DNAman软件设计引物,对EgAgB8/3编码分泌型多肽片段的核酸序列进行PCR扩增,测序鉴定其正确性后定向连入原核表达质粒pTWIN1上,并转化至大肠杆菌ER2566,IPTG诱导表达CBD-intein

  13. Molecular biodiversity of cassava begomoviruses in Tanzania: evolution of cassava geminiviruses in Africa and evidence for East Africa being a center of diversity of cassava geminiviruses

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    Aveling TAS

    2005-03-01

    Full Text Available Abstract Cassava is infected by numerous geminiviruses in Africa and India that cause devastating losses to poor farmers. We here describe the molecular diversity of seven representative cassava mosaic geminiviruses (CMGs infecting cassava from multiple locations in Tanzania. We report for the first time the presence of two isolates in East Africa: (EACMCV-[TZ1] and EACMCV-[TZ7] of the species East African cassava mosaic Cameroon virus, originally described in West Africa. The complete nucleotide sequence of EACMCV-[TZ1] DNA-A and DNA-B components shared a high overall sequence identity to EACMCV-[CM] components (92% and 84%. The EACMCV-[TZ1] and -[TZ7] genomic components have recombinations in the same genome regions reported in EACMCV-[CM], but they also have additional recombinations in both components. Evidence from sequence analysis suggests that the two strains have the same ancient origin and are not recent introductions. EACMCV-[TZ1] occurred widely in the southern part of the country. Four other CMG isolates were identified: two were close to the EACMV-Kenya strain (named EACMV-[KE/TZT] and EACMV-[KE/TZM] with 96% sequence identity; one isolate, TZ10, had 98% homology to EACMV-UG2Svr and was named EACMV-UG2 [TZ10]; and finally one isolate was 95% identical to EACMV-[TZ] and named EACMV-[TZ/YV]. One isolate of African cassava mosaic virus with 97% sequence identity with other isolates of ACMV was named ACMV-[TZ]. It represents the first ACMV isolate from Tanzania to be sequenced. The molecular variability of CMGs was also evaluated using partial B component nucleotide sequences of 13 EACMV isolates from Tanzania. Using the sequences of all CMGs currently available, we have shown the presence of a number of putative recombination fragments that are more prominent in all components of EACMV than in ACMV. This new knowledge about the molecular CMG diversity in East Africa, and in Tanzania in particular, has led us to hypothesize about the

  14. 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.

  15. 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

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    Yaunori eNoguchi

    2016-03-01

    Full Text Available 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 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

  16. Efficacy of submucosal injection of different solutions inclusive blood components on mucosa elevation for endoscopic resection

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    Al-Taie OH

    2012-04-01

    Full Text Available Oliver H Al-Taie1, Yildiz Bauer2, Christoph G Dietrich3, Wolfgang Fischbach21Department of Internal Medicine, Sankt Elisabeth-Hospital, Gütersloh, 2Department of Internal Medicine II, Klinikum Aschaffenburg, Aschaffenburg, 3Department of Internal Medicine, Bethlehem-Hospital, Stolberg, GermanyBackground: Endoscopic resection has become the standard treatment for noninvasive gastrointestinal malignancies. In flat mucosal tumors, normal saline is frequently used for submucosal fluid injection in order to reduce the risk of complications during endoscopic resection. Recent studies have demonstrated longer-lasting mucosa elevation by injection of agents such as hyaluronic acid or glyceol, rather than normal saline. We investigated the efficacy of different blood components in comparison with other solutions for use as a submucosal fluid cushion.Methods: Normal saline, sodium hyaluronate, glyceol, hydroxyethyl starch, serum, plasma, and whole blood were evaluated for their effectiveness in creating a submucosal cushion. One milliliter of each solution was injected into the submucosa of 5 × 5 cm specimens of resected porcine stomach. Mucosa elevation was measured before and up to 60 minutes after injection.Results: The shortest duration of mucosa elevation was observed after injection of normal saline, glyceol, and 0.125% hyaluronic acid. A significantly longer duration was obtained after injection of hydroxyethyl starch, 0.25% and 0.5% hyaluronic acid, serum, and plasma. However, whole blood generated a longer-lasting mucosa elevation than all other agents.Conclusion: The results of the current study suggest that whole blood is more effective in generating long-lasting mucosa elevation than any other commonly used solution. Because autologous blood is readily available at almost no cost, this seems to be an optimal agent for creating the mucosa elevation needed for endoscopic resection. Further in vivo studies in humans are needed to clarify the

  17. Comets, meteors, and eclipses: Art and science in early Renaissance Italy

    Science.gov (United States)

    Olson, R. J. M.; Pasachoff, J. M.

    2002-11-01

    We discuss eight trecento (fourteenth century) paintings containing depictions of astronomical events to reveal the revolutionary advances made in both astronomy and naturalistic painting in early Renaissance Italy, noting that an artistic interest in naturalism predisposed these pioneering painters to make their scientific observations. In turn, the convincing representations of their observations of astronomical phenomena in works of art rendered their paintings more believable, convincing. Padua was already a renowned center for mathematics and nascent astronomy (which was separating from astrology) when Enrico Scrovegni commissioned the famous Florentine artist Giotto di Bondone to decorate his lavish family chapel (circa 1301-1303). Giotto painted a flaming comet in lieu of the traditional Star of Bethlehem in the Adoration of the Magi scene. Moreover, he painted a historical apparition that he recently had observed with a great accuracy even by modern standards. Halley's Comet of 1301 (Olson, 1979). While we do not know the identity of the artist's theological advisor, we discuss the possibility that Pietro d'Abano, the Paduan medical doctor and "astronomer" who wrote on comets, might have been influential. We also compare Giotto's blazing comet with two others painted by the artist's shop in San Francesco at Assisi (before 1316) and account for the differences. In addition, we discuss Giotto's pupil, Taddeo Gaddi, reputed to have been partially blinded by a solar eclipse, whose calamity may find expression in his frescoes in Santa Croce, Florence (1328-30; 1338?). Giotto also influenced the Sienese painter Pietro Lorenzetti, two of whose Passion cycle frescoes at Assisi (1316-20) contain dazzling meteor showers that reveal the artist's observed astronomical phenomena, such as the "radiant" effect of meteor showers, first recorded by Alexander von Humboldt in 1799 and only accepted in the nineteenth century. Lorenzetti also painted sporadic, independent

  18. Comets, Meteors, and Eclipses: Art and Science in Early Renaissance Italy (Invited)

    Science.gov (United States)

    Olson, R. J. M.; Pasachoff, J. M.

    1999-09-01

    We discuss several topics relating artists and their works with actual astronomical events in early Renaissance Italy to reveal the revolutionary advances made in both astronomy and naturalistic painting. Padua, where Galileo would eventually hold a chair at the University, was already by the fourteenth century (trecento) a renowned center for mathematics and nascent astronomy (which was separating from astrology). It is no wonder that when Enrico Scrovegni commissioned the famous Florentine artist Giotto di Bondone to decorate his lavish family chapel (c. 1303) that in the scene of the Adoration of the Magi Giotto painted a flaming comet in lieu of the traditional Star of Bethlehem. Moreover, he painted an historical apparition he recently had observed with a great understanding of its scientific structure: Halley's Comet of 1301 (since Olson's first publication of this idea in Scientific American we have expanded the argument in several articles and talks). While we do not know the identity of the artist's theological advisor, we discuss the possibility that Pietro d'Abano, the Paduan medical doctor and ``astronomer" who wrote on comets, might have been influential. We also compare Giotto's blazing comet with two others painted by the artist's shop in San Francesco at Assisi (before 1316) and account for the differences. In addition, we tackle the question how Giotto's pupil, Taddeo Gaddi, who is documented as having been partially blinded by lengthy unprotected observation of the partial phase of an annular solar eclipse, reflects his observations in his frescoes in Santa Croce, Florence (1328-30). Giotto also influenced the Sienese painter Pietro Lorenzetti, two of whose Passion cycle frescoes at Assisi (1316-20), contain dazzling meteor showers that hold important symbolic meanings in the cyle's argument but more importantly reveal that the artist observed astronomical phenomena, such as the ``radiant" effect, which was first recorded by Alexander von Humboldt

  19. Combination of radar and daily precipitation data to estimate meaningful sub-daily point precipitation extremes

    Science.gov (United States)

    Bárdossy, András; Pegram, Geoffrey

    2017-01-01

    Bethlehem from 1998 to 2003, whose scan at 1.5 km above ground [CAPPI] overlapped a dense (10 km spacing) set of 45 pluviometers recording in the same 6-year period. This valuable set of data was obtained from each of 37 selected radar pixels [1 km square in plan] which contained a pluviometer not masked out by the radar foot-print. The pluviometer data were also aggregated to daily totals, for the same purpose. The extremes obtained using disaggregation methods were compared to the observed extremes in a cross validation procedure. The unusual and novel goal was not to obtain the reproduction of the precipitation matching in space and time, but to obtain frequency distributions of the point extremes, which we found to be stable.

  20. 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

  1. CARACTERÍSTICAS QUE EVIDENCIAN EL IMPACTO EDUCATIVO Y CULTURAL, A CAUSA DEL FENÓMENO DEL DESPLAZAMIENTO FORZADO EN CÚCUTA

    Directory of Open Access Journals (Sweden)

    Hugo Alexander Vega Riaño

    2015-06-01

    of the displaced population and the Pastor of Bethlehem the city of Cucuta, Colombia neighborhoods. The object of study focuses on how the forced displacement transgresses in education and cul- ture in families and children. The study is approached from ethnography, allowing us to contextualize the phenomenon, addressing a theoretical foundation from an anthropological perspective and a shortcut to the subject study population approach: parents, children, subjects of displacement.

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

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    McCook JP

    2015-08-01

    Full Text Available 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 component in anti-aging cosmeceuticals, and has been shown to have anti-hyaluronidase activity. In this study we evaluated various commercial lots of sodium copper chlorophyllin complex to identify the primary small molecule constituents, and to test various sodium copper chlorophyllin complexes and their small molecule analog compounds for hyaluronidase inhibitory activity in vitro. Ascorbate analogs were tested in combination with copper chlorophyllin complexes for potential additive or synergistic activity. Materials and methods: For hyaluronidase activity assays, dilutions of test materials were evaluated for hydrolytic activity of hyaluronidase by precipitation of non-digested hyaluronate by measuring related turbidity at 595 nm. High-performance liquid chromatography and mass spectroscopy was used to analyze and identify the primary small molecule constituents in various old and new commercial lots of sodium copper chlorophyllin complex. Results: The most active small molecule component of sodium copper chlorophyllin complex was disodium copper isochlorin e4, followed by oxidized disodium copper isochlorin e4. Sodium copper chlorophyllin complex and copper isochlorin e4 disodium salt had hyaluronidase inhibitory activity down to 10 µg/mL. The oxidized form of copper isochlorin e4 disodium salt had substantial hyaluronidase inhibitory activity at 100 µg/mL but not at 10 µg/mL. Ascorbate derivatives did not enhance the hyaluronidase inhibitory activity of

  3. BiPAP呼吸机正压给氧对增强肺癌放疗疗效的随机对照试验%Effect of positive pressure ventilation on radiosensitivity of human pr imary lung cancers

    Institute of Scientific and Technical Information of China (English)

    熊玮; 沈寒放; 张蔚东

    2001-01-01

    Objective To investigate the effect of positive pressure ventilation on radiosensitivity of the patients suffering from primary lung cancer. Methods Thirty cases of lung cancer were randomly divided into two groups: ① combining therapy group: patients treated with posi tive pressure ventilation using BiPAP respirator and radiotherapy;② simple radi otherapy group. The changes of PaO2, SaO2, blood white cells and the cellula r immunological function were observed before and after treatment. Resul ts Nasal or naso-facial positive pressure ventilation with BiPAP respi rator increased PaO2 significantly with the maximum of 7.5 kPa, SaO2 was ma inteined at above 95%. No significant change for the cellular immunological func tion was found in the combining therapy group (P>0.05)and only one patient with leukopenia(6.7%). But in the radiotherapy group, the lymphocyte transfor mation efficiency and the ratio of CD4/CD8 were obviously decreased(P0.05),而对照组在放疗后淋巴细胞转化率、CD4及CD4/CD8比值明显降低(P<0.01)。③治疗组仅1例出现白细胞减少( 6.7%),而对照组有5例出现白细胞明显降低(33.3%),需用升白药物才能继续完成放疗。胃肠道反应发生较对照组明显减少。结论 BiPAP呼吸机经鼻面罩正压通气给氧辅助肺癌放疗有一定疗效,可明显提高机体氧分压,对骨髓及细胞免疫功能有一定保护作用,亦能减少放疗所引起的毒副作用。

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

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    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. Palliative care situation in Palestinian Authority.

    Science.gov (United States)

    Shawawra, Mousa; Khleif, Amal Dweib

    2011-04-01

    Palliative care is a very new concept in Palestine. In fact, it is still not applicable or provided within the Palestinian health care system. However, Al-Sadeel Society had organized a one day workshop in Bethlehem on November 2008 for the health professionals from the governmental and non-governmental sectors to initiate and introduce the idea of palliative care for the first time in Palestine. The general population of Palestine is approximately 2.4 millions (2007), with a life expectancy of 74.3 years of age, the death rate is 3.7 per 1000 population, having 8,910 deaths a year. Deaths due to cancer were 2,305 in five years (1999-2003), where 5,542 new cases were newly diagnosed in the same period. Health services available for cancer patients are hospital units either in patient or day care units. According to the ministry of health (MOH) statistics there are 75 beds in oncology departments in MOH hospitals; represent 2.7% of the total number of beds available, and 60 beds in daily care departments with an occupancy rate at 231.8%. There is no hospice or bereavement follow up care available for patients or their families. Despite the fact that the Palestinian culture is one of the cultures that respect and care for the elderly, but at the end of life, when the load of symptoms is high, most of the patient are care for at hospitals, and usually dye there, because the families are not able to care for their patients, and as there is no system for home care available for the Palestinian patients, and if it is available it is available in limited places and on private bases that are expensive and not affordable to the majority of patients, gross domestic product (GPD) per capita= 1,100 as 2007 estimates). We conducted a needs assessment survey within the only four facilities that provide care for the oncology patients in the West Bank and were filled by the direct health care providers. The results were expressing the fact that there is no palliative care service

  6. 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.

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    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

  7. Highly Efficient Preparation of Recombinant PACAP Derivate RDB and Preliminary Study on Its Improving Fat Cells Insulin Resistance%PACAP衍生多肽RDB的高效制备及其改善胰岛素抵抗作用的初步研究

    Institute of Scientific and Technical Information of China (English)

    罗天杰; 马义; 叶祖禄; 徐文娜; 饶磊; 洪岸

    2013-01-01

    为了制备垂体腺苷酸环化酶激活肽(PACAP)衍生多肽RDB并初步研究其改善脂肪细胞胰岛素抵抗的作用,利用基因工程技术,选用大肠杆菌偏爱密码子,以重叠延伸PCR方法合成RDB基因序列,定向插入到高效表达载体pKYB-MCS中,用大肠杆菌ER2566进行表达,融合蛋白经Chitin-Beads柱纯化后,利用β-巯基乙醇诱导蛋白内含肽的N端自剪切,释放目的肽,再用HPLC制备纯度较高的PACAP衍生多肽RDB,实现RDB的高效制备;利用制备的重组肽RDB研究其对胰岛素抵抗3T3-L1脂肪细胞的改善作用及初步机制.制备的重组肽RDB的Mr为3.990 k,纯度大于95%,其产率为17.3 mg/L发酵产物;制备的RDB可明显促进胰岛素抵抗的3T3-L1脂肪细胞的葡萄糖利用及信号分子IRS-1的表达.结果表明在确立的优化条件下可高效制备纯度较高的PACAP衍生多肽RDB(纯度≥95%),RDB能改善胰岛素抵抗3T3-L1脂肪细胞的胰岛素敏感性,其作用机制与胰岛素信号通路相关蛋白的表达有关.%To express the recombinant Pituitary adenylate cyclase-activating polypeptide(PACAP)derivate RDB and preliminary study on its role of improving fat cells insulin resistance,Using gene engineering technology,by PCR technology synthesizing the gene of RDB with preference codon of E. coli and the RDB gene was inserted into high efficiency expression vector pKYB-MCS. Expressed fusion proteins in E. coli ER2566 were purified with Chitin-Beads column. Fusion proteins binding on Chitin-Beads were cut on N-terminus of intein due to the induction ofβ-mercaptoethanol and the target peptide was released,and then highly purity PACAP derivate RDB was purified by HPLC,realized the highly efficient RDB preparation,the recombinant peptide RDB was used to study on the improving function in insulin resistance 3T3-L1 adipocytes. The molecular weight of RDB is 3.990k,its purity is greater than 95% and the yield of the RDB was 17. 3mg /L (fermentation

  8. 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

  9. 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