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Sample records for integrase a4 helix

  1. An unusual helix turn helix motif in the catalytic core of HIV-1 integrase binds viral DNA and LEDGF.

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    Hayate Merad

    Full Text Available BACKGROUND: Integrase (IN of the type 1 human immunodeficiency virus (HIV-1 catalyzes the integration of viral DNA into host cellular DNA. We identified a bi-helix motif (residues 149-186 in the crystal structure of the catalytic core (CC of the IN-Phe185Lys variant that consists of the alpha(4 and alpha(5 helices connected by a 3 to 5-residue turn. The motif is embedded in a large array of interactions that stabilize the monomer and the dimer. PRINCIPAL FINDINGS: We describe the conformational and binding properties of the corresponding synthetic peptide. This displays features of the protein motif structure thanks to the mutual intramolecular interactions of the alpha(4 and alpha(5 helices that maintain the fold. The main properties are the binding to: 1- the processing-attachment site at the LTR (long terminal repeat ends of virus DNA with a K(d (dissociation constant in the sub-micromolar range; 2- the whole IN enzyme; and 3- the IN binding domain (IBD but not the IBD-Asp366Asn variant of LEDGF (lens epidermal derived growth factor lacking the essential Asp366 residue. In our motif, in contrast to the conventional HTH (helix-turn-helix, it is the N terminal helix (alpha(4 which has the role of DNA recognition helix, while the C terminal helix (alpha(5 would rather contribute to the motif stabilization by interactions with the alpha(4 helix. CONCLUSION: The motif, termed HTHi (i, for inverted emerges as a central piece of the IN structure and function. It could therefore represent an attractive target in the search for inhibitors working at the DNA-IN, IN-IN and IN-LEDGF interfaces.

  2. The HIV-1 integrase α4-helix involved in LTR-DNA recognition is also a highly antigenic peptide element.

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    Sandy Azzi

    Full Text Available Monoclonal antibodies (MAbas constitute remarkable tools to analyze the relationship between the structure and the function of a protein. By immunizing a mouse with a 29mer peptide (K159 formed by residues 147 to 175 of the HIV-1 integrase (IN, we obtained a monoclonal antibody (MAba4 recognizing an epitope lying in the N-terminal portion of K159 (residues 147-166 of IN. The boundaries of the epitope were determined in ELISA assays using peptide truncation and amino acid substitutions. The epitope in K159 or as a free peptide (pep-a4 was mostly a random coil in solution, while in the CCD (catalytic core domain crystal, the homologous segment displayed an amphipathic helix structure (α4-helix at the protein surface. Despite this conformational difference, a strong antigenic crossreactivity was observed between pep-a4 and the protein segment, as well as K156, a stabilized analogue of pep-a4 constrained into helix by seven helicogenic mutations, most of them involving hydrophobic residues. We concluded that the epitope is freely accessible to the antibody inside the protein and that its recognition by the antibody is not influenced by the conformation of its backbone and the chemistry of amino acids submitted to helicogenic mutations. In contrast, the AA →Glu mutations of the hydrophilic residues Gln148, Lys156 and Lys159, known for their interactions with LTRs (long terminal repeats and inhibitors (5CITEP, for instance, significantly impaired the binding of K156 to the antibody. Moreover, we found that in competition ELISAs, the processed and unprocessed LTR oligonucleotides interfered with the binding of MAba4 to IN and K156, confirming that the IN α4-helix uses common residues to interact with the DNA target and the MAba4 antibody. This also explains why, in our standard in vitro concerted integration assays, MAba4 strongly impaired the IN enzymatic activity.

  3. Reversible Dimerization of Acid-Denatured ACBP Controlled by Helix A4

    DEFF Research Database (Denmark)

    Fieber, Wolfgang; Kragelund, Birthe Brandt; Meldal, Morten Peter;

    2005-01-01

    of dimers and revealed a cooperative stabilization of helix A4 in this process. This emphasizes its special role in the structure formation in the denatured state of ACBP. No dimers are formed in the presence of guanidine hydrochloride, which underlines the fundamental difference between the nature...

  4. Hamming distance geometry of a protein conformational space. Application to the clustering of a 4 ns molecular dynamics trajectory of the HIV-1 integrase catalytic core

    CERN Document Server

    Laboulais, C; Le Bret, M; Gabarro-Arpa, J; Laboulais, Cyril; Ouali, Mohammed; Bret, Marc Le; Gabarro-Arpa, Jacques

    2001-01-01

    Protein structures can be encoded into binary sequences, these are used to define a Hamming distance in conformational space: the distance between two different molecular conformations is the number of different bits in their sequences. Each bit in the sequence arises from a partition of conformational space in two halves. Thus, the information encoded in the binary sequences is also used to characterize the regions of conformational space visited by the system. We apply this distance and their associated geometric structures, to the clustering and analysis of conformations sampled during a 4 ns molecular dynamics simulation of the HIV-1 integrase catalytic core. The cluster analysis of the simulation shows a division of the trajectory into two segments of 2.6 and 1.4 ns length, which are qualitatively different: the data points to the fact that equilibration is only reached at the end of the first segment. Some length of the paper is devoted to compare the Hamming distance to the r.m.s. deviation measure. Th...

  5. The membrane localization domains of two distinct bacterial toxins form a 4-helix-bundle in solution.

    Science.gov (United States)

    Hisao, Grant S; Brothers, Michael C; Ho, Mengfei; Wilson, Brenda A; Rienstra, Chad M

    2017-03-01

    Membrane localization domain (MLD) was first proposed for a 4-helix-bundle motif in the crystal structure of the C1 domain of Pasteurella multocida toxin (PMT). This structure motif is also found in the crystal structures of several clostridial glycosylating toxins (TcdA, TcdB, TcsL, and TcnA). The Ras/Rap1-specific endopeptidase (RRSP) module of the multifunctional autoprocessing repeats-in-toxins (MARTX) toxin produced by Vibrio vulnificus has sequence homology to the C1-C2 domains of PMT, including a putative MLD. We have determined the solution structure for the MLDs in PMT and in RRSP using solution state NMR. We conclude that the MLDs in these two toxins assume a 4-helix-bundle structure in solution. © 2016 The Protein Society.

  6. Integrase and integration: biochemical activities of HIV-1 integrase

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    Deprez Eric

    2008-12-01

    Full Text Available Abstract Integration of retroviral DNA is an obligatory step of retrovirus replication because proviral DNA is the template for productive infection. Integrase, a retroviral enzyme, catalyses integration. The process of integration can be divided into two sequential reactions. The first one, named 3'-processing, corresponds to a specific endonucleolytic reaction which prepares the viral DNA extremities to be competent for the subsequent covalent insertion, named strand transfer, into the host cell genome by a trans-esterification reaction. Recently, a novel specific activity of the full length integrase was reported, in vitro, by our group for two retroviral integrases (HIV-1 and PFV-1. This activity of internal cleavage occurs at a specific palindromic sequence mimicking the LTR-LTR junction described into the 2-LTR circles which are peculiar viral DNA forms found during viral infection. Moreover, recent studies demonstrated the existence of a weak palindromic consensus found at the integration sites. Taken together, these data underline the propensity of retroviral integrases for binding symmetrical sequences and give perspectives for targeting specific sequences used for gene therapy.

  7. Biochemical characterization of novel retroviral integrase proteins.

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    Allison Ballandras-Colas

    Full Text Available Integrase is an essential retroviral enzyme, catalyzing the stable integration of reverse transcribed DNA into cellular DNA. Several aspects of the integration mechanism, including the length of host DNA sequence duplication flanking the integrated provirus, which can be from 4 to 6 bp, and the nucleotide preferences at the site of integration, are thought to cluster among the different retroviral genera. To date only the spumavirus prototype foamy virus integrase has provided diffractable crystals of integrase-DNA complexes, revealing unprecedented details on the molecular mechanisms of DNA integration. Here, we characterize five previously unstudied integrase proteins, including those derived from the alpharetrovirus lymphoproliferative disease virus (LPDV, betaretroviruses Jaagsiekte sheep retrovirus (JSRV, and mouse mammary tumor virus (MMTV, epsilonretrovirus walleye dermal sarcoma virus (WDSV, and gammaretrovirus reticuloendotheliosis virus strain A (Rev-A to identify potential novel structural biology candidates. Integrase expressed in bacterial cells was analyzed for solubility, stability during purification, and, once purified, 3' processing and DNA strand transfer activities in vitro. We show that while we were unable to extract or purify accountable amounts of WDSV, JRSV, or LPDV integrase, purified MMTV and Rev-A integrase each preferentially support the concerted integration of two viral DNA ends into target DNA. The sequencing of concerted Rev-A integration products indicates high fidelity cleavage of target DNA strands separated by 5 bp during integration, which contrasts with the 4 bp duplication generated by a separate gammaretrovirus, the Moloney murine leukemia virus (MLV. By comparing Rev-A in vitro integration sites to those generated by MLV in cells, we concordantly conclude that the spacing of target DNA cleavage is more evolutionarily flexible than are the target DNA base contacts made by integrase during integration

  8. Prediction of reorganization free energies for biological electron transfer: a comparative study of Ru-modified cytochromes and a 4-helix bundle protein.

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    Tipmanee, Varomyalin; Oberhofer, Harald; Park, Mina; Kim, Kwang S; Blumberger, Jochen

    2010-12-01

    The acceleration of electron transfer (ET) rates in redox proteins relative to aqueous solutes can be attributed to the protein's ability to reduce the nuclear response or reorganization upon ET, while maintaining sufficiently high electronic coupling. Quantitative predictions of reorganization free energy remain a challenge, both experimentally and computationally. Using density functional calculations and molecular dynamics simulation with an electronically polarizable force field, we report reorganization free energies for intraprotein ET in four heme-containing ET proteins that differ in their protein fold, hydrophilicity, and solvent accessibility of the electron-accepting group. The reorganization free energies for ET from the heme cofactors of cytochrome c and b(5) to solvent exposed Ru-complexes docked to histidine residues at the surface of these proteins fall within a narrow range of 1.2-1.3 eV. Reorganization free energy is significantly lowered in a designed 4-helix bundle protein where both redox active cofactors are protected from the solvent. For all ET reactions investigated, the major components of reorganization are the solvent and the protein, with the solvent contributing close to or more than 50% of the total. In three out of four proteins, the protein reorganization free energy can be viewed as a collective effect including many residues, each of which contributing a small fraction. These results have important implications for the design of artificial electron transport proteins. They suggest that reorganization free energy may in general not be effectively controlled by single point mutations, but to a large extent by the degree of solvent exposure of the ionizable cofactors.

  9. HIV-2 integrase variation in integrase inhibitor-naive adults in Senegal, West Africa.

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    Geoffrey S Gottlieb

    Full Text Available BACKGROUND: Antiretroviral therapy for HIV-2 infection is hampered by intrinsic resistance to many of the drugs used to treat HIV-1. Limited studies suggest that the integrase inhibitors (INIs raltegravir and elvitegravir have potent activity against HIV-2 in culture and in infected patients. There is a paucity of data on genotypic variation in HIV-2 integrase that might confer intrinsic or transmitted INI resistance. METHODS: We PCR amplified and analyzed 122 HIV-2 integrase consensus sequences from 39 HIV-2-infected, INI-naive adults in Senegal, West Africa. We assessed genetic variation and canonical mutations known to confer INI-resistance in HIV-1. RESULTS: No amino acid-altering mutations were detected at sites known to be pivotal for INI resistance in HIV-1 (integrase positions 143, 148 and 155. Polymorphisms at several other HIV-1 INI resistance-associated sites were detected at positions 72, 95, 125, 154, 165, 201, 203, and 263 of the HIV-2 integrase protein. CONCLUSION: Emerging genotypic and phenotypic data suggest that HIV-2 is susceptible to the new class of HIV integrase inhibitors. We hypothesize that intrinsic HIV-2 integrase variation at "secondary" HIV-1 INI-resistance sites may affect the genetic barrier to HIV-2 INI resistance. Further studies will be needed to assess INI efficacy as part of combination antiretroviral therapy in HIV-2-infected patients.

  10. Different Pathways Leading to Integrase Inhibitors Resistance

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    Thierry, Eloïse; Deprez, Eric; Delelis, Olivier

    2017-01-01

    Integrase strand-transfer inhibitors (INSTIs), such as raltegravir (RAL), elvitegravir, or dolutegravir (DTG), are efficient antiretroviral agents used in HIV treatment in order to inhibit retroviral integration. By contrast to RAL treatments leading to well-identified mutation resistance pathways at the integrase level, recent clinical studies report several cases of patients failing DTG treatment without clearly identified resistance mutation in the integrase gene raising questions for the mechanism behind the resistance. These compounds, by impairing the integration of HIV-1 viral DNA into the host DNA, lead to an accumulation of unintegrated circular viral DNA forms. This viral DNA could be at the origin of the INSTI resistance by two different ways. The first one, sustained by a recent report, involves 2-long terminal repeat circles integration and the second one involves expression of accumulated unintegrated viral DNA leading to a basal production of viral particles maintaining the viral information. PMID:28123383

  11. Multifunctional facets of retrovirus integrase

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    Duane; P; Grandgenett; Krishan; K; Pandey; Sibes; Bera; Hideki; Aihara

    2015-01-01

    The retrovirus integrase(IN) is responsible for integration of the reverse transcribed linear c DNA into the host DNA genome. First, IN cleaves a dinucleotide from the 3’ OH blunt ends of the viral DNA exposing the highly conserved CA sequence in the recessed ends. IN utilizes the 3’ OH ends to catalyze the concerted integration of the two ends into opposite strands of the cellular DNA producing 4 to 6 bp staggered insertions, depending on the retrovirus species. The staggered ends are repaired by host cell machinery that results in a permanent copy of the viral DNA in the cellular genome. Besides integration, IN performs other functions in the replication cycle of several studied retroviruses. The proper organization of IN within the viral internal core is essential for the correct maturation of the virus. IN plays a major role in reverse transcription by interacting directly with the reverse transcriptase and by binding to the viral capsid protein and a cellular protein. Recruitment of several other host proteins into the viral particle are also promoted by IN. IN assists with the nuclear transport of the preintegration complex across the nuclear membrane. With several retroviruses, IN specifically interacts with different host protein factors that guide the preintegration complex to preferentially integrate the viral genome into specific regions of the host chromosomal target. Human gene therapy using retrovirus vectors is directly affected by the interactions of IN with these host factors. Inhibitors directed against the human immunodeficiency virus(HIV) IN bind within the active site of IN containing viral DNA ends thus preventing integration and subsequent HIV/AIDS.

  12. Raltegravir: first in class HIV integrase inhibitor

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    Zelalem Temesgen

    2008-06-01

    Full Text Available Zelalem Temesgen1, Dawd S Siraj21Mayo Clinic, Rochester, MN, USA; 2East Carolina University Greenville, NC, USAAbstract: On October 16, 2007, the US Food and Drug Administration (FDA approved raltegravir for treatment of human immunodeficiency virus (HIV-1 infection in combination with other antiretroviral agents in treatment-experienced adult patients who have evidence of viral replication and HIV-1 strains resistant to multiple antiretroviral agents. Raltegravir is first in a novel class of antiretroviral drugs known as integrase inhibitors. It has demonstrated potent anti HIV activity in both antiretroviral treatment-naïve and experienced patients. The most common adverse events reported with raltegravir during phase 2 and 3 clinical trials were diarrhea, nausea, and headache. Laboratory abnormalities include mild elevations in liver transaminases and creatine phosphokinase.Keywords: raltegravir, HIV, antiretroviral agents, integrase inhibitors

  13. The hunt for HIV-1 integrase inhibitors.

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    Lataillade, Max; Kozal, Michael J

    2006-07-01

    Currently, there are three distinct mechanistic classes of antiretrovirals: inhibitors of the HIV- 1 reverse transcriptase and protease enzymes and inhibitors of HIV entry, including receptor and coreceptor binding and cell fusion. A new drug class that inhibits the HIV-1 integrase enzyme (IN) is in development and may soon be available in the clinic. IN is an attractive drug target because it is essential for a stable and productive HIV-1 infection and there is no mammalian homologue of IN. Inhibitors of integrase enzyme (INI) block the integration of viral double-stranded DNA into the host cell's chromosomal DNA. HIV-1 integration has many potential steps that can be inhibited and several new compounds that target specific integration steps have been identified by drug developers. Recently, two INIs, GS-9137 and MK-0518, demonstrated promising early clinical trial results and have been advanced into later stage trials. In this review, we describe how IN facilitates HIV-1 integration, the needed enzyme cofactors, and the resultant byproducts created during integration. Furthermore, we review the different INIs under development, their mechanism of actions, site of IN inhibition, potency, resistance patterns, and discuss the early clinical trial results.

  14. Discovery and analysis of inhibitors of the human immunodeficiency integrase.

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    Hazuda, D; Felock, P J; Hastings, J C; Pramanik, B; Wolfe, A L

    1997-05-01

    An essential step in the replication of retroviruses is the integration of a DNA copy of the viral genome into the genome of the host cell. Integration encompasses a series of ordered endonucleolytic and DNA strand transfer reactions catalyzed by the viral enzyme, integrase. The requirement for integrase activity in the propagation of HIV-1 in cell culture defines the enzyme as a potential target for chemotherapeutic intervention. We have therefore developed a non-radioisotopic microtiter plate assay which can be used to identify novel inhibitors of integrase from random chemical screens and for the bioassay driven isolation of inhibitors from natural products. This assay uncouples various steps in the reaction pathway and therefore can be exploited to characterize inhibitors. In this monograph we describe a series of modifications to the method which facilitate such mechanistic studies using as an example a series of previously described integrase inhibitors.

  15. Pyridoxine hydroxamic acids as novel HIV-integrase inhibitors.

    Science.gov (United States)

    Stranix, Brent R; Wu, Jinzi J; Milot, Guy; Beaulieu, Françis; Bouchard, Jean-Emanuel; Gouveia, Kristine; Forte, André; Garde, Seema; Wang, Zhigang; Mouscadet, Jean-François; Delelis, Olivier; Xiao, Yong

    2016-02-15

    A series of pyridoxine hydroxamic acid analog bearing a 5-aryl-spacers were synthesized. Evaluation of these novel HIV integrase complex inhibitors revealed compounds with high potency against wild-type HIV virus.

  16. Detection of integron integrase genes on King George Island, Antarctica

    Institute of Scientific and Technical Information of China (English)

    Vernica Antelo; Hctor Romero; Silvia Batista

    2015-01-01

    The presence and diversity of class 1 integrase gene (intI) sequences were evaluated by PCR using previously designed primers. Two clone libraries were constructed from DNA in sediment and microbial mat samples collected on Fildes Peninsula, King George Island, Antarctica.The libraries constructed from samples collected at Halfthree Point (HP) and Norma Cove (NC) contained 62 and 36 partial intI sequences, respectively. These sequences clustered into 10 different groups with <95% amino acid identity. Alignment of the deduced amino acid sequences with those from recognized integron-encoded integrases demonstrated the presence of highly conserved motifs characteristic of intI integrases. The HP library contained 42 nucleotide sequences identical to the class 1 intI gene found in a collection of trimethoprim-resistant (Tmpr) Antarctic Enterobacter sp. isolates, previously collected in the same area. These integrons, located on plasmids, had a genetic organization similar to that of pKOX105 from Klebsiella oxytoca. The 20 remaining HP and NC library sequences were similar to integrase sequences previously determined in a metagenomic analysis of environmental samples. We have demonstrated the presence of integron integrase genes in Antarctic sediment samples. About half these genes were very similar to the class 1 integrons found in human-associated microbiota, suggesting that they originated from human-dominated ecosystems. The remaining integrase genes were probably associated with endemic bacteria.

  17. Double-helix stellarator

    Energy Technology Data Exchange (ETDEWEB)

    Moroz, P.E.

    1997-09-01

    A new stellarator configuration, the Double-Helix Stellarator (DHS), is introduced. This novel configuration features a double-helix center post as the only helical element of the stellarator coil system. The DHS configuration has many unique characteristics. One of them is the extreme low plasma aspect ratio, A {approx} 1--1.2. Other advantages include a high enclosed volume, appreciable rotational transform, and a possibility of extreme-high-{beta} MHD equilibria. Moreover, the DHS features improved transport characteristics caused by the absence of the magnetic field ripple on the outboard of the torus. Compactness, simplicity and modularity of the coil system add to the DHS advantages for fusion applications.

  18. Waardecreatie in triple helix : Recepten voor triple helix samenwerking

    NARCIS (Netherlands)

    Vos, P.M.; Vries, F. de

    2016-01-01

    Om innovaties in het veiligheidsdomein te realiseren worden triple helix samenwerkingen gezien als een belangrijke motor. Een triple helix samenwerking is een tijdelijk samenwerkingsverband tussen drie of meer organisaties die middelen, risico’s en opbrengsten delen om individuele organisatiedoelen,

  19. COP9 signalosome subunit 6 binds and inhibits avian leukosis virus integrase.

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    Wang, Zhanxin; Xu, Aotian; Hou, Xinhui; Chen, Fuyong; Cao, Weisheng; Yu, Jieshi; Liao, Ming; Tang, Jun

    2014-10-24

    The retroviral integrase plays an essential role in the integration of reverse-transcribed retroviral cDNA into the host cell genome, and serves as an important target for anti-viral therapeutics. In this study, we identified the COP9 signalosome subunit 6 (CSN6) as a novel avian leukosis virus (ALV) integrase binding protein. Co-immunoprecipitation and GST pull-down assays showed that CSN6 bound to ALV integrase likely through direct interaction of CSN6 to the catalytic core of the integrase. We further demonstrated CSN6 inhibited integrase activity in vitro; knockdown of CSN6 in DF-1 promoted ALV production. These results indicated that CSN6 may be a negative regulator of ALV replication by binding to and inhibiting integrase. Our findings provided the insight into the integrase-based host defense system and may have implications in the development of integrase-based anti-viral strategies.

  20. Characterization of natural polymorphic sites of the HIV-1 integrase before the introduction of HIV-1 integrase inhibitors in Germany

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    Karolin Meixenberger

    2014-11-01

    Full Text Available Introduction: The aim of our study was to analyze the occurrence and evolution of HIV-1 integrase polymorphisms during the HIV-1 epidemic in Germany prior to the introduction of the first integrase inhibitor raltegravir in 2007. Materials and Methods: Plasma samples from drug-naïve HIV-1 infected individuals newly diagnosed between 1986 and 2006 were used to determine PCR-based population sequences of the HIV-1 integrase (amino acids 1–278. The HIV-1 subtype was determined using the REGA HIV-1 subtyping tool. We calculated the frequency of amino acids at each position of the HIV-1 integrase in 337 subtype B strains for the time periods 1986–1989, 1991–1994, 1995–1998, 1999–2002, and 2003–2006. Positions were defined as polymorphic if amino acid variation was >1% in any period. Logistic regression was used to identify trends in amino acid variation over time. Resistance-associated mutations were identified according to the IAS 2013 list and the HIVdb, ANRS and GRADE algorithms. Results: Overall, 56.8% (158/278 amino acid positions were polymorphic and 15.8% (25/158 of these positions exhibited a significant trend in amino acid variation over time. Proportionately, most polymorphic positions (63.3%, 31/49 were detected in the N-terminal zinc finger domain of the HIV-1 integrase. Motifs and residues essential for HIV-1 integrase activity were little polymorphic, but within the minimal non-specific DNA binding region I220-D270 up to 18.1% amino acid variation was noticed, including four positions with significant amino acid variation over time (S230, D232, D256, A265. No major resistance mutations were identified, and minor resistance mutations were rarely observed without trend over time. E157Q considered by HIVdb, ANRS, and GRADE algorithms was the most frequent resistance-associated polymorphism with an overall prevalence of 2.4%. Conclusions: Detailed knowledge of the evolutionary variation of HIV-1 integrase polymorphisms is

  1. Crystal structure of the bacteriophage P2 integrase catalytic domain.

    Science.gov (United States)

    Skaar, Karin; Claesson, Magnus; Odegrip, Richard; Högbom, Martin; Haggård-Ljungquist, Elisabeth; Stenmark, Pål

    2015-11-30

    Bacteriophage P2 is a temperate phage capable of integrating its DNA into the host genome by site-specific recombination upon lysogenization. Integration and excision of the phage genome requires P2 integrase, which performs recognition, cleavage and joining of DNA during these processes. This work presents the high-resolution crystal structure of the catalytic domain of P2 integrase, and analysis of the structure-function relationship of several previously identified non-functional P2 integrase mutants. The DNA binding area is characterized by a large positively charged patch, harboring key residues. The structure reveals potential for large dimer flexibility, likely essential for rearrangement of DNA strands upon integration and excision of the phage DNA.

  2. Ion-mediated nucleic acid helix-helix interactions.

    Science.gov (United States)

    Tan, Zhi-Jie; Chen, Shi-Jie

    2006-07-15

    Salt ions are essential for the folding of nucleic acids. We use the tightly bound ion (TBI) model, which can account for the correlations and fluctuations for the ions bound to the nucleic acids, to investigate the electrostatic free-energy landscape for two parallel nucleic acid helices in the solution of added salt. The theory is based on realistic atomic structures of the helices. In monovalent salt, the helices are predicted to repel each other. For divalent salt, while the mean-field Poisson-Boltzmann theory predicts only the repulsion, the TBI theory predicts an effective attraction between the helices. The helices are predicted to be stabilized at an interhelix distance approximately 26-36 A, and the strength of the attractive force can reach -0.37 k(B)T/bp for helix length in the range of 9-12 bp. Both the stable helix-helix distance and the strength of the attraction are strongly dependent on the salt concentration and ion size. With the increase of the salt concentration, the helix-helix attraction becomes stronger and the most stable helix-helix separation distance becomes smaller. For divalent ions, at very high ion concentration, further addition of ions leads to the weakening of the attraction. Smaller ion size causes stronger helix-helix attraction and stabilizes the helices at a shorter distance. In addition, the TBI model shows that a decrease in the solvent dielectric constant would enhance the ion-mediated attraction. The theoretical findings from the TBI theory agree with the experimental measurements on the osmotic pressure of DNA array as well as the results from the computer simulations.

  3. Cell type differences in activity of the Streptomyces bacteriophage phiC31 integrase.

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    Maucksch, Christof; Aneja, Manish Kumar; Hennen, Elisabeth; Bohla, Alexander; Hoffmann, Florian; Elfinger, Markus; Rosenecker, Joseph; Rudolph, Carsten

    2008-10-01

    Genomic integration by the Streptomyces bacteriophage C31 integrase is a promising tool for non-viral gene therapy of various genetic disorders. We investigated the C31 integrase recombination activity in T cell derived cell lines, primary T lymphocytes and CD34(+) haematopoietic stem cells in comparison to mesenchymal stem cells and cell lines derived from lung-, liver- and cervix-tissue. In T cell lines, enhanced long-term expression above control was observed only with high amounts of integrase mRNA. Transfections of C31 integrase plasmids were not capable of mediating enhanced long-term transgene expression in T cell lines. In contrast, moderate to high efficiency could be detected in human mesenchymal stem cells, human lung, liver and cervix carcinoma cell lines. Up to 100-fold higher levels of recombination product was found in C31 integrase transfected A549 lung than Jurkat T cells. When the C31 integrase activity was normalized to the intracellular integrase mRNA levels, a 16-fold difference was found. As one possible inhibitor of the C31 integrase, we found 3- to 5-fold higher DAXX levels in Jurkat than in A549 cells, which could in addition to other yet unknown factors explain the observed discrepancy of C31 integrase activity.

  4. Folding pathways of a helix-turn-helix model protein

    CERN Document Server

    Hoffmann, D

    1997-01-01

    A small model polypeptide represented in atomic detail is folded using Monte Carlo dynamics. The polypeptide is designed to have a native conformation similar to the central part of the helix-turn-helix protein ROP. Starting from a beta-strand conformation or two different loop conformations of the protein glutamine synthetase, six trajectories are generated using the so-called window move in dihedral angle space. This move changes conformations locally and leads to realistic, quasi-continuously evolving trajectories. Four of the six trajectories end in stable native-like conformations. Their folding pathways show a fast initial development of a helix-bend-helix motif, followed by a dynamic behaviour predicted by the diffusion-collision model of Karplus and Weaver. The phenomenology of the pathways is consistent with experimental results.

  5. Dialysis purification of integrase-DNA complexes provides high-resolution atomic force microscopy images: dimeric recombinant HIV-1 integrase binding and specific looping on DNA.

    Directory of Open Access Journals (Sweden)

    Tatsuaki Tsuruyama

    Full Text Available It remains difficult to obtain high-resolution atomic force microscopy images of HIV-1 integrase bound to DNA in a dimeric or tetrameric fashion. We therefore constructed specific target DNAs to assess HIV-1 integrase binding and purified the complex by dialysis prior to analysis. Our resulting atomic force microscopy analyses indicated precise size of binding human immunodeficiency virus type 1 (HIV-1 recombinant integrase in a tetrameric manner, inducing formation of a loop-like or figure-eight-like secondary structure in the target DNA. Our findings regarding the target DNA secondary structure provide new insights into the intermediate states of retroviral integration.

  6. Dialysis purification of integrase-DNA complexes provides high-resolution atomic force microscopy images: dimeric recombinant HIV-1 integrase binding and specific looping on DNA.

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    Tsuruyama, Tatsuaki; Nakai, Tonau; Ohmori, Rei; Ozeki, Munetaka; Tamaki, Keiji; Yoshikawa, Kenichi

    2013-01-01

    It remains difficult to obtain high-resolution atomic force microscopy images of HIV-1 integrase bound to DNA in a dimeric or tetrameric fashion. We therefore constructed specific target DNAs to assess HIV-1 integrase binding and purified the complex by dialysis prior to analysis. Our resulting atomic force microscopy analyses indicated precise size of binding human immunodeficiency virus type 1 (HIV-1) recombinant integrase in a tetrameric manner, inducing formation of a loop-like or figure-eight-like secondary structure in the target DNA. Our findings regarding the target DNA secondary structure provide new insights into the intermediate states of retroviral integration.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-11-10

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

  8. Modeling the HIV-1 Intasome: A Prototype View of the Target of Integrase Inhibitors

    Directory of Open Access Journals (Sweden)

    Robert Craigie

    2010-12-01

    Full Text Available The HIV-1 integrase enzyme is essential for integrating the viral DNA into the host chromosome. Infection is aborted in the absence of integration, making integrase an attractive antiviral target. Recently approved inhibitors of integrase bind tightly to integrase assembled in a nucleoprotein complex with the viral DNA ends (intasome, but have only low affinity for free integrase. High-resolution structures of HIV-1 intasomes are therefore required to understand the detailed mechanisms of inhibition and resistance. Although the structure of the HIV-1 intasome has not yet been determined, the structure of the related prototype foamy virus (PFV intasome was recently solved. A new study [1] exploits the PFV structure to model the HIV-1 intasome. The model provides the most reliable picture to date of the active site region of the HIV-1 intasome and is an important advance in studies of inhibition of this essential HIV-1 enzyme.

  9. Design, synthesis and biological activity of aromatic diketone derivatives as HIV-1 integrase inhibitors.

    Science.gov (United States)

    Hu, Liming; Li, Zhipeng; Wang, Zhanyang; Liu, Gengxin; He, Xianzhuo; Wang, Xiaoli; Zeng, Chengchu

    2015-01-01

    A series of aromatic diketone derivatives were designed and synthesized as potential HIV-1 integrase (IN) inhibitors and evaluated to determine their ability to inhibit the strand transfer process of HIV-1 integrase. The results indicate that (Z)-1-(3-acetyl-2-hydroxy-4,6-dimethoxyphenyl)-3-hydroxy-3-(substituted)phenylprop-2-en-1-one (5a-5d) can moderately inhibit HIV-1 integrase. The cyclization and condensation products (6a-6c and 7e-7f) of compounds 5a-5d show poor inhibitory activity against HIV-1 integrase. The molecular docking results indicate that the different types of compounds act on HIV-1 integrase in different ways, and these results can explain the differences in the inhibitory activities.

  10. Inibidores da HIV-integrase: potencial abordagem farmacológica para tratamento da AIDS HIV-integrase inhibitors: potential pharmacological approach for AIDS therapy

    Directory of Open Access Journals (Sweden)

    Eduardo Borges de Melo

    2006-06-01

    Full Text Available AIDS has the HIV as its etiological agent. Researches has been done to find new pharmacological agents to be used in therapy, because of problems of resistance and side effects. The HIV-integrase inhibitors are some of those new agents that are being studied. This updating focusses on the fundamental information about HIV and HIV-integrase and the main methods being used to develop these new drugs, with examples for each case.

  11. Use of Integrase-Minus Lentiviral Vector for Transient Expression

    Directory of Open Access Journals (Sweden)

    Hossein Azadeh

    2012-01-01

    Full Text Available Objective: Lentivirus-derived vectors are among the most promising viral vectors for gene therapy which is currently available, but their use in clinical practice is limited due to associated risk of insertional mutagenesis. Gene targeting is an ideal method for gene therapy, but it has low efficiency in comparison to viral vector methods. In this study, we are going to design and construct an integrase-minus lentiviral vector. This vector is suitable for transient expression of gene and gene targeting with viral vector.Materials and Methods: In this experimental study, three missense mutations were induced in the catalytic domain of Integrase gene in the pLP1 plasmid and resulted D64V, D116A and E152G changes in the amino acid sequence through site directed mutagenesis. The pLenti6.2-GW/EmGFP transfer vector, associated with native and mutated packaging mix, was transfected into 293T cell line. In order to titer the lentivirus stock, the viruses were harvested. Finally, the viruses transduced into COS-7 cell line to assess green fluorescent protein (GFP gene expression by a fluorescence microscopy.Results: Recombinant and wild lentiviruses titer was about 5~8×106 transducing units/ml in COS-7 cell line. The number of GFP-positive cells transduced with native viruses was decreased slightly during two weeks after viral transduction. In contrast, in the case of integrase-minus viruses, a dramatic decrease in the number of GFP positive cells was observed.Conclusion: This study was conducted to overcome the integration of lentiviral genome into a host genome. Nonintegrating lentiviral vectors can be used for transient gene expression and gene targeting if a Target gene cassette is placed in the lentivirus gene structure. This combination method decreases disadvantages of both processes, such as random integration of lentiviruses and low efficiency of gene targeting.

  12. Indole-based allosteric inhibitors of HIV-1 integrase.

    Science.gov (United States)

    Patel, Pratiq A; Kvaratskhelia, Nina; Mansour, Yara; Antwi, Janet; Feng, Lei; Koneru, Pratibha; Kobe, Mathew J; Jena, Nivedita; Shi, Guqin; Mohamed, Mosaad S; Li, Chenglong; Kessl, Jacques J; Fuchs, James R

    2016-10-01

    Employing a scaffold hopping approach, a series of allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) have been synthesized based on an indole scaffold. These compounds incorporate the key elements utilized in quinoline-based ALLINIs for binding to the IN dimer interface at the principal LEDGF/p75 binding pocket. The most potent of these compounds displayed good activity in the LEDGF/p75 dependent integration assay (IC50=4.5μM) and, as predicted based on the geometry of the five- versus six-membered ring, retained activity against the A128T IN mutant that confers resistance to many quinoline-based ALLINIs.

  13. Structural and Functional Insights into Foamy Viral Integrase

    Directory of Open Access Journals (Sweden)

    Cha-Gyun Shin

    2013-07-01

    Full Text Available Successful integration of retroviral DNA into the host chromosome is an essential step for viral replication. The process is mediated by virally encoded integrase (IN and orchestrated by 3'-end processing and the strand transfer reaction. In vitro reaction conditions, such as substrate specificity, cofactor usage, and cellular binding partners for such reactions by the three distinct domains of prototype foamy viral integrase (PFV-IN have been described well in several reports. Recent studies on the three‑dimensional structure of the interacting complexes between PFV-IN and DNA, cofactors, binding partners, or inhibitors have explored the mechanistic details of such interactions and shown its utilization as an important target to develop anti-retroviral drugs. The presence of a potent, non-transferable nuclear localization signal in the PFV C-terminal domain extends its use as a model for investigating cellular trafficking of large molecular complexes through the nuclear pore complex and also to identify novel cellular targets for such trafficking. This review focuses on recent advancements in the structural analysis and in vitro functional aspects of PFV-IN.

  14. Effect of HIV-1 Subtype C integrase mutations implied using molecular modeling and docking data

    Science.gov (United States)

    Sachithanandham, Jaiprasath; Konda Reddy, Karnati; Solomon, King; David, Shoba; Kumar Singh, Sanjeev; Vadhini Ramalingam, Veena; Alexander Pulimood, Susanne; Cherian Abraham, Ooriyapadickal; Rupali, Pricilla; Sridharan, Gopalan; Kannangai, Rajesh

    2016-01-01

    The degree of sequence variation in HIV-1 integrase genes among infected patients and their impact on clinical response to Anti retroviral therapy (ART) is of interest. Therefore, we collected plasma samples from 161 HIV-1 infected individuals for subsequent integrase gene amplification (1087 bp). Thus, 102 complete integrase gene sequences identified as HIV-1 subtype-C was assembled. This sequence data was further used for sequence analysis and multiple sequence alignment (MSA) to assess position specific frequency of mutations within pol gene among infected individuals. We also used biophysical geometric optimization technique based molecular modeling and docking (Schrodinger suite) methods to infer differential function caused by position specific sequence mutations towards improved inhibitor selection. We thus identified accessory mutations (usually reduce susceptibility) leading to the resistance of some known integrase inhibitors in 14% of sequences in this data set. The Stanford HIV-1 drug resistance database provided complementary information on integrase resistance mutations to deduce molecular basis for such observation. Modeling and docking analysis show reduced binding by mutants for known compounds. The predicted binding values further reduced for models with combination of mutations among subtype C clinical strains. Thus, the molecular basis implied for the consequence of mutations in different variants of integrase genes of HIV-1 subtype C clinical strains from South India is reported. This data finds utility in the design, modification and development of a representative yet an improved inhibitor for HIV-1 integrase.

  15. Absence of primary integrase resistance mutations in HIV type 1-infected patients in Venezuela.

    Science.gov (United States)

    Rangel, Héctor R; Garzaro, Domingo; Fabbro, Rona; Martinez, Nahir; Ossenkop, John; Torres, Jaime R; Gutiérrez, Cristina R; Pujol, Flor H

    2010-08-01

    The preexistence of mutations to integrase inhibitors in HIV-1-infected Venezuelan patients was evaluated. The integrase region of the HIV-1 genome was amplified by nested-PCR and sequenced in 57 isolates from both naive (n = 24) and treated patients who received protease and/or reverse transcriptase inhibitors (PI and RTI, n = 33), but were never exposed to integrase inhibitors. Only one primary integrase resistance mutation, not conferring drug resistance by itself, was found among these patients, although several minor viral mutations, equally distributed among naive and PI- and RTI-treated patients, were also found. In the limited number of samples, no relation was found among the presence of resistance mutations to PI or RTI and the presence of minor mutations to integrase. The absence of resistance to integrase inhibitors may be related to the recent introduction of these drugs in our country. The availability of in-house assays allows for a more comprehensive surveillance of drug resistance to integrase inhibitors in Venezuela.

  16. Interrogating HIV integrase for compounds that bind- a SAMPL challenge

    Science.gov (United States)

    Peat, Thomas S.; Dolezal, Olan; Newman, Janet; Mobley, David; Deadman, John J.

    2014-04-01

    Tremendous gains and novel methods are often developed when people are challenged to do something new or difficult. This process is enhanced when people compete against each other-this can be seen in sport as well as in science and technology (e.g. the space race). The SAMPL challenges, like the CASP challenges, aim to challenge modellers and software developers to develop new ways of looking at molecular interactions so the community as a whole can progress in the accurate prediction of these interactions. In order for this challenge to occur, data must be supplied so the prospective test can be done. We have supplied unpublished data related to a drug discovery program run several years ago on HIV integrase for the SAMPL4 challenge. This paper describes the methods used to obtain these data and the chemistry involved.

  17. Design of second generation HIV-1 integrase inhibitors.

    Science.gov (United States)

    Deng, Jinxia; Dayam, Raveendra; Al-Mawsawi, Laith Q; Neamati, Nouri

    2007-01-01

    The prospect of HIV-1 integrase (IN) as a therapeutically viable retroviral drug target is on the verge of realization. The observed preclinical and clinical performance of beta-diketo containing and naphthyridine carboxamide compounds provides direct proof for the clinical application of IN inhibition. These validated lead compounds are useful in the design and development of second generation IN inhibitors. The results from preclinical and clinical studies on the first generation IN inhibitors reiterate a demand for novel second generation inhibitors with improved pharmacokinetic and metabolic properties. Pharmacophore-based drug design techniques facilitate the discovery of novel compounds on the basis of validated lead compounds specific for a drug target. In this article we have comprehensively reviewed the application of pharmacophore-based drug design methods in the field of IN inhibitor discovery.

  18. HIV-1 integrase inhibitor resistance and its clinical implications.

    Science.gov (United States)

    Blanco, Jose-Luis; Varghese, Vici; Rhee, Soo-Yon; Gatell, Jose M; Shafer, Robert W

    2011-05-01

    With the approval in 2007 of the first integrase inhibitor (INI), raltegravir, clinicians became better able to suppress virus replication in patients infected with human immunodeficiency virus type 1 (HIV-1) who were harboring many of the most highly drug-resistant viruses. Raltegravir also provided clinicians with additional options for first-line therapy and for the simplification of regimens in patients with stable virological suppression. Two additional INIs in advanced clinical development-elvitegravir and S/GSK1349572-may prove equally versatile. However, the INIs have a relatively low genetic barrier to resistance in that 1 or 2 mutations are capable of causing marked reductions in susceptibility to raltegravir and elvitegravir, the most well-studied INIs. This perspective reviews the genetic mechanisms of INI resistance and their implications for initial INI therapy, the treatment of antiretroviral-experienced patients, and regimen simplification.

  19. Gated rotation mechanism of site-specific recombination by  C31 integrase

    National Research Council Canada - National Science Library

    Olorunniji, F. J; Buck, D. E; Colloms, S. D; McEwan, A. R; Smith, M. C. M; Stark, W. M; Rosser, S. J

    2012-01-01

    Integrases, such as that of the Streptomyces temperate bacteriophage [phi]C31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. [phi...

  20. The use of integrase inhibitors in treatment-experienced patients

    Directory of Open Access Journals (Sweden)

    Gatell Jose M

    2009-11-01

    Full Text Available Abstract Raltegravir, the first approved HIV-1 integrase inhibitor, is able to block the strand transfer step of the HIV proviral DNA integration process into the cellular host DNA. The selected dosage for the pivotal phase III studies (subsequently approved by the regulatory agencies was 400 mg bid by oral route with or without food. Raltegravir has a week effect (either inhibition or induction on the hepatic cytochrone P450 activity. There is not need of dose adjustments in renal insufficiency or in mild-to-moderate hepatic impairment. The emerging paradigm in the field of salvage therapy was to achieve a viral load below limit of detection in almost all patients. Pretty soon it became apparent that this was feasible in more than 70-90% of patients. Raltegravir proved to be pivotal for this new paradigm. Raltegravir vs placebo both with an optimized background therapy has been tested for salvage therapy in the 005 and in the BENCHMRK studies (018 and 019. In all three studies proved to be superior to the placebo at 24, 48 and 96 weeks. Tolerance was remarkably good and virological failure was often associated with selection of integrase gene resistance mutations following the Y143C/H/R, Q148H/K/R o less frequently the NI55H paths. Finally, in the two SWITCHMRK studies non-inferiority vs Lopinavir/r could not be demonstrated in virogically suppressed patients with an stable cART containing Lopinavir/r. Most likely explanation was the presence of archived resistance mutationts to background therapy leading to a functional monotherapy with raltegravir.

  1. Small molecule inhibitors of the LEDGF site of human immunodeficiency virus integrase identified by fragment screening and structure based design.

    Directory of Open Access Journals (Sweden)

    Thomas S Peat

    Full Text Available A fragment-based screen against human immunodeficiency virus type 1 (HIV integrase led to a number of compounds that bound to the lens epithelium derived growth factor (LEDGF binding site of the integrase catalytic core domain. We determined the crystallographic structures of complexes of the HIV integrase catalytic core domain for 10 of these compounds and quantitated the binding by surface plasmon resonance. We demonstrate that the compounds inhibit the interaction of LEDGF with HIV integrase in a proximity AlphaScreen assay, an assay for the LEDGF enhancement of HIV integrase strand transfer and in a cell based assay. The compounds identified represent a potential framework for the development of a new series of HIV integrase inhibitors that do not bind to the catalytic site of the enzyme.

  2. Molecular structure of the collagen triple helix.

    Science.gov (United States)

    Brodsky, Barbara; Persikov, Anton V

    2005-01-01

    The molecular conformation of the collagen triple helix confers strict amino acid sequence constraints, requiring a (Gly-X-Y)(n) repeating pattern and a high content of imino acids. The increasing family of collagens and proteins with collagenous domains shows the collagen triple helix to be a basic motif adaptable to a range of proteins and functions. Its rodlike domain has the potential for various modes of self-association and the capacity to bind receptors, other proteins, GAGs, and nucleic acids. High-resolution crystal structures obtained for collagen model peptides confirm the supercoiled triple helix conformation, and provide new information on hydrogen bonding patterns, hydration, sidechain interactions, and ligand binding. For several peptides, the helix twist was found to be sequence dependent, and such variation in helix twist may serve as recognition features or to orient the triple helix for binding. Mutations in the collagen triple-helix domain lead to a variety of human disorders. The most common mutations are single-base substitutions that lead to the replacement of one Gly residue, breaking the Gly-X-Y repeating pattern. A single Gly substitution destabilizes the triple helix through a local disruption in hydrogen bonding and produces a discontinuity in the register of the helix. Molecular information about the collagen triple helix and the effect of mutations will lead to a better understanding of function and pathology.

  3. Metabolism, excretion, and mass balance of the HIV-1 integrase inhibitor dolutegravir in humans.

    Science.gov (United States)

    Castellino, Stephen; Moss, Lee; Wagner, David; Borland, Julie; Song, Ivy; Chen, Shuguang; Lou, Yu; Min, Sherene S; Goljer, Igor; Culp, Amanda; Piscitelli, Stephen C; Savina, Paul M

    2013-08-01

    The pharmacokinetics, metabolism, and excretion of dolutegravir, an unboosted, once-daily human immunodeficiency virus type 1 integrase inhibitor, were studied in healthy male subjects following single oral administration of [(14)C]dolutegravir at a dose of 20 mg (80 μCi). Dolutegravir was well tolerated, and absorption of dolutegravir from the suspension formulation was rapid (median time to peak concentration, 0.5 h), declining in a biphasic fashion. Dolutegravir and the radioactivity had similar terminal plasma half-lives (t1/2) (15.6 versus 15.7 h), indicating metabolism was formation rate limited with no long-lived metabolites. Only minimal association with blood cellular components was noted with systemic radioactivity. Recovery was essentially complete (mean, 95.6%), with 64.0% and 31.6% of the dose recovered in feces and urine, respectively. Unchanged dolutegravir was the predominant circulating radioactive component in plasma and was consistent with minimal presystemic clearance. Dolutegravir was extensively metabolized. An inactive ether glucuronide, formed primarily via UGT1A1, was the principal biotransformation product at 18.9% of the dose excreted in urine and the principal metabolite in plasma. Two minor biotransformation pathways were oxidation by CYP3A4 (7.9% of the dose) and an oxidative defluorination and glutathione substitution (1.8% of the dose). No disproportionate human metabolites were observed.

  4. Microfluidic 3D Helix Mixers

    Directory of Open Access Journals (Sweden)

    Georgette B. Salieb-Beugelaar

    2016-10-01

    Full Text Available Polymeric microfluidic systems are well suited for miniaturized devices with complex functionality, and rapid prototyping methods for 3D microfluidic structures are increasingly used. Mixing at the microscale and performing chemical reactions at the microscale are important applications of such systems and we therefore explored feasibility, mixing characteristics and the ability to control a chemical reaction in helical 3D channels produced by the emerging thread template method. Mixing at the microscale is challenging because channel size reduction for improving solute diffusion comes at the price of a reduced Reynolds number that induces a strictly laminar flow regime and abolishes turbulence that would be desired for improved mixing. Microfluidic 3D helix mixers were rapidly prototyped in polydimethylsiloxane (PDMS using low-surface energy polymeric threads, twisted to form 2-channel and 3-channel helices. Structure and flow characteristics were assessed experimentally by microscopy, hydraulic measurements and chromogenic reaction, and were modeled by computational fluid dynamics. We found that helical 3D microfluidic systems produced by thread templating allow rapid prototyping, can be used for mixing and for controlled chemical reaction with two or three reaction partners at the microscale. Compared to the conventional T-shaped microfluidic system used as a control device, enhanced mixing and faster chemical reaction was found to occur due to the combination of diffusive mixing in small channels and flow folding due to the 3D helix shape. Thus, microfluidic 3D helix mixers can be rapidly prototyped using the thread template method and are an attractive and competitive method for fluid mixing and chemical reactions at the microscale.

  5. Multiple helix ecosystems for sustainable competitiveness

    CERN Document Server

    Ferreira, João; Farinha, Luís; Fernandes, Nuno

    2016-01-01

    This book discusses the main issues, challenges, opportunities, and trends involving the interactions between academia, industry, government and society. Specifically, it aims to explore how these interactions enhance the ways in which companies deliver products and services in order to achieve sustainable competitiveness in the marketplace. Sustainable competitiveness has been widely discussed by academics and practitioners, considering the importance of protecting the environment while sustaining the economic goals of organizations. The Quintuple Helix innovation model is a framework for facilitating knowledge, innovation and sustainable competitive advantage. It embeds the Triple and the Quadruple Helix models by adding a fifth helix, the “natural environment.” The Triple Helix model focuses on the university-industry-government triad, while the Quadruple adds civil society (the media- and culture-driven public) as a fourth helix. The Quintuple Helix model facilitates research, public policy, and pract...

  6. Dolutegravir Interactions with HIV-1 Integrase-DNA: Structural Rationale for Drug Resistance and Dissociation Kinetics

    Science.gov (United States)

    DeAnda, Felix; Hightower, Kendra E.; Nolte, Robert T.; Hattori, Kazunari; Yoshinaga, Tomokazu; Kawasuji, Takashi; Underwood, Mark R.

    2013-01-01

    Signature HIV-1 integrase mutations associated with clinical raltegravir resistance involve 1 of 3 primary genetic pathways, Y143C/R, Q148H/K/R and N155H, the latter 2 of which confer cross-resistance to elvitegravir. In accord with clinical findings, in vitro drug resistance profiling studies with wild-type and site-directed integrase mutant viruses have shown significant fold increases in raltegravir and elvitegravir resistance for the specified viral mutants relative to wild-type HIV-1. Dolutegravir, in contrast, has demonstrated clinical efficacy in subjects failing raltegravir therapy due to integrase mutations at Y143, Q148 or N155, which is consistent with its distinct in vitro resistance profile as dolutegravir’s antiviral activity against these viral mutants is equivalent to its activity against wild-type HIV-1. Kinetic studies of inhibitor dissociation from wild-type and mutant integrase-viral DNA complexes have shown that dolutegravir also has a distinct off-rate profile with dissociative half-lives substantially longer than those of raltegravir and elvitegravir, suggesting that dolutegravir’s prolonged binding may be an important contributing factor to its distinct resistance profile. To provide a structural rationale for these observations, we constructed several molecular models of wild-type and clinically relevant mutant HIV-1 integrase enzymes in complex with viral DNA and dolutegravir, raltegravir or elvitegravir. Here, we discuss our structural models and the posited effects that the integrase mutations and the structural and electronic properties of the integrase inhibitors may have on the catalytic pocket and inhibitor binding and, consequently, on antiviral potency in vitro and in the clinic. PMID:24146996

  7. Dolutegravir interactions with HIV-1 integrase-DNA: structural rationale for drug resistance and dissociation kinetics.

    Directory of Open Access Journals (Sweden)

    Felix DeAnda

    Full Text Available Signature HIV-1 integrase mutations associated with clinical raltegravir resistance involve 1 of 3 primary genetic pathways, Y143C/R, Q148H/K/R and N155H, the latter 2 of which confer cross-resistance to elvitegravir. In accord with clinical findings, in vitro drug resistance profiling studies with wild-type and site-directed integrase mutant viruses have shown significant fold increases in raltegravir and elvitegravir resistance for the specified viral mutants relative to wild-type HIV-1. Dolutegravir, in contrast, has demonstrated clinical efficacy in subjects failing raltegravir therapy due to integrase mutations at Y143, Q148 or N155, which is consistent with its distinct in vitro resistance profile as dolutegravir's antiviral activity against these viral mutants is equivalent to its activity against wild-type HIV-1. Kinetic studies of inhibitor dissociation from wild-type and mutant integrase-viral DNA complexes have shown that dolutegravir also has a distinct off-rate profile with dissociative half-lives substantially longer than those of raltegravir and elvitegravir, suggesting that dolutegravir's prolonged binding may be an important contributing factor to its distinct resistance profile. To provide a structural rationale for these observations, we constructed several molecular models of wild-type and clinically relevant mutant HIV-1 integrase enzymes in complex with viral DNA and dolutegravir, raltegravir or elvitegravir. Here, we discuss our structural models and the posited effects that the integrase mutations and the structural and electronic properties of the integrase inhibitors may have on the catalytic pocket and inhibitor binding and, consequently, on antiviral potency in vitro and in the clinic.

  8. Nucleic acid amplification of HIV-1 integrase sequence subtypes CRF01_AE and B for development of HIV anti-integrase drug resistance genotyping assay

    Science.gov (United States)

    Adlar, F. R.; Bela, B.

    2017-08-01

    To anticipate the potential use of anti-integrase drugs in Indonesia for treatment of HIV-1 infection, the development of a drug resistance genotyping assay for anti-integrase is crucial in identifying the genetic drug resistance profile of Indonesian HIV-1 strains. This experiment aimed to amplify a target region in the integrase gene of Indonesian HIV-1 subtypes CRF01_AE and B that contain genetic mutations known to confer resistance to anti-integrase drug. Eleven archived plasma samples from individuals living with HIV-1 were obtained from the Virology and Cancer Pathobiology Research Center for Health Service (VCPRC FKUI-RSCM) laboratory. One of the plasma samples contained HIV-1 subtype B, and the remaining plasma samples contained subtype CRF01_AE. The target regions for all samples were amplified through RT-PCR, with an annealing temperature of 55 °C, using the primer pair AE_POL 4086F and AE_POL 5232R that were designed by VCPRC FKUI-RSCM. The results of this experiment show that 18.2% (2/11) of the samples were successfully amplified using the one-step RT-PCR. While the primer pair was effective in amplifying the target region in the integrase gene sequence for subtype B (100%; 1/1), it had a low efficacy (10%, 1/10) for subtype CRF01_AE. In conclusion, the primer pair can be used to amplify the target region in Indonesian HIV-1 strain subtypes CRF01_AE and B. However, optimization of the PCR condition and an increased number of samples would help to determine an accurate representation of the efficacy of the primer pair.

  9. Transient gene expression mediated by integrase-defective retroviral vectors.

    Science.gov (United States)

    Yu, Seung Shin; Dan, Kazuyuki; Chono, Hideto; Chatani, Emi; Mineno, Junichi; Kato, Ikunoshin

    2008-04-18

    Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells.

  10. Structural basis for functional tetramerization of lentiviral integrase.

    Directory of Open Access Journals (Sweden)

    Stephen Hare

    2009-07-01

    Full Text Available Experimental evidence suggests that a tetramer of integrase (IN is the protagonist of the concerted strand transfer reaction, whereby both ends of retroviral DNA are inserted into a host cell chromosome. Herein we present two crystal structures containing the N-terminal and the catalytic core domains of maedi-visna virus IN in complex with the IN binding domain of the common lentiviral integration co-factor LEDGF. The structures reveal that the dimer-of-dimers architecture of the IN tetramer is stabilized by swapping N-terminal domains between the inner pair of monomers poised to execute catalytic function. Comparison of four independent IN tetramers in our crystal structures elucidate the basis for the closure of the highly flexible dimer-dimer interface, allowing us to model how a pair of active sites become situated for concerted integration. Using a range of complementary approaches, we demonstrate that the dimer-dimer interface is essential for HIV-1 IN tetramerization, concerted integration in vitro, and virus infectivity. Our structures moreover highlight adaptable changes at the interfaces of individual IN dimers that allow divergent lentiviruses to utilize a highly-conserved, common integration co-factor.

  11. In vitro selection of G-rich RNA aptamers that target HIV-1 integrase

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Aptamers that interact with various HIV-1 proteins,such as reverse transcriptase,Rev,Tat protein,and nuclear capsule protein,have been prepared through SELEX (systematic evolution of ligands by ex-ponential enrichment) technique. However,there are few reports about the DNA or RNA aptamers that target HIV-1 integrase. In this investigation,we selected alternative RNA aptamers specific for the HIV-1 integrase by using a different binding buffer containing 10 mmol·L-1 MgCl2 and 100 mmol·L-1 KCl. Aptamer IN1,IN2,IN3 had similar and the highest Kd values from 145 to 239 nmol·L-1. Structural studies showed that they formed similar stem-loop structure. Deletion of any stem structure resulted in diminished affinity. In addition,structure probing study with antisense DNA indicated that the stem-loop structure in the random region was critical for integrase binding. Although aptamer IN1 failed to form G-quartet structure,it might directly interact with the DDE motif of integrase,which is the virus DNA-binding site,because G-quadruplex T40214 competitively inhibited the interaction between IN1 and integrase. Together,this study generated a novel RNA aptamer IN1,which could be useful in basic research and anti-HIV drug screening.

  12. Monoclonal antibodies against human immunodeficiency virus type 1 integrase: epitope mapping and differential effects on integrase activities in vitro.

    Science.gov (United States)

    Nilsen, B M; Haugan, I R; Berg, K; Olsen, L; Brown, P O; Helland, D E

    1996-01-01

    Human immunodeficiency virus type 1 (HIV-1) integrase (IN) catalyzes the integration of viral DNA into the host chromosome, an essential step in retroviral replication. As a tool to study the structure and function of this enzyme, monoclonal antibodies (MAbs) against HIV-1 IN were produced. Epitope mapping demonstrated that the 17 MAbs obtained could be divided into seven different groups, and the selection of MAbs representing these groups were tested for their effect on in vitro activities of IN. Four groups of MAbs recognized epitopes within the region of amino acids (aa) 1 to 16, 17 to 38, or 42 to 55 in and around the conserved HHCC motif near the N terminus of IN. MAbs binding to these epitopes inhibited end processing and DNA joining and either stimulated or had little effect on disintegration and reintegration activities of IN. Two MAbs binding to epitopes within the region of aa 56 to 102 in the central core or aa 186 to 250 in the C-terminal half of the protein showed only minor effects on the in vitro activities of IN. Three Mabs which recognized on epitope within the region of aa262 to 271 of HIV-1 IN cross-reacted with HIV-2 IN. MAbs binding to this epitope clearly inhibited end processing and DNA joining and stimulated or had little effect on disintegration. In contrast to the N-terminal-specific MAbs, these C-terminal-specific MAbs abolished reintegration activity of IN. PMID:8627677

  13. Square Helix TWT for THz Frequencies

    DEFF Research Database (Denmark)

    Kotiranta, Mikko; Krozer, Viktor; Zhurbenko, Vitaliy

    2010-01-01

    A helix slow-wave structure with a square shape has been designed for use in traveling-wave tubes operating at terahertz frequencies. The square shape makes it compatible with microfabrication methods. A 3-D particle-in-cell simulation of a traveling-wave tube with such a helix indicates a gain...

  14. Kevlar: Transitioning Helix from Research to Practice

    Science.gov (United States)

    2015-04-01

    KEVLAR : TRANSITIONING HELIX FROM RESEARCH TO PRACTICE UNIVERSITY OF VIRGINIA APRIL 2015 FINAL TECHNICAL REPORT...DATES COVERED (From - To) FEB 2013 – NOV 2014 4. TITLE AND SUBTITLE KEVLAR : TRANSITIONING HELIX FROM RESEARCH TO PRACTICE 5a. CONTRACT NUMBER...possible exploitation. Our technology, called Kevlar , includes key security technologies are protective transformations and targeted recovery. The

  15. Thioamides in the collagen triple helix.

    Science.gov (United States)

    Newberry, Robert W; VanVeller, Brett; Raines, Ronald T

    2015-06-14

    To probe noncovalent interactions within the collagen triple helix, backbone amides were replaced with a thioamide isostere. This subtle substitution is the first in the collagen backbone that does not compromise thermostability. A triple helix with a thioamide as a hydrogen bond donor was found to be more stable than triple helices assembled from isomeric thiopeptides.

  16. Helix-packing motifs in membrane proteins.

    Science.gov (United States)

    Walters, R F S; DeGrado, W F

    2006-09-12

    The fold of a helical membrane protein is largely determined by interactions between membrane-imbedded helices. To elucidate recurring helix-helix interaction motifs, we dissected the crystallographic structures of membrane proteins into a library of interacting helical pairs. The pairs were clustered according to their three-dimensional similarity (rmsd universe of common transmembrane helix-pairing motifs is relatively simple. The largest cluster, which comprises 29% of the library members, consists of an antiparallel motif with left-handed packing angles, and it is frequently stabilized by packing of small side chains occurring every seven residues in the sequence. Right-handed parallel and antiparallel structures show a similar tendency to segregate small residues to the helix-helix interface but spaced at four-residue intervals. Position-specific sequence propensities were derived for the most populated motifs. These structural and sequential motifs should be quite useful for the design and structural prediction of membrane proteins.

  17. Inhibition of HIV-1 Integrase gene expression by 10-23 DNAzyme

    Indian Academy of Sciences (India)

    Nirpendra Singh; Atul Ranjan; Souvik Sur; Ramesh Chandra; Vibha Tandon

    2012-07-01

    HIV Integrase (IN) is an enzyme that is responsible for the integration of the proviral genome into the human genome, and this integration step is the first step of the virus hijacking the human cell machinery for its propagation and replication. 10-23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage. We have designed three novel DNAzymes, DIN54, DIN116, and DIN152, against HIV-1 Integrase gene using Mfold software and evaluated them for target site cleavage activity on the in vitro transcribed mRNA. All DNAzymes were tested for its inhibition of expression of HIV Integrase protein in the transiently transfected cell lines. DIN116 and DIN152 inhibited IN-EGFP expression by 80% and 70% respectively.

  18. Using ΦC31 integrase to mediate insertion of DNA in Xenopus embryos.

    Science.gov (United States)

    Li, You E; Allen, Bryan G; Weeks, Daniel L

    2012-01-01

    The two most common methods used to generate transgenic Xenopus embryos, restriction enzyme-mediated insertion, and I-SceI meganuclease take advantage of relatively common but spatially unpredictable double-stranded breaks in sperm, egg, or early embryo genomes. These methods also tend to insert multimeric copies of the transgene. An alternative is to use bacteriophage- or transposon-derived integrase or recombinase to mediate more site-specific insertion of the transgene. The use of phiC31 integrase requires a defined sequence for insertion and is compatible with insertion of a single copy of the transgene. We describe the protocol we use to facilitate phiC31 integrase transgene insertion including the use of insulator sequences to reduce position effect disruption of transgene activity.

  19. Subtype diversity associated with the development of HIV-1 resistance to integrase inhibitors.

    Science.gov (United States)

    Brenner, Bluma G; Lowe, Matthew; Moisi, Daniela; Hardy, Isabelle; Gagnon, Simon; Charest, Hugues; Baril, Jean Guy; Wainberg, Mark A; Roger, Michel

    2011-05-01

    We used genotypic and phylogenetic analysis to determine integrase diversity among subtypes, and studied natural polymorphisms and mutations implicated in resistance to integrase inhibitors (INI) in treatment-naïve persons (n = 220) and -experienced individuals (n = 24). Phylogenetics revealed 7 and 10% inter-subtype diversity in the integrase and reverse transcriptase (RT)/protease regions, respectively. Integrase sequencing identified a novel A/B recombinant in which all viruses in a male-sex-male (MSM) transmission cluster (n = 12) appeared to possess subtype B in integrase and subtype A in the remainder of the pol region. Natural variations and signature polymorphisms were observed at codon positions 140, 148, 151, 157, and 160 among HIV subtypes. These variations predicted higher genetic barriers to G140S and G140C in subtypes C, CRF02_AG, and A/CRF01_AE, as well as higher genetic barriers toward acquisition of V151I in subtypes CRF02_AG and A/CRF01_AE. The E157Q and E160Q mutational motif was observed in 35% of INI-naïve patients harboring subtype C infections, indicating intra-subtype variations. Thirteen patients failed raltegravir (RAL)-containing regimens within 8 ± 1 months, in association with the major Q148K/R/H and G140A/S (n = 8/24) or N155H (n = 5/24) mutational pathways. Of note, the remaining patients on RAL regimens for 14 ± 3 months harbored no or only minor integrase mutations/polymorphisms (T66I, T97A, H114P, S119P, A124S, G163R, I203M, R263K). These results demonstrate the importance of understanding subtype variability in the development of resistance to INIs.

  20. A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector.

    Directory of Open Access Journals (Sweden)

    Shigeyuki Yamaguchi

    Full Text Available The production of cells capable of expressing gene(s of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.

  1. A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector.

    Science.gov (United States)

    Yamaguchi, Shigeyuki; Kazuki, Yasuhiro; Nakayama, Yuji; Nanba, Eiji; Oshimura, Mitsuo; Ohbayashi, Tetsuya

    2011-02-24

    The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.

  2. Identification of minority resistance mutations in the HIV-1 integrase coding region using next generation sequencing

    DEFF Research Database (Denmark)

    Fonager, Jannik; Larsson, Jonas T; Hussing, Christian

    2015-01-01

    BACKGROUND: The current widely applied standard method to screen for HIV-1 genotypic resistance is based on Sanger population sequencing (Sseq), which does not allow for the identification of minority variants (MVs) below the limit of detection for the Sseq-method in patients receiving integrase......: raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG). STUDY DESIGN: NGS and Sseq were used to analyze RT-PCR products of the HIV-1 integrase coding region from six patients and in serial samples from two patients. NGS sequences were assembled and analyzed using the low frequency variant detection...

  3. HIV integrase inhibitors: from diketoacids to heterocyclic templates: a history of HIV integrase medicinal chemistry at Merck West Point and Merck Rome (IRBM).

    Science.gov (United States)

    Egbertson, Melissa S

    2007-01-01

    Replication of the human immunodeficiency virus (HIV) is dependent upon the enzyme HIV integrase (IN), one of three essential enzymes encoded in the viral genome. HIV-1 IN catalyzes the insertion of the proviral DNA into the host genome (strand transfer). HIV-1 IN therefore presents an attractive chemotherapeutic target for the treatment of HIV infection and AIDS that could provide patients and physicians with an additional option for treatment. Assays were developed to identify inhibitors of IN strand transfer. Diketoacid lead compounds were explored and developed into a variety of heterocyclic templates that are potent inhibitors of integrase strand transfer with suitable medicinal chemical properties for treating HIV infection and AIDS. The 1,6-naphthyridine L-870810 (Antiviral activity in cells IC(95) NHS = 102 nM, n=237), was shown to be efficacious in reducing viral RNA by 1.7 log units after doses of 400mg BID to HIV infected patients. Optimization of physical properties led to L-900564, an inhibitor of HIV IN that has excellent cell potency in the presence of protein (Antiviral activity in cells IC(95) NHS = 16 nM, n=15), excellent activity against mutants raised to HIV integrase inhibitors, and a very good pharmacokinetic profile.

  4. In vitro selection of G-rich RNA aptamers that target HIV-1 integrase

    Institute of Scientific and Technical Information of China (English)

    LIU YingChun; ZHANG Yan; YE GuoZhu; YANG ZhenJun; ZHANG LiangRen; ZHANG LiHe

    2008-01-01

    Aptamers that interact with various HIV-1 proteins, such as reverse transcriptase, Rev, Tat protein, and nuclear capsule protein, have been prepared through SELEX (systematic evolution of ligands by ex-ponential enrichment) technique. However, there are few reports about the DNA or RNA aptamers that target HIV-1 integrase. In this investigation, we selected alternative RNA aptamers specific for the HIV-1 Aptamer IN1, IN2, IN3 had similar and the highest Kd values from 145 to 239 nmol. L-1. Structural studies showed that they formed similar stem-loop structure. Deletion of any stem structure resulted in diminished affinity. In addition, structure probing study with antisense DNA indicated that the stem-loop structure in the random region was critical for integrase binding. Although aptamer IN1 failed to form G-quartet structure, it might directly interact with the DDE motif of integrase, which is the virus DNA-binding site, because G-quadruplex T40214 competitively inhibited the interaction between IN1 and integrase. Together, this study generated a novel RNA aptamer IN1, which could be useful in basic research and anti-HIV drug screening.

  5. Regulated gene insertion by steroid-induced PhiC31 integrase.

    Science.gov (United States)

    Sharma, Nynne; Moldt, Brian; Dalsgaard, Trine; Jensen, Thomas G; Mikkelsen, Jacob Giehm

    2008-06-01

    Nonviral integration systems are widely used genetic tools in transgenesis and play increasingly important roles in strategies for therapeutic gene transfer. Methods to efficiently regulate the activity of transposases and site-specific recombinases have important implications for their spatiotemporal regulation in live transgenic animals as well as for studies of their applicability as safe vectors for genetic therapy. In this report, strategies for posttranslational induction of a variety of gene-inserting proteins are investigated. An engineered hormone-binding domain, derived from the human progesterone receptor, hPR891, and specifically recognized by the synthetic steroid mifepristone, is fused to the Sleeping Beauty, Frog Prince, piggyBac and Tol2 transposases as well as to the Flp and PhiC31 recombinases. By analyzing mifepristone-directed inducibility of gene insertion in cultured human cells, efficient posttranslational regulation of the Flp recombinase and the PhiC31 integrase is documented. In addition, fusion of the PhiC31 integrase with the ER(T2) modified estrogen receptor hormone-binding domain results in a protein, which is inducible by a factor of 22-fold and retains 75% of the activity of the wild-type protein. These inducible PhiC31 integrase systems are important new tools in transgenesis and in safety studies of the PhiC31 integrase for gene therapy applications.

  6. Gated rotation mechanism of site-specific recombination by ϕC31 integrase.

    Science.gov (United States)

    Olorunniji, Femi J; Buck, Dorothy E; Colloms, Sean D; McEwan, Andrew R; Smith, Margaret C M; Stark, W Marshall; Rosser, Susan J

    2012-11-27

    Integrases, such as that of the Streptomyces temperate bacteriophage ϕC31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. ϕC31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a "subunit rotation" mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining att sites, and that many rounds of subunit rotation can occur before the strands are religated. We have analyzed the mechanism of ϕC31 integrase-mediated recombination in a topologically constrained experimental system using hybrid "phes" recombination sites, each of which comprises a ϕC31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180° rotation. However, multiple processive "360° rotation" rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory "gating" mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round.

  7. Cancer-specific binary expression system activated in mice by bacteriophage HK022 Integrase

    DEFF Research Database (Denmark)

    Elias, Amer; Spector, Itay; Sogolovsky-Bard, Ilana

    2016-01-01

    Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression of a ...

  8. Cancer-specific binary expression system activated in mice by bacteriophage HK022 Integrase

    DEFF Research Database (Denmark)

    Elias, Amer; Spector, Itay; Sogolovsky-Bard, Ilana;

    2016-01-01

    Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression...

  9. Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase.

    Science.gov (United States)

    Collis, C M; Hall, R M

    1992-01-01

    Deletion of individual antibiotic resistance genes found within the variable region of integrons is demonstrated. Evidence for gene duplications and rearrangements resulting from the insertion of gene units at new locations is also presented. Deletion, duplication, and rearrangement occur only in the presence of the integron-encoded DNA integrase. These events are precise and involve loss or gain of one or more complete insert units or gene cassettes. This confirms the recent definition of gene cassettes as consisting of the gene coding sequences, all except the last 7 bases of the 59-base element found at the 3' end of the gene, and the core site located 5' to the gene (Hall et al., Mol. Microbiol. 5:1941-1959, 1991) and demonstrates that individual gene cassettes are functional units which can be independently mobilized. Both deletions and duplications can be generated by integrase-mediated cointegrate formation followed by integrase-mediated resolution involving a different pair of sites. However, deletion occurs 10 times more frequently than duplication, and we propose that the majority of deletion events are likely to involve integrase-dependent excision of the gene unit to generate a circular gene cassette. The implications of these findings in understanding the evolution of integrons and the spread of antibiotic resistance genes in bacterial populations is discussed. Images PMID:1311297

  10. Rosalind Franklin and the Double Helix

    Science.gov (United States)

    Elkin, Lynne Osman

    2003-03-01

    Although she made essential contributions toward elucidating the structure of DNA, Rosalind Franklin is known to many only as seen through the distorting lens of James Watson's book, The Double Helix.

  11. The triple helix perspective of innovation systems

    NARCIS (Netherlands)

    Leydesdorff, L.; Zawdie, G.

    2010-01-01

    Alongside the neo-institutional model of networked relations among universities, industries, and governments, the triple helix can be provided with a neo-evolutionary interpretation as three selection environments operating upon one another: markets, organisations and technological opportunities. Ho

  12. Implications of integrase inhibitors for HIV-infected transplantation recipients: raltegravir and dolutegravir (S/GSK 1349572).

    Science.gov (United States)

    Waki, Kayo; Sugawara, Yasuhiko

    2011-01-01

    In the modern era of highly active antiretroviral therapy (HAART), reluctance to perform transplantation (Tx) in HIV-infected individuals is no longer justified. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) or protease inhibitors (PIs), the current first line regimens of HAART, are metabolized by the cytochrome P450 family (CYP3A4). Most NNRTIs induce CYP3A4, whereas PIs inhibit it. Calcinuerin inhibitors (CNIs), which are mandatory for Tx, need the same enzyme complex for their clearance. Therefore, a significant drug-drug interaction (DDI) is encountered between current HAART and CNIs. This results in extreme difficulty in adjusting the optimal dose of CNIs, for which the therapeutic range is narrow. Of interest, integrase inhibitors (INIs) - novel, potent anti-HIV drugs - are mainly metabolized by uridine diphosphate glucuronosyltransferase (UGT) 1A1 and do not induce or inhibit CYP3A4. DDI is presumably absent when NNTRIs or PIs are replaced by INIs. Raltegravir (RAL), a first generation INI, has been introduced into kidney and liver Tx. There is increasing evidence that rejection is well controlled without renal impairment due to CNI over-exposure while persistent, robust suppression of HIV is achieved. Global phase III clinical trials of dolutegravir (DTG), a second generation INI, are currently in progress. In vitro data has suggested that DTG may be less prone to resistance than RAL (referred to as having a higher genetic barrier). The time has come to extensively discuss the implications of INIs in Tx for HIV positive patients.

  13. Influence of Mg2+ ions on the interaction between 3,5-dicaffeoylquinic acid and HTLV-I integrase

    OpenAIRE

    2012-01-01

    Objective. Using molecular simulation, we studied the influence of Mg2+ ions on the binding mode of HTLV-I Integrase (IN) catalytic domain (modeled by homology) with the 3,5- Dicaffeoylquinic Acid (DCQA). HTLV-I Integrase homology model was built using template-like crystallographic data of the IN catalytic domain solved for Avian Sarcoma Virus (VSA, pdb: 1VSD). Materials and methods. In order to analyze the role of Mg2+ in the interaction or coupling between 3,5-DCQA and Integrase, three mod...

  14. Cylindrical Helix Spline Approximation of Spatial Curves

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In this paper, we present a new method for approximating spatial curves with a G1 cylindrical helix spline within a prescribed tolerance. We deduce the general formulation of a cylindrical helix,which has 11 freedoms. This means that it needs 11 restrictions to determine a cylindrical helix. Given a spatial parametric curve segment, including the start point and the end point of this segment, the tangent and the principal normal of the start point, we can always find a cylindrical segment to interpolate the given direction and position vectors. In order to approximate the known parametric curve within the prescribed tolerance, we adopt the trial method step by step. First, we must ensure the helix segment to interpolate the given two end points and match the principal normal and tangent of the start point, and then, we can keep the deviation between the cylindrical helix segment and the known curve segment within the prescribed tolerance everywhere. After the first segment had been formed, we can construct the next segment. Circularly, we can construct the G1 cylindrical helix spline to approximate the whole spatial parametric curve within the prescribed tolerance. Several examples are also given to show the efficiency of this method.

  15. Peptides derived from HIV-1 integrase that bind Rev stimulate viral genome integration.

    Directory of Open Access Journals (Sweden)

    Aviad Levin

    Full Text Available BACKGROUND: The human immunodeficiency virus type 1 (HIV-1 integrase protein (IN, catalyzes the integration of viral DNA into the host cell genome. IN catalyzes the first step of the integration process, namely the 3'-end processing in which IN removes a pGT dinucleotide from the 3' end of each viral long terminal repeat (LTR. Following nuclear import of the viral preintegration complex, the host chromosomal DNA becomes accessible to the viral cDNA and the second step of the integration process, namely the strand-transfer step takes place. This ordered sequence of events, centered on integration, is mandatory for HIV replication. METHODOLOGY/PRINCIPAL FINDINGS: Using an integrase peptide library, we selected two peptides, designated INr-1 and INr-2, which interact with the Rev protein and probably mediate the Rev-integrase interaction. Using an in-vitro assay system, we show that INr-1 and INr-2 are able to abrogate the inhibitory effects exerted by Rev and Rev-derived peptides on integrase activity. Both INr-1 and INr-2 were found to be cell-permeable and nontoxic, allowing a study of their effect in HIV-1-infected cultured cells. Interestingly, both INr peptides stimulated virus infectivity as estimated by production of the viral P24 protein, as well as by determination of the appearance of newly formed virus particles. Furthermore, kinetics studies revealed that the cell-permeable INr peptides enhance the integration process, as was indeed confirmed by direct determination of viral DNA integration by real-time PCR. CONCLUSIONS/SIGNIFICANCE: The results of the present study raise the possibility that in HIV-infected cells, the Rev protein may be involved in the integration of proviral DNA by controlling/regulating the activity of the integrase. Release from such inhibition leads to stimulation of IN activity and multiple viral DNA integration events.

  16. QSAR analysis on benzodithiazine derivatives as HIV-1 in-tegrase inhibitors

    Institute of Scientific and Technical Information of China (English)

    Ravichandran V; Jain A; Mourya VK; Agrawal RK

    2009-01-01

    Objective:Inhibition of HIV-1 integrase is an important strategy for the treatment of HIV and AIDS.There-fore,HIV-1 integrase inhibitory activity of 3-aroyl-1,1-dioxo-1,4,2-benzodithiazines has been analyzed with different physicochemical parameters.Methods:In the present work,quantitative structure activity relation-ship studies were performed on a series of benzodithiazines as HIV-1 integrase inhibitors using the modeling software Win CAChe version 6.1.Multiple linear regression analysis was performed to derive quantitative structure activity relationship models which were further evaluated for statistical significance and predictive power by internal and external validation.Results:The best QSAR models were having good correlation coeffi-cient (r)with low standard error of estimation (SEE)and cross validated square of correlation coefficient (q2 ).The robustness of the models was checked by Y-randomization test and they were identified as good pre-dictive models.The model for HIV integrase (wt)inhibitory activity of benzodithiazines suggest that the in-crease of dipole moment (Z)of molecules leads to reduce 3'processing and strand transfer inhibitory activity, substitution with high electro positive groups is conducive for the 3'processing inhibitory activity,and the in-crease in heat of formation is favorable for 3'-processing and strand transfer inhibitory activity.Conclusion:The model for HIV integrase (C65s)inhibitory activity of benzodithiazines suggest that the increase of dipole moment (X)of molecules leads to reduce 3'processing and strand transfer inhibitory activity,and the substitu-tion with high hydrophobic groups is conducive for the 3'processing and strand transfer inhibitory.

  17. Challenges in Targeting a Basic Helix-Loop-Helix Transcription Factor with Hydrocarbon-Stapled Peptides

    NARCIS (Netherlands)

    Edwards, Amanda L; Meijer, Dimphna H; Guerra, Rachel M; Molenaar, Remco J; Alberta, John A; Bernal, Federico; Bird, Gregory H; Stiles, Charles D; Walensky, Loren D

    2016-01-01

    Basic helix-loop-helix (bHLH) transcription factors play critical roles in organism development and disease by regulating cell proliferation and differentiation. Transcriptional activity, whether by bHLH homo- or heterodimerization, is dependent on protein-protein and protein-DNA interactions mediat

  18. Biochemical analysis of the basic helix-loop-helix transcription factor Olig2

    NARCIS (Netherlands)

    Meijer, D.H.M.

    2014-01-01

    The basic helix-loop-helix (bHLH) transcription factors oligodendrocyte transcription factor 1 (Olig1) and Olig2 are structurally similar and, to a first approximation, coordinately expressed in the developing CNS and postnatal brain. Notwithstanding these similarities, it was apparent from early on

  19. Nanoparticle biofabrication using English ivy (Hedera helix

    Directory of Open Access Journals (Sweden)

    Burris Jason N

    2012-10-01

    Full Text Available Abstract Background English ivy (Hedera helix is well known for its adhesive properties and climbing ability. Essential to its ability to adhere to vertical surfaces is the secretion of a nanocomposite adhesive containing spherical nanoparticles, 60–85 nm in diameter, produced exclusively by root hairs present on adventitious roots. These organic nanoparticles have shown promise in biomedical and cosmetic applications, and represent a safer alternative to metal oxide nanoparticles currently available. Results It was discovered that the maximum adventitious root production was achieved by a 4 h application of 1 mg/ml indole-3 butyric acid (IBA to juvenile English ivy shoot segments cultured in custom vessels. After incubation of the shoots under continuous light at 83 μmol/m2 s at 20°C for 2 weeks, the adventitious roots were harvested from the culture system and it was possible to isolate 90 mg of dry weight nanoparticles per 12 g of roots. The nanoparticle morphology was characterized by atomic force microscopy, and found to be similar to previous studies. Conclusions An enhanced system for the production of English ivy adventitious roots and their nanoparticles by modifying GA7 Magenta boxes and identifying the optimal concentration of IBA for adventitious root growth was developed. This system is the first such platform for growing and harvesting organic nanoparticles from plants, and represents an important step in the development of plant-based nanomanufacturing. It is a significant improvement on the exploitation of plant systems for the formation of metallic nanoparticles, and represents a pathway for the generation of bulk ivy nanoparticles for translation into biomedical applications.

  20. Nanoparticle biofabrication using English ivy (Hedera helix).

    Science.gov (United States)

    Burris, Jason N; Lenaghan, Scott C; Zhang, Mingjun; Stewart, C Neal

    2012-10-24

    English ivy (Hedera helix) is well known for its adhesive properties and climbing ability. Essential to its ability to adhere to vertical surfaces is the secretion of a nanocomposite adhesive containing spherical nanoparticles, 60-85 nm in diameter, produced exclusively by root hairs present on adventitious roots. These organic nanoparticles have shown promise in biomedical and cosmetic applications, and represent a safer alternative to metal oxide nanoparticles currently available. It was discovered that the maximum adventitious root production was achieved by a 4 h application of 1 mg/ml indole-3 butyric acid (IBA) to juvenile English ivy shoot segments cultured in custom vessels. After incubation of the shoots under continuous light at 83 μmol/m2 s at 20°C for 2 weeks, the adventitious roots were harvested from the culture system and it was possible to isolate 90 mg of dry weight nanoparticles per 12 g of roots. The nanoparticle morphology was characterized by atomic force microscopy, and found to be similar to previous studies. An enhanced system for the production of English ivy adventitious roots and their nanoparticles by modifying GA7 Magenta boxes and identifying the optimal concentration of IBA for adventitious root growth was developed. This system is the first such platform for growing and harvesting organic nanoparticles from plants, and represents an important step in the development of plant-based nanomanufacturing. It is a significant improvement on the exploitation of plant systems for the formation of metallic nanoparticles, and represents a pathway for the generation of bulk ivy nanoparticles for translation into biomedical applications.

  1. Prokaryotic transcription regulators: more than just the helix-turn-helix motif.

    Science.gov (United States)

    Huffman, Joy L; Brennan, Richard G

    2002-02-01

    Over the past two years, the structures of many prokaryotic transcriptional regulators have been solved, and several of them have revealed the structural mechanism of gene regulation. The crystal structure of BmrR-TPP-DNA reveals a novel mechanism of transcription activation, whereby the drug-bound protein activates the bmr promoter by local DNA unwinding and base pair disruption. Myristoyl-CoA induces FadR by a three-helix pushing mechanism, whereas TetR employs a helical pendulum motion to regulate expression. The structures of AbrB, and DNA complexes of Rob and MuR unveil a novel DNA-binding motif, 'the looped-hinge helix', and new uses of the helix-turn-helix and winged helix motifs in DNA binding.

  2. Optimization of Streptomyces bacteriophage phi C31 integrase system to prevent post integrative gene silencing in pulmonary type II cells.

    Science.gov (United States)

    Aneja, Manish Kumar; Geiger, Johannes; Imker, Rabea; Uzgun, Senta; Kormann, Michael; Hasenpusch, Guenther; Maucksch, Christof; Rudolph, Carsten

    2009-12-31

    phi C31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study aimed at optimizing elements of phi C31 integrase system for alveolar type II cells. Luciferase and beta-galactosidase activities were measured at different time points post transfection. 5-Aza-2'deoxycytidine (AZA) and trichostatin A (TSA) were used to inhibit DNA methyltransferase and histone deacetylase complex (HDAC) respectively. In A549 cells, expression of the integrase using a CMV promoter resulted in highest integrase activity, whereas in MLE12 cells, both CAG and CMV promoter were equally effective. Effect of polyA site was observed only in A549 cells, where replacement of SV40 polyA by bovine growth hormone (BGH) polyA site resulted in an enhancement of integrase activity. Addition of a C-terminal SV40 nuclear localization signal (NLS) did not result in any significant increase in integrase activity. Long-term expression studies with AZA and TSA, provided evidence for post-integrative gene silencing. In MLE12 cells, both DNA methylases and HDACs played a significant role in silencing, whereas in A549 cells, it could be attributed majorly to HDAC activity. Donor plasmids comprising cellular promoters ubiquitin B (UBB), ubiquitin C (UCC) and elongation factor 1 alpha (EF1 alpha) in an improved backbone prevented post-integrative gene silencing. In contrast to A549 and MLE12 cells, no silencing could be observed in human bronchial epithelial cells, BEAS-2B. Donor plasmid coding for murine erythropoietin under the EF1 alpha promoter when combined with phi C31 integrase resulted in higher long-term erythropoietin expression and subsequently higher hematocrit levels in mice after intravenous delivery to the lungs. These results provide evidence for cell specific post integrative gene silencing with C31 integrase and demonstrate the pivotal role of donor plasmid in long-term expression attained with this system.

  3. The collagen triple-helix structure.

    Science.gov (United States)

    Brodsky, B; Ramshaw, J A

    1997-03-01

    Recent advances, principally through the study of peptide models, have led to an enhanced understanding of the structure and function of the collagen triple helix. In particular, the first crystal structure has clearly shown the highly ordered hydration network critical for stabilizing both the molecular conformation and the interactions between triple helices. The sequence dependent nature of the conformational features is also under active investigation by NMR and other techniques. The triple-helix motif has now been identified in proteins other than collagens, and it has been established as being important in many specific biological interactions as well as being a structural element. The nature of recognition and the degree of specificity for interactions involving triple helices may differ from globular proteins. Triple-helix binding domains consist of linear sequences along the helix, making them amenable to characterization by simple model peptides. The application of structural techniques to such model peptides can serve to clarify the interactions involved in triple-helix recognition and binding and can help explain the varying impact of different structural alterations found in mutant collagens in diseased states.

  4. Triple helix interactions for eco-innovation

    DEFF Research Database (Denmark)

    Hermann, Roberto Rivas; Riisgaard, Henrik; Remmen, Arne

    Authority with insights from consultants, universities and donnor agencies. The proximity of the science park to the canal, has hitherto not yielded with the creation of a “green cluster”, which could be a precedent to promote eco-innovations. These findings suggest that, Triple Helix interactions...... the role of science parks in promoting eco-innovation. This study uses qualitative data gathered in two units of analysis: Panama Canal Authority and City of Knowledge Science Park. The study examines how Triple Helix interactions have built the regional system of eco-innovation at the Panama Canal...... are not institutionalized but take place through adhoc projects. Further, science parks could become mediators in Triple Helix interactions between industry, universities and governments....

  5. Cancer-specific binary expression system activated in mice by bacteriophage HK022 Integrase.

    Science.gov (United States)

    Elias, Amer; Spector, Itay; Sogolovsky-Bard, Ilana; Gritsenko, Natalia; Rask, Lene; Mainbakh, Yuli; Zilberstein, Yael; Yagil, Ezra; Kolot, Mikhail

    2016-04-27

    Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression of a cytotoxic gene. In the present study we developed a new cancer specific binary expression system activated by the Integrase (Int) of the lambdoid phage HK022. We demonstrate the validity of this system by the specific expression of a luciferase (luc) reporter in human embryonic kidney 293T (HEK293T) cells and in a lung cancer mouse model. Due to the absence viral vectors and of cytotoxicity the Int based binary system offers advantages over previously described counterparts and may therefore be developed into a safer cancer cell killing system.

  6. Development of a novel in vitro assay for the evaluation of integron DNA integrase activity

    Directory of Open Access Journals (Sweden)

    Fatemeh Tohidi

    2016-05-01

    Full Text Available Integrons play an important role in multidrug resistance. The integron platform codes for integrase (intI that is required for gene cassette integration through site-specific recombination. The recombination crossover occurs between the G and TT nucleotides in non-palindromic attI and palindromic attC sites. The aim of this study was to establish an efficient in vitro assay for integrase purification and activity detection. To this end, the intI gene was cloned into the pET-22b plasmid. Then, the resulting recombinant plasmid was transformed into Escherichia coli Origami™ strain. The recombinant protein expression was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE and western blot assays. The recombinant intI protein was purified by nickel–nitrilotriacetic acid (Ni–NTA affinity chromatography, and its activity was measured by a newly introduced assay. Briefly, specific primers for each side of attI and attC were used, thereby, a polymerase chain reaction would be performed, if a fused plasmid containing both attI and attC sites was created upon recombination. SDS-PAGE and western blotting confirmed the presence of a 38-kDa recombinant protein. Optimum conditions were established for the measurement of the integrase activity and a new model assay was conducted to analyse the recombination activity in vitro. Although the electrophoretic mobility shift assay is an efficient and reliable method, the newly introduced assay provided new or enhanced capability to determine the integrase activity, suggesting that there is no need for expensive and advanced equipment.

  7. The systemic effects of sclerostin overexpression using ΦC31 integrase in mice.

    Science.gov (United States)

    Zhang, Dongdong; Park, Bo Mi; Kang, Myengmo; Nam, HeeJin; Kim, Eun Jin; Bae, ChuHyun; Lim, Sung Kil

    2016-04-01

    Sclerostin, encoded by the Sost gene, is mainly produced by osteocytes in bone and antagonizes the Wnt/β-catenin signaling pathway, which is a requisite for bone formation. Currently, human anti-sclerostin antibodies are being tested in phase III clinical trials. In addition, serum sclerostin levels are reported to be associated with bone mineral density and fracture risk in normal individuals; however, the correlation between serum sclerostin and bone mass remains controversial. To study the effects of the continuous exposure of exogenous sclerostin on bone, a ΦC31 integrase system, which has the characteristics of site-specificity and efficiency, was applied for the delivery of the Sost gene in this study. We injected Sost-attB plasmid with or without ΦC31 integrase plasmid into the mouse tail vein using a hydrodynamic-based method. The site-specific integration of the Sost gene into the mouse genome was confirmed by examining a pseudo-attP site on the hepatic genomic DNA. Sclerostin was expressed in the hepatocytes, secreted into the blood flow, and maintained at high concentrations in the mice with both Sost-attB plasmid and ΦC31 integrase plasmid injections, which was observed by serial measurement. Moreover, the mice with long-term high levels of serum sclerostin showed trabecular bone loss on micro-CT analysis. Peripheral B cell populations were not affected. Our results suggested that sclerostin could be expressed in the liver and sustained successfully at high levels in the blood by using the ΦC31 integrase system, leading to trabecular bone loss. These findings may help to further ascertain the effects of sclerostin introduced exogenously on the skeleton.

  8. phiC31 integrase-mediated site-specific recombination in barley.

    Directory of Open Access Journals (Sweden)

    Eszter Kapusi

    Full Text Available The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP, which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F(1, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity.

  9. Development of conditionally replicating integrase defective lentiviral vectors for Epstein-Barr virus gene therapy

    OpenAIRE

    Blasi, Maria

    2012-01-01

    Integrase defective lentiviral vectors show promise for achieving gene expression without integration, preserving some benefits of LVs, whereas reducing the potentially detrimental risk of insertional mutagenesis. Numerous reports supported the ability of these vectors to confer long-term gene expression in slowly dividing cell types for potentially corrective gene expression. These reports also highlighted additional applications of these vectors as delivery platforms for alternate integrati...

  10. Optimization Design of Helix Pitch for Efficiency Enhancement in the Helix Travelling Wave Tubes

    Institute of Scientific and Technical Information of China (English)

    DUAN Zhao-Yun; GONG Yu-Bin; L(U) Ming-Yi; WEI Yan-Yu; WANG Wen-Xiang

    2008-01-01

    @@ The output section of a helix travelling wave tube usually contains a helix pitch taper for high rf electron efficiency.By keeping the rf field as synchronous as possible with the decelerating electron beam bunches,the rf field can extract much more energy from the beam,and thus the maximum electron efficiency can be realized.Recently,a global simulated annealing algorithm has been employed to design the helix pitch profile so as to improve the electron efficiency as much as possible.From the numerical results,it is concluded that the electron efficiency can be enhanced by about 4%-8%.

  11. Chirality-specific lift forces of helix under shear flows: Helix perpendicular to shear plane.

    Science.gov (United States)

    Zhang, Qi-Yi

    2017-02-01

    Chiral objects in shear flow experience a chirality-specific lift force. Shear flows past helices in a low Reynolds number regime were studied using slender-body theory. The chirality-specific lift forces in the vorticity direction experienced by helices are dominated by a set of helix geometry parameters: helix radius, pitch length, number of turns, and helix phase angle. Its analytical formula is given. The chirality-specific forces are the physical reasons for the chiral separation of helices in shear flow. Our results are well supported by the latest experimental observations.

  12. The superintegron integrase and the cassette promoters are co-regulated in Vibrio cholerae.

    Directory of Open Access Journals (Sweden)

    Evelyne Krin

    Full Text Available Chromosome 2 of Vibrio cholerae carries a chromosomal superintegron, composed of an integrase, a cassette integration site (attI and an array of mostly promoterless gene cassettes. We determined the precise location of the promoter, Pc, which drives the transcription of the first cassettes of the V. cholerae superintegron. We found that cassette mRNA starts 65 bp upstream of the attI site, so that the inversely oriented promoters Pc and Pint (integrase promoter partly overlap, allowing for their potential co-regulation. Pint was previously shown to be induced during the SOS response and is further controlled by the catabolite repression cAMP-CRP complex. We found that cassette expression from Pc was also controlled by the cAMP-CRP complex, but is not part of the SOS regulon. Pint and Pc promoters were both found to be induced in rich medium, at high temperature, high salinity and at the end of exponential growth phase, although at very different levels and independently of sigma factor RpoS. All these results show that expression from the integrase and cassette promoters can take place at the same time, thus leading to coordinated excisions and integrations within the superintegron and potentially coupling cassette shuffling to immediate selective advantage.

  13. Structural Properties of HIV Integrase. Lens Epithelium-derived Growth Factor Oligomers

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, K.; Diamond, T; Hwang, Y; Bushman, F; Van Duyne, G

    2010-01-01

    Integrase (IN) is the catalytic component of the preintegration complex, a large nucleoprotein assembly critical for the integration of the retroviral genome into a host chromosome. Although partial crystal structures of human immunodeficiency virus IN alone and its complex with the integrase binding domain of the host factor PSIP1/lens epithelium-derived growth factor (LEDGF)/p75 are available, many questions remain regarding the properties and structures of LEDGF-bound IN oligomers. Using analytical ultracentrifugation, multiangle light scattering, and small angle x-ray scattering, we have established the oligomeric state, stoichiometry, and molecular shapes of IN {center_dot} LEDGF complexes in solution. Analyses of intact IN tetramers bound to two different LEDGF truncations allow for placement of the integrase binding domain by difference analysis. Modeling of the small angle x-ray scattering envelopes using existing structural data suggests domain arrangements in the IN oligomers that support and extend existing biochemical data for IN {center_dot} LEDGF complexes and lend new insights into the quaternary structure of LEDGF-bound IN tetramers. These IN oligomers may be involved in stages of the viral life cycle other than integration, including assembly, budding, and early replication.

  14. Increasing Deaths Due to Malignancy in HIV+ Patients is Associated with Integrase Inhibitor Therapy

    Institute of Scientific and Technical Information of China (English)

    Daniel O. Griffin[1,2,3; Arif Dharsee[1; Juan Carlos Rico[1; Joseph McGowan[1

    2014-01-01

    Despite reductions in AIDS-related deaths, registries show HIV+ patients are still dying at a younger age than HIV-peers. Although overall mortality has declined in this population, a growing percent of deaths are due to malignancy. Since the data demonstrating the growing percentage of deaths due to malignancy in the HIV+ population is derived from registries, the study explores whether the subset dying from malignancy has particular characteristics that can be seen in a well-characterized cohort. In the well-characterized HIV+ cohort, the percentage of deaths due to cancer was seen to increase over four years (2010-2013) from 21% to 24% to 38% to 40%. The mean CD4-count of those who died from malignancy was 252+/-42 and 333+/-36 in patients with death from other causes. The viral load was not suppressed in 26% of patients dying from malignancy. Of patients on integrase inhibitor therapy, 48% of deaths were due to malignancy while in patients not on this therapy, 10% of deaths were due to malignancy (relative risk = 4.8). In HIV+ patients, a low CD4-count, failure to achieve viral suppression, and use of integrase inhibitors were associated with malignancy as the cause of death. The association of a specific therapy, integrase inhibition, with malignancy is seen in the study.

  15. Postexposure protection of macaques from vaginal SHIV infection by topical integrase inhibitors.

    Science.gov (United States)

    Dobard, Charles; Sharma, Sunita; Parikh, Urvi M; West, Rolieria; Taylor, Andrew; Martin, Amy; Pau, Chou-Pong; Hanson, Debra L; Lipscomb, Jonathan; Smith, James; Novembre, Francis; Hazuda, Daria; Garcia-Lerma, J Gerardo; Heneine, Walid

    2014-03-12

    Coitally delivered microbicide gels containing antiretroviral drugs are important for HIV prevention. However, to date, microbicides have contained entry or reverse transcriptase inhibitors that block early steps in virus infection and thus need to be given as a preexposure dose that interferes with sexual practices and may limit compliance. Integrase inhibitors block late steps after virus infection and therefore are more suitable for post-coital dosing. We first determined the kinetics of strand transfer in vitro and confirmed that integration begins about 6 hours after infection. We then used a repeat-challenge macaque model to assess efficacy of vaginal gels containing integrase strand transfer inhibitors when applied before or after simian/human immunodeficiency virus (SHIV) challenge. We showed that gel containing the strand transfer inhibitor L-870812 protected two of three macaques when applied 30 min before SHIV challenge. We next evaluated the efficacy of 1% raltegravir gel and demonstrated its ability to protect macaques when applied 3 hours after SHIV exposure (five of six protected; P test). Breakthrough infections showed no evidence of drug resistance in plasma or vaginal secretions despite continued gel dosing after infection. We documented rapid vaginal absorption reflecting a short pharmacological lag time and noted that vaginal, but not plasma, virus load was substantially reduced in the breakthrough infection after raltegravir gel treatment. We provide a proof of concept that topically applied integrase inhibitors protect against vaginal SHIV infection when administered shortly before or 3 hours after virus exposure.

  16. Rapid Optimization of Engineered Metabolic Pathways with Serine Integrase Recombinational Assembly (SIRA).

    Science.gov (United States)

    Merrick, C A; Wardrope, C; Paget, J E; Colloms, S D; Rosser, S J

    2016-01-01

    Metabolic pathway engineering in microbial hosts for heterologous biosynthesis of commodity compounds and fine chemicals offers a cheaper, greener, and more reliable method of production than does chemical synthesis. However, engineering metabolic pathways within a microbe is a complicated process: levels of gene expression, protein stability, enzyme activity, and metabolic flux must be balanced for high productivity without compromising host cell viability. A major rate-limiting step in engineering microbes for optimum biosynthesis of a target compound is DNA assembly, as current methods can be cumbersome and costly. Serine integrase recombinational assembly (SIRA) is a rapid DNA assembly method that utilizes serine integrases, and is particularly applicable to rapid optimization of engineered metabolic pathways. Using six pairs of orthogonal attP and attB sites with different central dinucleotide sequences that follow SIRA design principles, we have demonstrated that ΦC31 integrase can be used to (1) insert a single piece of DNA into a substrate plasmid; (2) assemble three, four, and five DNA parts encoding the enzymes for functional metabolic pathways in a one-pot reaction; (3) generate combinatorial libraries of metabolic pathway constructs with varied ribosome binding site strengths or gene orders in a one-pot reaction; and (4) replace and add DNA parts within a construct through targeted postassembly modification. We explain the mechanism of SIRA and the principles behind designing a SIRA reaction. We also provide protocols for making SIRA reaction components and practical methods for applying SIRA to rapid optimization of metabolic pathways.

  17. Helix reactor: great potential for flow chemistry

    NARCIS (Netherlands)

    Geerdink, P.; Runstraat, A. van den; Roelands, C.P.M.; Goetheer, E.L.V.

    2009-01-01

    The Helix reactor is highly suited for precise reaction control based on good hydrodynamics. The hydrodynamics are controlled by the Dean vortices, which create excellent heat transfer properties, approach plug flow and avoid turbulence. The flexibility of this reactor has been demonstrated using a

  18. A triple helix-loop-helix/basic helix-loop-helix cascade controls cell elongation downstream of multiple hormonal and environmental signaling pathways in Arabidopsis.

    Science.gov (United States)

    Bai, Ming-Yi; Fan, Min; Oh, Eunkyoo; Wang, Zhi-Yong

    2012-12-01

    Environmental and endogenous signals, including light, temperature, brassinosteroid (BR), and gibberellin (GA), regulate cell elongation largely by influencing the expression of the paclobutrazol-resistant (PRE) family helix-loop-helix (HLH) factors, which promote cell elongation by interacting antagonistically with another HLH factor, IBH1. However, the molecular mechanism by which PREs and IBH1 regulate gene expression has remained unknown. Here, we show that IBH1 interacts with and inhibits a DNA binding basic helix-loop-helix (bHLH) protein, HBI1, in Arabidopsis thaliana. Overexpression of HBI1 increased hypocotyl and petiole elongation, whereas dominant inactivation of HBI1 and its homologs caused a dwarf phenotype, indicating that HBI1 is a positive regulator of cell elongation. In vitro and in vivo experiments showed that HBI1 directly bound to the promoters and activated two EXPANSIN genes encoding cell wall-loosening enzymes; HBI1's DNA binding and transcriptional activities were inhibited by IBH1, but the inhibitory effects of IBH1 were abolished by PRE1. The results indicate that PREs activate the DNA binding bHLH factor HBI1 by sequestering its inhibitor IBH1. Altering each of the three factors affected plant sensitivities to BR, GA, temperature, and light. Our study demonstrates that PREs, IBH1, and HBI1 form a chain of antagonistic switches that regulates cell elongation downstream of multiple external and endogenous signals.

  19. Molecular recognition in helix-loop-helix and helix-loop-helix-leucine zipper domains. Design of repertoires and selection of high affinity ligands for natural proteins.

    Science.gov (United States)

    Ciarapica, Roberta; Rosati, Jessica; Cesareni, Gianni; Nasi, Sergio

    2003-04-04

    Helix-loop-helix (HLH) and helix-loop-helix-leucine zipper (HLHZip) are dimerization domains that mediate selective pairing among members of a large transcription factor family involved in cell fate determination. To investigate the molecular rules underlying recognition specificity and to isolate molecules interfering with cell proliferation and differentiation control, we assembled two molecular repertoires obtained by directed randomization of the binding surface in these two domains. For this strategy we selected the Heb HLH and Max Zip regions as molecular scaffolds for the randomization process and displayed the two resulting molecular repertoires on lambda phage capsids. By affinity selection, many domains were isolated that bound to the proteins Mad, Rox, MyoD, and Id2 with different levels of affinity. Although several residues along an extended surface within each domain appeared to contribute to dimerization, some key residues critically involved in molecular recognition could be identified. Furthermore, a number of charged residues appeared to act as switch points facilitating partner exchange. By successfully selecting ligands for four of four HLH or HLHZip proteins, we have shown that the repertoires assembled are rather general and possibly contain elements that bind with sufficient affinity to any natural HLH or HLHZip molecule. Thus they represent a valuable source of ligands that could be used as reagents for molecular dissection of functional regulatory pathways.

  20. Helix aspersa Müller in Holland

    NARCIS (Netherlands)

    Vernhout, J.H.

    1912-01-01

    In „Nachrichtsblatt der Deutschen Malakozool. Gesellsch. 1910, p. 134” Mr. V. Franz (Helgoland) gives a note on the occurrence of Helix aspersa in Holland at Vlissingen. He suggests that the animals have been transported fortuitously to that locality. Now I will not discuss the possibility this havi

  1. HIV-1 integrase modulates the interaction of the HIV-1 cellular cofactor LEDGF/p75 with chromatin

    Directory of Open Access Journals (Sweden)

    Garcia-Rivera Jose A

    2011-04-01

    Full Text Available Abstract Background Chromatin binding plays a central role in the molecular mechanism of LEDGF/p75 in HIV-1 DNA integration. Conflicting results have been reported in regards to the relevance of the LEDGF/p75 chromatin binding element PWWP domain in its HIV-1 cofactor activity. Results Here we present evidence that re-expression of a LEDGF/p75 mutant lacking the PWWP domain (ΔPWWP rescued HIV-1 infection in cells verified to express background levels of endogenous LEDGF/p75 that do not support efficient HIV-1 infection. The HIV-1 cofactor activity of LEDGF/p75 ΔPWWP was similar to that of LEDGF/p75 wild type (WT. A possible molecular explanation for the nonessential role of PWWP domain in the HIV-1 cofactor activity of LEDGF/p75 comes from the fact that coexpression of HIV-1 integrase significantly restored the impaired chromatin binding activity of LEDGF/p75 ΔPWWP. However, integrase failed to promote chromatin binding of a non-chromatin bound LEDGF/p75 mutant that lacks both the PWWP domain and the AT hook motifs (ΔPWWP/AT and that exhibits negligible HIV-1 cofactor activity. The effect of integrase on the chromatin binding of LEDGF/p75 requires the direct interaction of these two proteins. An HIV-1 integrase mutant, unable to interact with LEDGF/p75, failed to enhance chromatin binding, whereas integrase wild type did not increase the chromatin binding strength of a LEDGF/p75 mutant lacking the integrase binding domain (ΔIBD. Conclusions Our data reveal that the PWWP domain of LEDGF/p75 is not essential for its HIV-1 cofactor activity, possibly due to an integrase-mediated increase of the chromatin binding strength of this LEDGF/p75 mutant.

  2. HIV-1 integrase modulates the interaction of the HIV-1 cellular cofactor LEDGF/p75 with chromatin.

    Science.gov (United States)

    Astiazaran, Paulina; Bueno, Murilo Td; Morales, Elisa; Kugelman, Jeffrey R; Garcia-Rivera, Jose A; Llano, Manuel

    2011-04-21

    Chromatin binding plays a central role in the molecular mechanism of LEDGF/p75 in HIV-1 DNA integration. Conflicting results have been reported in regards to the relevance of the LEDGF/p75 chromatin binding element PWWP domain in its HIV-1 cofactor activity. Here we present evidence that re-expression of a LEDGF/p75 mutant lacking the PWWP domain (ΔPWWP) rescued HIV-1 infection in cells verified to express background levels of endogenous LEDGF/p75 that do not support efficient HIV-1 infection. The HIV-1 cofactor activity of LEDGF/p75 ΔPWWP was similar to that of LEDGF/p75 wild type (WT). A possible molecular explanation for the nonessential role of PWWP domain in the HIV-1 cofactor activity of LEDGF/p75 comes from the fact that coexpression of HIV-1 integrase significantly restored the impaired chromatin binding activity of LEDGF/p75 ΔPWWP. However, integrase failed to promote chromatin binding of a non-chromatin bound LEDGF/p75 mutant that lacks both the PWWP domain and the AT hook motifs (ΔPWWP/AT) and that exhibits negligible HIV-1 cofactor activity. The effect of integrase on the chromatin binding of LEDGF/p75 requires the direct interaction of these two proteins. An HIV-1 integrase mutant, unable to interact with LEDGF/p75, failed to enhance chromatin binding, whereas integrase wild type did not increase the chromatin binding strength of a LEDGF/p75 mutant lacking the integrase binding domain (ΔIBD). Our data reveal that the PWWP domain of LEDGF/p75 is not essential for its HIV-1 cofactor activity, possibly due to an integrase-mediated increase of the chromatin binding strength of this LEDGF/p75 mutant.

  3. Thermodynamics of helix-coil transitions studied by multicanonical algorithms

    CERN Document Server

    Okamoto, Y; Okamoto, Yuko; Hansmann, Ulrich H.E.

    1995-01-01

    Thermodynamics of helix-coil transitions in amino-acid homo-oligomers are studied by the recently proposed multicanonical algorithms. Homo-oligomers of length 10 are considered for three characteristic amino acids, alanine (helix former), valine (helix indifferent), and glycine (helix breaker). For alanine other lengths (15 and 20) are also considered in order to examine the length dependence. From one multicanonical production run with completely random initial conformations, we have obtained the lowest-energy conformations and various thermodynamic quantities (average helicity, Zimm-Bragg $s$ and $\\sigma$ parameters, free energy differences between helix and coil states, etc.) as functions of temperature. The results confirm the fact that alanine is helix-forming, valine is helix-indifferent, and glycine is helix-breaking.

  4. Relaxed Constraint and Evolutionary Rate Variation between Basic Helix-Loop-Helix Floral Anthocyanin Regulators in Ipomoea

    National Research Council Canada - National Science Library

    Streisfeld, Matthew A; Rausher, Mark D

    2007-01-01

    .... We cloned the full-length coding region from 2 basic helix-loop-helix transcription factors from several species of Ipomoea with diverse flower colors and determined the selective forces operating on them...

  5. HelixFlex: bioinspired maneuverable instrument for skull base surgery.

    Science.gov (United States)

    Gerboni, Giada; Henselmans, Paul W J; Arkenbout, Ewout A; van Furth, Wouter R; Breedveld, Paul

    2015-12-01

    Endoscopic endonasal surgery is currently regarded as the 'gold standard' for operating on pituitary gland tumors, and is becoming more and more accepted for treatment of other skull base lesions. However, endoscopic surgical treatment of most skull base pathologies, including certain pituitary tumors, is severely impaired by current instruments lack of maneuverability. Especially, gaining access to, and visibility of, difficult-to-reach anatomical corners without interference with surrounding neurovascular structures or other instruments, is a challenge. In this context there is the need for instruments that are able to provide a stable shaft position, while both the orientation and the position of the end-effector can be independently controlled. Current instruments that allow for this level of maneuverability are usually mechanically complex, and hence less suitable for mass production. This study therefore focuses on the development of a new actuation technique that allows for the required maneuverability while reducing the construction complexity. This actuation technique, referred to as multi-actuation, integrates multiple cable routings into a single steerable structure. Multi-actuation has been successfully integrated and tested in a handheld prototype instrument called HelixFlex. HelixFlex contains a 4 degrees of freedom maneuverable 5.8 mm (diameter) tip and shows promising results concerning its maneuverability and potential rigidity.

  6. DNA cleavage is independent of synapsis during Streptomyces phage phiBT1 integrase-mediated site-specific recombination.

    Science.gov (United States)

    Zhang, Lin; Wang, Lu; Wang, Jin; Ou, Xijun; Zhao, Guoping; Ding, Xiaoming

    2010-10-01

    Bacteriophage-encoded serine recombinases have great potential in genetic engineering but their catalytic mechanisms have not been adequately studied. Integration of ϕBT1 and ϕC31 via their attachment (att) sites is catalyzed by integrases of the large serine recombinase subtype. Both ϕBT1 and ϕC31 integrases were found to cleave single-substrate att sites without synaptic complex formation, and ϕBT1 integrase relaxed supercoiled DNA containing a single integration site. Systematic mutation of the central att site dinucleotide revealed that cleavage was independent of nucleotide sequence, but rejoining was crucially dependent upon complementarity of the cleavage products. Recombination between att sites containing dinucleotides with antiparallel complementarity led to antiparallel recombination. Integrase-substrate pre-incubation experiments revealed that the enzyme can form an attP-integrase tetramer complex that then captures naked attB DNA, and suggested that two alternative assembly pathways can lead to synaptic complex formation.

  7. The Quintuple Helix innovation model: Global warming as a challenge and driver for innovation

    OpenAIRE

    Carayannis, Elias G.; Thorsten D. Barth; Campbell David F. J.

    2012-01-01

    The Triple Helix innovation model focuses on university-industry-government relations. The Quadruple Helix embeds the Triple Helix by adding as a fourth helix the media-based and culture-based public and civil society. The Quintuple Helix innovation model is even broader and more comprehensive by contextualizing the Quadruple Helix and by additionally adding the helix (and perspective) of the natural environments of society. The Triple Helix acknowledges explicitly the importance of higher ed...

  8. Helix Scan: A Scan Design for Diagnosis

    Institute of Scientific and Technical Information of China (English)

    WANG Fei; HU Yu; LI Xiaowei

    2007-01-01

    Scan design is a widely used design-for-testability technique to improve test quality and efficiency. For the scan-designed circuit, test and diagnosis of the scan chain and the circuit is an important process for silicon debug and yield learning. However, conventional scan designs and diagnosis methods abort the subsequent diagnosis process after diagnosing the scan chain if the scan chain is faulty. In this work, we propose a design-for-diagnosis scan strategy called helix scan and a diagnosis algorithm to address this issue. Unlike previous proposed methods, helix scan has the capability to carry on the diagnosis process without losing information when the scan chain is faulty. What is more, it simplifies scan chain diagnosis and achieves high diagnostic resolution as well as accuracy. Experimental results demonstrate the effectiveness of our design.

  9. FPGA helix tracking algorithm for PANDA

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Yutie; Galuska, Martin; Gessler, Thomas; Kuehn, Wolfgang; Lange, Jens Soeren; Muenchow, David; Spruck, Bjoern [II. Physikalisches Institut, Giessen University (Germany); Ye, Hua [Institute of High Energy Physics, Beijing (China); Collaboration: PANDA-Collaboration

    2015-07-01

    The PANDA detector is a general-purpose detector for physics with high luminosity cooled antiproton beams, planed to operate at the FAIR facility in Darmstadt, Germany. The central detector includes a silicon Micro Vertex Detector (MVD) and a Straw Tube Tracker (STT). Without any hardware trigger, large amounts of raw data are streaming into the data acquisition system. The data reduction task is performed in the online system by reconstruction algorithms programmed on FPGAs (Field Programmable Gate Arrays) as first level and on a farm of GPUs or PCs as a second level. One important part in the system is the online track reconstruction. In this presentation, an online tracking algorithm for helix tracking reconstruction in the solenoidal field is shown. The tracking algorithm is composed by two parts, a road finding module followed by an iterative helix parameter calculation module. A performance study using C++ and the status of the VHDL implementation are presented.

  10. Genetic diversity on the integrase region of the pol gene among HIV type 1-infected patients naive for integrase inhibitors in São Paulo City, Brazil.

    Science.gov (United States)

    Arruda, Liã Bárbara; Fonseca, Luiz Augusto M; Duarte, Alberto J S; Casseb, Jorge

    2010-01-01

    The presence of mutations associated with integrase inhibitor (INI) resistance among INI-naive patients may play an important clinical role in the use of those drugs Samples from 76 HIV-1-infected subjects naive to INIs were submitted to direct sequencing. No differences were found between naive (25%) subjects and subjects on HAART (75%). No primary mutation associated with raltegravir or elvitegravir resistance was found. However, 78% of sequences showed at least one accessory mutation associated with resistance. The analysis of the 76 IN sequences showed a high polymorphic level on this region among Brazilian HIV-1-infected subjects, including a high prevalence of aa substitutions related to INI resistance. The impact of these findings remains unclear and further studies are necessary to address these questions.

  11. The double helix and the 'wronged heroine'.

    Science.gov (United States)

    Maddox, Brenda

    2003-01-23

    In 1962, James Watson, Francis Crick and Maurice Wilkins received the Nobel prize for the discovery of the structure of DNA. Notably absent from the podium was Rosalind Franklin, whose X-ray photographs of DNA contributed directly to the discovery of the double helix. Franklin's premature death, combined with misogynist treatment by the male scientific establishment, cast her as a feminist icon. This myth overshadowed her intellectual strength and independence both as a scientist and as an individual.

  12. Nanoparticle biofabrication using English ivy (Hedera helix)

    OpenAIRE

    Burris Jason N; Lenaghan Scott C; Zhang Mingjun; Stewart C

    2012-01-01

    Abstract Background English ivy (Hedera helix) is well known for its adhesive properties and climbing ability. Essential to its ability to adhere to vertical surfaces is the secretion of a nanocomposite adhesive containing spherical nanoparticles, 60–85 nm in diameter, produced exclusively by root hairs present on adventitious roots. These organic nanoparticles have shown promise in biomedical and cosmetic applications, and represent a safer alternative to metal oxide nanoparticles currently ...

  13. Dolutegravir (S/GSK1349572) exhibits significantly slower dissociation than raltegravir and elvitegravir from wild-type and integrase inhibitor-resistant HIV-1 integrase-DNA complexes.

    Science.gov (United States)

    Hightower, Kendra E; Wang, Ruolan; Deanda, Felix; Johns, Brian A; Weaver, Kurt; Shen, Yingnian; Tomberlin, Ginger H; Carter, H Luke; Broderick, Timothy; Sigethy, Scott; Seki, Takahiro; Kobayashi, Masanori; Underwood, Mark R

    2011-10-01

    The integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations. DTG dissociates slowly from a wild-type IN-DNA complex at 37°C with an off-rate of 2.7 × 10(-6) s(-1) and a dissociative half-life (t(1/2)) of 71 h, significantly longer than the half-lives for RAL (8.8 h) and ELV (2.7 h). Prolonged binding (t(1/2), at least 5 h) was observed for DTG with IN-DNA complexes containing E92, Y143, Q148, and N155 substitutions. The addition of a second substitution to either Q148 or N155 typically resulted in an increase in the off-rate compared to that with the single substitution. For all of the IN substitutions tested, the off-rate of DTG from IN-DNA complexes was significantly slower (from 5 to 40 times slower) than the off-rate of RAL or ELV. These data are consistent with the potential for DTG to have a higher genetic barrier to resistance, provide evidence that the INI off-rate may be an important component of the mechanism of INI resistance, and suggest that the slow dissociation of DTG may contribute to its distinctive resistance profile.

  14. Damping effect of helix-like pili

    CERN Document Server

    Zakrisson, Johan; Axner, Ove; Andersson, Magnus

    2014-01-01

    Biopolymers are vital structures for many living organisms; for a variety of bacteria, adhesion polymers play a crucial role for the initiation of colonization. Some bacteria express, on their surface, attachment organelles (pili) that comprise subunits formed into stiff helix-like structures that possess unique biomechanical properties. These helix-like structures possess a high degree of flexibility that gives the biopolymers a unique extendibility. This has been considered beneficial for piliated bacteria adhering to host surfaces in the presence of a fluid flow. We show in this work that helix-like pili have the ability to act as efficient dampers of force that can, for a limited time, lower the load on the force-mediating adhesin-receptor bond on the tip of an individual pilus. The model presented is applied to bacteria adhering with a single pilus of either of the two most common types expressed by uropathogenic Escherichia coli, P or type 1 pili, subjected to realistic flows. The results indicate that ...

  15. Effect of attC structure on cassette excision by integron integrases

    Directory of Open Access Journals (Sweden)

    Larouche André

    2011-02-01

    Full Text Available Abstract Background Integrons are genetic elements able to integrate and disseminate genes as cassettes by a site-specific recombination mechanism. These elements contain a gene coding for an integrase that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a gene cassette. Integron integrases (IntIs bind specifically to the bottom strand of attC sites. The extrahelical bases resulting from folding of attC bottom strands are important for the recognition by integrases. These enzymes are directly involved in the accumulation and formation of new cassette arrangements in the variable region of integrons. Thus, it is important to better understand interactions between IntIs and their substrates. Results We compared the ability of five IntIs to carry out excision of several cassettes flanked by different attC sites. The results showed that for most cassettes, IntI1 was the most active integrase. However, IntI2*179E and SonIntIA could easily excise cassettes containing the attCdfrA1 site located upstream, whereas IntI1 and IntI3 had only a weak excision activity for these cassettes. Analysis of the secondary structure adopted by the bottom strand of attCdfrA1 has shown that the identity of the extrahelical bases and the distance between them (A-N7-8-C differ from those of attCs contained in the cassettes most easily excisable by IntI1 (T-N6-G. We used the attCdfrA1 site upstream of the sat2 gene cassette as a template and varied the identity and spacing between the extrahelical bases in order to determine how these modifications influence the ability of IntI1, IntI2*179E, IntI3 and SonIntIA to excise cassettes. Our results show that IntI1 is more efficient in cassette excision using T-N6-G or T-N6-C attCs while IntI3 recognizes only a limited range of attCs. IntI2*179E and SonIntIA are more tolerant of changes to the identity and spacing of extrahelical

  16. Screening of potential pseudo att sites of Streptomyces phage ΦC31 integrase in the human genome

    Institute of Scientific and Technical Information of China (English)

    Zhi-peng HU; Lu-sheng CHEN; Cai-yan JIA; Huan-zhang ZHU; Wei WANG; Jiang ZHONG

    2013-01-01

    Aim:ΦC31 integrase mediates site-specific recombination between two short sequences,attP and attB,in phage and bacterial genomes,which is a promising tool in gene regulation-based therapy since the zinc finger structure is probably the DNA recognizing domain that can further be engineered.The aim of this study was to screen potential pseudo att sites of ΦC31 integrase in the human genome,and evaluate the risks of its application in human gene therapy.Methods:TFBS (transcription factor binding sites) were found on the basis of reported pseudo att sites using multiple motif-finding tools,including AlignACE,BioProspector,Consensus,MEME,and Weeder.The human genome with the proposed motif was scanned tc find the potential pseudo att sites of ΦC31 integrase.Results:The possible recognition motif of ΦC31 integrase was identified,which was composed of two co-occurrence conserved elements that were reverse complement to each other flanking the core sequence TTG.In the human genome,a total of 27924 potential pseudo att sites of ΦC31 integrase were found,which were distributed in each human chromosome with high-risk specificity values in the chromosomes 16,17,and 19.When the risks of the sites were evaluate more rigorously,53 hits were discovered,and some of them were just the vital functional genes or regulatory regions,such as ACYP2,AKR1B1,DUSP4,etc.Conclusion:The results provide clues for more comprehensive evaluation of the risks of using ΦC31 integrase in human gene therapy and for drug discovery.

  17. BF integrase genes of HIV-1 circulating in Sao Paulo, Brazil, with a recurrent recombination region.

    Directory of Open Access Journals (Sweden)

    Atila Iamarino

    Full Text Available Although some studies have shown diversity in HIV integrase (IN genes, none has focused particularly on the gene evolving in epidemics in the context of recombination. The IN gene in 157 HIV-1 integrase inhibitor-naïve patients from the São Paulo State, Brazil, were sequenced tallying 128 of subtype B (23 of which were found in non-B genomes, 17 of subtype F (8 of which were found in recombinant genomes, 11 integrases were BF recombinants, and 1 from subtype C. Crucially, we found that 4 BF recombinant viruses shared a recurrent recombination breakpoint region between positions 4900 and 4924 (relative to the HXB2 that includes 2 gRNA loops, where the RT may stutter. Since these recombinants had independent phylogenetic origin, we argue that these results suggest a possible recombination hotspot not observed so far in BF CRF in particular, or in any other HIV-1 CRF in general. Additionally, 40% of the drug-naïve and 45% of the drug-treated patients had at least 1 raltegravir (RAL or elvitegravir (EVG resistance-associated amino acid change, but no major resistance mutations were found, in line with other studies. Importantly, V151I was the most common minor resistance mutation among B, F, and BF IN genes. Most codon sites of the IN genes had higher rates of synonymous substitutions (dS indicative of a strong negative selection. Nevertheless, several codon sites mainly in the subtype B were found under positive selection. Consequently, we observed a higher genetic diversity in the B portions of the mosaics, possibly due to the more recent introduction of subtype F on top of an ongoing subtype B epidemics and a fast spread of subtype F alleles among the B population.

  18. Effects of etravirine on the pharmacokinetics of the integrase inhibitor S/GSK1265744.

    Science.gov (United States)

    Ford, Susan L; Gould, Elizabeth; Chen, Shuguang; Lou, Yu; Dumont, Etienne; Spreen, William; Piscitelli, Stephen

    2013-01-01

    HIV integrase inhibitors such as raltegravir and elvitegravir halt HIV progression, but treatment-emergent resistance and cross-resistance have been observed. The nonnucleoside reverse transcriptase inhibitor etravirine (ETR) may be used in combination with integrase inhibitors in patients with drug resistance. This single-center, open-label, two-period, single-sequence crossover study evaluated the effects of ETR coadministration on the pharmacokinetic profile of S/GSK1265744, an investigational integrase inhibitor in phase 2 studies. Healthy subjects received 30 mg of S/GSK1265744 alone once daily for 10 days (period 1) and in combination with 200 mg of ETR twice daily for 14 days (period 2). Serial plasma samples for pharmacokinetic analyses were collected on day 10 during period 1 and on day 14 during period 2. All treatments were well tolerated. Etravirine had no effects on S/GSK1265744 geometric mean ratios of the area under the curve from time zero until the end of the dosing interval (1.01; 90% confidence interval [CI], 0.956 to 1.06), of the maximum observed plasma concentration (1.04; 90% CI, 0.987 to 1.09), or of the plasma concentration at the end of the dosing interval (0.999; 90% CI, 0.942 to 1.06). Etravirine pharmacokinetics (PK) parameters observed following coadministration with S/GSK1265744 were in the range of historical values reported for ETR alone in healthy subjects. These results indicate that 30 mg of S/GSK1265744 for 10 days as monotherapy followed by an additional 14 days in combination with ETR was well tolerated in healthy subjects and that no dose adjustment of S/GSK1265744 is required when it is coadministered with ETR.

  19. Thermal stability of collagen triple helix.

    Science.gov (United States)

    Xu, Yujia

    2009-01-01

    Chief among the challenges of characterizing the thermal stability of the collagen triple helix are the lack of the reversibility of the thermal transition and the presence of multiple folding-unfolding steps during the thermal transition which rarely follows the simple two-state, all-or-none mechanism. Despite of the difficulties inherited in the quantitative depiction of the thermal transition of collagen, biophysical studies combined with proteolysis and mutagenesis approaches using full-chain collagens, short synthetic peptides, and recombinant collagen fragments have revealed molecular features of the thermal unfolding of the subdomains of collagen and led to a better understanding of the diverse biological functions of this versatile protein. The subdomain of collagen generally refers to a segment of the long, rope-like triple helical molecule that can unfold cooperatively as an independent unit whose properties (their size, location, and thermal stability) are considered essential for the molecular recognition during the self-assembly of collagen and during the interactions of collagen with other macromolecules. While the unfolding of segments of the triple helix at temperatures below the apparent melting temperature of the molecule has been used to interpret much of the features of the thermal unfolding of full-chain collagens, the thermal studies of short, synthetic peptides have firmly established the molecular basis of the subdomains by clearly demonstrating the close dependence of the thermal stability of a triple helix on the constituent amino acid residues at the X and the Y positions of the characteristic Gly-X-Y repeating sequence patterns of the triple helix. Studies using recombinant collagen fragments further revealed that in the context of the long, linear molecule, the stability of a segment of the triple helix is also modulated by long-range impact of the local interactions such as the interchain salt bridges. Together, the combined approaches

  20. Integrase strand transfer inhibitor-associated diabetes mellitus: A case report.

    Science.gov (United States)

    Fong, Peter S; Flynn, Devon M; Evans, Christopher D; Korthuis, P Todd

    2017-05-01

    Integrase strand transfer inhibitors (INSTIs) have become integral antiretroviral therapy (ART) agents for treating HIV infection. We report the case of a 44-year-old male with a history of hemophilia A who developed diabetes mellitus four months after switching from abacavir, lamivudine, and efavirenz to abacavir, lamivudine, and raltegravir. Hemoglobin A1C normalized without further need for exogenous insulin after raltegravir was switched back to efavirenz. In this case report, we will review a possible mechanism for INSTI-induced hyperglycemia and/or diabetes mellitus.

  1. 3-Hydroxypyrimidine-2,4-diones as an inhibitor scaffold of HIV integrase.

    Science.gov (United States)

    Tang, Jing; Maddali, Kasthuraiah; Metifiot, Mathieu; Sham, Yuk Y; Vince, Robert; Pommier, Yves; Wang, Zhengqiang

    2011-04-14

    Integrase (IN) represents a clinically validated target for the development of antivirals against human immunodeficiency virus (HIV). Inhibitors with a novel structure core are essential for combating resistance associated with known IN inhibitors (INIs). We have previously disclosed a novel dual inhibitor scaffold of HIV IN and reverse transcriptase (RT). Here we report the complete structure-activity relationship (SAR), molecular modeling, and resistance profile of this inhibitor type on IN inhibition. These studies support an antiviral mechanism of dual inhibition against both IN and RT and validate 3-hydroxypyrimidine-2,4-diones as an IN inhibitor scaffold.

  2. Ferrocenyl chalcone difluoridoborates inhibit HIV-1 integrase and display low activity towards cancer and endothelial cells.

    Science.gov (United States)

    Monserrat, Jean-Philippe; Al-Safi, Rasha I; Tiwari, Keshri Nath; Quentin, Lionel; Chabot, Guy G; Vessières, Anne; Jaouen, Gérard; Neamati, Nouri; Hillard, Elizabeth A

    2011-10-15

    We report here the discovery of a potent series of HIV-1 integrase (IN) inhibitors based on the ferrocenyl chalcone difluoridoborate structure. Ten new compounds have been synthesized and were generally found to have similar inhibitory activities against the IN 3' processing and strand transfer (ST) processes. IC(50) values were found to be in the low micromolar range, and significantly lower than those found for the non-coordinated ferrocenyl chalcones and other ferrocene molecules. The ferrocenyl chalcone difluoridoborates furthermore exhibited low cytotoxicity against cancer cells and low morphological activity against epithelial cells.

  3. Creation of Tenecteplase-Producing CHO Cell Line Using Site-Specific Integrase from the Phage φC31

    OpenAIRE

    2010-01-01

    Objective: The aim of this study was to produce a stable CHO cell line expressing tenecteplase.Materials and Methods: In the first step, the tenecteplase coding sequence was clonedin a pDB2 vector containing attB recognition sites for the phage φC31 integrase. Then,using lipofection, the CHO cells were co-transfected with constructed recombinant plasmidencoding tenecteplase and attB recognition sites and the integrase coding sequencecontaining pCMV-Int plasmid. As the recombinant plasmid cont...

  4. The evaluation of statins as potential inhibitors of the LEDGF/p75-HIV-1 integrase interaction.

    Science.gov (United States)

    Harrison, Angela T; Kriel, Frederik H; Papathanasopoulos, Maria A; Mosebi, Salerwe; Abrahams, Shaakira; Hewer, Raymond

    2015-03-01

    Lovastatin was identified through virtual screening as a potential inhibitor of the LEDGF/p75-HIV-1 integrase interaction. In an AlphaScreen assay, lovastatin inhibited the purified recombinant protein-protein interaction (IC50 = 1.97 ± 0.45 μm) more effectively than seven other tested statins. None of the eight statins, however, yielded antiviral activity in vitro, while only pravastatin lactone yielded detectable inhibition of HIV-1 integrase strand transfer activity (31.65% at 100 μm). A correlation between lipophilicity and increased cellular toxicity of the statins was observed.

  5. Loop-to-helix transition in the structure of multidrug regulator AcrR at the entrance of the drug-binding cavity

    Energy Technology Data Exchange (ETDEWEB)

    Manjasetty, Babu A.; Halavaty, Andrei S.; Luan, Chi-Hao; Osipiuk, Jerzy; Mulligan, Rory; Kwon, Keehwan; Anderson, Wayne F.; Joachimiak, Andrzej

    2016-04-01

    Multidrug transcription regulator AcrR from Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 belongs to the tetracycline repressor family, one of the largest groups of bacterial transcription factors. The crystal structure of dimeric AcrR was determined and refined to 1.56 Å resolution. The tertiary and quaternary structures of AcrR are similar to those of its homologs. The multidrug binding site was identified based on structural alignment with homologous proteins and has a di(hydroxyethyl)ether molecule bound. Residues from helices a4 and a7 shape the entry into this binding site. The structure of AcrR reveals that the extended helical conformation of helix a4 is stabilized by the hydrogen bond between Glu67 (helix a4) and Gln130 (helix a7). Based on the structural comparison with the closest homolog structure, the Escherichia coli AcrR, we propose that this hydrogen bond is responsible for control of the loop-to-helix transition within helix a4. This local conformational switch of helix a4 may be a key step in accessing the multidrug binding site and securing ligands at the binding site. Solution smallmolecule binding studies suggest that AcrR binds ligands with their core chemical structure resembling the tetracyclic ring of cholesterol.

  6. Expanding the diversity of oenococcal bacteriophages: insights into a novel group based on the integrase sequence.

    Science.gov (United States)

    Jaomanjaka, Fety; Ballestra, Patricia; Dols-lafargue, Marguerite; Le Marrec, Claire

    2013-09-01

    Temperate bacteriophages are a contributor of the genetic diversity in the lactic acid bacterium Oenococcus oeni. We used a classification scheme for oenococcal prophages based on integrase gene polymorphism, to analyze a collection of Oenococcus strains mostly isolated in the area of Bordeaux, which represented the major lineages identified through MLST schemes in the species. Genome sequences of oenococcal prophages were clustered into four integrase groups (A to D) which were related to the chromosomal integration site. The prevalence of each group was determined and we could show that members of the intB- and intC-prophage groups were rare in our panel of strains. Our study focused on the so far uncharacterized members of the intD-group. Various intD viruses could be easily isolated from wine samples, while intD lysogens could be induced to produce phages active against two permissive O. oeni isolates. These data support the role of this prophage group in the biology of O. oeni. Global alignment of three relevant intD-prophages revealed significant conservation and highlighted a number of unique ORFs that may contribute to phage and lysogen fitness.

  7. Pyrroloaryls and pyrroloheteroaryls: Inhibitors of the HIV fusion/attachment, reverse transcriptase and integrase.

    Science.gov (United States)

    Patel, Rahul V; Park, Se Won

    2015-09-01

    Heterocyclic compounds execute a very important role in drug design and discovery. This article provides the basic milestones of the research for pyrroloaryl and pyrroloheteroaryl based components targeting HIV viral replication cycle. Anti-HIV activity is elaborated for several classes of pyrrolo-compounds as pyrrolopyridines, pyrrolopyrimidines, pyrrolopyridazines, pyrrolobenzodiazepinones, pyrrolobenzothiazepines, pyrrolobenzoxazepinones, pyrrolophenanthridines, pyrroloquinoxalines, pyrrolotriazines, pyrroloquinolines, pyrrolopyrazinones, pyrrolothiatriazines, arylthiopyrroles and pyrrolopyrazolones targeting two essential HIV enzymes, reverse transcriptase and integrase as well as attachment/fusion of HIV virons to the host CD-4 cell. Such attempts were resulted in a discovery of highly potent anti-HIV agents suitable for clinical trials, for example, BMS-378806, BMS-585248, BMS-626529, BMS-663068, BMS-488043 and BMS-663749, etc. as anti-HIV attachment agents, triciribine, QX432, BI-1 and BI-2 as HIV RT inhibitors which are in preclinical or clinical development. Mechanism of action of compounds presented in this article towards the suppression of HIV attachment/fusion as well as against the activities of HIV enzymes reverse transcriptase and integrase has been discussed. Relationships of new compounds' molecular framework and HIV viral target has been overviewed in order to facilitate further construction of promising anti-HIV agents in future drug discovery process.

  8. Computational and synthetic approaches for developing Lavendustin B derivatives as allosteric inhibitors of HIV-1 integrase

    Science.gov (United States)

    Agharbaoui, Fatima E.; Hoyte, Ashley C.; Ferro, Stefania; Gitto, Rosaria; Buemi, Maria Rosa; Fuchs, James R.; Kvaratskhelia, Mamuka; De Luca, Laura

    2017-01-01

    Through structure-based virtual screening and subsequent activity assays of selected natural products, Lavendustin B was previously identified as an inhibitor of HIV-1 integrase (IN) interaction with its cognate cellular cofactor, lens epithelium-derived growth factor (LEDGF/p75). In order to improve the inhibitory potency we have employed in silico-based approaches. Particularly, a series of new analogues was designed and docked into the LEDGF/p75 binding pocket of HIV-1 IN. To identify promising leads we used the Molecular Mechanics energies combined with the Generalized Born and Surface Area continuum solvation (MM-GBSA) method, molecular dynamics simulations and analysis of hydrogen bond occupancies. On the basis of these studies, six analogues of Lavendustine B, containing the benzylamino-hydroxybenzoic scaffold, were selected for synthesis and structure activity-relationship (SAR) studies. Our results demonstrated a good correlation between computational and experimental data, and all six analogues displayed an improved potency for inhibiting IN binding to LEDGF/p75 in vitro to respect to the parent compound Lavendustin B. Additionally, these analogs show to inhibit weakly LEDGF/p75-independent IN catalytic activity suggesting a multimodal allosteric mechanism of action. Nevertheless, for the synthesized compounds similar profiles for HIV-1 inhibition and cytoxicity were highlighted. Taken together, our studies elucidated the mode of action of Lavendustin B analogs and provided a path for their further development as a new promising class of HIV-1 integrase inhibitors. PMID:27517812

  9. Lead Screening for HIV-1 Integrase (IN Inhibited by Traditional Chinese Medicine

    Directory of Open Access Journals (Sweden)

    Tzu-Chieh Hung

    2014-01-01

    Full Text Available Human immunodeficiency virus causes the acquired immunodeficiency syndrome (AIDS and becomes a serious world-wide problem because of this disease's rapid propagation and incurability. Integrase strand transfer inhibitors (INSTIs supports HIV have rapid drug resistance for antitreatment. Screening the traditional Chinese medicine (TCM database by simulating molecular docking and molecular dynamics may select molecular compounds to inhibit INSTIs against HIV drug resistance. (S-cathinone and (1S,2S-norpseudoephedrine are selected based on structure and ligand-based drugs are designed and then get higher bioactivity predicted score from SVM than Raltegravir and other TCM compounds. The molecular dynamics are helpful in the analysis and detection of protein-ligand interactions. According to the docking poses, hydrophobic interactions and hydrogen bond variations define the main regions of important amino acids in integrase. In addition to the detection of TCM compound efficacy, we suggest (1S,2S-norpseudoephedrine is better than the others based on the analysis of interaction and the effect on the structural variation.

  10. Virtual screening of the SAMPL4 blinded HIV integrase inhibitors dataset

    Science.gov (United States)

    Colas, Claire; Iorga, Bogdan I.

    2014-04-01

    Several combinations of docking software and scoring functions were evaluated for their ability to predict the binding of a dataset of potential HIV integrase inhibitors. We found that different docking software were appropriate for each one of the three binding sites considered (LEDGF, Y3 and fragment sites), and the most suitable two docking protocols, involving Glide SP and Gold ChemScore, were selected using a training set of compounds identified from the structural data available. These protocols could successfully predict respectively 20.0 and 23.6 % of the HIV integrase binders, all of them being present in the LEDGF site. When a different analysis of the results was carried out by removing all alternate isomers of binders from the set, our predictions were dramatically improved, with an overall ROC AUC of 0.73 and enrichment factor at 10 % of 2.89 for the prediction obtained using Gold ChemScore. This study highlighted the ability of the selected docking protocols to correctly position in most cases the ortho-alkoxy-carboxylate core functional group of the ligands in the corresponding binding site, but also their difficulties to correctly rank the docking poses.

  11. Computational and synthetic approaches for developing Lavendustin B derivatives as allosteric inhibitors of HIV-1 integrase.

    Science.gov (United States)

    Agharbaoui, Fatima E; Hoyte, Ashley C; Ferro, Stefania; Gitto, Rosaria; Buemi, Maria Rosa; Fuchs, James R; Kvaratskhelia, Mamuka; De Luca, Laura

    2016-11-10

    Through structure-based virtual screening and subsequent activity assays of selected natural products, Lavendustin B was previously identified as an inhibitor of HIV-1 integrase (IN) interaction with its cognate cellular cofactor, lens epithelium-derived growth factor (LEDGF/p75). In order to improve the inhibitory potency we have employed in silico-based approaches. Particularly, a series of new analogues was designed and docked into the LEDGF/p75 binding pocket of HIV-1 IN. To identify promising leads we used the Molecular Mechanics energies combined with the Generalized Born and Surface Area continuum solvation (MM-GBSA) method, molecular dynamics simulations and analysis of hydrogen bond occupancies. On the basis of these studies, six analogues of Lavendustine B, containing the benzylamino-hydroxybenzoic scaffold, were selected for synthesis and structure activity-relationship (SAR) studies. Our results demonstrated a good correlation between computational and experimental data, and all six analogues displayed an improved potency for inhibiting IN binding to LEDGF/p75 in vitro to respect to the parent compound Lavendustin B. Additionally, these analogs show to inhibit weakly LEDGF/p75-independent IN catalytic activity suggesting a multimodal allosteric mechanism of action. Nevertheless, for the synthesized compounds similar profiles for HIV-1 inhibition and cytoxicity were highlighted. Taken together, our studies elucidated the mode of action of Lavendustin B analogs and provided a path for their further development as a new promising class of HIV-1 integrase inhibitors.

  12. HIV-1 integrase strand-transfer inhibitors: design, synthesis and molecular modeling investigation.

    Science.gov (United States)

    De Luca, Laura; De Grazia, Sara; Ferro, Stefania; Gitto, Rosaria; Christ, Frauke; Debyser, Zeger; Chimirri, Alba

    2011-02-01

    This study is focused on a new series of benzylindole derivatives with various substituents at the benzene-fused ring, suggested by our 3D pharmacophore model developed for HIV-1 integrase inhibitors (INIs). All synthesized compounds proved to be active in the nanomolar range (6-35 nM) on the strand-transfer step (ST). In particular, derivative 4-[1-(4-fluorobenzyl)-5,7-dimethoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic acid (8e), presenting the highest best-fit value on pharmacophore model, showed a potency comparable to that of clinical INSTIs GS 9137 (1) and MK-0518 (2). The binding mode of our molecules has been investigated using the recently published crystal structure of the complex of full-length integrase from the prototype foamy virus in complex with its cognate DNA (PFV-IN/DNA). The results highlighted the ability of derivative 8e to assume the same binding mode of MK-0518 and GS 9137.

  13. Fragment Based Strategies for Discovery of Novel HIV-1 Reverse Transcriptase and Integrase Inhibitors.

    Science.gov (United States)

    Latham, Catherine F; La, Jennifer; Tinetti, Ricky N; Chalmers, David K; Tachedjian, Gilda

    2016-01-01

    Human immunodeficiency virus (HIV) remains a global health problem. While combined antiretroviral therapy has been successful in controlling the virus in patients, HIV can develop resistance to drugs used for treatment, rendering available drugs less effective and limiting treatment options. Initiatives to find novel drugs for HIV treatment are ongoing, although traditional drug design approaches often focus on known binding sites for inhibition of established drug targets like reverse transcriptase and integrase. These approaches tend towards generating more inhibitors in the same drug classes already used in the clinic. Lack of diversity in antiretroviral drug classes can result in limited treatment options, as cross-resistance can emerge to a whole drug class in patients treated with only one drug from that class. A fresh approach in the search for new HIV-1 drugs is fragment-based drug discovery (FBDD), a validated strategy for drug discovery based on using smaller libraries of low molecular weight molecules (<300 Da) screened using primarily biophysical assays. FBDD is aimed at not only finding novel drug scaffolds, but also probing the target protein to find new, often allosteric, inhibitory binding sites. Several fragment-based strategies have been successful in identifying novel inhibitory sites or scaffolds for two proven drug targets for HIV-1, reverse transcriptase and integrase. While any FBDD-generated HIV-1 drugs have yet to enter the clinic, recent FBDD initiatives against these two well-characterised HIV-1 targets have reinvigorated antiretroviral drug discovery and the search for novel classes of HIV-1 drugs.

  14. Chatter Prediction for Variable Pitch and Variable Helix Milling

    Directory of Open Access Journals (Sweden)

    Yong Wang

    2015-01-01

    Full Text Available Regenerative chatter is a self-excited vibration that can occur during milling, which shortens the lifetime of the tool and results in unacceptable surface quality. In this paper, an improved semidiscretization method for modeling and simulation with variable pitch and variable helix milling is proposed. Because the delay between each flute varies along the axial depth of the tool in milling, the cutting tool is discrete into some axial layers to simplify calculation. A comparison of the predicted and observed performance of variable pitch and variable helix against uniform pitch and uniform helix milling is presented. It is shown that variable pitch and variable helix milling can obtain larger stable cutting area than uniform pitch and uniform helix milling. Thus, it is concluded that variable pitch and variable helix milling are an effective way for suppressing chatter.

  15. Control of Collagen Triple Helix Stability by Phosphorylation.

    Science.gov (United States)

    Acevedo-Jake, Amanda M; Ngo, Daniel H; Hartgerink, Jeffrey D

    2017-03-10

    The phosphorylation of the collagen triple helix plays an important role in collagen synthesis, assembly, signaling, and immune response, although no reports detailing the effect this modification has on the structure and stability of the triple helix exist. Here we investigate the changes in stability and structure resulting from the phosphorylation of collagen. Additionally, the formation of pairwise interactions between phosphorylated residues and lysine is examined. In all tested cases, phosphorylation increases helix stability. When charged-pair interactions are possible, stabilization via phosphorylation can play a very large role, resulting inasmuch as a 13.0 °C increase in triple helix stability. Two-dimensional NMR and molecular modeling are used to study the local structure of the triple helix. Our results suggest a mechanism of action for phosphorylation in the regulation of collagen and also expand upon our understanding of pairwise amino acid stabilization of the collagen triple helix.

  16. Site-specific unfolding thermodynamics of a helix-turn-helix protein.

    Science.gov (United States)

    Amunson, Krista E; Ackels, Loren; Kubelka, Jan

    2008-07-01

    The thermal unfolding of a 40-residue helix-turn-helix subdomain of the P22 viral coat protein was investigated using circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) with site-specific 13C isotopic labeling. Helix-turn-helix is the simplest alpha-helical structural motif that combines both secondary and tertiary structural elements. The CD of individual helical fragments reveals that the P22 subdomain is stabilized by tertiary interhelical interactions. Overall the temperature-dependent CD and FTIR data can be described by a three-state process with a partially folded intermediate. However, the analysis of the site-specific 13C IR signals reveals distinct unfolding thermodynamics for each of the labeled sites. The thermodynamic parameters of the thermal unfolding of each of the labeled segments were obtained using singular value decomposition in combination with target transformation and global fitting. The P22 subdomain unfolds from the N-terminus toward the helical segments near the turn. Our results show that as few as two 13C labeled residues can be detected in a 40 residue protein and provide local, site-specific structural information about protein unfolding, which is not resolved by standard, nonsite-specific spectroscopic probes.

  17. A Classification of Basic Helix-Loop-Helix Transcription Factors of Soybean

    Directory of Open Access Journals (Sweden)

    Karen A. Hudson

    2015-01-01

    Full Text Available The complete genome sequence of soybean allows an unprecedented opportunity for the discovery of the genes controlling important traits. In particular, the potential functions of regulatory genes are a priority for analysis. The basic helix-loop-helix (bHLH family of transcription factors is known to be involved in controlling a wide range of systems critical for crop adaptation and quality, including photosynthesis, light signalling, pigment biosynthesis, and seed pod development. Using a hidden Markov model search algorithm, 319 genes with basic helix-loop-helix transcription factor domains were identified within the soybean genome sequence. These were classified with respect to their predicted DNA binding potential, intron/exon structure, and the phylogeny of the bHLH domain. Evidence is presented that the vast majority (281 of these 319 soybean bHLH genes are expressed at the mRNA level. Of these soybean bHLH genes, 67% were found to exist in two or more homeologous copies. This dataset provides a framework for future studies on bHLH gene function in soybean. The challenge for future research remains to define functions for the bHLH factors encoded in the soybean genome, which may allow greater flexibility for genetic selection of growth and environmental adaptation in this widely grown crop.

  18. Identification of amino acids in HIV-2 integrase involved in site-specific hydrolysis and alcoholysis of viral DNA termini

    NARCIS (Netherlands)

    D.C. van Gent (Dik); A.A. Groeneger; R.H. Plassterk

    1993-01-01

    textabstractThe human immunodeficiency virus integrase (HIV IN) protein cleaves two nucleotides off the 3' end of viral DNA and subsequently integrates the viral DNA into target DNA. IN exposes a specific phosphodiester bond near the viral DNA end to nucleophilic attack by water or

  19. Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by PhiC31 integrase

    Energy Technology Data Exchange (ETDEWEB)

    Iri-Sofla, Farnoush Jafari [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Rahbarizadeh, Fatemeh, E-mail: rahbarif@modares.ac.ir [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Ahmadvand, Davoud [Center of Pharmaceutical Nanotechnology and Nanotoxicology, Department of Pharmaceutics and Analytical Chemistry, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen O (Denmark); Rasaee, Mohammad J. [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of)

    2011-11-01

    The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3{zeta}/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of Fc{gamma}RII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.

  20. Identification of amino acids in HIV-2 integrase involved in site-specific hydrolysis and alcoholysis of viral DNA termini

    NARCIS (Netherlands)

    D.C. van Gent (Dik); A.A. Groeneger; R.H. Plassterk

    1993-01-01

    textabstractThe human immunodeficiency virus integrase (HIV IN) protein cleaves two nucleotides off the 3' end of viral DNA and subsequently integrates the viral DNA into target DNA. IN exposes a specific phosphodiester bond near the viral DNA end to nucleophilic attack by water or

  1. Design, Synthesis and Anti-HIV Integrase Evaluation of 4-Oxo-4H-quinolizine-3-carboxylic Acid Derivatives

    Directory of Open Access Journals (Sweden)

    Li-Ming Hu

    2009-02-01

    Full Text Available 4-Oxo-4H-quinolizine-3-carboxylic acid derivatives bearing sulfamido, carboxylamido, benzimidazole and benzothiazole substituents have been designed and synthesized. The structures of these new compounds were confirmed by 1H-NMR, 13C- NMR, IR and ESI (or HRMS spectra. Compounds were screened for possible HIV integrase inhibitory activity.

  2. The bridge helix coordinates movements of modules in RNA polymerase

    Directory of Open Access Journals (Sweden)

    Landick Robert

    2010-11-01

    Full Text Available Abstract The RNA polymerase 'bridge helix' is a metastable α-helix that spans the leading edge of the enzyme active-site cleft. A new study published in BMC Biology reveals surprising tolerance to helix-disrupting changes in a region previously thought crucial for translocation, and suggests roles for two hinge-like segments of the bridge helix in coordinating modules that move during the nucleotide-addition cycle. See Research article: http://www.biomedcentral.com/1741-7007/8/134

  3. HELIX: The High Energy Light Isotope Experiment

    Science.gov (United States)

    Wakely, Scott

    This is the lead proposal for a new suborbital program, HELIX (High-Energy Light Isotope eXperiment), designed to make measurements of the isotopic composition of light cosmic-ray nuclei from ~200 MeV/nuc to ~10 GeV/nuc. Past measurements of this kind have provided profound insights into the nature and origin of cosmic rays, revealing, for instance, information on acceleration and confinement time scales, and exposing some conspicuous discrepancies between solar and cosmic-ray abundances. The most detailed information currently available comes from the ACE/CRIS mission, but is restricted to energies below a few 100 MeV/nuc. HELIX aims at extending this energy range by over an order of magnitude, where, in most cases, no measurements of any kind exist, and where relativistic time dilation affects the apparent lifetime of radioactive clock nuclei. The HELIX measurements will provide essential information for understanding the propagation history of cosmic rays in the galaxy. This is crucial for properly interpreting several intriguing anomalies reported in recent cosmic-ray measurements, pertaining to the energy spectra of protons, helium, and heavier nuclei, and to the anomalous rise in the positron fraction at higher energy. HELIX employs a high-precision magnet spectrometer to provide measurements which are not achievable by any current or planned instrument. The superconducting magnet originally used for the HEAT payload in five successful high-altitude flights will be combined with state-of-the-art detectors to measure the charge, time-of-flight, magnetic rigidity, and velocity of cosmic-ray particles with high precision. The instrumentation includes plastic scintillators, silicon-strip detectors repurposed from Fermilab's CDF detector, a high-performance gas drift chamber, and a ring-imaging Cherenkov counter employing aerogel radiators and silicon photomultipliers. To reduce cost and technical risk, the HELIX program will be structured in two stages. The first

  4. Synthesis, antiinflammatory and HIV-1 integrase inhibitory activities of 1,2-bis[5-thiazolyl]ethane-1,2-dione derivatives

    Directory of Open Access Journals (Sweden)

    Franklin P

    2009-01-01

    Full Text Available Based on principles of pharmacophore delineation and drug designing, compounds containing diketofunctionallity namely 1,2-bis[5-thiazolyl]ethane-1,2-diones were designed and synthesized as antiinflammatory agents. The compounds were evaluated in carrageenan-induced rat-paw edema method. G-3, G-6, G-17, G-20, G-23, G-22, L-708 and 906 showed good antiinflammatory activity. In addition as diketo functionality containing compounds are reported to have HIV-1 integrase inhibitory property, and these compounds contains diketo functionality, so these compounds were screened in assay for HIV-1 integrase inhibition. Few compounds showed weak HIV-1 integrase Inhibitory activity.

  5. Importance of Hydrophilic Hydration and Intramolecular Interactions in the Thermodynamics of Helix-Coil Transition and Helix-Helix Assembly in a Deca-Alanine Peptide.

    Science.gov (United States)

    Tomar, Dheeraj S; Weber, Valéry; Pettitt, B Montgomery; Asthagiri, D

    2016-01-14

    For a model deca-alanine peptide the cavity (ideal hydrophobic) contribution to hydration favors the helix state over extended states and the paired helix bundle in the assembly of two helices. The energetic contributions of attractive protein-solvent interactions are separated into quasi-chemical components consisting of a short-range part arising from interactions with solvent in the first hydration shell and the remaining long-range part that is well described by a Gaussian. In the helix-coil transition, short-range attractive protein-solvent interactions outweigh hydrophobic hydration and favor the extended coil states. Analysis of enthalpic effects shows that it is the favorable hydration of the peptide backbone that favors the unfolded state. Protein intramolecular interactions favor the helix state and are decisive in favoring folding. In the pairing of two helices, the cavity contribution outweighs the short-range attractive protein-water interactions. However, long-range, protein-solvent attractive interactions can either enhance or reverse this trend depending on the mutual orientation of the helices. In helix-helix assembly, change in enthalpy arising from change in attractive protein-solvent interactions favors disassembly. In helix pairing as well, favorable protein intramolecular interactions are found to be as important as hydration effects. Overall, hydrophilic protein-solvent interactions and protein intramolecular interactions are found to play a significant role in the thermodynamics of folding and assembly in the system studied.

  6. Novel 3′-Processing Integrase Activity Assay by Real-Time PCR for Screening and Identification of HIV-1 Integrase Inhibitors

    Directory of Open Access Journals (Sweden)

    Supachai Sakkhachornphop

    2015-01-01

    Full Text Available The 3′-end processing (3′P of each viral long terminal repeat (LTR during human immunodeficiency virus type-1 (HIV-1 integration is a vital step in the HIV life cycle. Blocking the 3′P using 3′P inhibitor has recently become an attractive strategy for HIV-1 therapeutic intervention. Recently, we have developed a novel real-time PCR based assay for the detection of 3′P activity in vitro. The methodology usually involves biotinylated HIV-1 LTR, HIV-1 integrase (IN, and specific primers and probe. In this novel assay, we designed the HIV-1 LTR substrate based on a sequence with a homology to HIV-1 LTR labeled at its 3′ end with biotin on the sense strand. Two nucleotides at the 3′ end were subsequently removed by IN activity. Only two nucleotides labeled biotin were captured on an avidin-coated tube; therefore, inhibiting the binding of primers and probe results in late signals in the real-time PCR. This novel assay has successfully detected both the 3′P activity of HIV-1 IN and the anti-IN activity by Raltegravir and sodium azide agent. This real-time PCR assay has been shown to be effective and inexpensive for a high-throughput screening of novel IN inhibitors.

  7. HIV Type 1 Integrase Natural Polymorphisms in Viral Variants Circulating in FSU Countries.

    Science.gov (United States)

    Lapovok, Ilya; Laga, Vita; Kazennova, Elena; Bobkova, Marina

    2017-08-15

    Natural variability of integrase (IN) across HIV-1 variants may influence the emergence of resistant viruses. The most apparent explanation of these fact is the IN polymorphism and the associated differences in codon usage, which, in turn, influence the probability and the terms of DRMs acquisition. Possible mechanisms by which polymorphisms affect DRMs emergence remain disputed and should still be clarified because these substitutions may be associated with a reduced activity of some INSTIs and may impact on ART regimen choice depending of HIV-1 subtype. The aim of this work was to assess the prevalence of naturally occurring polymorphisms within the HIV-1 integrase gene, which might influence the susceptibility to INSTIs, among the patients from Russia and former USSR countries, according to HIV-1 subtypes. A study involved 506 HIV-1 IN sequences of INSTI-naive patients from Russia, Ukraine, Armenia, Kyrgyzstan, Kazakhstan, Uzbekistan, Belarus, and Georgia. Among them, 194 sequences were newly obtained in this study and 312 were downloaded from Los-Alamos database. The proviral DNA was sequenced using an in-house PCR protocol designed on the basis of a well-conserved integrase region in order to detect all HIV-1 variants. The phylogenetic analyses based on IN population sequencing found subtype A6 being the most prevalent (259) (51.2%) in the collection studied, followed by subtype G (36) (7.1%), AG-recombinants (148) (29.3%), subtype B (50) (9.9%), and CRF03_AB (5) (1,0%). The major INSTI resistance-associated mutations (DRMs) were found only in two A6 samples. The prevalence of minor/accessory substitutions depended on HIV-1 variants, while the most notable findings were L74I in subtype A6 (93.1%) and E157Q in subtype B (44.0%). Most of minor DRMs and polymorphic substitutions were concentrated in the central catalytic domain of the IN molecule. Both the DDE triad and HHCC zinc binding motifs were fully conserved. The results of the study suggest a very low

  8. "Special Issue": Regional Dimensions of the Triple Helix Model

    Science.gov (United States)

    Todeva, Emanuela; Danson, Mike

    2016-01-01

    This paper introduces the rationale for the special issue and its contributions, which bridge the literature on regional development and the Triple Helix model. The concept of the Triple Helix at the sub-national, and specifically regional, level is established and examined, with special regard to regional economic development founded on…

  9. Regional Dimensions of the Triple Helix Model: Setting the Context

    Science.gov (United States)

    Todeva, Emanuela; Danson, Mike

    2016-01-01

    This paper introduces the rationale for the special issue and its contributions, which bridge the literature on regional development and the Triple Helix model. The concept of the Triple Helix at the sub-national, and specifically regional, level is established and examined, with special regard to regional economic development founded on…

  10. Government and Governance of Regional Triple Helix Interactions

    Science.gov (United States)

    Danson, Mike; Todeva, Emanuela

    2016-01-01

    This conceptual paper contributes to the discussion of the role of regional government and regional Triple Helix constellations driving economic development and growth within regional boundaries. The impact of regionalism and subsidiarity on regional Triple Helix constellations, and the questions of governmentality, governance and institutional…

  11. Time-programmed helix inversion in phototunable liquid crystals.

    Science.gov (United States)

    Asshoff, Sarah J; Iamsaard, Supitchaya; Bosco, Alessandro; Cornelissen, Jeroen J L M; Feringa, Ben L; Katsonis, Nathalie

    2013-05-14

    Doping cholesteric liquid crystals with photo-responsive molecules enables controlling the colour and polarisation of the light they reflect. However, accelerating the rate of relaxation of these photo-controllable liquid crystals remains challenging. Here we show that the relaxation rate of the cholesteric helix is fully determined by helix inversion of the molecular dopants.

  12. An FPGA helix tracking algorithm for PANDA

    Energy Technology Data Exchange (ETDEWEB)

    Muenchow, David; Galuska, Martin; Gessler, Thomas; Kuehn, Wolfgang; Lange, Jens Soeren; Liang, Yutie; Liu, Ming; Spruck, Bjoern [Justus Liebig University Giessen (Germany); Spataro, Stefano [University of Torino (Italy)

    2010-07-01

    An online track finder for the PANDA experiment at the future FAIR facility was developed and tested. The central Panda tracking detectors for charged particles will consist of a silicon based micro vertex detector (MVD, 5-7 hits/track) and possibly of a straw tube tracker (STT, 15 double layers of straws). Due to the solenoidal magnetic field, tracks of charged particles can be parametrized by a helix (if neglecting energy loss). The algorithm works in several steps. Perpendicular to the beam direction the projection of the tracks is equivalent to a circle. Thus, first a conformal transformation will be used to convert the circles to straight lines. Second, a Hough transform is used to find the straight lines by a peak finding algorithm. Along the beam direction, a different Hough transformation is used. As the algorithm was developed for an FPGA, it uses lookup tables. Possible FPGA implementation is discussed.

  13. FPGA helix tracking algorithm for PANDA

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Yutie; Galuska, Martin; Gessler, Thomas; Hu, Jifeng; Kuehn, Wolfgang; Lange, Jens Soeren; Muenchow, David; Spruck, Bjoern [II. Physikalisches, Giessen University (Germany); Ye, Hua [II. Physikalisches, Giessen University (Germany); Institute of High Energy Physics, Beijing (China); Collaboration: PANDA-Collaboration

    2014-07-01

    The PANDA detector is a general-purpose detector for physics with high luminosity cooled antiproton beams, planed to operate at the FAIR facility in Darmstadt, Germany. The central detector includes a silicon Micro Vertex Detector (MVD) and a Straw Tube Tracker (STT). Without any hardware trigger, large amounts of raw data are streaming into the data acquisition system. The data reduction task is performed in the online system by reconstruction algorithms programmed in VHDL (Very High Speed Integrated Circuit Hardware Description Language) on FPGAs (Field Programmable Gate Arrays) as first level and on a farm of GPUs or PCs as a second level. One important part in the system is the online track reconstruction. In this presentation, an online tracking finding algorithm for helix track reconstruction in the solenoidal field is shown. A performance study using C++ and the status of the VHDL implementation are presented.

  14. The Triple Helix Perspective of Innovation Systems

    CERN Document Server

    Leydesdorff, Loet

    2010-01-01

    Alongside the neo-institutional model of networked relations among universities, industries, and governments, the Triple Helix can be provided with a neo-evolutionary interpretation as three selection environments operating upon one another: markets, organizations, and technological opportunities. How are technological innovation systems different from national ones? The three selection environments fulfill social functions: wealth creation, organization control, and organized knowledge production. The main carriers of this system-industry, government, and academia-provide the variation both recursively and by interacting among them under the pressure of competition. Empirical case studies enable us to understand how these evolutionary mechanisms can be expected to operate in historical instance. The model is needed for distinguishing, for example, between trajectories and regimes.

  15. The Triple Helix of the Organizational Knowledge

    Directory of Open Access Journals (Sweden)

    Contantin BRĂTIANU

    2013-09-01

    Full Text Available The purpose of this paper is to present the inner triple helix dynamics of the organizationalknowledge. This is a new perspective of the classical view of tacit knowledge– explicit knowledge dyad of the organizational knowledge promoted by Nonaka and hisco-workers. The new perspective is based on the metaphor that organizational knowledge isa "eld rather than a stock, or stocks and flows. It is a complex metaphor using the thermodynamicsprinciples. The organizational knowledge is composed of three different "elds: cognitiveknowledge, emotional knowledge and spiritual knowledge. These "elds are nonuniform,nonhomogeneous and they interact in a dynamic way. Cognitive "eld contains knowledgeabout what is, emotional "eld contains knowledge about how we feel, and the spiritual "eldcontains knowledge about people’s aspirations and life values. This new perspective opens anew opportunity in understanding the challenges for the 21st century management.

  16. Anti-HIV-1 integrase activity of medicinal plants used as self medication by AIDS patients

    Directory of Open Access Journals (Sweden)

    Sopa Kummee

    2006-07-01

    Full Text Available The extracts of selected medicinal plants used as self medication by AIDS patients were investigated for their inhibitory activities against HIV-1 integrase (HIV-1 IN using the multiplate integration assay (MIA. Of these, the water extract of Eclipta prostrata (whole plant exhibited the most potent inhibitory activity with an IC50 value of 4.8 μg/ml, followed by the methanol extract of Eclipta prostrata (whole plant, IC50 = 21.1 μg/ ml, the water extract of Barleria lupulina (stem, IC50 = 26.4 μg/ml, the chloroform extract of Barleria lupulina (stem, IC50 = 33.0 μg/ml, the methanol extract of Barleria lupulina (stem, IC50 = 38.2 μg/ml and the chloroform extract of Piper betle (leaf, IC50 = 39.3 μg/ml, respectively.

  17. Genetic Innovation in Vertebrates: Gypsy Integrase Genes and Other Genes Derived from Transposable Elements

    Directory of Open Access Journals (Sweden)

    Domitille Chalopin

    2012-01-01

    Full Text Available Due to their ability to drive DNA rearrangements and to serve as a source of new coding and regulatory sequences, transposable elements (TEs are considered as powerful evolutionary agents within genomes. In this paper, we review the mechanism of molecular domestication, which corresponds to the formation of new genes derived from TE sequences. Many genes derived from retroelements and DNA transposons have been identified in mammals and other vertebrates, some of them fulfilling essential functions for the development and survival of their host organisms. We will particularly focus on the evolution and expression of Gypsy integrase (GIN genes, which have been formed from ancient event(s of molecular domestication and have evolved differentially in some vertebrate sublineages. What we describe here is probably only the tip of the evolutionary iceberg, and future genome analyses will certainly uncover new TE-derived genes and biological functions driving genetic innovation in vertebrates and other organisms.

  18. Raltegravir, elvitegravir, and metoogravir: the birth of "me-too" HIV-1 integrase inhibitors

    Directory of Open Access Journals (Sweden)

    Neamati Nouri

    2009-03-01

    Full Text Available Abstract Merck's MK-0518, known as raltegravir, has recently become the first FDA-approved HIV-1 integrase (IN inhibitor and has since risen to blockbuster drug status. Much research has in turn been conducted over the last few years aimed at recreating but optimizing the compound's interactions with the protein. Resulting me-too drugs have shown favorable pharmacokinetic properties and appear drug-like but, as expected, most have a highly similar interaction with IN to that of raltegravir. We propose that, based upon conclusions drawn from our docking studies illustrated herein, most of these me-too MK-0518 analogues may experience a low success rate against raltegravir-resistant HIV strains. As HIV has a very high mutational competence, the development of drugs with new mechanisms of inhibitory action and/or new active substituents may be a more successful route to take in the development of second- and third-generation IN inhibitors.

  19. Investigating the role of metal chelation in HIV-1 integrase strand transfer inhibitors.

    Science.gov (United States)

    Bacchi, Alessia; Carcelli, Mauro; Compari, Carlotta; Fisicaro, Emilia; Pala, Nicolino; Rispoli, Gabriele; Rogolino, Dominga; Sanchez, Tino W; Sechi, Mario; Sinisi, Valentina; Neamati, Nouri

    2011-12-22

    HIV-1 integrase (IN) has been validated as an attractive target for the treatment of HIV/AIDS. Several studies have confirmed that the metal binding function is a crucial feature in many of the reported IN inhibitors. To provide new insights on the metal chelating mechanism of IN inhibitors, we prepared a series of metal complexes of two ligands (HL1 and HL2), designed as representative models of the clinically used compounds raltegravir and elvitegravir. Potentiometric measurements were conducted for HL2 in the presence of Mg(II), Mn(II), Co(II), and Zn(II) in order to delineate a metal speciation model. We also determined the X-ray structures of both of the ligands and of three representative metal complexes. Our results support the hypothesis that several selective strand transfer inhibitors preferentially chelate one cation in solution and that the metal complexes can interact with the active site of the enzyme.

  20. Hexa Helix: Modified Quad Helix Appliance to Correct Anterior and Posterior Crossbites in Mixed Dentition

    Directory of Open Access Journals (Sweden)

    Syed Mohammed Yaseen

    2012-01-01

    Full Text Available Among the commonly encountered dental irregularities which constitute developing malocclusion is the crossbite. During primary and mixed dentition phase, the crossbite is seen very often and if left untreated during these phases then a simple problem may be transformed into a more complex problem. Different techniques have been used to correct anterior and posterior crossbites in mixed dentition. This case report describes the use of hexa helix, a modified version of quad helix for the management of anterior crossbite and bilateral posterior crossbite in early mixed dentition. Correction was achieved within 15 weeks with no damage to the tooth or the marginal periodontal tissue. The procedure is a simple and effective method for treating anterior and bilateral posterior crossbites simultaneously.

  1. Resistance mutations against dolutegravir in HIV integrase impair the emergence of resistance against reverse transcriptase inhibitors.

    Science.gov (United States)

    Oliveira, Maureen; Mesplède, Thibault; Quashie, Peter K; Moïsi, Daniela; Wainberg, Mark A

    2014-03-27

    Among 1222 antiretroviral-naive patients who received dolutegravir (DTG) as part of first-line therapy, none has developed resistance against this compound after 48-96 weeks of follow-up. Moreover, only four occurrences of virological failure with resistance mutations have been documented in previously drug-experienced patients who received DTG as a first time integrase inhibitor as a component of a second-line regimen. The R263K integrase resistance mutation was observed in two of these individuals who received suboptimal background regimens. We have previously selected mutations at position R263K, G118R, H51Y, and E138K as being associated with low-level resistance to DTG. Now, we sought to investigate the facility with which resistance on the part of R263K-containing viruses might develop. We tested the ability of DTG-resistant viruses containing either the R263K or G118R and/or H51Y mutations to develop further resistance against several reverse transcriptase inhibitors during in-vitro selection experiments. Our results show that DTG-resistant viruses are impaired in their ability to acquire further resistance to each of nevirapine and lamivudine as a consequence of their relative inability to develop resistance mutations associated with these two compounds. Our findings provide an explanation for the fact that no individual has yet progressed to virological failure with resistance mutations associated with dolutegravir in clinical trials in which patients received dolutegravir together with an optimized background regimen.

  2. The discovery of S/GSK1265744: a carbamoyl pyridone HIV-1 integrase inhibitor

    Directory of Open Access Journals (Sweden)

    H Murai

    2012-11-01

    Full Text Available Background: HIV-1 integrase is a virally encoded enzyme essential for lentiviral replication. Assiduous medicinal chemistry efforts culminated in the discovery of raltegravir, the first marketed HIV-1 integrase inhibitor (INI. However, there is significant opportunity for improvement including overall dose burden, dosing interval and potency against resistant viruses. Our molecular design approach used a two-metal binding pharmacophore strategy and succeeded in identification of carbamoyl pyridone HIV-1 INIs. This enriched core scaffold has abundant structural features expanding the opportunity to control drug properties, leading to the discovery of S/GSK1265744. Methods: The carbamoyl pyridone scaffold was derivatized and evaluated for antiviral activity against wild-type virus (±HSA along with key INI-resistant mutants. Animal pharmacokinetic profiles including a key measure of the trough drug concentration over protein-adjusted antiviral potency (C24/PAIC50 along with in vitro DMPK properties, were used along with the virological data for compound selection. Results: The carbamoyl pyridone series inhibitors exhibited potent antiviral profiles with promising DMPK properties. S/GSK1265744 demonstrated good coverage of C24 over PAIC50 predicting low mg unboosted once daily dosing, now validated in phase 2 clinical studies. These preclinical data along with a long human T1/2 of ~30 hours in oral tablet study supports S/GSK1265744 as a long acting parenteral agent for once-monthly or less frequent dosing. Conclusions: A medicinal chemistry approach utilizing key viral mutants in combination with C24/PAIC50 has allowed for discovery of S/GSK1265744. This agent is currently in phase 2 development evaluating a novel, long-acting parenteral route of administration and may enable new approaches to HIV therapy and prevention.

  3. NMR-based approach to measure the free energy of transmembrane helix-helix interactions.

    Science.gov (United States)

    Mineev, Konstantin S; Lesovoy, Dmitry M; Usmanova, Dinara R; Goncharuk, Sergey A; Shulepko, Mikhail A; Lyukmanova, Ekaterina N; Kirpichnikov, Mikhail P; Bocharov, Eduard V; Arseniev, Alexander S

    2014-01-01

    Knowledge of the energetic parameters of transmembrane helix-helix interactions is necessary for the establishment of a structure-energy relationship for α-helical membrane domains. A number of techniques have been developed to measure the free energies of dimerization and oligomerization of transmembrane α-helices, and all of these have their advantages and drawbacks. In this study we propose a methodology to determine the magnitudes of the free energy of interactions between transmembrane helices in detergent micelles. The suggested approach employs solution nuclear magnetic resonance (NMR) spectroscopy to determine the population of the oligomeric states of the transmembrane domains and introduces a new formalism to describe the oligomerization equilibrium, which is based on the assumption that both the dimerization of the transmembrane domains and the dissociation of the dimer can occur only upon the collision of detergent micelles. The technique has three major advantages compared with other existing approaches: it may be used to analyze both weak and relatively strong dimerization/oligomerization processes, it works well for the analysis of complex equilibria, e.g. when monomer, dimer and high-order oligomer populations are simultaneously present in the solution, and it can simultaneously yield both structural and energetic characteristics of the helix-helix interaction under study. The proposed methodology was applied to investigate the oligomerization process of transmembrane domains of fibroblast growth factor receptor 3 (FGFR3) and vascular endothelium growth factor receptor 2 (VEGFR2), and allowed the measurement of the free energy of dimerization of both of these objects. In addition the proposed method was able to describe the multi-state oligomerization process of the VEGFR2 transmembrane domain.

  4. Mutations in TWIST, a basic helix-loop-helix transcription factor, in Saethre-Chotzen syndrome.

    Science.gov (United States)

    Howard, T D; Paznekas, W A; Green, E D; Chiang, L C; Ma, N; Ortiz de Luna, R I; Garcia Delgado, C; Gonzalez-Ramos, M; Kline, A D; Jabs, E W

    1997-01-01

    Saethre-Chotzen syndrome is one of the most common autosomal dominant disorders of craniosynostosis in humans and is characterized by craniofacial and limb anomalies. The locus for Saethre-Chotzen syndrome maps to chromosome 7p21-p22. We have evaluated TWIST, a basic helix-loop-helix transcription factor, as a candidate gene for this condition because its expression pattern and mutant phenotypes in Drosophila and mouse are consistent with the Saethre-Chotzen phenotype. We mapped TWIST to human chromosome 7p21-p22 and mutational analysis reveals nonsense, missense, insertion and deletion mutations in patients. These mutations occur within the basic DNA binding, helix I and loop domains, or result in premature termination of the protein. Studies in Drosophila indicate that twist may affect the transcription of fibroblast growth factor receptors (FGFRs), another gene family implicated in human craniosynostosis. The emerging cascade of molecular components involved in craniofacial and limb development now includes TWIST, which may function as an upstream regulator of FGFRs.

  5. In vivo and in vitro characterization of site-specific recombination of a novel serine integrase from the temperate phage EFC-1

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Bohyun; Kim, Inki; Nam, Ja-Ae [Asan Institute for Life Sciences, Asan Medical Center, College of Medicine, University of Ulsan, 86 Asanbyeoungwon-gil, Songpa-gu, Seoul 138-736 (Korea, Republic of); Chang, Hyo-Ihl [College of Life Sciences & Biotechnology, Korea University, 5-1 Anam-dong, Seongbuk-gu, Seoul 136-701 (Korea, Republic of); Ha, Chang Hoon, E-mail: chhoonha@amc.seoul.kr [Asan Institute for Life Sciences, Asan Medical Center, College of Medicine, University of Ulsan, 86 Asanbyeoungwon-gil, Songpa-gu, Seoul 138-736 (Korea, Republic of)

    2016-04-22

    EFC-1 integrase is a site-specific recombinase that belongs to the large family of serine recombinase. In previously study, we isolated the temperate phage EFC-1, and characterized its genomic sequence. Within its genome, Orf28 was predicted encode a 464 amino acid of a putative integrase gene. In this study, EFC-1 integrase was characterized in vitro and in vivo. In vitro assay was performed using purified His-tag fusion integrase. Also, to identify which serine is involved in the catalytic domain, we used site-directed mutagenesis and analyzed by a recombination assay in vitro. In vivo assay involved PCR and confocal microscopy in HEK293 cells, and determined the minimal lengths of attP and attB sites. According to our results, the EFC-1 integrase-mediated recombination was site-specific and unidirectional system in vitro and in vivo. Serine 21 of EFC-1 integrase plays a major role in the catalytic domain, and minimal sizes of attB and attP was defined 48 and 54 bp. Our finding may help develop a useful tool for gene therapy and gene delivery system. - Highlights: • EFC-1 integrase-mediated recombination was site-specific and unidirectional system. • Serine 21 of EFC-1 integrase plays a major role in the catalytic domain. • The functional minimal sizes of attB and attP was defined 48 and 54 bp.

  6. Can synergy in Triple Helix relations be quantified? A review of the development of the Triple Helix indicator

    NARCIS (Netherlands)

    Leydesdorff, L.; Park, H.W.

    2014-01-01

    Triple Helix arrangements of bi- and trilateral relations can be considered as adaptive ecosystems. During the last decade, we have further developed a Triple Helix indicator of synergy as reduction of uncertainty in niches that can be shaped among three or more sets of relations. Reduction of uncer

  7. Differential regulation of granulopoiesis by the basic helix-loop-helix transcriptional inhibitors Id1 and Id2

    NARCIS (Netherlands)

    Buitenhuis, M; van Deutekom, HWM; Verhagen, LP; Castor, A; Jacobsen, SEW; Lammers, JWJ; Koenderman, L; Coffer, PJ

    2005-01-01

    Inhibitor of DNA binding (Id) proteins function as inhibitors of members of the basic helix-loop-helix family of transcription factors and have been demonstrated to play an important role in regulating lymphopoiesis. However, the role of these proteins in regulation of myelopoiesis is currently

  8. Concentration-temperature superposition of helix folding rates in gelatin.

    Science.gov (United States)

    Gornall, J L; Terentjev, E M

    2007-07-13

    Using optical rotation as the primary technique, we have characterized the kinetics of helix renaturation in water solutions of gelatin. By covering a wide range of solution concentrations we identify a universal exponential dependence of folding rate on concentration and quench temperature. We demonstrate a new concentration-temperature superposition of data at all temperatures and concentrations, and build the corresponding master curve. The normalized rate constant is consistent with helix lengthening. Nucleation of the triple helix occurs rapidly and contributes less to the helical onset than previously thought.

  9. Electrostatic free energy landscapes for nucleic acid helix assembly

    OpenAIRE

    Tan, Zhi-Jie; Chen, Shi-Jie

    2006-01-01

    Metal ions are crucial for nucleic acid folding. From the free energy landscapes, we investigate the detailed mechanism for ion-induced collapse for a paradigm system: loop-tethered short DNA helices. We find that Na + and Mg2+ play distinctive roles in helix–helix assembly. High [Na+] (>0.3 M) causes a reduced helix–helix electrostatic repulsion and a subsequent disordered packing of helices. In contrast, Mg2+ of concentration >1 mM is predicted to induce helix–helix attraction and results i...

  10. Novel DNA sensor system for highly sensitive and quantitative retrovirus detection using virus encoded integrase as a biomarker

    DEFF Research Database (Denmark)

    Wang, Jing; Liu, Jiangnan; Thomsen, Jonas

    2017-01-01

    In the current study we describe a novel DNA sensor system that allows the detection of single catalytic DNA integration events mediated by retrovirus encoded integrase (IN) present in viral particles. This is achieved by rolling circle amplification mediated conversion of enzymatic reactions...... type of diagnostic tools suitable for early (within hours of infection) detection of HIV, which would be valuable for prevention strategies as well as for efficient treatment....

  11. Silver conical helix broadband plasmonic nanoantenna

    Science.gov (United States)

    Sobhkhiz, Nader; Moshaii, Ahmad

    2014-01-01

    The discrete dipole approximation method is used to investigate the optical extinction spectra and the electric field enhancement of Ag conical helix (CH) nanostructures. Based on an expected similarity between the radio frequency response of the antenna with the infrared and the visible response of the nanoantenna, the Ag CH nanostructures were designed as a broadband nanoantenna. It is shown that with engineering the structure parameters of the CH nanostructure the plasmonic response of the nanostructure can be designed for a desirable application. In addition, the change of the substrate material for the nanohelix growth is shown to have infinitesimal effect on the resonance peaks of the conical nanohelix. However, varying the surrounding medium can lead to considerable red-shifting of the plasmonic resonance peaks (up to 230 nm). Calculations of the near field around the helical nanoantenna show that the smaller and the larger sides of the CH are related to the plasmonic resonance peaks at low and high wavelengths, respectively. The calculation result for the extinction spectrum has also been compared with similar experimental data for a 2-pitch Ag conical nanohelix and a relatively good agreement between the numerical calculation and the experiment has been obtained.

  12. A highly efficient site-specific integration strategy using combination of homologous recombination and the ΦC31 integrase.

    Science.gov (United States)

    Ou, Hailong; Huang, Ying; Ma, Qingwen; Ren, Zhaorui; Huang, Shuzhen; Zeng, Fanyi; Zeng, Yitao

    2013-09-20

    The introduction of double-strand breaks (DSBs) at target sites could greatly enhance homologous recombination, and engineered nucleases, such as zinc finger and transcription activator-like effector nucleases, have been successfully developed for making such breaks. In this study, we present a highly efficient site-specific integration strategy based on homologous recombination and ΦC31 integrase. An attB sequence was introduced at the homologous arm of an insertion targeting vector. DSBs at the target locus and donor were then simultaneously generated by the ΦC31 integrase when co-transfected with the donor vector, consequently stimulating homologous recombination. The results demonstrated that our strategy is feasible and the efficiency at the BF4 target site, which we previously identified in the bovine genome, was as high as 93%. The frequency at another site (BF10) was almost two-fold greater in comparison to the vector without homologous arms. This technology requires no sophisticated nuclease design efforts, and the off-target effect is reduced by ΦC31 integrase compared to the use of engineered nucleases, thereby offering a simple and safe way to effectively express a donor gene at a desired locus. This development has great potential value, especially in transgenesis or gene therapy applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. A Profile of Native Integration Sites Used by φC31 Integrase in the Bovine Genome

    Institute of Scientific and Technical Information of China (English)

    Lijuan Qu; Qingwen Ma; Zaiwei Zhou; Haiyan Ma; Ying Huang; Shuzhen Huang; Fanyi Zeng; Yitao Zeng

    2012-01-01

    The Streptomyces phage φC31 integrase can efficiently target attB-beadng transgenes to endogenous pseudo attP sites within mammalian genomes.To better understand the activity of φC31 integrase in the bovine genome,DNA sequences of 44 integration events were analyzed,and 32 pseudo attP sites were identified.The majority of these sites share a sequence motif that contains inverted repeats and has similarities to wild-type attP site.Genomic DNA flanking these sites typically contained repetitive sequence elements,such as short and long interspersed repetitive elements.These sequence features indicate that DNA sequence recognition plays an important role in guiding φC31-mediated site-specific integration.In addition,BF27 integration hotspot sites were identified in the bovine genome,which accounted for 13.6% of all isolated integration events and mapped to an intron of the deleted in liver cancer 1 (DLC1) gene.Also we found that the pseudo attP sites in the bovine genome had other features in common with those in the human genome.This study represents the first time that the sequence features of pseudo attP sites in the bovine genome were analyzed.We conclude that this sitespecific integrase system has great potential for applied modifications of the bovine genome.

  14. Secondary mutations in viruses resistant to HIV-1 integrase inhibitors that restore viral infectivity and replication kinetics.

    Science.gov (United States)

    Nakahara, Koichiro; Wakasa-Morimoto, Chiaki; Kobayashi, Masanori; Miki, Shigeru; Noshi, Takeshi; Seki, Takahiro; Kanamori-Koyama, Mikiko; Kawauchi, Shinobu; Suyama, Akemi; Fujishita, Toshio; Yoshinaga, Tomokazu; Garvey, Edward P; Johns, Brian A; Foster, Scott A; Underwood, Mark R; Sato, Akihiko; Fujiwara, Tamio

    2009-02-01

    Passage of HIV-1 in the presence of integrase inhibitors (INIs) generates resistant viruses that have mutations in the integrase region. Integrase-resistant mutations Q148K and Q148R were identified as primary mutations with the passage of HIV-1 IIIB in the presence of INIs S-1360 or S/GSK-364735, respectively. Secondary amino acid substitutions E138K or G140S were observed when passage with INI was continued. The role of these mutations was investigated with molecular clones. Relative to Q148K alone, Q148K/E138K had 2- and >6-fold increases in resistance to S-1360 and S/GSK-364735, respectively, and the double mutant had slightly better infectivity and replication kinetics. In contrast, Q148K/G140S and Q148R/E138K had nearly equivalent or slightly reduced fold resistance to the INI compared with their respective Q148 primary mutants, and had increases in infectivity and replication kinetics. Recovery of these surrogates of viral fitness coincided with the recovery of integration efficiency of viral DNA into the host cell chromosome for these double mutants. These data show that recovery of viral integration efficiency can be an important factor for the emergence and maintenance of INI-resistant mutations.

  15. Creation of Tenecteplase-Producing CHO Cell Line Using Site-Specific Integrase from the Phage φC31

    Directory of Open Access Journals (Sweden)

    Khadijeh Karbalaie

    2010-01-01

    Full Text Available Objective: The aim of this study was to produce a stable CHO cell line expressing tenecteplase.Materials and Methods: In the first step, the tenecteplase coding sequence was clonedin a pDB2 vector containing attB recognition sites for the phage φC31 integrase. Then,using lipofection, the CHO cells were co-transfected with constructed recombinant plasmidencoding tenecteplase and attB recognition sites and the integrase coding sequencecontaining pCMV-Int plasmid. As the recombinant plasmid contained the neomycin resistancegene (neo, stable cells were then selected using G418 as an antibiotic. Stabletransformed cells were assessed using genomic PCR and RT-PCR. Finally, the functionalityof tenecteplase was evaluated on the cell culture media.Results: our results indicated that tenecteplase coding sequence was inserted into theCHO cell genome and was successfully expressed. Moreover, tenecteplase activity assessmentindicated the presence of our functional tenecteplase in the cell culture medium.Conclusion: Considering the data obtained from this study, φC31 integrase can be usedfor the production of a stable cell line and it be used to introduce ectopic genes into mammaliancells.

  16. Role of hydration and intramolecular interactions in the helix-coil transition and helix-helix assembly in a deca-alanine peptide

    CERN Document Server

    Tomar, Dheeraj S; Pettitt, B M; Asthagiri, D

    2015-01-01

    For a model deca-alanine peptide the cavity (ideal hydrophobic) contribution to hydration favors the helix state in the coil-to-helix transition and the paired helix bundle in the assembly of two helices. The energetic contributions of attractive protein-solvent interactions are separated into a short-range part arising from interactions with solvent in the first hydration shell and the remaining long-range part. In the helix-coil transition, short-range attractive protein-solvent interactions outweigh hydrophobic hydration and favor the unfolded coil states. Analysis of enthalpic effects shows that it is the favorable hydration of the peptide backbone that favors the unfolded state. Protein intramolecular interactions favor the helix state and are decisive in folding. In the pairing of two helices, the cavity contribution outweighs short-range attractive protein-water interactions. However, long-range, protein-solvent attractive interactions can either enhance or reverse this trend depending on the mutual or...

  17. Data on diverse roles of helix perturbations in membrane proteins

    Directory of Open Access Journals (Sweden)

    Ashish Shelar

    2016-12-01

    Full Text Available The various structural variations observed in TM helices of membrane proteins have been deconstructed into 9 distinct types of helix perturbations. These perturbations are defined by the deviation of TM helices from the predominantly observed linear α-helical conformation, to form 310- and π-helices, as well as adopting curved and kinked geometries. The data presented here supplements the article ‘Helix perturbations in Membrane Proteins Assist in Inter-helical Interactions and Optimal Helix Positioning in the Bilayer’ (A. Shelar, M. Bansal, 2016 [1]. This data provides strong evidence for the role of various helix perturbations in influencing backbone torsion angles of helices, mediating inter-helical interactions, oligomer formation and accommodation of hydrophobic residues within the bilayer. The methodology used for creation of various datasets of membrane protein families (Sodium/Calcium exchanger and Heme Copper Oxidase has also been mentioned.

  18. Structural alignment of RNA with triple helix structure.

    Science.gov (United States)

    Wong, Thomas K F; Yiu, S M

    2012-04-01

    Structural alignment is useful in identifying members of ncRNAs. Existing tools are all based on the secondary structures of the molecules. There is evidence showing that tertiary interactions (the interaction between a single-stranded nucleotide and a base-pair) in triple helix structures are critical in some functions of ncRNAs. In this article, we address the problem of structural alignment of RNAs with the triple helix. We provide a formal definition to capture a simplified model of a triple helix structure, then develop an algorithm of O(mn(3)) time to align a query sequence (of length m) with known triple helix structure with a target sequence (of length n) with an unknown structure. The resulting algorithm is shown to be useful in identifying ncRNA members in a simulated genome.

  19. Development of a receptor model for efficient in silico screening of HIV-1 integrase inhibitors.

    Science.gov (United States)

    Quevedo, Mario A; Ribone, Sergio R; Briñón, Margarita C; Dehaen, Wim

    2014-07-01

    Integrase (IN) is a key viral enzyme for the replication of the type-1 human immunodeficiency virus (HIV-1), and as such constitutes a relevant therapeutic target for the development of anti-HIV agents. However, the lack of crystallographic data of HIV IN complexed with the corresponding viral DNA has historically hindered the application of modern structure-based drug design techniques to the discovery of new potent IN inhibitors (INIs). Consequently, the development and validation of reliable HIV IN structural models that may be useful for the screening of large databases of chemical compounds is of particular interest. In this study, four HIV-1 IN homology models were evaluated respect to their capability to predict the inhibition potency of a training set comprising 36 previously reported INIs with IC50 values in the low nanomolar to the high micromolar range. Also, 9 inactive structurally related compounds were included in this training set. In addition, a crystallographic structure of the IN-DNA complex corresponding to the prototype foamy virus (PFV) was also evaluated as structural model for the screening of inhibitors. The applicability of high throughput screening techniques, such as blind and ligand-guided exhaustive rigid docking was assessed. The receptor models were also refined by molecular dynamics and clustering techniques to assess protein sidechain flexibility and solvent effect on inhibitor binding. Among the studied models, we conclude that the one derived from the X-ray structure of the PFV integrase exhibited the best performance to rank the potencies of the compounds in the training set, with the predictive power being further improved by explicitly modeling five water molecules within the catalytic side of IN. Also, accounting for protein sidechain flexibility enhanced the prediction of inhibition potencies among the studied compounds. Finally, an interaction fingerprint pattern was established for the fast identification of potent IN

  20. Class II integrase mutants with changes in putative nuclear localization signals are primarily blocked at a postnuclear entry step of human immunodeficiency virus type 1 replication.

    Science.gov (United States)

    Lu, Richard; Limón, Ana; Devroe, Eric; Silver, Pamela A; Cherepanov, Peter; Engelman, Alan

    2004-12-01

    Integrase has been implicated in human immunodeficiency virus type 1 (HIV-1) nuclear import. Integrase analyses, however, can be complicated by the pleiotropic nature of mutations: whereas class I mutants are integration defective, class II mutants display additional assembly and/or reverse transcription defects. We previously determined that HIV-1(V165A), originally reported as defective for nuclear import, was a class II mutant. Here we analyzed mutants containing changes in other putative nuclear localization signals, including (186)KRK(188)/(211)KELQKQITK(219) and Cys-130. Previous work established HIV-1(K186Q), HIV-1(Q214L/Q216L), and HIV-1(C130G) as replication defective, but phenotypic classification was unclear and nuclear import in nondividing cells was not addressed. Consistent with previous reports, most of the bipartite mutants studied here were replication defective. These mutants as well as HIV-1(V165A) synthesized reduced cDNA levels, but a normal fraction of mutant cDNA localized to dividing and nondividing cell nuclei. Somewhat surprisingly, recombinant class II mutant proteins were catalytically active, and class II Vpr-integrase fusion proteins efficiently complemented class I mutant virus. Since a class I Vpr-integrase mutant efficiently complemented class II mutant viruses under conditions in which class II Vpr-integrases failed to function, we conclude that classes I and II define two distinct complementation groups and suggest that class II mutants are primarily defective at a postnuclear entry step of HIV-1 replication. HIV-1(C130G) was also defective for reverse transcription, but Vpr-integrase(C130G) did not efficiently complement class I mutant HIV-1. Since HIV-1(C130A) grew like the wild type, we conclude that Cys-130 is not essential for replication and speculate that perturbation of integrase structure contributed to the pleiotropic HIV-1(C130G) phenotype.

  1. Impact of hydrodynamic injection and phiC31 integrase on tumor latency in a mouse model of MYC-induced hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Lauren E Woodard

    Full Text Available BACKGROUND: Hydrodynamic injection is an effective method for DNA delivery in mouse liver and is being translated to larger animals for possible clinical use. Similarly, phiC31 integrase has proven effective in mediating long-term gene therapy in mice when delivered by hydrodynamic injection and is being considered for clinical gene therapy applications. However, chromosomal aberrations have been associated with phiC31 integrase expression in tissue culture, leading to questions about safety. METHODOLOGY/PRINCIPAL FINDINGS: To study whether hydrodynamic delivery alone, or in conjunction with delivery of phiC31 integrase for long-term transgene expression, could facilitate tumor formation, we used a transgenic mouse model in which sustained induction of the human C-MYC oncogene in the liver was followed by hydrodynamic injection. Without injection, mice had a median tumor latency of 154 days. With hydrodynamic injection of saline alone, the median tumor latency was significantly reduced, to 105 days. The median tumor latency was similar, 106 days, when a luciferase donor plasmid and backbone plasmid without integrase were administered. In contrast, when active or inactive phiC31 integrase and donor plasmid were supplied to the mouse liver, the median tumor latency was 153 days, similar to mice receiving no injection. CONCLUSIONS/SIGNIFICANCE: Our data suggest that phiC31 integrase does not facilitate tumor formation in this C-MYC transgenic mouse model. However, in groups lacking phiC31 integrase, hydrodynamic injection appeared to contribute to C-MYC-induced hepatocellular carcinoma in adult mice. Although it remains to be seen to what extent these findings may be extrapolated to catheter-mediated hydrodynamic delivery in larger species, they suggest that caution should be used during translation of hydrodynamic injection to clinical applications.

  2. Hedera helix L. and damages in Tlos Ancient City

    Directory of Open Access Journals (Sweden)

    Elinç, Z.K.

    2013-03-01

    Full Text Available There are various plant types in Tlos Ancient City of Fethiye district in the Province of Mugla, a city where different residential ruins of Lycia Civilization starting from Classical Age until Byzantine Period. Tlos is an important city in West-Lycia and is situated right on the control point of Lycia Way. Hedera helix L. is one of the plants living in this area, which attracts the attention as it mostly harms the ancient ruins. One of the most important reasons why Hedera helix L. is growing commonly in this region is the perfect ecological circumstances for the growth of this plant of the location where this ancient city is situated in. Additionally the fact that the ruins of the city are left on their fate, is another perfect circumstance for the Hedera helix L. to grow. Climbing or creeping stems of Hedera helix L. stick easily to the objects it touches and encircle them. Due to this characteristic, the walls of the ancient city are covered by this plant. Nevertheless, Hedera helix L. does not only harm the ancient constructions and natural rocks but also woody plants. The harm caused by dried out or cut Hedera helix L. are more than the harm caused by them when they were untouched. The subject of this study is to prove the shape and level of the harm caused by Hedera helix L. on ancient ruins of Tlos. At the same time, this study will underline the fighting methods against Hedera helix L. by comparing similar studies in other countries. Knowledge collected after this study will offer an insight into the excavation and restoration studies undertaken in all Mediterranean countries.

  3. The Triple Helix of University-Industry-Government Relations

    OpenAIRE

    Leydesdorff, Loet

    2012-01-01

    Etzkowitz & Leydesdorff (2000) further elaborated the Triple Helix of University-Industry-Government Relations (cf. Etzkowitz & Leydesdorff, 1995; Lowe, 1982) into a model for studying knowledge-based economies. A series of workshops, conferences, and special issues of journals have developed under this title since 1996. In various countries, the Triple Helix concept has also been used as an operational strategy for regional development and to further the knowledge-based economy. This short r...

  4. Slow Wave Characteristics of Helix Structure with Elliptical Cross Section

    Institute of Scientific and Technical Information of China (English)

    XIE Jian-Xiang; WEI Yan-Yu; GONG Yu-Bin; Fu Cheng-Fang; YUE Ling-Na; WANG Wen-Xiang

    2007-01-01

    We present a novel helix slow wave structure with an elliptical cross section shielded by an elliptical waveguide.The rf characteristics including dispersion properties,interaction impedance of zero mode in this structure have been studied in detail.The theoretical results reveal that weaker dispersion even abnormal dispersion characteristics is obtained with the increasing eccentricity of the elliptical waveguide,while the interaction impedance is enhanced by enlarging the eccentricity of elliptical helix.

  5. A directional nucleation-zipping mechanism for triple helix formation.

    Science.gov (United States)

    Alberti, Patrizia; Arimondo, Paola B; Mergny, Jean-Louis; Garestier, Thérèse; Hélène, Claude; Sun, Jian-Sheng

    2002-12-15

    A detailed kinetic study of triple helix formation was performed by surface plasmon resonance. Three systems were investigated involving 15mer pyrimidine oligonucleotides as third strands. Rate constants and activation energies were validated by comparison with thermodynamic values calculated from UV-melting analysis. Replacement of a T.A base pair by a C.G pair at either the 5' or the 3' end of the target sequence allowed us to assess mismatch effects and to delineate the mechanism of triple helix formation. Our data show that the association rate constant is governed by the sequence of base triplets on the 5' side of the triplex (referred to as the 5' side of the target oligopurine strand) and provides evidence that the reaction pathway for triple helix formation in the pyrimidine motif proceeds from the 5' end to the 3' end of the triplex according to the nucleation-zipping model. It seems that this is a general feature for all triple helices formation, probably due to the right-handedness of the DNA double helix that provides a stronger base stacking at the 5' than at the 3' duplex-triplex junction. Understanding the mechanism of triple helix formation is not only of fundamental interest, but may also help in designing better triple helix-forming oligonucleotides for gene targeting and control of gene expression.

  6. Challenges in Targeting a Basic Helix-Loop-Helix Transcription Factor with Hydrocarbon-Stapled Peptides.

    Science.gov (United States)

    Edwards, Amanda L; Meijer, Dimphna H; Guerra, Rachel M; Molenaar, Remco J; Alberta, John A; Bernal, Federico; Bird, Gregory H; Stiles, Charles D; Walensky, Loren D

    2016-11-18

    Basic helix-loop-helix (bHLH) transcription factors play critical roles in organism development and disease by regulating cell proliferation and differentiation. Transcriptional activity, whether by bHLH homo- or heterodimerization, is dependent on protein-protein and protein-DNA interactions mediated by α-helices. Thus, α-helical decoys have been proposed as potential targeted therapies for pathologic bHLH transcription. Here, we developed a library of stabilized α-helices of OLIG2 (SAH-OLIG2) to test the capacity of hydrocarbon-stapled peptides to disrupt OLIG2 homodimerization, which drives the development and chemoresistance of glioblastoma multiforme, one of the deadliest forms of human brain cancer. Although stapling successfully reinforced the α-helical structure of bHLH constructs of varying length, sequence-specific dissociation of OLIG2 dimers from DNA was not achieved. Re-evaluation of the binding determinants for OLIG2 self-association and stability revealed an unanticipated role of the C-terminal domain. These data highlight potential pitfalls in peptide-based targeting of bHLH transcription factors given the liabilities of their positively charged amino acid sequences and multifactorial binding determinants.

  7. A genome-wide survey on basic helix-loop-helix transcription factors in giant panda.

    Directory of Open Access Journals (Sweden)

    Chunwang Dang

    Full Text Available The giant panda (Ailuropoda melanoleuca is a critically endangered mammalian species. Studies on functions of regulatory proteins involved in developmental processes would facilitate understanding of specific behavior in giant panda. The basic helix-loop-helix (bHLH proteins play essential roles in a wide range of developmental processes in higher organisms. bHLH family members have been identified in over 20 organisms, including fruit fly, zebrafish, mouse and human. Our present study identified 107 bHLH family members being encoded in giant panda genome. Phylogenetic analyses revealed that they belong to 44 bHLH families with 46, 25, 15, 4, 11 and 3 members in group A, B, C, D, E and F, respectively, while the remaining 3 members were assigned into "orphan". Compared to mouse, the giant panda does not encode seven bHLH proteins namely Beta3a, Mesp2, Sclerax, S-Myc, Hes5 (or Hes6, EBF4 and Orphan 1. These results provide useful background information for future studies on structure and function of bHLH proteins in the regulation of giant panda development.

  8. An exploration of alternative visualisations of the basic helix-loop-helix protein interaction network

    Directory of Open Access Journals (Sweden)

    Amoutzias Grigoris D

    2007-08-01

    Full Text Available Abstract Background Alternative representations of biochemical networks emphasise different aspects of the data and contribute to the understanding of complex biological systems. In this study we present a variety of automated methods for visualisation of a protein-protein interaction network, using the basic helix-loop-helix (bHLH family of transcription factors as an example. Results Network representations that arrange nodes (proteins according to either continuous or discrete information are investigated, revealing the existence of protein sub-families and the retention of interactions following gene duplication events. Methods of network visualisation in conjunction with a phylogenetic tree are presented, highlighting the evolutionary relationships between proteins, and clarifying the context of network hubs and interaction clusters. Finally, an optimisation technique is used to create a three-dimensional layout of the phylogenetic tree upon which the protein-protein interactions may be projected. Conclusion We show that by incorporating secondary genomic, functional or phylogenetic information into network visualisation, it is possible to move beyond simple layout algorithms based on network topology towards more biologically meaningful representations. These new visualisations can give structure to complex networks and will greatly help in interpreting their evolutionary origins and functional implications. Three open source software packages (InterView, TVi and OptiMage implementing our methods are available.

  9. Hydroxyproline Ring Pucker Causes Frustration of Helix Parameters in the Collagen Triple Helix

    Science.gov (United States)

    Ying Chow, W.; Bihan, Dominique; Forman, Chris J.; Slatter, David A.; Reid, David G.; Wales, David J.; Farndale, Richard W.; Duer, Melinda J.

    2015-07-01

    Collagens, the most abundant proteins in mammals, are defined by their triple-helical structures and distinctive Gly-Xaa-Yaa repeating sequence, where Xaa is often proline and Yaa, hydroxyproline (Hyp/O). It is known that hydroxyproline in the Yaa position stabilises the triple helix, and that lack of proline hydroxylation in vivo leads to dysfunctional collagen extracellular matrix assembly, due to a range of factors such as a change in hydration properties. In addition, we note that in model peptides, when Yaa is unmodified proline, the Xaa proline has a strong propensity to adopt an endo ring conformation, whilst when Yaa is hydroxyproline, the Xaa proline adopts a range of endo and exo conformations. Here we use a combination of solid-state NMR spectroscopy and potential energy landscape modelling of synthetic triple-helical collagen peptides to understand this effect. We show that hydroxylation of the Yaa proline causes the Xaa proline ring conformation to become metastable, which in turn confers flexibility on the triple helix.

  10. Dermatitis from common ivy (Hedera helix L. subsp. helix) in Europe: past, present, and future.

    Science.gov (United States)

    Paulsen, Evy; Christensen, Lars P; Andersen, Klaus E

    2010-04-01

    Common ivy (Hedera helix subsp. helix) is a well-known native and ornamental plant in Europe. Reports on contact dermatitis from ivy have regularly appeared since 1899. Recently, it has been suggested that allergic contact dermatitis from the plant may be under-diagnosed, partly due to lack of commercial patch test allergens. The objective of the article is to present the results of aimed patch testing with the main common ivy allergen, falcarinol, during a 16-year period and review the newer literature. Consecutive patients tested with falcarinol 0.03% petrolatum from May 1993 to May 2009 were included. Cases published since 1987 were retrieved from the PubMed database. One hundred and twenty-seven Danish patients were tested with falcarinol and 10 (7.9%) tested positive. Seven were occupationally sensitized. Between 1994 and 2009, 28 new cases of contact dermatitis from ivy were reported, 2 of which were occupational. Only 11 of the 28 patients were tested with pure allergens. Falcarinol is not only widely distributed in the ivy family, but also in the closely related Apiaceae. Sensitization may occur in childhood or in adults pruning ivy plants or handling them in an occupational setting. In view of the ubiquity of falcarinol-containing plants and the relatively high prevalence of positive reactions in aimed patch testing, falcarinol should be the next plant allergen to be commercially available and included in the plant series worldwide.

  11. Hydroxyproline Ring Pucker Causes Frustration of Helix Parameters in the Collagen Triple Helix.

    Science.gov (United States)

    Chow, W Ying; Bihan, Dominique; Forman, Chris J; Slatter, David A; Reid, David G; Wales, David J; Farndale, Richard W; Duer, Melinda J

    2015-07-29

    Collagens, the most abundant proteins in mammals, are defined by their triple-helical structures and distinctive Gly-Xaa-Yaa repeating sequence, where Xaa is often proline and Yaa, hydroxyproline (Hyp/O). It is known that hydroxyproline in the Yaa position stabilises the triple helix, and that lack of proline hydroxylation in vivo leads to dysfunctional collagen extracellular matrix assembly, due to a range of factors such as a change in hydration properties. In addition, we note that in model peptides, when Yaa is unmodified proline, the Xaa proline has a strong propensity to adopt an endo ring conformation, whilst when Yaa is hydroxyproline, the Xaa proline adopts a range of endo and exo conformations. Here we use a combination of solid-state NMR spectroscopy and potential energy landscape modelling of synthetic triple-helical collagen peptides to understand this effect. We show that hydroxylation of the Yaa proline causes the Xaa proline ring conformation to become metastable, which in turn confers flexibility on the triple helix.

  12. An integrase of endogenous retrovirus is involved in maternal mitochondrial DNA inheritance of the mouse.

    Science.gov (United States)

    Hayashida, Kenji; Omagari, Katsuhisa; Masuda, Jun-Ichi; Kohno, Shigeru

    2008-02-01

    The mechanism of maternal mitochondrial DNA (mtDNA) inheritance in animals can be said to be the selective elimination of sperm mtDNA via the elimination factor of the egg and a sperm mitochondria-specific factor. In 2005, we clarified that t-tpis (Spag1 isoform 1) is a mitochondria-specific translocator and the sperm factor, and furthermore estimated that the elimination factors of the egg are the divalent cation-dependent endonuclease and s-tpis (Spag1 isoform 2 and isoform 3) as the elimination system-specific chaperone [K. Hayashida, K. Omagari, J. Masuda, H. Hazama, Y. Kadokawa, K. Ohba, S. Kohno, The sperm mitochondria-specific translocator has a key role in maternal mitochondrial inheritance, Cell Biol. Int. 29 (2005) 472-481]. This time, using a recombinant Spag1 isoform 1 protein, a pull-down assay of ovary cytosol was performed and the elimination factors searched for. Surprisingly, an endogenous retroviral integrase fragment (Eri15) was identified using mass spectrometry of the electrophoresis band of the pull-down protein. Eri15 was detected as a complex of approximately 500kDa with Spag1 isoform 2 or isoform 3 in native PAGE of the ovary cytosol. This strongly suggested that Eri15 is selectively transported into the sperm mitochondria matrix by Spag1 isoform 2 and 3 via Spag1 isoform 1 and that sperm mtDNA is destroyed, thus causing the establishment of maternal mtDNA inheritance.

  13. Structural Basis for the Recognition Between HIV-1 Integrase and Transcriptional Coactivator p75

    Energy Technology Data Exchange (ETDEWEB)

    Cherepanov,P.; Ambrosio, A.; Rahman, S.; Ellenberger, T.; Engelman, A.

    2005-01-01

    Integrase (IN) is an essential retroviral enzyme, and human transcriptional coactivator p75, which is also referred to as lens epithelium-derived growth factor (LEDGF), is the dominant cellular binding partner of HIV-1 IN. Here, we report the crystal structure of the dimeric catalytic core domain of HIV-1 IN complexed to the IN-binding domain of LEDGF. Previously identified LEDGF hotspot residues anchor the protein to both monomers at the IN dimer interface. The principal structural features of IN that are recognized by the host factor are the backbone conformation of residues 168-171 from one monomer and a hydrophobic patch that is primarily comprised of {alpha}-helices 1 and 3 of the second IN monomer. Inspection of diverse retroviral primary and secondary sequence elements helps to explain the apparent lentiviral tropism of the LEDGF-IN interaction. Because the lethal phenotypes of HIV-1 mutant viruses unable to interact with LEDGF indicate that IN function is highly sensitive to perturbations of the structure around the LEDGF-binding site, we propose that small molecule inhibitors of the protein-protein interaction might similarly disrupt HIV-1 replication.

  14. Mechanism of action of the HIV-1 integrase inhibitory peptide LEDGF 361-370.

    Science.gov (United States)

    Hayouka, Zvi; Levin, Aviad; Maes, Michal; Hadas, Eran; Shalev, Deborah E; Volsky, David J; Loyter, Abraham; Friedler, Assaf

    2010-04-01

    The HIV-1 integrase protein (IN) mediates integration of the viral cDNA into the host genome and is a target for anti-HIV drugs. We have recently described a peptide derived from residues 361-370 of the IN cellular partner protein LEDGF/p75, which inhibited IN catalytic activity in vitro and HIV-1 replication in cells. Here we performed a comprehensive study of the LEDGF 361-370 mechanism of action in vitro, in cells and in vivo. Alanine scan, fluorescence anisotropy binding studies, homology modeling and NMR studies demonstrated that all residues in LEDGF 361-370 contribute to IN binding and inhibition. Kinetic studies in cells showed that LEDGF 361-370 specifically inhibited integration of viral cDNA. Thus, the full peptide was chosen for in vivo studies, in which it inhibited the production of HIV-1 RNA in mouse model. We conclude that the full LEDGF 361-370 peptide is a potent HIV-1 inhibitor and may be used for further development as an anti-HIV lead compound.

  15. Study on the drug resistance and the binding mode of HIV-1 integrase with LCA inhibitor

    Institute of Scientific and Technical Information of China (English)

    HU; JianPing; CHANG; Shan; CHEN; WeiZu; WANG; CunXin

    2007-01-01

    Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is an essential enzyme in the lifecycle of this virus and also an important target for the study of anti-HIV drugs. The binding mode of the wild type IN core domain and its G140S mutant with L-Chicoric acid (LCA) inhibitor were investigated by using multiple conformation molecular docking and molecular dynamics (MD) simulation. Based on the binding modes, the drug resistance mechanism was explored for the G140S mutant of IN with LCA. The results indicate that the binding site of the G140S mutant of IN core domain with LCA is different from that of the core domain of the wild type IN, which leads to the partial loss of inhibition potency of LCA. The flexibility of the IN functional loop region and the interactions between Mg2+ ion and the three key residues (i.e., D64, D116, E152) stimulate the biological operation of IN. The drug resistance also lies in several other important effects, such as the repulsion between LCA and E152 in the G140S mutant core domain, the weakening of K159 binding with LCA and Y143 pointing to the pocket of the G140S mutant. All of the above simulation results agree well with experimental data, which provide us with some helpful information for designing the drug of anti-HIV based on the structure of IN.

  16. The putative U94 integrase is dispensable for human herpesvirus 6 (HHV-6) chromosomal integration.

    Science.gov (United States)

    Wallaschek, Nina; Gravel, Annie; Flamand, Louis; Kaufer, Benedikt B

    2016-08-01

    Human herpesvirus 6 (HHV-6) can integrate its genome into the telomeres of host chromosomes and is present in the germline of about 1 % of the human population. HHV-6 encodes a putative integrase U94 that possesses all molecular functions required for recombination including DNA-binding, ATPase, helicase and nuclease activity, and was hypothesized by many researchers to facilitate integration ever since the discovery of HHV-6 integration. However, analysis of U94 in the virus context has been hampered by the lack of reverse-genetic systems and efficient integration assays. Here, we addressed the role of U94 and the cellular recombinase Rad51 in HHV-6 integration. Surprisingly, we could demonstrate that HHV-6 efficiently integrated in the absence of U94 using a new quantitative integration assay. Additional inhibition of the cellular recombinase Rad51 had only a minor impact on virus integration. Our results shed light on this complex integration mechanism that includes factors beyond U94 and Rad51.

  17. Porcine endogenous retrovirus-A/C: biochemical properties of its integrase and susceptibility to raltegravir.

    Science.gov (United States)

    Demange, Antonin; Yajjou-Hamalian, Halima; Gallay, Kathy; Luengo, Catherine; Beven, Véronique; Leroux, Aurélie; Confort, Marie-Pierre; Al Andary, Elsy; Gouet, Patrice; Moreau, Karen; Ronfort, Corinne; Blanchard, Yannick

    2015-10-01

    Porcine endogenous retroviruses (PERVs) are present in the genomes of pig cells. The PERV-A/C recombinant virus can infect human cells and is a major risk of zoonotic disease in the case of xenotransplantation of pig organs to humans. Raltegravir (RAL) is a viral integrase (IN) inhibitor used in highly active antiretroviral treatment. In the present study, we explored the potential use of RAL against PERV-A/C. We report (i) a three-dimensional model of the PERV-A/C intasome complexed with RAL, (ii) the sensitivity of PERV-A/C IN to RAL in vitro and (iii) the sensitivity of a PERV-A/C-IRES-GFP recombinant virus to RAL in cellulo. We demonstrated that RAL is a potent inhibitor against PERV-A/C IN and PERV-A/C replication with IC50s in the nanomolar range. To date, the use of retroviral inhibitors remains the only way to control the risk of zoonotic PERV infection during pig-to-human xenotransplantation.

  18. Design of inhibitors of the HIV-1 integrase core domain using virtual screening.

    Science.gov (United States)

    Regon, Preetom; Gogoi, Dhrubajyoti; Rai, Ashok Kumar; Bordoloi, Manabjyoti; Bezbaruah, Rajib Lochan

    2014-01-01

    Acquired immunodeficiency syndrome (AIDS) is a disease of the human immune system caused by the human immunodeficiency virus (HIV). The integrase (IN) enzyme of HIV interacts with several cellular and viral proteins during the integration process. Thus, it represents an appropriate target for antiretroviral drugs (ARVs). We performed virtual screening of database compounds and designed analogues using Elvitegravir (EVG) as a standard compound. The 378 screened compounds were retrieved from ZINC, ChemSpider, PubChem, and ChemBank Chemical Databases based on chemical similarity and literature searches related to the structure of EVG. The Physiochemical properties, Bioactivity, Toxicity and Absorption, Distribution, Metabolism and Excretion of Molecules (ADME) of these compounds were predicted and docking Experiments were conducted using Molegro Virtual Docker software. The docking and ADME suggested very significant results in regard to EVG. The MolDock and Rerank scores were used to analyze the results. The compounds ZINC26507991 (-84.22), Analogue 9 (-68.49), ZINC20731658 (-66.79), ZINC00210363 (-43.44) showed better binding orientation with IN receptor model with respect to EVG (182.52). The ZINC26507991 has showed significant ADME result.

  19. Class 1 integrase, sulfonamide and tetracycline resistance genes in wastewater treatment plant and surface water.

    Science.gov (United States)

    Makowska, Nicoletta; Koczura, Ryszard; Mokracka, Joanna

    2016-02-01

    Wastewater treatment plants are considered hot spots for multiplication and dissemination of antibiotic-resistant bacteria and resistance genes. In this study, we determined the presence of class 1 integron integrase and genes conferring resistance to tetracyclines and sulfonamides in the genomes of culturable bacteria isolated from a wastewater treatment plant and the river that receives the treated wastewater. Moreover, using PCR-based metagenomic approach, we quantified intI1, tet and sul genes. Wastewater treatment caused the decrease in the total number of culturable heterotrophs and bacteria resistant to tetracycline and sulfonamides, along with the decrease in the number of intI1, sul and tet gene copies per ml, with significant reduction of tet(B). On the other hand, the treatment process increased both the frequency of tetracycline- and sulfonamide-resistant bacteria and intI1-positive strains, and the relative abundance of all quantified antibiotic resistance genes (ARGs) and intI1 gene; in the case of tet(A) and sul2 significantly. The discharge of treated wastewater increased the number of intI1, tet and sul genes in the receiving river water both in terms of copy number per ml and relative abundance. Hence, despite the reduction of the number of ARGs and ARBs, wastewater treatment selects for bacteria with ARGs in effluent.

  20. Selection of diverse and clinically relevant integrase inhibitor-resistant human immunodeficiency virus type 1 mutants.

    Science.gov (United States)

    Kobayashi, Masanori; Nakahara, Koichiro; Seki, Takahiro; Miki, Shigeru; Kawauchi, Shinobu; Suyama, Akemi; Wakasa-Morimoto, Chiaki; Kodama, Makoto; Endoh, Takeshi; Oosugi, Eiichi; Matsushita, Yoshihiro; Murai, Hitoshi; Fujishita, Toshio; Yoshinaga, Tomokazu; Garvey, Edward; Foster, Scott; Underwood, Mark; Johns, Brian; Sato, Akihiko; Fujiwara, Tamio

    2008-11-01

    Resistance passage studies were conducted with five INIs (integrase inhibitors) that have been tested in clinical trials to date: a new naphthyridinone-type INI S/GSK-364735, raltegravir, elvitegravir, L-870,810 and S-1360. In establishing the passage system and starting from concentrations several fold above the EC(50) value, resistance mutations against S-1360 and related diketoacid-type compounds could be isolated from infected MT-2 cell cultures from day 14 to 28. Q148R and F121Y were the two main pathways of resistance to S/GSK-364735. Q148R/K and N155H, which were found in patients failing raltegravir treatment in Phase IIb studies, were observed during passage with raltegravir with this method. The fold resistance of 40 mutant molecular clones versus wild type virus was compared with these five INIs. The overall resistance pattern of S/GSK-364735 was similar to that of raltegravir and other INIs. However, different fold resistances of particular mutations were noted among different INIs, reflecting a potential to develop INIs with distinctly different resistant profiles.

  1. Methods for the Analyses of Inhibitor-Induced Aberrant Multimerization of HIV-1 Integrase

    Science.gov (United States)

    Kessl, Jacques J.; Sharma, Amit; Kvaratskhelia, Mamuka

    2016-01-01

    HIV-1 integrase (IN) is an important therapeutic target as its function is essential for the viral lifecycle. The discovery of multifunctional allosteric IN inhibitors or ALLINIs, which potently impair viral replication by promoting aberrant, higher order IN multimerization as well as inhibit IN interactions with its cellular cofactor, LEDGF/p75, has opened new venues to exploit IN multimerization as a therapeutic target. Furthermore, the recent discovery of multimerization selective IN inhibitors or MINIs, has provided new investigational probes to study the direct effects of aberrant IN multimerization in vitro and in infected cells. Here we describe three complementary methods designed to detect and quantify the effects of these new classes of inhibitors on IN multimerization. These methods include a homogenous time-resolved fluorescence-based assay which allows for measuring EC50 values for the inhibitor-induced aberrant IN multimerization, a dynamic light scattering-based assay which allows for monitoring the formation and sizes of oligomeric IN particles in a time-dependent manner, and a chemical cross-linking-based assay of interacting IN subunits which allows for the determination of IN oligomers in viral particles. PMID:26714710

  2. Design of inhibitors of the HIV-1 integrase core domain using virtual screening

    Science.gov (United States)

    Regon, Preetom; Gogoi, Dhrubajyoti; Rai, Ashok Kumar; Bordoloi, Manabjyoti; Bezbaruah, Rajib Lochan

    2014-01-01

    Acquired immunodeficiency syndrome (AIDS) is a disease of the human immune system caused by the human immunodeficiency virus (HIV). The integrase (IN) enzyme of HIV interacts with several cellular and viral proteins during the integration process. Thus, it represents an appropriate target for antiretroviral drugs (ARVs). We performed virtual screening of database compounds and designed analogues using Elvitegravir (EVG) as a standard compound. The 378 screened compounds were retrieved from ZINC, ChemSpider, PubChem, and ChemBank Chemical Databases based on chemical similarity and literature searches related to the structure of EVG. The Physiochemical properties, Bioactivity, Toxicity and Absorption, Distribution, Metabolism and Excretion of Molecules (ADME) of these compounds were predicted and docking Experiments were conducted using Molegro Virtual Docker software. The docking and ADME suggested very significant results in regard to EVG. The MolDock and Rerank scores were used to analyze the results. The compounds ZINC26507991 (-84.22), Analogue 9 (-68.49), ZINC20731658 (-66.79), ZINC00210363 (-43.44) showed better binding orientation with IN receptor model with respect to EVG (182.52). The ZINC26507991 has showed significant ADME result. PMID:24616558

  3. Effect of Hedera helix on lung histopathology in chronic asthma.

    Directory of Open Access Journals (Sweden)

    Arzu Babayigit Hocaoglu

    2012-12-01

    Full Text Available Hedera helix  is widely used to treat bronchial asthma for many years. However, effects of this herb on lung histopathology is still far from clear. We aimed to determine the effect of oral administration of Hedera helix on lung histopathology in a murine model of chronic asthma.BALB/c  mice  were  divided  into  four  groups;   I  (Placebo,  II  (Hedera  helix, III (Dexamethasone and IV (Control. All mice except controls were sensitized and challenged with ovalbumin. Then, mice in group I received saline, group II 100 mg/kg Hedera helix and group III 1 mg/kg  dexamethasone via orogastic gavage once daily for one week. Airway histopathology was evaluated by using light and electron microscopy in all groups.Goblet  cell numbers and thicknesses of basement membrane were found  significantly lower in group II, but there was no statistically significant difference in terms of number of mast cells, thicknesses of epithelium and subepithelial smooth muscle layers between group I and II. When Hedera helix and dexamethasone groups were compared with each other, thickness of epithelium, subepithelial muscle layers, number of mast cells and goblet cells of group III were significantly ameliorated when compared with the group II.Although Hedera helix administration reduced only goblet cell counts and the thicknesses of basement membrane  in the  asthmatic airways, dexamethasone ameliorated all histopathologic parameters except thickness of  basement  membrane  better  than  Hedera helix.

  4. Structure, stability and folding of the alpha-helix.

    Science.gov (United States)

    Doig, A J; Andrew, C D; Cochran, D A; Hughes, E; Penel, S; Sun, J K; Stapley, B J; Clarke, D T; Jones, G R

    2001-01-01

    Pauling first described the alpha-helix nearly 50 years ago, yet new features of its structure continue to be discovered, using peptide model systems, site-directed mutagenesis, advances in theory, the expansion of the Protein Data Bank and new experimental techniques. Helical peptides in solution form a vast number of structures, including fully helical, fully coiled and partly helical. To interpret peptide results quantitatively it is essential to use a helix/coil model that includes the stabilities of all these conformations. Our models now include terms for helix interiors, capping, side-chain interactions, N-termini and 3(10)-helices. The first three amino acids in a helix (N1, N2 and N3) and the preceding N-cap are unique, as their amide NH groups do not participate in backbone hydrogen bonding. We surveyed their structures in proteins and measured their amino acid preferences. The results are predominantly rationalized by hydrogen bonding to the free NH groups. Stabilizing side-chain-side-chain energies, including hydrophobic interactions, hydrogen bonding and polar/non-polar interactions, were measured accurately in helical peptides. Helices in proteins show a preference for having approximately an integral number of turns so that their N- and C-caps lie on the same side. There are also strong periodic trends in the likelihood of terminating a helix with a Schellman or alpha L C-cap motif. The kinetics of alpha-helix folding have been studied with stopped-flow deep ultraviolet circular dichroism using synchrotron radiation as the light source; this gives a far superior signal-to-noise ratio than a conventional instrument. We find that poly(Glu), poly(Lys) and alanine-based peptides fold in milliseconds, with longer peptides showing a transient overshoot in helix content.

  5. Polyproline and triple helix motifs in host-pathogen recognition.

    Science.gov (United States)

    Berisio, Rita; Vitagliano, Luigi

    2012-12-01

    Secondary structure elements often mediate protein-protein interactions. Despite their low abundance in folded proteins, polyproline II (PPII) and its variant, the triple helix, are frequently involved in protein-protein interactions, likely due to their peculiar propensity to be solvent-exposed. We here review the role of PPII and triple helix in mediating hostpathogen interactions, with a particular emphasis to the structural aspects of these processes. After a brief description of the basic structural features of these elements, examples of host-pathogen interactions involving these motifs are illustrated. Literature data suggest that the role played by PPII motif in these processes is twofold. Indeed, PPII regions may directly mediate interactions between proteins of the host and the pathogen. Alternatively, PPII may act as structural spacers needed for the correct positioning of the elements needed for adhesion and infectivity. Recent investigations have highlighted that collagen triple helix is also a common target for bacterial adhesins. Although structural data on complexes between adhesins and collagen models are rather limited, experimental and theoretical studies have unveiled some interesting clues of the recognition process. Interestingly, very recent data show that not only is the triple helix used by pathogens as a target in the host-pathogen interaction but it may also act as a bait in these processes since bacterial proteins containing triple helix regions have been shown to interact with host proteins. As both PPII and triple helix expose several main chain non-satisfied hydrogen bond acceptors and donors, both elements are highly solvated. The preservation of the solvation state of both PPII and triple helix upon protein-protein interaction is an emerging aspect that will be here thoroughly discussed.

  6. Hierarchical self-assembly of designed 2x2-alpha-helix bundle proteins on Au(111) surfaces

    DEFF Research Database (Denmark)

    Wackerbarth, Hainer; Tofteng, A.P.; Jensen, K.J.

    2006-01-01

    Self-assembled monolayers of biomolecules on atomically planar surfaces offer the prospect of complex combinations of controlled properties, e. g., for bioelectronics. We have prepared a novel hemi-4-alpha-helix bundle protein by attaching two alpha-helical peptides to a cyclo-dithiothreitol (cyclo......-DTT) template. The protein was de novo designed to self-assemble in solution to form a 4-alpha-helix bundle, whereas the disulfide moiety enables the formation of a self-assembled monolayer on a Au(111) surface by opening of the disulfide, thus giving rise to a two-step self-assembly process. The 2 x 2-alpha......-helix bundle protein and its template were studied by X-ray photo electron spectroscopy (XPS), electrochemical methods, and electrochemical in situ scanning tunneling microscopy (in situ STM). XPS showed that the cyclo-DTT opens on adsorption to a gold surface with the integrity of the 2 x 2- R-helix bundle...

  7. TOWARDS UNDERSTANDING OF HELIX B BASED CONFORMATIONAL DISEASES IN SERPIN

    Directory of Open Access Journals (Sweden)

    Mohamad Aman Jairajpuri

    2012-12-01

    Full Text Available Serine protease inhibitors (serpins are a unique family of protease inhibitors that are prone to polymer formation due to their metastable nature and a complex inhibition mechanism that involves large scale conformational change. Helix B is in the shutter region near the strand 2A and strand 3A of �-sheet A, where reactive centre loop inserts during the serpin inhibition mechanism. Helix B region in serpins is a mutation hotspot for naturally occurring variants that result in pathological conditions due to polymerization. Helix B residues are completely buried in the native state and loop inserted latent state but not in the inhibitory loop inserted cleaved conformation. Native to cleaved transition during inhibition forms a large cavity in the shutter region, which invariably is the largest cavity in most serpins in native state. In a recent paper we had for the first time hypothesized that exposure of helix B at the N-terminal end is important for smooth insertion of the reactive center loop during serpin inhibition mechanism. It is therefore possible that natural variant that induces conformational deformation of helix B probably alter the cavity size which increases the rate of loop-sheet interaction between the monomers resulting in increased polymerization.

  8. The Other Double Helix--The Fascinating Chemistry of Starch

    Science.gov (United States)

    Hancock, Robert D.; Tarbet, Bryon J.

    2000-08-01

    Current textbooks deal only briefly with the chemistry of starch. A short review with 21 references is presented, describing the structure of starch and indicating the double helix structure of A-type and B-type starch. The structure of the starch granule is examined, pointing out the existence of growth rings of alternating crystalline and noncrystalline starch, with growing amylopectin molecules extending from the hilum (point of origin) to the surface of the starch granule. The swelling of starch granules in water, above the gelatinization temperature of about 60 °C, is discussed. The process of gelatinization involves unraveling of the starch helix and a manyfold increase in volume of the starch granule as water is imbibed and bound to the unraveled starch polymer by hydrogen bonding. Baking bread or pastries causes unraveling of the starch helix, and the process by which these products become stale corresponds primarily to the re-forming of the starch helix. The importance of this phenomenon in food science is discussed. The absorption of nonpolar linear molecules such as I2, or linear nonpolar portions of molecules such as n-butanol or fats and phospholipids, by the C-type helix of starch is examined. The way in which starch is structurally modified to retard staling is discussed in relation to food technology.

  9. Sample Extraction Bsaed on Helix Scattering for Polarimetric SAR Calibratio

    Science.gov (United States)

    Chang, Y.; Yang, J.; Li, P.; Zhao, L.; Shi, L.

    2017-09-01

    Polarimetric calibration (PolCAL) of Synthetic Aperture Radar (SAR) images is a significant preprocessing for further applications. Since the reflection symmetry property of distributed objects can provide stable constraints for PolCAL. It is reasonable to extract these reference samples before calibration. The helix scattering generally appears in complex urban area and disappears for a natural scatterer, making it a good measure to extract distributed objects. In this paper, a novel technique that extracts reflecting symmetry samples is proposed by using helix scattering. The helix scattering information is calculated by Yamaguchi four-component decomposition algorithm. An adaptive threshold selection algorithm based on generalized Gaussian distribution is also utilized to scale the helix scattering components automatically, getting rid of the problem of various numerical range. The extracting results will be taken as PolCAL reference samples and the Quegan method are utilized to calibrate these PolSAR images. A C-band airborne PolSAR data was taken as examples to evaluate its ability in improving calibration precision. Traditional method i.e. extracting samples with span power was also evaluated as contrast experiment. The results showed that the samples extracting method based on helix scattering can improve the Polcal precision preferably.

  10. HIV-1 integrase inhibitors are substrates for the multidrug transporter MDR1-P-glycoprotein

    Directory of Open Access Journals (Sweden)

    Cara Andrea

    2007-03-01

    Full Text Available Abstract Background The discovery of diketoacid-containing derivatives as inhibitors of HIV-1 Integrase (IN (IN inhibitors, IINs has played a major role in validating this enzyme as an important target for antiretroviral therapy. Since the in vivo efficacy depends on access of these drugs to intracellular sites where HIV-1 replicates, we determined whether the IINs are recognized by the multidrug transporter MDR1-P-glycoprotein (P-gp thereby reducing their intracellular accumulation. To address the effect of IINs on drug transport, nine quinolonyl diketo acid (DKA derivatives active on the HIV-1 IN strand transfer (ST step and with EC50 ranging from 1.83 to >50 μm in cell-based assays were tested for their in vitro interaction with P-gp in the CEM-MDR cell system. IINs were investigated for the inhibition and induction of the P-gp function and expression as well as for multidrug resistance (MDR reversing ability. Results The HIV-1 IINs act as genuine P-gp substrates by inhibiting doxorubicin efflux and inducing P-gp functional conformation changes as evaluated by the modulation of UIC2 mAb epitope. Further, IINs chemosensitize MDR cells to vinblastine and induce P-gp expression in drug sensitive revertants of CEM-MDR cells. Conclusion To our knowledge, this is the first demonstration that HIV-1 IINs are P-gp substrates. This biological property may influence the absorption, distribution and elimination of these novels anti HIV-1 compounds.

  11. In vitro DNA tethering of HIV-1 integrase by the transcriptional coactivator LEDGF/p75.

    Science.gov (United States)

    McNeely, Melissa; Hendrix, Jelle; Busschots, Katrien; Boons, Eline; Deleersnijder, Angélique; Gerard, Melanie; Christ, Frauke; Debyser, Zeger

    2011-07-29

    Although LEDGF/p75 is believed to act as a cellular cofactor of lentiviral integration by tethering integrase (IN) to chromatin, there is no good in vitro model to analyze this functionality. We designed an AlphaScreen assay to study how LEDGF/p75 modulates the interaction of human immunodeficiency virus type 1 IN with DNA. IN bound with similar affinity to DNA mimicking the long terminal repeat or to random DNA. While LEDGF/p75 bound DNA strongly, a mutant of LEDGF/p75 with compromised nuclear localization signal (NLS)/AT hook interacted weakly, and the LEDGF/p75 PWWP domain did not interact, corroborating previous reports on the role of NLS and AT hooks in charge-dependent DNA binding. LEDGF/p75 stimulated IN binding to DNA 10-fold to 30-fold. Stimulation of IN-DNA binding required a direct interaction between IN and the C-terminus of LEDGF/p75. Addition of either the C-terminus of LEDGF/p75 (amino acids 325-530) or LEDGF/p75 mutated in the NLS/AT hooks interfered with IN binding to DNA. Our results are consistent with an in vitro model of LEDGF/p75-mediated tethering of IN to DNA. The inhibition of IN-DNA interaction by the LEDGF/p75 C-terminus may provide a novel strategy for the inhibition of HIV IN activity and may explain the potent inhibition of HIV replication observed after the overexpression of C-terminal fragments in cell culture.

  12. Efficient and specific internal cleavage of a retroviral palindromic DNA sequence by tetrameric HIV-1 integrase.

    Directory of Open Access Journals (Sweden)

    Olivier Delelis

    Full Text Available BACKGROUND: HIV-1 integrase (IN catalyses the retroviral integration process, removing two nucleotides from each long terminal repeat and inserting the processed viral DNA into the target DNA. It is widely assumed that the strand transfer step has no sequence specificity. However, recently, it has been reported by several groups that integration sites display a preference for palindromic sequences, suggesting that a symmetry in the target DNA may stabilise the tetrameric organisation of IN in the synaptic complex. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the ability of several palindrome-containing sequences to organise tetrameric IN and investigated the ability of IN to catalyse DNA cleavage at internal positions. Only one palindromic sequence was successfully cleaved by IN. Interestingly, this symmetrical sequence corresponded to the 2-LTR junction of retroviral DNA circles-a palindrome similar but not identical to the consensus sequence found at integration sites. This reaction depended strictly on the cognate retroviral sequence of IN and required a full-length wild-type IN. Furthermore, the oligomeric state of IN responsible for this cleavage differed from that involved in the 3'-processing reaction. Palindromic cleavage strictly required the tetrameric form, whereas 3'-processing was efficiently catalysed by a dimer. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the restriction-like cleavage of palindromic sequences may be a general physiological activity of retroviral INs and that IN tetramerisation is strongly favoured by DNA symmetry, either at the target site for the concerted integration or when the DNA contains the 2-LTR junction in the case of the palindromic internal cleavage.

  13. Optimization of mucosal responses after intramuscular immunization with integrase defective lentiviral vector.

    Directory of Open Access Journals (Sweden)

    Alessandra Rossi

    Full Text Available Many infectious agents infiltrate the host at the mucosal surfaces and then spread systemically. This implies that an ideal vaccine should induce protective immune responses both at systemic and mucosal sites to counteract invasive mucosal pathogens. We evaluated the in vivo systemic and mucosal antigen-specific immune response induced in mice by intramuscular administration of an integrase defective lentiviral vector (IDLV carrying the ovalbumin (OVA transgene as a model antigen (IDLV-OVA, either alone or in combination with sublingual adjuvanted OVA protein. Mice immunized intramuscularly with OVA and adjuvant were compared with IDLV-OVA immunization. Mice sublingually immunized only with OVA and adjuvant were used as a positive control of mucosal responses. A single intramuscular dose of IDLV-OVA induced functional antigen-specific CD8+ T cell responses in spleen, draining and distal lymph nodes and, importantly, in the lamina propria of the large intestine. These results were similar to those obtained in a prime-boost regimen including one IDLV immunization and two mucosal boosts with adjuvanted OVA or vice versa. Remarkably, only in groups vaccinated with IDLV-OVA, either alone or in prime-boost regimens, the mucosal CD8+ T cell response persisted up to several months from immunization. Importantly, following IDLV-OVA immunization, the mucosal boost with protein greatly increased the plasma IgG response and induced mucosal antigen-specific IgA in saliva and vaginal washes. Overall, intramuscular administration of IDLV followed by protein boosts using the sublingual route induced strong, persistent and complementary systemic and mucosal immune responses, and represents an appealing prime-boost strategy for immunization including IDLV as a delivery system.

  14. Prevalence of Integrase Strand Transfer Inhibitors (INSTI) Resistance Mutations in Taiwan

    Science.gov (United States)

    Chang, Sui-Yuan; Lin, Pi-Han; Cheng, Chien-Lin; Chen, Mao-Yuan; Sun, Hsin-Yun; Hsieh, Szu-Min; Sheng, Wang-Huei; Su, Yi-Ching; Su, Li-Hsin; Chang, Shu-Fang; Liu, Wen-Chun; Hung, Chien-Ching; Chang, Shan-Chwen

    2016-01-01

    Antiretroviral therapy containing an integrase strand transfer inhibitor (INSTI) plus two NRTIs has become the recommended treatment for antiretroviral-naive HIV-1-infected patients in the updated guidelines. We aimed to determine the prevalence of INSTI-related mutations in Taiwan. Genotypic resistance assays were performed on plasma from ARV-naïve patients (N = 948), ARV-experienced but INSTI-naive patients (N = 359), and raltegravir-experienced patients (N = 63) from 2006 to 2015. Major INSTI mutations were defined according to the IAS-USA list and other substitutions with a Stanford HIVdb score ≧ 10 to at least one INSTI were defined as minor mutations. Of 1307 HIV-1 samples from patients never exposed to INSTIs, the overall prevalence of major resistance mutations to INSTIs was 0.9% (n = 12), with an increase to 1.2% in 2013. Of these 12 sequences, 11 harboured Q148H/K/R, one Y143R, and none N155H. Of 30 sequences (47.6%) with INSTI-resistant mutations from raltegravir-experienced patients, 17 harboured Q148H/K/R, 8 N155H, and 6 Y143C/R. Other than these major mutations, the prevalence of minor mutations were 5.3% and 38.1%, respectively, in ARV-naive and raltegravir-experienced patients. The overall prevalence of INSTI mutations remains low in Taiwan. Surveillance of INSTI resistance is warranted due to circulation of polymorphisms contributing to INSTI resistance and expected increasing use of INSTIs. PMID:27779200

  15. Anti-HIV-1 integrase and anti-allergic activities of Bauhinia strychnifolia

    Directory of Open Access Journals (Sweden)

    Kingkan Bunluepuech

    2013-12-01

    Full Text Available A stem ethanol extract of Bauhinia strychnifolia and its compounds were investigated for their anti-HIV-1 integrase (IN and anti-allergic activities. From bioassay-guided isolation, five compounds including quercetin (1, 3,5,7,3',5' pentahydroxyflavanonol-3-O-α-L-rhamnopyranoside (2, 3,5,7-trihydroxychromone-3-α-L-rhamnopyranoside (3 and a mixture of β-sitosterol (4 and stigmasterol (5 were isolated. Of the tested samples, compound 1 (quercetin showed the highest activity against HIV-1 IN with an IC50 value of 15.2 µM, followed by 3 (3,5,7-trihydroxychromone-3-α-L-rhamnopyranoside, 4+5 (mixture of β-sitosterol and stigmasterol and 2 (3,5,7,3',5'-pentahydroxyflavanonol-3-O-α-L-rhamnopyranoside with % inhibition of 28.2, 26.2 and 6.7 at 100 µM, respectively. With regard to anti-allergic activity, quercetin (1 possessed the highest anti-allergic activity with an IC50 of 8.1 µM, followed by 3 (3,5,7-trihydroxychromone-3-α-L-rhamnopyranoside and 4+5 (mixture of β-sitosterol and stigmasterol with IC50 values of 52.1 and 77.5 µM, respectively. Whereas compound 2 (3,5,7,3',5'-pentahydroxyflavanonol-3-O-α-L-rhamnopyranoside was inactive. The present study is the first report of chemical constituents and biological activities of Bauhinia strychnifolia.

  16. Universal kinetics of helix-coil transition in gelatin.

    Science.gov (United States)

    Gornall, J L; Terentjev, E M

    2008-03-01

    By covering a much wider concentration range than previous studies we find a very unusual exponential dependence of the rate of helix formation on concentration of gelatin in water and ethylene glycol solutions. By applying a procedure of concentration-temperature superposition we build a master curve describing the initial renaturation rates in both solvents. The growth of the normalized helical fraction chi(t) is a first-order process, with a rate constant consistent with cis-trans isomerization, in most situations. We propose that association of three separate chains to form a triple helical nucleus occurs rapidly and contributes less to the helical onset than previously thought. The measured helix content is a result of lengthening of the triple helix after nucleation, by zipping from the associated nuclei.

  17. Stabilization of magnetic helix in exchange-coupled thin films.

    Science.gov (United States)

    Dzemiantsova, L V; Meier, G; Röhlsberger, R

    2015-11-05

    Based on micromagnetic simulations, we report on a novel magnetic helix in a soft magnetic film that is sandwiched between and exchange-coupled to two hard magnetic layers with different anisotropies. We show that such a confined helix stays stable without the presence of an external magnetic field. The magnetic stability is determined by the energy minimization and is a result of an internal magnetic field created by the exchange interaction. We show that this internal field stores a magnetic energy density of a few kJ/m(3). We also find that it dramatically modifies ferromagnetic resonances, such that the helix can be used as a ferromagnetic resonance filter and a fast acting attenuator.

  18. The Knowledge-Based Economy and the Triple Helix Model

    CERN Document Server

    Leydesdorff, Loet

    2012-01-01

    1. Introduction - the metaphor of a "knowledge-based economy"; 2. The Triple Helix as a model of the knowledge-based economy; 3. Knowledge as a social coordination mechanism; 4. Neo-evolutionary dynamics in a Triple Helix of coordination mechanism; 5. The operation of the knowledge base; 6. The restructuring of knowledge production in a KBE; 7. The KBE and the systems-of-innovation approach; 8. The KBE and neo-evolutionary theories of innovation; 8.1 The construction of the evolving unit; 8.2 User-producer relations in systems of innovation; 8.3 'Mode-2' and the production of scientific knowledge; 8.4 A Triple Helix model of innovations; 9. Empirical studies and simulations using the TH model; 10. The KBE and the measurement; 10.1 The communication of meaning and information; 10.2 The expectation of social structure; 10.3 Configurations in a knowledge-based economy

  19. Integrase inhibitor (INI) genotypic resistance in treatment-naive and raltegravir-experienced patients infected with diverse HIV-1 clades

    Science.gov (United States)

    Doyle, Tomas; Dunn, David T.; Ceccherini-Silberstein, Francesca; De Mendoza, Carmen; Garcia, Frederico; Smit, Erasmus; Fearnhill, Esther; Marcelin, Anne-Genevieve; Martinez-Picado, Javier; Kaiser, Rolf; Geretti, Anna Maria

    2015-01-01

    Objectives The aim of this study was to characterize the prevalence and patterns of genotypic integrase inhibitor (INI) resistance in relation to HIV-1 clade. Methods The cohort comprised 533 INI-naive subjects and 255 raltegravir recipients with viraemia who underwent integrase sequencing in routine care across Europe, including 134/533 (25.1%) and 46/255 (18.0%), respectively, with non-B clades (A, C, D, F, G, CRF01, CRF02, other CRFs, complex). Results No major INI resistance-associated mutations (RAMs) occurred in INI-naive subjects. Among raltegravir recipients with viraemia (median 3523 HIV-1 RNA copies/mL), 113/255 (44.3%) had one or more major INI RAMs, most commonly N155H (45/255, 17.6%), Q148H/R/K + G140S/A (35/255, 13.7%) and Y143R/C/H (12/255, 4.7%). In addition, four (1.6%) raltegravir recipients showed novel mutations at recognized resistance sites (E92A, S147I, N155D, N155Q) and novel mutations at other integrase positions that were statistically associated with raltegravir exposure (K159Q/R, I161L/M/T/V, E170A/G). Comparing subtype B with non-B clades, Q148H/R/K occurred in 42/209 (20.1%) versus 2/46 (4.3%) subjects (P = 0.009) and G140S/A occurred in 36/209 (17.2%) versus 1/46 (2.2%) subjects (P = 0.005). Intermediate- to high-level cross-resistance to twice-daily dolutegravir was predicted in 40/255 (15.7%) subjects, more commonly in subtype B versus non-B clades (39/209, 18.7% versus 1/46, 2.2%; P = 0.003). A glycine (G) to serine (S) substitution at integrase position 140 required one nucleotide change in subtype B and two nucleotide changes in all non-B clades. Conclusions No major INI resistance mutations occurred in INI-naive subjects. Reduced occurrence of Q148H/R/K + G140S/A was seen in non-B clades versus subtype B, and was explained by the higher genetic barrier to the G140S mutation observed in all non-B clades analysed. PMID:26311843

  20. A Method for Producing Transgenic Cells Using a Multi-Integrase System on a Human Artificial Chromosome Vector

    OpenAIRE

    Shigeyuki Yamaguchi; Yasuhiro Kazuki; Yuji Nakayama; Eiji Nanba; Mitsuo Oshimura; Tetsuya Ohbayashi

    2011-01-01

    The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-spec...

  1. Prediction of transmembrane helix orientation in polytopic membrane proteins

    Directory of Open Access Journals (Sweden)

    Liang Jie

    2006-06-01

    Full Text Available Abstract Background Membrane proteins compose up to 30% of coding sequences within genomes. However, their structure determination is lagging behind compared with soluble proteins due to the experimental difficulties. Therefore, it is important to develop reliable computational methods to predict structures of membrane proteins. Results We present a method for prediction of the TM helix orientation, which is an essential step in ab initio modeling of membrane proteins. Our method is based on a canonical model of the heptad repeat originally developed for coiled coils. We identify the helical surface patches that interface with lipid molecules at an accuracy of about 88% from the sequence information alone, using an empirical scoring function LIPS (LIPid-facing Surface, which combines lipophilicity and conservation of residues in the helix. We test and discuss results of prediction of helix-lipid interfaces on 162 transmembrane helices from 18 polytopic membrane proteins and present predicted orientations of TM helices in TRPV1 channel. We also apply our method to two structures of homologous cytochrome b6f complexes and find discrepancy in the assignment of TM helices from subunits PetG, PetN and PetL. The results of LIPS calculations and analysis of packing and H-bonding interactions support the helix assignment found in the cytochrome b6f structure from green alga but not the assignment of TM helices in the cyanobacterium b6f structure. Conclusion LIPS calculations can be used for the prediction of helix orientation in ab initio modeling of polytopic membrane proteins. We also show with the example of two cytochrome b6f structures that our method can identify questionable helix assignments in membrane proteins. The LIPS server is available online at http://gila.bioengr.uic.edu/lab/larisa/lips.html.

  2. A statistically derived parameterization for the collagen triple-helix.

    Science.gov (United States)

    Rainey, Jan K; Goh, M Cynthia

    2002-11-01

    The triple-helix is a unique secondary structural motif found primarily within the collagens. In collagen, it is a homo- or hetero-tripeptide with a repeating primary sequence of (Gly-X-Y)(n), displaying characteristic peptide backbone dihedral angles. Studies of bulk collagen fibrils indicate that the triple-helix must be a highly repetitive secondary structure, with very specific constraints. Primary sequence analysis shows that most collagen molecules are primarily triple-helical; however, no high-resolution structure of any entire protein is yet available. Given the drastic morphological differences in self-assembled collagen structures with subtle changes in assembly conditions, a detailed knowledge of the relative locations of charged and sterically bulky residues in collagen is desirable. Its repetitive primary sequence and highly conserved secondary structure make collagen, and the triple-helix in general, an ideal candidate for a general parameterization for prediction of residue locations and for the use of a helical wheel in the prediction of residue orientation. Herein, a statistical analysis of the currently available high-resolution X-ray crystal structures of model triple-helical peptides is performed to produce an experimentally based parameter set for predicting peptide backbone and C(beta) atom locations for the triple-helix. Unlike existing homology models, this allows easy prediction of an entire triple-helix structure based on all existing high-resolution triple-helix structures, rather than only on a single structure or on idealized parameters. Furthermore, regional differences based on the helical propensity of residues may be readily incorporated. The parameter set is validated in terms of the predicted bond lengths, backbone dihedral angles, and interchain hydrogen bonding.

  3. Parametric-Resonance Ionization Cooling in Twin-Helix.

    Energy Technology Data Exchange (ETDEWEB)

    V.S. Morozov, Ya.S. Derbenev, A. Afanasev, R.P. Johnson, Erdelyi. B., J.A. Maloney

    2011-09-01

    Parametric-resonance Ionization Cooling (PIC) is proposed as the final 6D cooling stage of a highluminosity muon collider. For the implementation of PIC, we developed an epicyclic twin-helix channel with correlated optics. Wedge-shaped absorbers immediately followed by short rf cavities are placed into the twin-helix channel. Parametric resonances are induced in both planes using helical quadrupole harmonics. We demonstrate resonant dynamics and cooling with stochastic effects off using GEANT4/G4beamline. We illustrate compensation of spherical aberrations and benchmark COSY Infinity, a powerful tool for aberration analysis and compensation.

  4. DNA-like double helix formed by peptide nucleic acid

    DEFF Research Database (Denmark)

    Wittung, P; Nielsen, Peter E.; Buchardt, O;

    1994-01-01

    Although the importance of the nucleobases in the DNA double helix is well understood, the evolutionary significance of the deoxyribose phosphate backbone and the contribution of this chemical entity to the overall helical structure and stability of the double helix is not so clear. Peptide nucleic...... acid (PNA) is a DNA analogue with a backbone consisting of N-(2-aminoethyl)glycine units (Fig. 1) which has been shown to mimic DNA in forming Watson-Crick complementary duplexes with normal DNA. Using circular dichroism spectroscopy we show here that two complementary PNA strands can hybridize to one...

  5. Type VIa β-turn-fused helix N-termini: A novel helix N-cap motif containing cis proline.

    Science.gov (United States)

    Dasgupta, Rubin; Ganguly, Himal K; Modugula, E K; Basu, Gautam

    2017-01-01

    Helix N-capping motifs often form hydrogen bonds with terminal amide groups which otherwise would be free. Also, without an amide hydrogen, proline (trans) is over-represented at helix N-termini (N1 position) because this naturally removes the need to hydrogen bond one terminal amide. However, the preference of cisPro, vis-à-vis helix N-termini, is not known. We show that cisPro (αR or PPII ) often appears at the N-cap position (N0) of helices. The N-cap cisPro(αR ) is associated with a six-residue sequence motif - X(-2) -X(-1) -cisPro-X(1) -X(2) -X(3) - with preference for Glu/Gln at X(-1) , Phe/Tyr/Trp at X(1) and Ser/Thr at X(3) . The motif, formed by the fusion of a helix and a type VIa β-turn, contains a hydrogen bond between the side chain of X(-1) and the side chain/backbone of X(3) , a α-helical hydrogen bond between X(-2) and X(2) and stacking interaction between cisPro and an aromatic residue at X(1) . NMR experiments on peptides containing the motif and its variants showed that local interactions associated with the motif, as found in folded proteins, were not enough to significantly tilt the cis/trans equilibrium towards cisPro. This suggests that some other evolutionary pressure must select the cisPro motif (over transPro) at helix N-termini. Database analysis showed that >C = O of the pre-cisPro(αR ) residue at the helix N-cap, directed opposite to the N→C helical axis, participates in long-range interactions. We hypothesize that the cisPro(αR ) motif is preferred at helix N-termini because it allows the helix to participate in long-range interactions that may be structurally and functionally important.

  6. Solution structure of the ETS domain from murine Ets-1: a winged helix-turn-helix DNA binding motif.

    OpenAIRE

    Donaldson, L W; Petersen, J.M.; Graves, B J; McIntosh, L. P.

    1996-01-01

    Ets-1 is the prototypic member of the ets family of transcription factors. This family is characterized by the conserved ETS domain that mediates specific DNA binding. Using NMR methods, we have determined the structure of a fragment of murine Ets-1 composed of the 85 residue ETS domain and a 25 amino acid extension that ends at its native C-terminus. The ETS domain folds into a helix-turn-helix motif on a four-stranded anti-parallel beta-sheet scaffold. This structure places Ets-1 in the win...

  7. LEDGIN-mediated Inhibition of Integrase-LEDGF/p75 Interaction Reduces Reactivation of Residual Latent HIV.

    Science.gov (United States)

    Vranckx, Lenard S; Demeulemeester, Jonas; Saleh, Suha; Boll, Annegret; Vansant, Gerlinde; Schrijvers, Rik; Weydert, Caroline; Battivelli, Emilie; Verdin, Eric; Cereseto, Anna; Christ, Frauke; Gijsbers, Rik; Debyser, Zeger

    2016-06-01

    Persistence of latent, replication-competent Human Immunodeficiency Virus type 1 (HIV-1) provirus is the main impediment towards a cure for HIV/AIDS (Acquired Immune Deficiency Syndrome). Therefore, different therapeutic strategies to eliminate the viral reservoirs are currently being explored. We here propose a novel strategy to reduce the replicating HIV reservoir during primary HIV infection by means of drug-induced retargeting of HIV integration. A novel class of integration inhibitors, referred to as LEDGINs, inhibit the interaction between HIV integrase and the LEDGF/p75 host cofactor, the main determinant of lentiviral integration site selection. We show for the first time that LEDGF/p75 depletion hampers HIV-1 reactivation in cell culture. Next we demonstrate that LEDGINs relocate and retarget HIV integration resulting in a HIV reservoir that is refractory to reactivation by different latency-reversing agents. Taken together, these results support the potential of integrase inhibitors that modulate integration site targeting to reduce the likeliness of viral rebound.

  8. Talent Development as a University Mission: The Quadruple Helix

    Science.gov (United States)

    Holm-Nielsen, Lauritz B.; Thorn, Kristian; Olesen, Jeppe Dorup; Huey, Tina

    2013-01-01

    In this paper, the authors discuss the rationale behind making talent development at the PhD, post-doctoral and early career levels an equal fourth pillar of the university's mission, alongside the more traditional pillars of the triple helix. Using Denmark and Aarhus University as a case study, the paper describes how increased institutional…

  9. Design of GEO helix tourist orbit based on perturbation compensation

    Science.gov (United States)

    Xu, Yanli; Zhou, Haijun; Dai, Huayu

    2017-05-01

    Constrained by country area and technology level, GEO target and environment's detection become practical difficulty which restrict development of our country's space technology. Helix tourist orbit is introduced; orbit formation effect from perturbation of nonsphericfigure of the Earth, the solar and lunar attraction is analyzed; orbit design method based on perturbation compensation is put forward.

  10. The Triple Helix Model and the Knowledge-Based Economy

    NARCIS (Netherlands)

    Leydesdorff, L.; Meyer, M.

    2010-01-01

    The Triple Helix model of university-industry-government relations can be generalized from a neo-institutional model of networks of relations to a neo-evolutionary model of how three selection environments operate upon one another. Two selection mechanisms operating upon each other can mutually

  11. Alpha- and beta-hemocyanin of Helix pomatia

    NARCIS (Netherlands)

    Dijk, Jan

    1971-01-01

    This thesis deals with several aspects of the protein structure of Alpha- and Beta -hemocyanin of Helix pomatia. a- and 0-hemocyanin possess a virtually identical amino acid composition; it closely resembles the amino acid com- positions of hemocyanins of other MOLLUSCA and ARTHROPODA. All hemocyani

  12. The Triple Helix Model and the Knowledge-Based Economy

    NARCIS (Netherlands)

    Leydesdorff, L.; Meyer, M.

    2010-01-01

    The Triple Helix model of university-industry-government relations can be generalized from a neo-institutional model of networks of relations to a neo-evolutionary model of how three selection environments operate upon one another. Two selection mechanisms operating upon each other can mutually shap

  13. Collagen model peptides: Sequence dependence of triple-helix stability.

    Science.gov (United States)

    Persikov, A V; Ramshaw, J A; Brodsky, B

    2000-01-01

    The triple helix is a specialized protein motif, found in all collagens as well as in noncollagenous proteins involved in host defense. Peptides will adopt a triple-helical conformation if the sequence contains its characteristic features of Gly as every third residue and a high content of Pro and Hyp residues. Such model peptides have proved amenable to structural studies by x-ray crystallography and NMR spectroscopy, suitable for thermodynamic and kinetic analysis, and a valuable tool in characterizing the binding activities of the collagen triple helix. A systematic approach to understanding the amino acid sequence dependence of the collagen triple helix has been initiated, based on a set of host-guest peptides of the form, (Gly-Pro-Hyp)(3)-Gly-X-Y-(Gly-Pro-Hyp)(4). Comparison of their thermal stabilities has led to a propensity scale for the X and Y positions, and the additivity of contributions of individual residues is now under investigation. The local and global stability of the collagen triple helix is normally modulated by the residues in the X and Y positions, with every third position occupied by Gly in fibril-forming collagens. However, in collagen diseases, such as osteogenesis imperfecta, a single Gly may be substituted by another residue. Host-guest studies where the Gly is replaced by various amino acids suggest that the identity of the residue in the Gly position affects the degree of destabilization and the clinical severity of the disease.

  14. Spontaneous Helix Hand Reversal and Tendril Perversion in Climbing Plants

    Science.gov (United States)

    Goriely, Alain; Tabor, Michael

    1998-02-01

    The helix hand reversal exhibited by the tendrils of climbing plants when attached to a support is investigated. Modeled as a thin elastic rod with intrinsic curvature, a linear and nonlinear stability analysis shows the problem to be a paradigm for curvature induced morphogenesis in which symmetry breaking is constrained by a global invariant.

  15. Targeting intracellular bacteria with an extended cationic amphiphilic polyproline helix.

    Science.gov (United States)

    Nepal, Manish; Thangamani, Shankar; Seleem, Mohamed N; Chmielewski, Jean

    2015-06-07

    An extended cationic and amphiphilic polyproline helix (CAPH) is described with a dual mode of action: effective cell penetration of human macrophages, and potent antimicrobial activity in vitro against both Gram-positive and negative pathogens, including Acinetobacter baumannii, Escherichia coli O157 and Bacillus anthracis. This dual action was successfully combined to clear pathogenic bacteria (Brucella and Salmonella) residing within macrophages.

  16. Organizing product innovation: hierarchy, market or triple-helix networks?

    Science.gov (United States)

    Fitjar, Rune Dahl; Gjelsvik, Martin; Rodríguez-Pose, Andrés

    This paper assesses the extent to which the organization of the innovation effort in firms, as well as the geographical scale at which this effort is pursued, affects the capacity to benefit from product innovations. Three alternative modes of organization are studied: hierarchy, market and triple-helix-type networks. Furthermore, we consider triple-helix networks at three geographical scales: local, national and international. These relationships are tested on a random sample of 763 firms located in five urban regions of Norway which reported having introduced new products or services during the preceding 3 years. The analysis shows that firms exploiting internal hierarchy or triple-helix networks with a wide range of partners managed to derive a significantly higher share of their income from new products, compared to those that mainly relied on outsourcing within the market. In addition, the analysis shows that the geographical scale of cooperation in networks, as well as the type of partner used, matters for the capacity of firms to benefit from product innovation. In particular, firms that collaborate in international triple-helix-type networks involving suppliers, customers and R&D institutions extract a higher share of their income from product innovations, regardless of whether they organize the processes internally or through the network.

  17. A review of fast circle and helix fitting

    CERN Document Server

    Frühwirth, R; Waltenberger, W; Wroldsen, J

    2003-01-01

    Circle and helix fitting is of paramount importance in the data analysis of LHC experiments. We review several approaches to exact but fast fitting, including a recent development based on the projection of the measured points onto a second-order surface in space (a sphere or a paraboloid). We present results of a comparison with global and recursive linearized least-squares estimators.

  18. A catastrophe theory model of the conflict helix, with tests.

    Science.gov (United States)

    Rummel, R J

    1987-10-01

    Macro social field theory has undergone extensive development and testing since the 1960s. One of these has been the articulation of an appropriate conceptual micro model--called the conflict helix--for understanding the process from conflict to cooperation and vice versa. Conflict and cooperation are viewed as distinct equilibria of forces in a social field; the movement between these equilibria is a jump, energized by a gap between social expectations and power, and triggered by some minor event. Quite independently, there also has been much recent application of catastrophe theory to social behavior, but usually without a clear substantive theory and lacking empirical testing. This paper uses catastrophe theory--namely, the butterfly model--mathematically to structure the conflict helix. The social field framework and helix provide the substantive interpretation for the catastrophe theory; and catastrophe theory provides a suitable mathematical model for the conflict helix. The model is tested on the annual conflict and cooperation between India and Pakistan, 1948 to 1973. The results are generally positive and encouraging.

  19. Integrase inhibitors in late pregnancy and rapid HIV viral load reduction.

    Science.gov (United States)

    Rahangdale, Lisa; Cates, Jordan; Potter, JoNell; Badell, Martina L; Seidman, Dominika; Miller, Emilly S; Coleman, Jenell S; Lazenby, Gweneth B; Levison, Judy; Short, William R; Yawetz, Sigal; Ciaranello, Andrea; Livingston, Elizabeth; Duthely, Lunthita; Rimawi, Bassam H; Anderson, Jean R; Stringer, Elizabeth M

    2016-03-01

    Minimizing time to HIV viral suppression is critical in pregnancy. Integrase strand transfer inhibitors (INSTIs), like raltegravir, are known to rapidly suppress plasma HIV RNA in nonpregnant adults. There are limited data in pregnant women. We describe time to clinically relevant reduction in HIV RNA in pregnant women using INSTI-containing and non-INSTI-containing antiretroviral therapy (ART) options. We conducted a retrospective cohort study of pregnant HIV-infected women in the United States from 2009 through 2015. We included women who initiated ART, intensified their regimen, or switched to a new regimen due to detectable viremia (HIV RNA >40 copies/mL) at ≥20 weeks gestation. Among women with a baseline HIV RNA permitting 1-log reduction, we estimated time to 1-log RNA reduction using the Kaplan-Meier estimator comparing women starting/adding an INSTI in their regimen vs other ART. To compare groups with similar follow-up time, we also conducted a subgroup analysis limited to women with ≤14 days between baseline and follow-up RNA data. This study describes 101 HIV-infected pregnant women from 11 US clinics. In all, 75% (76/101) of women were not taking ART at baseline; 24 were taking non-INSTI containing ART, and 1 received zidovudine monotherapy. In all, 39% (39/101) of women started an INSTI-containing regimen or added an INSTI to their ART regimen. Among 90 women with a baseline HIV RNA permitting 1-log reduction, the median time to 1-log RNA reduction was 8 days (interquartile range [IQR], 7-14) in the INSTI group vs 35 days (IQR, 20-53) in the non-INSTI ART group (P < .01). In a subgroup of 39 women with first and last RNA measurements ≤14 days apart, median time to 1-log reduction was 7 days (IQR, 6-10) in the INSTI group vs 11 days (IQR, 10-14) in the non-INSTI group (P < .01). ART that includes INSTIs appears to induce more rapid viral suppression than other ART regimens in pregnancy. Inclusion of an INSTI may play a role in optimal reduction

  20. Lack of pharmacokinetic interaction between rilpivirine and integrase inhibitors dolutegravir and GSK1265744.

    Science.gov (United States)

    Ford, Susan L; Gould, Elizabeth; Chen, Shuguang; Margolis, David; Spreen, William; Crauwels, Herta; Piscitelli, Stephen

    2013-11-01

    Dolutegravir (DTG) and GSK1265744 are HIV integrase inhibitors (INIs) in clinical development. The oral formulation of rilpivirine (RPV), a nonnucleoside reverse transcriptase inhibitor (NNRTI), has been approved for treatment-naive HIV infection. Long-acting depot injections of GSK1265744 and RPV are also being developed. This study evaluated the potential for drug interactions between RPV and these INIs. This phase 1, open-label, two-cohort, three-period, single-sequence crossover study evaluated oral coadministration of RPV with DTG or GSK1265744. Healthy subjects received DTG (50 mg every 24 h for 5 days) or GSK1265744 (30 mg every 24 h for 12 days) in period 1 followed by a washout, RPV (25 mg every 24 h for 11 or 12 days) in period 2, immediately followed by RPV (25 mg every 24 h) plus DTG (50 mg every 24 h) for 5 days or GSK1265744 (30 mg every 24 h) for 12 days in period 3. Steady-state pharmacokinetic (PK) parameters were estimated using noncompartmental analysis of data collected on the last day of each period. The combinations of RPV and DTG (n = 16) and of RPV and GSK1265744 (n = 11) were well tolerated; no grade 3 or 4 adverse events (AEs) or AE-related discontinuations were observed. The 90% confidence intervals for the area under the curve from time zero until the end of the dosage interval [AUC0-τ] and maximum concentration of drug in serum (Cmax) geometric mean ratios were within 0.8 to 1.25. Following administration of DTG + RPV, DTG and RPV Cτ increased by 22% and 21%, respectively. Following administration of GSK1265744 + RPV, RPV Cτ decreased 8%. DTG and GSK1265744 can be administered with RPV without dosage adjustment for either agent. These results support coadministration of RPV with DTG or GSK1265744 as either oral or long-acting depot injection regimens. (This study has been registered at ClinicalTrials.gov under registration no. NCT01467531.).

  1. Cross-resistance profile determination of two second-generation HIV-1 integrase inhibitors using a panel of recombinant viruses derived from raltegravir-treated clinical isolates.

    Science.gov (United States)

    Van Wesenbeeck, L; Rondelez, E; Feyaerts, M; Verheyen, A; Van der Borght, K; Smits, V; Cleybergh, C; De Wolf, H; Van Baelen, K; Stuyver, L J

    2011-01-01

    The integrase inhibitor raltegravir (RAL) is currently used for the treatment of both treatment-naïve and treatment-experienced HIV-1-infected patients. Elvitegravir (EVG) is in late phases of clinical development. Since significant cross-resistance between RAL and EVG is observed, there is a need for second-generation integrase inhibitors (INIs) with a higher genetic barrier and limited cross-resistance to RAL/EVG. A panel of HIV-1 integrase recombinants, derived from plasma samples from raltegravir-treated patients (baseline and follow-up samples), were used to study the cross-resistance profile of two second-generation integrase inhibitors, MK-2048 and compound G. Samples with Q148H/R mutations had elevated fold change values with all compounds tested. Although samples with the Y143R/C mutation had reduced susceptibility to RAL, they remained susceptible to MK-2048 and compound G. Samples with the N155H mutation had no reduced susceptibility to compound G. In conclusion, our results allowed ranking of the INIs on the basis of the antiviral activities using recombinant virus stocks from RAL-treated patient viruses. The order according to decreasing susceptibility is compound G, MK-2048, and EVG.

  2. Monte Carlo Method Based QSAR Modeling of Coumarin Derivates as Potent HIV‐1 Integrase Inhibitors and Molecular Docking Studies of Selected 4‐phenyl Hydroxycoumarins

    Directory of Open Access Journals (Sweden)

    Veselinović Jovana

    2014-06-01

    Full Text Available In search for new and promising coumarin compounds as HIV-1 integrase inhibitors, chemoinformatic methods like quantitative structure-activity relationships (QSAR modeling and molecular docking have an important role since they can predict desired activity and propose molecule binding to enzyme.

  3. Surface simulation synthesis: a new strategy to spy alpha-helix structure.

    Science.gov (United States)

    Dong, Xiao-Nan; Chen, Yu; Chen, Ying-Hua

    2007-09-04

    In key proteins, there are always some alpha-helix structures, which play important role in the structure and functions. Many epitopes lie on the surface of alpha-helix. These epitopes are not easy to be recruited into the vaccine development, because they are conformation dependent epitopes. Can such epitopes on alpha-helix be mimicked synthetically? Our findings undoubtedly validate the feasibility of surface simulation synthesis with short linear peptide to mimic the antigenic side of alpha-helix structure.

  4. Npas4, a novel helix-loop-helix PAS domain protein, is regulated in response to cerebral ischemia

    DEFF Research Database (Denmark)

    Shamloo, Mehrdad; Soriano, Liza; von Schack, David

    2006-01-01

    Basic helix-loop-helix PAS domain proteins form a growing family of transcription factors. These proteins are involved in the process of adaptation to cellular stresses and environmental factors such as a change in oxygen concentration. We describe the identification and characterization of a rec......Basic helix-loop-helix PAS domain proteins form a growing family of transcription factors. These proteins are involved in the process of adaptation to cellular stresses and environmental factors such as a change in oxygen concentration. We describe the identification and characterization...... of a recently cloned PAS domain protein termed Npas4 in ischemic rat brain. Using gene expression profiling following middle cerebral artery occlusion, we showed that the Npas4 mRNA is differentially expressed in ischemic tissue. The full-length gene was cloned from rat brain and its spatial and temporal...... expression characterized with in situ hybridization and Northern blotting. The Npas4 mRNA is specifically expressed in the brain and is highly up-regulated in ischemic tissues following both focal and global cerebral ischemic insults. Immunohistochemistry revealed a strong expression in the limbic system...

  5. Archaeal MBF1 binds to 30S and 70S ribosomes via its helix-turn-helix domain

    NARCIS (Netherlands)

    Blombach, F.; Launay, H.; Snijders, A.P.; Zorraquino, V.; Wu, H.; Koning, de B.; Brouns, S.J.J.; Ettema, T.J.; Camilloni, C.; Cavalli, A.; Vendruscolo, M.; Dickman, M.J.; Cabrita, L.D.; Teana, La A.; Benelli, D.; Londei, P.; Christodoulou, J.; Oost, van der J.

    2014-01-01

    MBF1 (multi-protein bridging factor 1) is a protein containing a conserved HTH (helix–turn–helix) domain in both eukaryotes and archaea. Eukaryotic MBF1 has been reported to function as a transcriptional co-activator that physically bridges transcription regulators with the core transcription initia

  6. Iron-binding E3 ligase mediates iron response in plants by targeting basic helix-loop-helix transcription factors.

    Science.gov (United States)

    Selote, Devarshi; Samira, Rozalynne; Matthiadis, Anna; Gillikin, Jeffrey W; Long, Terri A

    2015-01-01

    Iron uptake and metabolism are tightly regulated in both plants and animals. In Arabidopsis (Arabidopsis thaliana), BRUTUS (BTS), which contains three hemerythrin (HHE) domains and a Really Interesting New Gene (RING) domain, interacts with basic helix-loop-helix transcription factors that are capable of forming heterodimers with POPEYE (PYE), a positive regulator of the iron deficiency response. BTS has been shown to have E3 ligase capacity and to play a role in root growth, rhizosphere acidification, and iron reductase activity in response to iron deprivation. To further characterize the function of this protein, we examined the expression pattern of recombinant ProBTS::β-GLUCURONIDASE and found that it is expressed in developing embryos and other reproductive tissues, corresponding with its apparent role in reproductive growth and development. Our findings also indicate that the interactions between BTS and PYE-like (PYEL) basic helix-loop-helix transcription factors occur within the nucleus and are dependent on the presence of the RING domain. We provide evidence that BTS facilitates 26S proteasome-mediated degradation of PYEL proteins in the absence of iron. We also determined that, upon binding iron at the HHE domains, BTS is destabilized and that this destabilization relies on specific residues within the HHE domains. This study reveals an important and unique mechanism for plant iron homeostasis whereby an E3 ubiquitin ligase may posttranslationally control components of the transcriptional regulatory network involved in the iron deficiency response.

  7. Structural and functional aspects of winged-helix domains at the core of transcription initiation complexes.

    Science.gov (United States)

    Teichmann, Martin; Dumay-Odelot, Hélène; Fribourg, Sébastien

    2012-01-01

    The winged helix (WH) domain is found in core components of transcription systems in eukaryotes and prokaryotes. It represents a sub-class of the helix-turn-helix motif. The WH domain participates in establishing protein-DNA and protein-protein-interactions. Here, we discuss possible explanations for the enrichment of this motif in transcription systems.

  8. Playing with peptides: how to build a supramolecular peptide nanostructure by exploiting helix···helix macrodipole interactions.

    Science.gov (United States)

    Gatto, E; Porchetta, A; Scarselli, M; De Crescenzi, M; Formaggio, F; Toniolo, C; Venanzi, M

    2012-02-07

    A novel method to build bicomponent peptide self-assembled monolayers (SAMs) has been developed, by exploiting helix···helix macrodipole interactions. In this work, a peptide-based self-assembled monolayer composed of two helical peptides was immobilized on a gold surface. Specifically, a pyrene-containing octapeptide, devoid of any sulfur atom (A8Pyr), and a hexapeptide, functionalized at the N-terminus with (S,R) lipoic acid, for binding to gold substrates (SSA4WA) via a Au-S linkage, have been employed. Both peptides investigated attain a helical structure, because they are almost exclusively formed by strongly folding inducer C(α)-tetrasubstituted α-amino acids. We demonstrate that the two peptides generate a stable supramolecular nanostructure (a densely packed bicomponent peptide monolayer), where A8Pyr is incorporated into the SSA4WA palisade by exploiting helix···helix macrodipole interactions. The presence of both peptides on the gold surface was investigated by spectroscopic and electrochemical techniques, while the morphology of the monolayer was analyzed by ultra high-vacuum scanning tunnelling microscopy. The composition of the bicomponent SAM on the surface was studied by a combination of electrochemical and spectroscopic techniques. In particular, the amount of Au-S linkages from the sulfur-containing peptides was quantified from reductive desorption of the peptide-based SAM, while the amount of A8Pyr was estimated by fluorescence spectroscopy. The antiparallel orientation of the A8Pyr and SSA4WA peptide chains minimizes the interaction energy between the helix dipoles, suggesting that this kind of electrostatic phenomenon is the driving force that stabilizes the bicomponent SAM.

  9. Helix-helix interconversion rates of short 13C-labeled helical peptides as measured by dynamic NMR spectroscopy.

    Science.gov (United States)

    Kubasik, Matthew; Kotz, James; Szabo, Christopher; Furlong, Theresa; Stace, Justin

    2005-06-05

    The rates at which a peptide hexamer and a peptide octamer interconvert between left- and right-handed helical forms in CD2Cl2 solution have been characterized by 13C dynamic NMR (DNMR) spectroscopy. The peptide esters studied are Fmoc-(Aib)n-OtBu (n = 6 and 8), where Fmoc is 9-fluorenylmethyoxycarbonyl and Aib is the strongly helix-forming residue alpha-aminoisobutyric acid. Because the Aib residue is itself achiral, homooligomers of this residue form a 50/50 mixture of enantiomeric 3(10)-helices in solution. It has been demonstrated (R.-P. Hummel, C. Toniolo, and G. Jung, Angewandte Chemie International Edition, 1987, Vol. 26, pp. 1150-1152) that oligomers of Aib interconvert on the millisecond timescale. We have performed lineshape analysis of 13C-NMR spectra collected for our peptides enriched with 13C at a single residue. Rate constants for the octamer range from 6 s(-1) at 196 K to about 56,500 s(-1) at 320 K. At all temperatures, the hexamer interconverts about three times faster than the octamer. Eyring plots of the data reveal experimentally indistinguishable DeltaH++ values for the hexamer and octamer of 37.8 +/- 0.6 and 37.6 +/- 0.4 kJ mol(-1) respectively. The difference in the rates of interconversion is dictated by entropic factors. The hexamer and octamer exhibit negative DeltaS++ values of -29.0(-1) +/- 2.5 and -37.3 +/- 1.7 J K(-1) mol(-1), respectively. A mechanism for the helix-helix interconversion is proposed. and calculated DeltaG++ values are compared to the estimate for a decamer undergoing a helix-helix interconversion.

  10. The hepatitis B virus ribonuclease H is sensitive to inhibitors of the human immunodeficiency virus ribonuclease H and integrase enzymes.

    Directory of Open Access Journals (Sweden)

    John E Tavis

    2013-01-01

    Full Text Available Nucleos(tide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(tide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(tide analogs. The HBV ribonuclease H (RNAseH is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 µM, the best compounds had low micromolar IC(50 values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 µM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug

  11. Design and synthesis of DNA four-helix bundles

    Energy Technology Data Exchange (ETDEWEB)

    Rangnekar, Abhijit; Gothelf, Kurt V [Department of Chemistry, Centre for DNA Nanotechnology (CDNA) and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C (Denmark); LaBean, Thomas H, E-mail: kvg@chem.au.dk, E-mail: thl@cs.duke.edu [Department of Chemistry, Duke University, Durham, NC 27708 (United States)

    2011-06-10

    The field of DNA nanotechnology has evolved significantly in the past decade. Researchers have succeeded in synthesizing tile-based structures and using them to form periodic lattices in one, two and three dimensions. Origami-based structures have also been used to create nanoscale structures in two and three dimensions. Design and construction of DNA bundles with fixed circumference has added a new dimension to the field. Here we report the design and synthesis of a DNA four-helix bundle. It was found to be extremely rigid and stable. When several such bundles were assembled using appropriate sticky-ends, they formed micrometre-long filaments. However, when creation of two-dimensional sheet-like arrays of the four-helix bundles was attempted, nanoscale rings were observed instead. The exact reason behind the nanoring formation is yet to be ascertained, but it provides an exciting prospect for making programmable circular nanostructures using DNA.

  12. Crowding effect on helix-coil transition: Beyond entropic stabilization

    Energy Technology Data Exchange (ETDEWEB)

    Koutsioubas, A.; Lairez, D.; Combet, S.; Longeville, S. [Laboratoire Leon Brillouin, CEA/CNRS UMR 12, CEA-Saclay, 91191 Gif-sur-Yvette Cedex (France); Fadda, G. C. [Laboratoire Leon Brillouin, CEA/CNRS UMR 12, CEA-Saclay, 91191 Gif-sur-Yvette Cedex (France); Universite Paris 13, UFR SMBH, 93017 Bobigny (France); Zalczer, G. [Service de Physique de l' Etat Condense, CEA-Saclay, 91191 Gif-sur-Yvette Cedex (France)

    2012-06-07

    We report circular dichroism measurements on the helix-coil transition of poly(L-glutamic acid) in solution with polyethylene glycol (PEG) as a crowding agent. The PEG solutions have been characterized by small angle neutron scattering and are well described by the picture of a network of mesh size {xi}, usual for semi-dilute chains in good solvent. We show that the increase of PEG concentration stabilizes the helices and increases the transition temperature. But more unexpectedly, we also notice that the increase of concentration of crowding agent reduces the mean helix extent at the transition, or in other words reduces its cooperativity. This result cannot be taken into account for by an entropic stabilization mechanism. Comparing the mean length of helices at the transition and the mesh size of the PEG network, our results strongly suggest two regimes: helices shorter or longer than the mesh size.

  13. Rational design of a triple helix-specific intercalating ligand.

    Science.gov (United States)

    Escudé, C; Nguyen, C H; Kukreti, S; Janin, Y; Sun, J S; Bisagni, E; Garestier, T; Hélène, C

    1998-03-31

    DNA triple helices offer new perspectives toward oligonucleotide-directed gene regulation. However, the poor stability of some of these structures might limit their use under physiological conditions. Specific ligands can intercalate into DNA triple helices and stabilize them. Molecular modeling and thermal denaturation experiments suggest that benzo[f]pyrido[3, 4-b]quinoxaline derivatives intercalate into triple helices by stacking preferentially with the Hoogsteen-paired bases. Based on this model, it was predicted that a benzo[f]quino[3,4-b]quinoxaline derivative, which possesses an additional aromatic ring, could engage additional stacking interactions with the pyrimidine strand of the Watson-Crick double helix upon binding of this pentacyclic ligand to a triplex structure. This compound was synthesized. Thermal denaturation experiments and inhibition of restriction enzyme cleavage show that this new compound can indeed stabilize triple helices with great efficiency and specificity and/or induce triple helix formation under physiological conditions.

  14. Triple Helix Formation in a Topologically Controlled DNA Nanosystem.

    Science.gov (United States)

    Yamagata, Yutaro; Emura, Tomoko; Hidaka, Kumi; Sugiyama, Hiroshi; Endo, Masayuki

    2016-04-11

    In the present study, we demonstrate single-molecule imaging of triple helix formation in DNA nanostructures. The binding of the single-molecule third strand to double-stranded DNA in a DNA origami frame was examined using two different types of triplet base pairs. The target DNA strand and the third strand were incorporated into the DNA frame, and the binding of the third strand was controlled by the formation of Watson-Crick base pairing. Triple helix formation was monitored by observing the structural changes in the incorporated DNA strands. It was also examined using a photocaged third strand wherein the binding of the third strand was directly observed using high-speed atomic force microscopy during photoirradiation. We found that the binding of the third strand could be controlled by regulating duplex formation and the uncaging of the photocaged strands in the designed nanospace.

  15. Soliton solutions for Davydov solitons in α-helix proteins

    Science.gov (United States)

    Taghizadeh, N.; Zhou, Qin; Ekici, M.; Mirzazadeh, M.

    2017-02-01

    The propagation equation for describing Davydov solitons in α-helix proteins has been investigated analytically. There are seven integration tools to extract analytical soliton solutions. They are the Ricatti equation expansion approach, ansatz scheme, improved extended tanh-equation method, the extend exp(-Ψ(τ)) -expansion method, the extended Jacobi elliptic function expansion method, the extended trial equation method and the extended G ' / G - expansion method.

  16. AND BUSINESS INCUBATORS INTO THE TRIPLE HELIX CONCEPT

    Directory of Open Access Journals (Sweden)

    Sizova, Y.S.

    2016-04-01

    Full Text Available The article’s author endeavors to create a practical model of the Russian economy’s development while integrating such tools of supporting and stimulating entrepreneurship as business incubators and research and technology parks into the Triple Helix concept. The article points out that configurations fusing together the nation-state, business and science/education, possess a greater potential for development.

  17. Chiral transformation: From single nanowire to double helix

    KAUST Repository

    Wang, Yong

    2011-12-21

    We report a new type of water-soluble ultrathin Au-Ag alloy nanowire (NW), which exhibits unprecedented behavior in a colloidal solution. Upon growth of a thin metal (Pd, Pt, or Au) layer, the NW winds around itself to give a metallic double helix. We propose that the winding originates from the chirality within the as-synthesized Au-Ag NWs, which were induced to untwist upon metal deposition. © 2011 American Chemical Society.

  18. Identification of small peptides inhibiting the integrase-LEDGF/p75 interaction through targeting the cellular co-factor.

    Science.gov (United States)

    Cavalluzzo, Claudia; Christ, Frauke; Voet, Arnout; Sharma, Ajendra; Singh, Brajendra Kumar; Zhang, Kam Y J; Lescrinier, Eveline; De Maeyer, Marc; Debyser, Zeger; Van der Eycken, Erik

    2013-10-01

    The integration of the viral DNA into the host genome is one of the essential steps in the HIV replication cycle. This process is mediated by the viral enzyme integrase (IN) and lens epithelium-derived growth factor (LEDGF/p75). LEDGF/p75 has been identified as a crucial cellular co-factor of integration that acts by tethering IN to the cellular chromatin. Recently, circular peptides were identified that bind to the C-terminal domain of IN and disrupt the interaction with LEDGF/p75. Starting from the circular peptides, we identified a short peptidic sequence able to inhibit the LEDGF/p75-IN interaction at low μM concentration through its binding to the IN binding site of LEDGF/p75. This discovery can lead to the synthesis of peptidomimetics with high anti-HIV activity targeting the cellular co-factor LEDGF/p75 and not the viral protein IN.

  19. New treatment options for HIV salvage patients: an overview of second generation PIs, NNRTIs, integrase inhibitors and CCR5 antagonists.

    Science.gov (United States)

    Hughes, Amelia; Barber, Tristan; Nelson, Mark

    2008-07-01

    Since 1996, the prognosis of those living with HIV and AIDS has improved significantly due to highly active antiretroviral therapy (HAART). Treatment failure can occur clinically, immunologically or virologically. Until recently, treatment options for those individuals harboring resistance to the three initial licensed classes of drug have been limited. These three classes are the nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs). New drugs are now available in these classes (second generation NNRTIs and novel PIs) as well as new classes of drugs, integrase inhibitors, CCR5 antagonists and fusion inhibitors. If these new drugs are used appropriately with other active antiretroviral agents, it is probable that antiretroviral therapy can achieve the optimum outcome of HIV therapy - durable suppression of HIV viraemia. This article is a review of currently available antiretroviral agents including the new classes and second generation drugs, resistance pathways and treatment options for salvage therapy.

  20. Partial Agonists Activate PPARgamma Using a Helix 12 Independent Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Bruning, J.B.; Chalmers, M.J.; Prasad, S.; Bushby, S.A.; Kamenecka, T.A.; He, Y.; Nettles, K.W.; Griffin, P.R.

    2009-05-28

    Binding to helix 12 of the ligand-binding domain of PPAR{gamma} is required for full agonist activity. Previously, the degree of stabilization of the activation function 2 (AF-2) surface was thought to correlate with the degree of agonism and transactivation. To examine this mechanism, we probed structural dynamics of PPAR{gamma} with agonists that induced graded transcriptional responses. Here we present crystal structures and amide H/D exchange (HDX) kinetics for six of these complexes. Amide HDX revealed each ligand induced unique changes to the dynamics of the ligand-binding domain (LBD). Full agonists stabilized helix 12, whereas intermediate and partial agonists did not at all, and rather differentially stabilized other regions of the binding pocket. The gradient of PPAR{gamma} transactivation cannot be accounted for solely through changes to the dynamics of AF-2. Thus, our understanding of allosteric signaling must be extended beyond the idea of a dynamic helix 12 acting as a molecular switch.

  1. Classical scattering of charged particles confined on an inhomogeneous helix

    CERN Document Server

    Zampetaki, A V; Krönke, S; Schmelcher, P

    2013-01-01

    We explore the effects arising due to the coupling of the center of mass and relative motion of two charged particles confined on an inhomogeneous helix with a locally modified radius. It is first proven that a separation of the center of mass and the relative motion is provided if and only if the confining manifold represents a homogeneous helix. In this case bound states of repulsively Coulomb interacting particles occur. For an inhomogeneous helix, the coupling of the center of mass and relative motion induces an energy transfer between the collective and relative motion, leading to dissociation of initially bound states in a scattering process. Due to the time reversal symmetry, a binding of the particles out of the scattering continuum is thus equally possible. We identify the regimes of dissociation for different initial conditions and provide an analysis of the underlying phase space via Poincar\\'e surfaces of section. Bound states inside the inhomogeneity as well as resonant states are identified.

  2. Unambiguous demonstration of triple-helix-directed gene modification.

    Science.gov (United States)

    Barre, F X; Ait-Si-Ali, S; Giovannangeli, C; Luis, R; Robin, P; Pritchard, L L; Helene, C; Harel-Bellan, A

    2000-03-28

    Triple-helix-forming oligonucleotides (TFOs), which can potentially modify target genes irreversibly, represent promising tools for antiviral therapies. However, their effectiveness on endogenous genes has yet to be unambiguously demonstrated. To monitor endogenous gene modification by TFOs in a yeast model, we inactivated an auxotrophic marker gene by inserting target sequences of interest into its coding region. The genetically engineered yeast cells then were treated with psoralen-linked TFOs followed by UV irradiation, thus generating highly mutagenic covalent crosslinks at the target site whose repair could restore gene function; the number of revertants and spectrum of mutations generated were quantified. Results showed that a phosphoramidate TFO indeed reaches its target sequence, forms crosslinks, and generates mutations at the expected site via a triplex-mediated mechanism: (i) under identical conditions, no mutations were generated by the same TFO at two other loci in the target strain, nor in an isogenic control strain carrying a modified target sequence incapable of supporting triple-helix formation; (ii) for a given target sequence, whether the triplex was formed in vivo on an endogenous gene or in vitro on an exogenous plasmid, the nature of the mutations generated was identical, and consistent with the repair of a psoralen crosslink at the target site. Although the mutation efficiency was probably too low for therapeutic applications, our results confirm the validity of the triple-helix approach and provide a means of evaluating the effectiveness of new chemically modified TFOs and analogs.

  3. A 20 GHz, 75 watt, helix TWT for space communications

    Science.gov (United States)

    Heney, J. F.; Tamashiro, R. N.

    A space-qualified, helix-type traveling wave tube is being developed for satellite communication systems in the frequency band of 17.7 to 21.2 GHz. The design approach stresses very high efficiency operation, but with very low distortion. The tube provides multi-mode operation, permitting CW saturated power output levels of 75, 40, and 7.5 W. Operation is also anticipated at 5 dB below these saturation levels to achieve the required low distortion levels. Advanced construction features include a five-stage depressed collector, a diamond supported helix slow-wave circuit, and a type M dispenser cathode. High reliability and long life (10 yr) are objectives of the tube design. Preliminary test results on early developmental models of this tube are very encouraging. An output power of 75 to 90 W has been achieved over the full bandwidth with about 40 dB of saturated gain. More importantly, the basic electronic efficiency of the interaction process has been increased from about 7.5-11 percent by the use of the diamond helix support compared to earlier tubes using BeO support rods. This effort is supported by NASA Lewis Research Center and is aimed toward application in the NASA Advanced Communications Satellite Technology Program.

  4. Structural dynamics of native and V260E mutant C-terminal domain of HIV-1 integrase

    Science.gov (United States)

    Sangeetha, Balasubramanian; Muthukumaran, Rajagopalan; Amutha, Ramaswamy

    2015-04-01

    The C-terminal domain (CTD) of HIV-1 integrase is a five stranded β-barrel resembling an SH3 fold. Mutational studies on isolated CTD and full-length IN have reported V260E mutant as either homo-dimerization defective or affecting the stability and folding of CTD. In this study, molecular dynamics simulation techniques were used to unveil the effect of V260E mutation on isolated CTD monomer and dimer. Both monomeric and dimeric forms of wild type and V260E mutant are highly stable during the simulated period. However, the stabilizing π-stacking interaction between Trp243 and Trp243' at the dimer interface is highly disturbed in CTD-V260E (>6 Å apart). The loss in entropy for dimerization is -30 and -25 kcal/mol for CTD-wt and CTD-V260E respectively signifying a weak hydrophobic interaction and its perturbation in CTD-V260E. The mutant Glu260 exhibits strong attraction/repulsion with all the basic/acidic residues of CTD. In addition to this, the dynamics of CTD-wild type and V260E monomers at 498 K was analyzed to elucidate the effect of V260E mutation on CTD folding. Increase in SASA and reduction in the number of contacts in CTD-V260E during simulation highlights the instability caused by the mutation. In general, V260E mutation affects both multimerization and protein folding with a pronounced effect on protein folding rather than multimerization. This study emphasizes the importance of the hydrophobic nature and SH3 fold of CTD in proper functioning of HIV integrase and perturbing this nature would be a rational approach toward designing more selective and potent allosteric anti-HIV inhibitors.

  5. Symmetrical 1-pyrrolidineacetamide showing anti-HIV activity through a new binding site on HIV-1 integrase

    Institute of Scientific and Technical Information of China (English)

    Li DU; Ya-xue ZHAO; Liu-meng YANG; Yong-tang ZHENG; Yun TANG; Xu SHEN; Hua-liang JIANG

    2008-01-01

    Aim:To characterize the functional and pharmacological features of a symmetrical 1-pyrrolidineacetamide,N,N'-(methylene-di-4,1-phenylene) bis-1-pyrrolidineacetamide,as a new anti-HIV compound which could competitively inhibit HIV-1 integrase (IN) binding to viral DNA.Methods:A surface plasma resonance (SPR)-based competitive assay was employed to determine the compound's inhibitory activity,and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell assay was used to qualify the antiviral activity.The potential binding sites were predicted by molecular modeling and determined by site-directed mutagenesis and a SPR binding assay.Results:l-pyrrolidineacetamide,N,N'-(methylene-di-4,1-phenylene) bis-1-pyrrolidineacetamide could competitively inhibit IN binding to viral DNA with a 50% inhibitory concentration (IC50) value of 7.29±0.68 μmol/L as investigated by SPR-based investigation.Another antiretroviral activity assay showed that this compound exhibited inhibition against HⅣ-Ⅰ(ⅢB) replication with a 50% effective concentration (EC50) value of 40.54 μmol/L in C8166 cells,and cytotoxicity with a cytotoxic concentration value of 173.84 μmol/L in mock-infected C8166 cells.Molecular docking predicted 3 potential residues as 1-pyrrolidineacetamide,N,N'-(methylene-di-4,1-phenylene)bis-1-pyrrolidineacetamide binding sites.The importance of 3 key amino acid residues (Lys103,Lys173,and Thr174) involved in the binding was further identified by site-directed mutagenesis and a SPR binding assay.Conclusion:This present work identified a new anti-HIV compound through a new IN-binding site which is expected to supply new potential drug-binding site information for HIV-1 integrase inhibitor discovery and development.

  6. High-throughput real-time assay based on molecular beacons for HIV-1 integrase 3'-processing reaction

    Institute of Scientific and Technical Information of China (English)

    Hong-qiu HE; Xiao-hui MA; Bin LIU; Xiao-yi ZHANG; Wei-zu CHEN; Cun-xin WANG; Shao-hui CHENG

    2007-01-01

    Aim: To develop a high-throughput real-time assay based on molecular beacons to monitor the integrase 3'-processing reaction in vitro and apply it to inhibitor screening.Methods: The recombinant human immunodeficiency virus (HIV)-1 integrase (IN) is incubated with a 38 mer oligonucleotide substrate, a sequence identical to the U5 end of HIV-1 long terminal repeats (LTR). Based on the fluores-cence properties of molecular beacons, the substrate is designed to form a stem-loop structure labeled with a fluorophore at the 5' end and a quencher at the 3'end.IN cleaves the terminal 3'-dinucleotide containing the quencher, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. To optimize this assay, tests were performed to investigate the effects of substrates, enzyme and the metal ion concentrations on the IN activity and optimal param-eters were obtained. Moreover, 2 IN inhibitors were employed to test the perfor-mance of this assay in antiviral compound screening.Results: The fluorescent intensity of the reaction mixture varies linearly with time and is proportional to the velocity of the 3'-processing reaction. Tests were performed and the results showed that the optimal rate was obtained for a reaction mixture containing 50 mg/L recom-binant HIV-1 IN, 400 nmol/L substrate, and 10 mmol/L Mn2+. The IN 3'-processing reaction under the optimal conditions showed a more than 18-fold increase in the fluorescence intensity compared to the enzyme-free control. The IC50 values of the IN inhibitors obtained in our assay were similar to the values obtained from a radiolabeled substrate assay.Conclusion: Our results demonstrated that this is a fast, reliable, and sensitive method to monitor HIV IN 3'-processing reaction and that it can be used for inhibitor screening.

  7. Pharmacovirological impact of an integrase inhibitor on human immunodeficiency virus type 1 cDNA species in vivo.

    Science.gov (United States)

    Goffinet, Christine; Allespach, Ina; Oberbremer, Lena; Golden, Pamela L; Foster, Scott A; Johns, Brian A; Weatherhead, Jason G; Novick, Steven J; Chiswell, Karen E; Garvey, Edward P; Keppler, Oliver T

    2009-08-01

    Clinical trials of the first approved integrase inhibitor (INI), raltegravir, have demonstrated a drop in the human immunodeficiency virus type 1 (HIV-1) RNA loads of infected patients that was unexpectedly more rapid than that with a potent reverse transcriptase inhibitor, and apparently dose independent. These clinical outcomes are not understood. In tissue culture, although their inhibition of integration is well documented, the effects of INIs on levels of unintegrated HIV-1 cDNAs have been variable. Furthermore, there has been no report to date on an INI's effect on these episomal species in vivo. Here, we show that prophylactic treatment of transgenic rats with the strand transfer INI GSK501015 reduced levels of viral integrants in the spleen by up to 99.7%. Episomal two-long-terminal-repeat (LTR) circles accumulated up to sevenfold in this secondary lymphoid organ, and this inversely correlated with the impact on the proviral burden. Contrasting raltegravir's dose-ranging study with HIV patients, titration of GSK501015 in HIV-infected animals demonstrated dependence of the INI's antiviral effect on its serum concentration. Furthermore, the in vivo 50% effective concentration calculated from these data best matched GSK501015's in vitro potency when serum protein binding was accounted for. Collectively, this study demonstrates a titratable, antipodal impact of an INI on integrated and episomal HIV-1 cDNAs in vivo. Based on these findings and known biological characteristics of viral episomes, we discuss how integrase inhibition may result in additional indirect antiviral effects that contribute to more rapid HIV-1 decay in HIV/AIDS patients.

  8. Detection of Integrase Gone and Analysis of Gone Cassette Structure of Variable Region in Acinetobactor Baumannii%鲍曼不动杆菌整合酶基因测定及可变区基因盒结构分析

    Institute of Scientific and Technical Information of China (English)

    马全玲; 武大伟; 魏殿军; 张坚磊; 胡静仪

    2011-01-01

    Objective: To investigate the distribution and prevalence of integrase gene of multidrug-resistance Acinetobacter baumannii in Tianjin, and to analyze the relationship between integron and multidrug-resisitance of Acinetobacter baumannii. Methods: Fifty-five multidrug- resistance Acinetobacter baumannn were collected from three hospitals in Tianjin.The drug sensitivation was detected by K-B test. The integrase genes were detected by PCR. The relationship of clinical isolates was analysed by multigene cluster typing. Results: There were 47 strains of Ⅰ class integrase genes, 23 contained variable regions, but no Ⅱ and Ⅲ class integrase gene in 55 multidrug- resistance Acinetobacter baumannii. The mam reason caused aminoglycosides resistance was the aacA4 and aadAI cassette in the variable region. Three clone strains were found in 55 multidrug-resistance Acinetohacter haumannii by multigene cluster typing. Conclusion: It was found that Ⅰ class integrase gene was existed extensively in multidrug- resistance Acinetobacter haumannii in Tianjin. All strains could be typed by multigene cluster typing.%目的:了解整合酶基因在天津地区多重耐药鲍曼不动杆菌株中的分布和流行情况,分析整合子与鲍曼不动杆菌多重耐药性的关系.方法:收集天津地区3家医院55株多重耐药鲍曼不动杆菌,以K-B法进行抗生素敏感试验,用PCR方法检测整合酶基因,结合以往检测的耐药基因,采用聚类法对55株多重耐药鲍曼不动杆菌进行菌株亲缘性分析.结果:55株多重耐药鲍曼不动杆菌共检出47株含有Ⅰ类整合酶基因,其中有23株检出可变区结构,未检出Ⅱ、Ⅲ类整合酶基因,可变区所携带的aacA4和aadA1基因盒是引起鲍曼不动杆菌对氨基糖苷类抗生素耐药的主要原因.55株多重耐药鲍曼不动杆菌共含有3个克隆株.结论:天津地区多重耐药鲍曼不动杆菌中主要存在Ⅰ类整合子,聚类分析方法可对所有菌株分型.

  9. Evaluation of a high-precision gear measuring machine for helix measurement using helix and wedge artifacts

    Science.gov (United States)

    Taguchi, Tetsuya; Kondo, Yohan

    2016-08-01

    High-precision gears are required for advanced motion and power transmission. The reliability of the measured value becomes important as the gear accuracy increases, and the establishment of a traceability system is needed. Therefore, a high-precision gear measuring machine (GMM) with a smaller uncertainty is expected to improve the gear calibration uncertainty. For this purpose, we developed a prototype of a high-precision GMM that adopts a direct drive mechanism and other features. Then, the high measurement capability of the developed GMM was verified using gear artifacts. Recently, some new measurement methods using simple shapes such as spheres and planes have been proposed as standards. We have verified the tooth profile measurement using a sphere artifact and reported the results that the developed GMM had a high capability in tooth profile measurement. Therefore, we attempted to devise a new evaluation method for helix measurement using a wedge artifact (WA) whose plane was treated as the tooth flank, and the high measurement capability of the developed GMM was verified. The results will provide a part of information to fully assess measurement uncertainty as our future work. This paper describes the evaluation results of the developed GMM for helix measurement using both a helix artifact and the WA, and discusses the effectiveness of the WA as a new artifact to evaluate the GMMs.

  10. Lipid solvation effects contribute to the affinity of Gly-xxx-Gly motif-mediated helix-helix interactions.

    Science.gov (United States)

    Johnson, Rachel M; Rath, Arianna; Melnyk, Roman A; Deber, Charles M

    2006-07-18

    Interactions between transmembrane helices are mediated by the concave Gly-xxx-Gly motif surface. Whether Gly residues per se are sufficient for selection of this motif has not been established. Here, we used the in vivo TOXCAT assay to measure the relative affinities of all 18 combinations of Gly, Ala, and Ser "small-xxx-small" mutations in glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP) homodimers. Affinity values were compared with the accessibility to a methylene-sized probe of the total surface area of each helix monomer as a measure of solvation by membrane components. A strong inverse correlation was found between nonpolar-group lipid accessibility and dimer affinity (R = 0.75 for GpA, p = 0.013, and R = 0.81 for MCP, p = 0.004), suggesting that lipid as a poor membrane protein solvent, conceptually analogous to water in soluble protein folding, can contribute to dimer stability and help to define helix-helix interfaces.

  11. Exploring the binding of d(GGGT)4 to the HIV-1 integrase: An approach to investigate G-quadruplex aptamer/target protein interactions.

    Science.gov (United States)

    Esposito, Veronica; Pirone, Luciano; Mayol, Luciano; Pedone, Emilia; Virgilio, Antonella; Galeone, Aldo

    2016-08-01

    The aptamer d(GGGT)4 (T30923 or T30695) forms a 5'-5' dimer of two stacked parallel G-quadruplexes, each characterized by three G-tetrads and three single-thymidine reversed-chain loops. This aptamer has been reported to exhibit anti-HIV activity by targeting the HIV integrase, a viral enzyme responsible for the integration of viral DNA into the host-cell genome. However, information concerning the aptamer/target interaction is still rather limited. In this communication we report microscale thermophoresis investigations on the interaction between the HIV-1 integrase and d(GGGT)4 aptamer analogues containing abasic sites singly replacing thymidines in the original sequence. This approach has allowed the identification of which part of the aptamer G-quadruplex structure is mainly involved in the interaction with the protein.

  12. HIV-1 group O integrase displays lower susceptibility to raltegravir and has a different mutational pathway for resistance than HIV-1 group M

    Directory of Open Access Journals (Sweden)

    Agnès Depatureaux

    2014-11-01

    Full Text Available Introduction: HIV-1 group O (HIV-O is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase gene, e.g. positions L74I, S153A, G163Q and T206S. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on susceptibility to integrase inhibitors and emergence of resistance associated mutations. Viruses and Methods: We cloned and purified integrase proteins from each of HIV-1 Group O clades A (HIV-O/A and B (HIV-O/B, a HIV-O divergent strain (HIV-O/Div, and HIV-1 group M (subtype B, HIV-M/B and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs in cell-free assays and in tissue culture, in the absence or presence of varying concentrations of several INSTIs. The inhibition constant (Ki and IC50 were calculated and compared for HIV-M and HIV-O integrases. Selections for resistance-related mutations were performed using cord blood mononuclear cells and increasing concentration of INSTIs. Results: HIV-O integrase and viruses were more susceptible to raltegravir (RAL in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. During selection, we observed different pathways of resistance depending on the drug and clade. Mutations selected in HIV-O can be classified as follows: (1 mutations described for HIV-M such as T97A, Q148R, V151A/I (RAL, T66I, E92Q, E157Q (EVG and M50I, R263K (DTG and (2 signature mutations for HIV-O (i.e. not described in HIV-M F121C (HIV-O/B for RAL, V75I (HIV-O/A for RAL and S153V (HIV-O/A for DTG. Only the HIV-O/Div selected the Q148R mutation for RAL and R263K+M50I for DTG, as previously described for HIV-M. None of the HIV-O viruses selected either N155H or Y143C. The selection of the specific S153V mutation could be explained at the nucleotide level: HIV-O at this position contains an alanine and substitution of alanine to

  13. Design and Synthesis of Bis-amide and Hydrazide-containing Derivatives of Malonic Acid as Potential HIV-1 Integrase Inhibitors

    Directory of Open Access Journals (Sweden)

    Nouri Neamati

    2008-10-01

    Full Text Available HIV-1 integrase (IN is an attractive and validated target for the development of novel therapeutics against AIDS. In the search for new IN inhibitors, we designed and synthesized three series of bis-amide and hydrazide-containing derivatives of malonic acid. We performed a docking study to investigate the potential interactions of the title compounds with essential amino acids on the IN active site.

  14. [Application of helix water jet to parotid surgery].

    Science.gov (United States)

    Zhang, Dong-Kun; Guo, Zhu-Ming; Zhang, Quan; Zeng, Zong-Yuan; Chen, Fu-Jin; Chen, Wen-Kuan; Li, Hao; Wang, Shun-Lan

    2008-01-01

    Dissecting the facial nerves safely is an important guarantee for the accomplishment of parotidectomy and reduction of postoperative complications. This study was to explore the application of helix water jet to parotidectomy. Clinical data of 43 patients with parotid tumors, who received operation with helix water jet from Feb. 2004 to Feb. 2005 at Cancer Center of Sun Yat-Sen University, were analyzed. Meanwhile, traditional techniques in parotidectomy was performed in 36 patients (control group). Duration of operation, postoperative drainage volume, postoperative hospitalization, and occurrence of postoperative complications, such as facial nerve dysfunction and salivary fistula, of the 2 groups were compared. The postoperative drainage volume was significantly lower in water jet group than in control group [(9.89+/-3.74) mL vs. (12.15+/-2.11) mL, P0.05], postoperative hospitalization [(6.39+/-1.38) days vs. (6.45+/-1.05) days, P>0.05] between the two groups. Of the 43 patients in water jet group, 6 (14.0%) had grade II facial nerve dysfunction and 1 (2.3%) had grade III facial nerve dysfunction; of the 36 patients in control group, 5 (13.9%) had grade II facial nerve dysfunction, 2 (5.6%) had grade III facial nerve dysfunction, 1 (2.8%) had grade IV facial nerve dysfunction and 1 (2.8%) had salivary fistula. There was no permanent facial nerve dysfunction occurred in both groups. There was no significant difference in the occurrence of complications between the two groups. Nine patients who retained nervus auricularis magnus suffered from slight numbness symptom of auricular lobule. Use of helix water jet in parotid surgery is safe and confers some advantages over conventional methods of parotid dissection.

  15. Helix dynamics in LacY: helices II/IV

    Science.gov (United States)

    Liu, Zhenyu; Madej, M. Gregor; Kaback, H. Ronald

    2011-01-01

    Biochemical and biophysical studies based upon crystal structures of both a mutant and wild-type lactose permease from Escherichia coli (LacY) in an inward-facing conformation have led to a model for the symport mechanism in which both sugar- and H+-binding sites are alternatively accessible to either side of the membrane. Previous findings indicate that the face of helix II with Asp68 is important for the conformational changes that occur during turnover. As shown here, replacement of Asp68 at the cytoplasmic end of helix II, particularly with Glu, abolishes active transport, but the mutants retain the ability to bind galactopyranoside. In the x-ray structure, Asp68 and Lys131 (helix IV) lie within ∼4.2 Å of each other. Although a double mutant with Cys replacements at both position 68 and 131 cross-links efficiently, single replacements for Lys131 exhibit very significant transport activity. Site-directed alkylation studies show that sugar binding by the Asp68 mutants causes closure of the cytoplasmic cavity, like wild-type LacY; but strikingly, the probability of opening the periplasmic pathway upon sugar binding is markedly reduced. Taken together with previous mutagenesis and cross-linking studies, the findings lead to a model in which replacement of Asp68 blocks a conformational transition involving helices II and IV that is important for opening the periplasmic cavity. Evidence is also presented suggesting that movements of helices II and IV are coupled functionally with movements in the pseudo-symmetrically paired helices VIII and X. PMID:20043916

  16. Broadband circular polarizers constructed using helix-like chiral metamaterials

    Science.gov (United States)

    Ji, Ruonan; Wang, Shao-Wei; Liu, Xingxing; Chen, Xiaoshuang; Lu, Wei

    2016-08-01

    In this paper, one kind of helix-like chiral metamaterial which can be realized by multiple conventional lithography or electron beam lithographic techniques is proposed to have a broadband bianisotropic optical response analogous to helical metamaterials. On the basis of twisted metamaterials, via tailoring the relative orientation within the lattice, the anisotropy of arcs is converted into magneto-electric coupling of closely spaced arc pairs, which leads to a broad bianisotropic optical response. By connecting the adjacent upper and lower arcs, the coupling of metasurface pairs is transformed into the coupling of the three-dimensional inclusions, and provides a much broader and higher bianisotropic optical response. For only a four-layer helix-like metamaterial, the maximum extinction ratio can reach 19.7. The operation band is in the wavelength range of 4.69 μm to 8.98 μm with an average extinction ratio of 6.9. And the transmittance for selective polarization is above 0.8 in the entire operation band. Such a structure is a promising candidate for integratable and scalable broadband circular polarizers, especially it has great potential to act as a broadband circular micropolarizer in the field of the full-Stokes division of focal plane polarimeters.In this paper, one kind of helix-like chiral metamaterial which can be realized by multiple conventional lithography or electron beam lithographic techniques is proposed to have a broadband bianisotropic optical response analogous to helical metamaterials. On the basis of twisted metamaterials, via tailoring the relative orientation within the lattice, the anisotropy of arcs is converted into magneto-electric coupling of closely spaced arc pairs, which leads to a broad bianisotropic optical response. By connecting the adjacent upper and lower arcs, the coupling of metasurface pairs is transformed into the coupling of the three-dimensional inclusions, and provides a much broader and higher bianisotropic optical

  17. Design of a Broadband Inverted Conical Quadrifilar Helix Antenna

    Directory of Open Access Journals (Sweden)

    Jingyan Mo

    2016-01-01

    Full Text Available This paper introduces the design of a broadband inverted conical circularly polarized quadrifilar helix antenna (QHA. The antenna has many good characteristics, including wide beam and broad bandwidth, which are achieved by utilizing inverted conical geometry and adjusting the dimensions of the inverted conical support. The antenna is fed by a wideband network to provide 90° phase difference between the four arms with constant amplitude. The antenna impedance and axial ratio bandwidth values are more than 39% and 31.5%, respectively. The measured results coincide well with the simulated ones, which verified the effectiveness of the proposed design.

  18. Genetic Analysis in Translational Medicine: The 2010 GOLDEN HELIX Symposium.

    Science.gov (United States)

    Patrinos, George P; Innocenti, Federico; Cox, Nancy; Fortina, Paolo

    2011-06-01

    The 2010 GOLDEN HELIX Symposium "Genetic Analysis in Translational Medicine" was held in Athens, Greece, 1-4 December 2010. The scientific program covered all aspects of this discipline, including genome-wide association studies, genomics of cancer and human disorders, molecular cytogenetics, advances in genomic technology, next-generation sequencing applications, pharmacogenomics, and bioinformatics. In addition, various topics on genetics and society and genetic analysis in clinical practice were discussed. We provide an overview of the plenary lectures and the topics discussed in the symposium.

  19. Folding Dynamics of an α Helix and a β Hairpin

    Science.gov (United States)

    Hofrichter, James

    1998-03-01

    What processes limit the rate at which proteins fold? In an effort to address this question we have begun to study the dynamics of the formation of loops, α helices and the minimal β structural element, a β hairpin, which must occur on the pathway from random coils to folded proteins. Because these processes occur on time scales of 10-5-10-9 seconds and experimental access to these time scales has been limited, the kinetics of these processes have not been extensively studied. The expectation is that a more complete understanding of the dynamics of these microprocesses will provide constraints on possible mechanisms for the overall folding of more complex structures. We have explored the kinetics of the helix-coil transition of a synthetic, 21-residue peptide: Ac-WAAAH^+(AAARA)_3A-NH2 and of the folding of a 16 residue β hairpin from protein G B1 using the nanosecond temperature jump technique. Both processes were studied by monitoring tryptophan fluorescence. In the helical peptide, the quantum yield of tryptophan decreases as a result of the interaction between tryptophan in position 1 with the protonated histidine in position 5. In the native conformation of the hairpin, it increases because it forms part of a hydrophobic cluster which stabilizes the native conformation (in a peptide in which a dansylated lysine is incorporated at the C-terminus the fluorescence is quenched). At 300 K, the relaxation time for the helix-coil transition is ~ 250 ns and that for the hairpin-coil transition is ~ 2.2 μs, about 10 times slower. The apparent activation energies are 6.8 kcal/mol for the helix and 10 kcal/mol for the hairpin. We have developed simple kinetic models for these processes which incorporate the sequence- and position-dependent properties known from equilibrium studies and the single-sequence approximation. These models provide a remarkably consistent picture of the dynamics, permitting us to extract information on both the microscopic rates for the c

  20. Some quality parameters of land snail meat - Helix pomatia

    Directory of Open Access Journals (Sweden)

    Tojagić Slobodan N.

    2004-01-01

    Full Text Available Considering the tradition in our regions to collect land snails (Helix pomatia for export, which is "disrupted" by social control resulting in limited possibilities to develop this attractive activity, there is a great interest lately for land snail breeding and fattening at farms. For this reason it is necessary to investigate systematically the possibilities to develop this activity in a longer period and in larger areas. The first investigations, although covering only nutritive and health safety aspects of the edible parts yielded the results presented in this work. Chemical composition, the content of some elements and organochlorine insecticides were followed as unavoidable in human living and environment.

  1. pH jump induced α-helix folding.

    Directory of Open Access Journals (Sweden)

    Donten M. L.

    2013-03-01

    Full Text Available pH can be used to impact the folding equilibrium of peptides and proteins. This fact is utilized, similarly to temperature jumps, in pH jump experiments employing laser time-resolved spectroscopy to study the function and structural dynamics of these molecules. Here the application of pH jumps in folding experiments was investigated. Experiments with poly-L-glutamic acid alpha-helix formation shown the critical aspects of pH jump experiments and yielded direct information about the folding kinetics monitored with the amide I IR band.

  2. Molecular dynamics simulation studies of the wild type and E92Q/N155H mutant of Elvitegravir-resistance HIV-1 integrase

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Qi [Shanghai Jiao Tong Univ., Shanghai (China). State Key Lab. of Microbial Metabolism and College of Life Science and Biotechnology; Cheng, Xiaolin [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Molecular Biophysics; Univ. of Tennessee, Knoxville, TN (United States). Dept. of Biochemistry and Cellular and Molecular Biology; Wei, Dongqing [Shanghai Jiao Tong Univ., Shanghai (China). State Key Lab. of Microbial Metabolism and College of Life Science and Biotechnology; Xu, Qin [Shanghai Jiao Tong Univ., Shanghai (China). State Key Lab. of Microbial Metabolism and College of Life Science and Biotechnology

    2014-11-06

    Although Elvitegravir (EVG) is a newly developed antiretrovirals drug to treat the acquired immunodeficiency syndrome (AIDS), drug resistance has already been found in clinic, such as E92Q/N155H and Q148H/G140S. Several structural investigations have already been reported to reveal the molecular mechanism of the drug resistance. As full length crystal structure for HIV-1 integrase is still unsolved, we use in this paper the crystal structure of the full length prototype foamy virus (PFV) in complex with virus DNA and inhibitor Elvitegravir as a template to construct the wild type and E92Q/N155H mutant system of HIV-1 integrase. Molecular dynamic simulations was used to revel the binding mode and the drug resistance of the EVG ligand in E92Q/N155H. Several important interactions were discovered between the mutated residues and the residues in the active site of the E92Q/N155H double mutant pattern, and cross correlation and clustering methods were used for detailed analysis. The results from the MD simulation studies will be used to guide the experimental efforts of developing novel inhibitors against drug-resistant HIV integrase mutants.

  3. Construction of a stepwise gene integration system by transient expression of actinophage R4 integrase in cyanobacterium Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Miura, Takamasa; Nishizawa, Akito; Nishizawa, Tomoyasu; Asayama, Munehiko; Takahashi, Hideo; Shirai, Makoto

    2014-08-01

    The integrase of actinophage R4, which belongs to the large serine-recombinase family, catalyzes site-specific recombination between two distinct attachment site sequences of the phage (attP) and actinomycete Streptomyces parvulus 2297 chromosome (attB). We previously reported that R4 integrase (Sre) catalyzed site-specific recombination both in vivo and in vitro. In the present study, a Sre-based system was developed for the stepwise site-specific integration of multiple genes into the chromosome of cyanobacterium Synechocystis sp. PCC 6803 (hereafter PCC 6803). A transgene-integrated plasmid with two attP sites and a non-replicative sre-containing plasmid were co-introduced into attB-inserted PCC 6803 cells. The transiently expressed Sre catalyzed highly efficient site-specific integration between one of the two attP sites on the integration plasmid and the attB site on the chromosome of PCC 6803. A second transgene-integrated plasmid with an attB site was integrated into the residual attP site on the chromosome by repeating site-specific recombination. The transformation frequencies (%) of the first and second integrations were approximately 5.1 × 10(-5) and 8.2 × 10(-5), respectively. Furthermore, the expression of two transgenes was detected. This study is the first to apply the multiple gene site-specific integration system based on R4 integrase to cyanobacteria.

  4. The close-packed triple helix as a possible new structural motif for collagen

    DEFF Research Database (Denmark)

    Bohr, Jakob; Olsen, Kasper

    2010-01-01

    The one-dimensional problem of selecting the triple helix with the highest volume fraction is solved and hence the condition for a helix to be close-packed is obtained. The close-packed triple helix is shown to have a pitch angle of v CP = 43.3°. Contrary to the conventional notion, we suggest...... that close packing form the underlying principle behind the structure of collagen, and the implications of this suggestion are considered. Further, it is shown that the unique zero-twist structure with no strain-twist coupling is practically identical to the close-packed triple helix. Some...

  5. Molecular modeling study on the allosteric inhibition mechanism of HIV-1 integrase by LEDGF/p75 binding site inhibitors.

    Directory of Open Access Journals (Sweden)

    Weiwei Xue

    Full Text Available HIV-1 integrase (IN is essential for the integration of viral DNA into the host genome and an attractive therapeutic target for developing antiretroviral inhibitors. LEDGINs are a class of allosteric inhibitors targeting LEDGF/p75 binding site of HIV-1 IN. Yet, the detailed binding mode and allosteric inhibition mechanism of LEDGINs to HIV-1 IN is only partially understood, which hinders the structure-based design of more potent anti-HIV agents. A molecular modeling study combining molecular docking, molecular dynamics simulation, and binding free energy calculation were performed to investigate the interaction details of HIV-1 IN catalytic core domain (CCD with two recently discovered LEDGINs BI-1001 and CX14442, as well as the LEDGF/p75 protein. Simulation results demonstrated the hydrophobic domain of BI-1001 and CX14442 engages one subunit of HIV-1 IN CCD dimer through hydrophobic interactions, and the hydrophilic group forms hydrogen bonds with HIV-1 IN CCD residues from other subunit. CX14442 has a larger tert-butyl group than the methyl of BI-1001, and forms better interactions with the highly hydrophobic binding pocket of HIV-1 IN CCD dimer interface, which can explain the stronger affinity of CX14442 than BI-1001. Analysis of the binding mode of LEDGF/p75 with HIV-1 IN CCD reveals that the LEDGF/p75 integrase binding domain residues Ile365, Asp366, Phe406 and Val408 have significant contributions to the binding of the LEDGF/p75 to HIV1-IN. Remarkably, we found that binding of BI-1001 and CX14442 to HIV-1 IN CCD induced the structural rearrangements of the 140 s loop and oration displacements of the side chains of the three conserved catalytic residues Asp64, Asp116, and Glu152 located at the active site. These results we obtained will be valuable not only for understanding the allosteric inhibition mechanism of LEDGINs but also for the rational design of allosteric inhibitors of HIV-1 IN targeting LEDGF/p75 binding site.

  6. Broadband circular polarizers constructed using helix-like chiral metamaterials.

    Science.gov (United States)

    Ji, Ruonan; Wang, Shao-Wei; Liu, Xingxing; Chen, Xiaoshuang; Lu, Wei

    2016-08-01

    In this paper, one kind of helix-like chiral metamaterial which can be realized by multiple conventional lithography or electron beam lithographic techniques is proposed to have a broadband bianisotropic optical response analogous to helical metamaterials. On the basis of twisted metamaterials, via tailoring the relative orientation within the lattice, the anisotropy of arcs is converted into magneto-electric coupling of closely spaced arc pairs, which leads to a broad bianisotropic optical response. By connecting the adjacent upper and lower arcs, the coupling of metasurface pairs is transformed into the coupling of the three-dimensional inclusions, and provides a much broader and higher bianisotropic optical response. For only a four-layer helix-like metamaterial, the maximum extinction ratio can reach 19.7. The operation band is in the wavelength range of 4.69 μm to 8.98 μm with an average extinction ratio of 6.9. And the transmittance for selective polarization is above 0.8 in the entire operation band. Such a structure is a promising candidate for integratable and scalable broadband circular polarizers, especially it has great potential to act as a broadband circular micropolarizer in the field of the full-Stokes division of focal plane polarimeters.

  7. Broadband Circular Polarizers Constructed by Helix-like Chiral Metamaterials

    CERN Document Server

    Ji, Ruonan; Liu, Xingxing; Chen, Xiaoshuang; Lu, Wei

    2016-01-01

    In this paper, a kind of helix-like chiral metamaterial, which can be realized with multiple conventional lithography or electron beam lithographic techniques, is proposed to achieve broadband bianisotropic optical response analogous to helical metamaterial. On the basis of twisted metamaterial, via tailoring the relative orientation within the lattice, the anisotropy of arc is converted into magneto-electric coupling of closely spaced arc pairs, which leads to a broad bianisotropic optical response. By connecting the adjacent upper and lower arcs, the coupling of metasurface pairs is transformed to the coupling of the three-dimensional inclusions, and provides a much broader and higher bianisotropic optical response. For only a four-layer helix-like metamaterial, the maximum extinction ratio can reach 19.7. The operation band is in the wavelength range from 4.69 {\\mu}m to 8.98 {\\mu}m with an average extinction ratio of 6.9. And the transmittance for selective polarization is above 0.8 in the entire operation...

  8. Structural Signatures and Membrane Helix 4 in GLUT1

    Science.gov (United States)

    Pascual, Juan M.; Wang, Dong; Yang, Ru; Shi, Lei; Yang, Hong; De Vivo, Darryl C.

    2008-01-01

    Exon IV of SLC2A1, a multiple facilitator superfamily (MFS) transporter gene, is particularly susceptible to mutations that cause GLUT1 deficiency syndrome, a human encephalopathy that results from decreased glucose flux through the blood-brain barrier. Genotyping of 100 patients revealed that in a third of them who harbor missense mutations in the GLUT1 transporter, transmembrane domain 4 (TM4), encoded by SLC2A1 exon IV, contains mutant residues that have the periodicity of one face of a kinked α-helix. Arg-126, located at the amino terminus of TM4, is the locus for most of the mutations followed by other arginine and glycine residues located elsewhere in the transporter but conserved among MFS proteins. The Arg-126 mutants were constructed and assayed for protein expression, targeting, and transport capacity in Xenopus oocytes. The role of charge at position 126, as well as its accessibility, was investigated in R126H by determining its activity as a function of extracellular pH. The results indicate that intracellular charges at the MFS TM2–3 and TM8–9 signature loops and flanking TMs 3, 5, and 6 are critical for the structure of GLUT1 as are TM glycines and that TM4, located at the catalytic core of MFS proteins, forms a helix that surfaces into the extracellular solution where another proton facilitates transport. PMID:18387950

  9. Transmembrane Helix Assembly by Max-Min Ant System Algorithm.

    Science.gov (United States)

    Sujaree, Kanon; Kitjaruwankul, Sunan; Boonamnaj, Panisak; Supunyabut, Chirayut; Sompornpisut, Pornthep

    2015-12-01

    Because of the rapid progress in biochemical and structural studies of membrane proteins, considerable attention has been given on developing efficient computational methods for solving low-to-medium resolution structures using sparse structural data. In this study, we demonstrate a novel algorithm, max-min ant system (MMAS), designed to find an assembly of α-helical transmembrane proteins using a rigid helix arrangement guided by distance constraints. The new algorithm generates a large variety with finite number of orientations of transmembrane helix bundle and finds the solution that is matched with the provided distance constraints based on the behavior of ants to search for the shortest possible path between their nest and the food source. To demonstrate the efficiency of the novel search algorithm, MMAS is applied to determine the transmembrane packing of KcsA and MscL ion channels from a limited distance information extracted from the crystal structures, and the packing of KvAP voltage sensor domain using a set of 10 experimentally determined constraints, and the results are compared with those of two popular used stochastic methods, simulated annealing Monte Carlo method and genetic algorithm. © 2015 John Wiley & Sons A/S.

  10. Genes and the physics of the DNA double-helix.

    Science.gov (United States)

    Yeramian, E

    2000-09-19

    The processing of the genetic information stored in the double-helical DNA implies the separation of the two strands, the physics of which is described by the helix-coil transition model. Is there a relationship between genetic maps and DNA physical stability maps that plot the sequence-specific propensity for the thermal disruption of the double-helix? Here, with appropriate methodological formulations, such maps are derived for a large set of sequences, including complete genomes. The superposition of the two maps leads to a contrasted picture with correlations ranging between two extremes: from almost perfect (with the genes precisely delineated as stable regions) to more or less complete unrelatedness. The simplest explanation for the results is that the observed striking correlations correspond to the relics of a primeval organisation of the genetic message, with the physics of DNA playing a role in the delimitation of coding regions. In order to trace the evolutionary fate of this signal further, a detailed study of the yeast complete genome is performed. In this study, the superposition of the genetic and physical stability maps is examined in the light of information concerning gene duplication. On the basis of this analysis it is concluded that the 'signature' associated with the supposed archaic signal is in the process of being erased, most probably because the underlying feature is no longer under selective pressure. There are many evolutionary implications for the results presented and for their proposed interpretations, notably concerning models of mutational dynamics in relation to erasure processes.

  11. Ab initio theory of helix <-> coil phase transition

    DEFF Research Database (Denmark)

    Yakubovich, Alexander V.; Solov'yov, Ilia; Solov'yov, Andrey V.

    2008-01-01

    In this paper, we suggest a theoretical method based on the statistical mechanics for treating the alpha-helix <-> random coil transition in alanine polypeptides. We consider this process as a first-order phase transition and develop a theory which is free of model parameters and is based solely ...... twisting. The suggested theory is general and with some modification can be applied for the description of phase transitions in other complex molecular systems (e.g. proteins, DNA, nanotubes, atomic clusters, fullerenes).......In this paper, we suggest a theoretical method based on the statistical mechanics for treating the alpha-helix random coil transition in alanine polypeptides. We consider this process as a first-order phase transition and develop a theory which is free of model parameters and is based solely...... on fundamental physical principles. It describes essential thermodynamical properties of the system such as heat capacity, the phase transition temperature and others from the analysis of the polypeptide potential energy surface calculated as a function of two dihedral angles, responsible for the polypeptide...

  12. Conformational preferences of substituted prolines in the collagen triple helix.

    Science.gov (United States)

    Mooney, Sean D; Kollman, Peter A; Klein, Teri E

    2002-07-05

    Researchers have recently questioned the role hydroxylated prolines play in stabilizing the collagen triple helix. To address these issues, we have developed new molecular mechanics parameters for the simulation of peptides containing 4(R)-fluoroproline (Flp), 4(R)-hydroxyproline (Hyp), and 4(R)-aminoproline (Amp). Simulations of peptides based on these parameters can be used to determine the components that stabilize hydroxyproline over proline in the triple helix. The dihedrals F-C-C-N, O-C-C-N, and N-C-C-N were built using a N-beta-ethyl amide model. One nanosecond simulations were performed on the trimers [(Pro-Pro-Gly)(10)](3), [(Pro-Hyp-Gly)(10)](3), [(Pro-Amp-Gly)(10)](3), [(Pro-Amp(1+)-Gly)(10)](3), and [(Pro-Flp-Gly)(10)](3) in explicit solvent. The results of our simulations suggest that pyrrolidine ring conformation is mediated by the strength of the gauche effect and classical electrostatic interactions.

  13. Biophysical studies of matrix metalloproteinase/triple-helix complexes.

    Science.gov (United States)

    Fields, Gregg B

    2014-01-01

    Several members of the zinc-dependent matrix metalloproteinase (MMP) family catalyze collagen degradation. The structures of MMPs, in solution and solid state and in the presence and absence of triple-helical collagen models, have been assessed by NMR spectroscopy, small-angle X-ray scattering, and X-ray crystallography. Structures observed in solution exhibit flexibility between the MMP catalytic (CAT) and hemopexin-like (HPX) domains, while solid-state structures are relatively compact. Evaluation of the maximum occurrence (MO) of MMP-1 conformations in solution found that, for all the high MO conformations, the CAT and HPX domains are not in tight contact, and the residues of the HPX domain reported to be responsible for the binding to the collagen triple-helix are solvent exposed. A mechanism for collagenolysis has been developed based on analysis of MMP solution structures. Information obtained from solid-state structures has proven valuable for analyzing specific contacts between MMPs and the collagen triple-helix.

  14. Effect of Ethanol on the Metabolic Characteristics of HIV-1 Integrase Inhibitor Elvitegravir and Elvitegravir/Cobicistat with CYP3A: An Analysis Using a Newly Developed LC-MS/MS Method.

    Science.gov (United States)

    Midde, Narasimha M; Rahman, Mohammad A; Rathi, Chetan; Li, Junhao; Meibohm, Bernd; Li, Weihua; Kumar, Santosh

    2016-01-01

    Elvitegravir (EVG), an integrase inhibitor for the treatment HIV infection, is increasingly becoming the part of first-line antiretroviral therapy (ART) regimen. EVG is mainly metabolized through cytochrome P450 (CYP) 3A4. Previously, we have shown that ethanol alters ART-CYP3A4 interactions with protease inhibitors thereby altering their metabolisms. However, as EVG is a fairly new class of drug, its kinetic characteristics and the effect of ethanol on EVG-CYPP3A4 interaction is poorly understood. In this study, we characterized EVG and cobicistat (COBI)-boosted EVG metabolism in human microsomes followed by ethanol-EVG, ethanol-COBI-EVG interaction with CYP3A. First, we developed and validated a simple, sensitive, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of EVG in the human liver microsomes. The lower limit of quantification for the drug was at 0.003 μM (1.34 ng/ml). Extraction yield, matrix effects, drug stability, and calibration curves for the proposed method were validated according to the FDA guidelines. Time dependent kinetics data showed that 20mM ethanol decreases the apparent half-life of EVG degradation by ~50% compared to EVG alone. Our substrate kinetic results revealed that ethanol mildly decreases the catalytic efficiency for EVG metabolism. Inhibition studies demonstrated that EVG inhibits CYP3A4, and 20 mM ethanol causes a decrease in the IC50 of EVG. However, in the presence of COBI we were unable to determine these parameters effectively because COBI, being a strong inhibitor of CYP3A4, blocked the EVG/ethanol-CYP3A4 interactions. Docking studies predicted a shift of EVG or COBI binding to the active site of CYP3A4 in the presence of ethanol. Taken together, these results suggest that ethanol interacts with microsomal CYP3A and alters EVG-CYP3A4 interaction thereby altering EVG metabolism and inhibition of CYP3A4 by EVG. This finding has clinical significance because alcohol use is

  15. Helix 69 of Escherichia coli 23S ribosomal RNA as a peptide nucleic acid target.

    Science.gov (United States)

    Kulik, Marta; Markowska-Zagrajek, Agnieszka; Wojciechowska, Monika; Grzela, Renata; Wituła, Tomasz; Trylska, Joanna

    2017-07-01

    A fragment of 23S ribosomal RNA (nucleotides 1906-1924 in E. coli), termed Helix 69, forms a hairpin that is essential for ribosome function. Helix 69 forms a conformationally flexible inter-subunit connection with helix 44 of 16S ribosomal RNA, and the nucleotide A1913 of Helix 69 influences decoding accuracy. Nucleotides U1911 and U1917 are post-transcriptionally modified with pseudouridines (Ψ) and U1915 with 3-methyl-Ψ. We investigated Helix 69 as a target for a complementary synthetic oligonucleotide - peptide nucleic acid (PNA). We determined thermodynamic properties of Helix 69 and its complexes with PNA and tested the performance of PNA targeted at Helix 69 in inhibiting translation in cell-free extracts and growth of E. coli cells. First, we examined the interactions of a PNA oligomer complementary to the G1907-A1919 fragment of Helix 69 with the sequences corresponding to human and bacterial species (with or without pseudouridine modifications). PNA invades the Helix 69 hairpin creating stable complexes and PNA binding to the pseudouridylated bacterial sequence is stronger than to Helix 69 without any modifications. Second, we confirmed the binding of PNA to 23S rRNA and 70S ribosomes. Third, we verified the efficiency of translation inhibition of these PNA oligomers in the cell-free translation/transcription E. coli system, which were in a similar range as tetracycline. Next, we confirmed that PNA conjugated to the (KFF)3K transporter peptide inhibited E. coli growth in micromolar concentrations. Overall, targeting Helix 69 with PNA or other sequence-specific oligomers could be a promising way to inhibit bacterial translation. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  16. Structure of bacteriophage [phi]29 head fibers has a supercoiled triple repeating helix-turn-helix motif

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Ye; Rossmann, Michael G. (Purdue)

    2011-12-22

    The tailed bacteriophage {phi}29 capsid is decorated with 55 fibers attached to quasi-3-fold symmetry positions. Each fiber is a homotrimer of gene product 8.5 (gp8.5) and consists of two major structural parts, a pseudohexagonal base and a protruding fibrous portion that is about 110 {angstrom} in length. The crystal structure of the C-terminal fibrous portion (residues 112-280) has been determined to a resolution of 1.6 {angstrom}. The structure is about 150 {angstrom} long and shows three distinct structural domains designated as head, neck, and stem. The stem region is a unique three-stranded helix-turn-helix supercoil that has not previously been described. When fitted into a cryoelectron microscope reconstruction of the virus, the head structure corresponded to a disconnected density at the distal end of the fiber and the neck structure was located in weak density connecting it to the fiber. Thin section studies of Bacillus subtilis cells infected with fibered or fiberless {phi}29 suggest that the fibers might enhance the attachment of the virions onto the host cell wall.

  17. Phylogeny, Functional Annotation, and Protein Interaction Network Analyses of the Xenopus tropicalis Basic Helix-Loop-Helix Transcription Factors

    Directory of Open Access Journals (Sweden)

    Wuyi Liu

    2013-01-01

    Full Text Available The previous survey identified 70 basic helix-loop-helix (bHLH proteins, but it was proved to be incomplete, and the functional information and regulatory networks of frog bHLH transcription factors were not fully known. Therefore, we conducted an updated genome-wide survey in the Xenopus tropicalis genome project databases and identified 105 bHLH sequences. Among the retrieved 105 sequences, phylogenetic analyses revealed that 103 bHLH proteins belonged to 43 families or subfamilies with 46, 26, 11, 3, 15, and 4 members in the corresponding supergroups. Next, gene ontology (GO enrichment analyses showed 65 significant GO annotations of biological processes and molecular functions and KEGG pathways counted in frequency. To explore the functional pathways, regulatory gene networks, and/or related gene groups coding for Xenopus tropicalis bHLH proteins, the identified bHLH genes were put into the databases KOBAS and STRING to get the signaling information of pathways and protein interaction networks according to available public databases and known protein interactions. From the genome annotation and pathway analysis using KOBAS, we identified 16 pathways in the Xenopus tropicalis genome. From the STRING interaction analysis, 68 hub proteins were identified, and many hub proteins created a tight network or a functional module within the protein families.

  18. In vivo analysis of the helix-turn-helix motif of the fushi tarazu homeo domain of Drosophila melanogaster.

    Science.gov (United States)

    Furukubo-Tokunaga, K; Müller, M; Affolter, M; Pick, L; Kloter, U; Gehring, W J

    1992-06-01

    We report a systematic mutational analysis of the helix-turn-helix motif (HTH) of the fushi tarazu (ftz) homeo domain (HD) of Drosophila. We started out by testing the function of chimeric ftz proteins containing either a part of the Sex combs reduced (Scr) or the muscle segment homeobox (msh) HDs. By complementation tests in transgenic flies, cotransfection assays in cultured Drosophila cells and in vitro DNA-binding assays, we have found that the ftz activity is retained in the ftz-Scr chimera but is lost in the ftz-msh chimera, which is defective in binding to an Antennapedia (Antp)-class target site. Further studies with a series of back-mutants of the ftz-msh chimera have revealed that a set of class-specific DNA backbone-contacting residues in the HTH, particularly Arg-28 and Arg-43, are required for efficient target site recognition and, hence, full ftz activity both in vitro and in vivo.

  19. A Dual Mechanism Controls Nuclear Localization in the Atypical Basic-Helix-Loop-Helix Protein PAR1 of Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Anahit Galstyan; Jordi Bou-Torrent; Irma Roig-Villanova; Jaime F. Martínez-García

    2012-01-01

    PAR1 is an atypical basic-helix-loop-helix (bHLH) protein that negatively regulates the shade avoidance syndrome in Arabidopsis thaliana acting as a transcriptional cofactor.Consistently with this function,PAR1 has to be in the nucleus to display biological activity.Previous structure-function analyses revealed that the N-terminal region of PAR1 drives the protein to the nucleus.However,truncated forms of PAR1 lacking this region still display biological activity,implying that PAR1 has additional mechanisms to localize into the nucleus.In this work,we compared the primary structure of PAR1 and various related and unrelated plant bHLH proteins,which led us to suggest that PAR1 contains a non-canonical nuclear localization signal (NLS) in the N-terminal region.By overexpressing truncated and mutated derivatives of PAR1,we have also investigated the importance of other regions of PAR1,such as the acidic and the extended HLH dimerization domains,for its nuclear localization.We found that,in the absence of the N-terminal region,a functional HLH domain is required for nuclear localization.Our results suggest the existence of a dual mechanism for PAR1 nuclear localization:(1) one mediated by the N-terminal non-consensus NLS and (2) a second one that involves interaction with other proteins via the dimerization domain.

  20. Caught red-handed: Rc encodes a basic helix-loop-helix protein conditioning red pericarp in rice.

    Science.gov (United States)

    Sweeney, Megan T; Thomson, Michael J; Pfeil, Bernard E; McCouch, Susan

    2006-02-01

    Rc is a domestication-related gene required for red pericarp in rice (Oryza sativa). The red grain color is ubiquitous among the wild ancestors of O. sativa, in which it is closely associated with seed shattering and dormancy. Rc encodes a basic helix-loop-helix (bHLH) protein that was fine-mapped to an 18.5-kb region on rice chromosome 7 using a cross between Oryza rufipogon (red pericarp) and O. sativa cv Jefferson (white pericarp). Sequencing of the alleles from both mapping parents as well as from two independent genetic stocks of Rc revealed that the dominant red allele differed from the recessive white allele by a 14-bp deletion within exon 6 that knocked out the bHLH domain of the protein. A premature stop codon was identified in the second mutant stock that had a light red pericarp. RT-PCR experiments confirmed that the Rc gene was expressed in both red- and white-grained rice but that a shortened transcript was present in white varieties. Phylogenetic analysis, supported by comparative mapping in rice and maize (Zea mays), showed that Rc, a positive regulator of proanthocyanidin, is orthologous with INTENSIFIER1, a negative regulator of anthocyanin production in maize, and is not in the same clade as rice bHLH anthocyanin regulators.

  1. A genome-wide survey on basic helix-loop-helix transcription factors in rat and mouse.

    Science.gov (United States)

    Zheng, Xiaodong; Zheng, X; Wang, Yong; Wang, Y; Yao, Qin; Yao, Q; Yang, Zhe; Yang, Z; Chen, Keping; Chen, K

    2009-04-01

    The basic helix-loop-helix (bHLH) proteins play essential roles in a wide range of developmental processes in higher organisms. bHLH family members have been identified in over 20 organisms, including nematode, fruit fly, and human. Our study identified 114 rat and 14 additional mouse bHLH members in rat and mouse genomes, respectively. Phylogenetic analyses revealed that both rat and mouse had 49, 26, 15, 4, 12, and 4 bHLH members in groups A, B, C, D, E, and F, respectively. Only the rat Mxi1 gene has two copies in the genome. All other rat bHLH genes and all mouse bHLH genes are single-copy genes. The chromosomal distribution pattern of mouse, rat, and human bHLH genes suggests the emergence of some bHLH genes through gene duplication, which probably happened at least before the divergence of vertebrates from invertebrates. The present study provides useful information for future studies using rat as a model animal for mammalian development.

  2. Structure of bacteriophage phi29 head fibers has a supercoiled triple repeating helix-turn-helix motif.

    Science.gov (United States)

    Xiang, Ye; Rossmann, Michael G

    2011-03-22

    The tailed bacteriophage 29 capsid is decorated with 55 fibers attached to quasi-3-fold symmetry positions. Each fiber is a homotrimer of gene product 8.5 (gp8.5) and consists of two major structural parts, a pseudohexagonal base and a protruding fibrous portion that is about 110 Å in length. The crystal structure of the C-terminal fibrous portion (residues 112-280) has been determined to a resolution of 1.6 Å. The structure is about 150 Å long and shows three distinct structural domains designated as head, neck, and stem. The stem region is a unique three-stranded helix-turn-helix supercoil that has not previously been described. When fitted into a cryoelectron microscope reconstruction of the virus, the head structure corresponded to a disconnected density at the distal end of the fiber and the neck structure was located in weak density connecting it to the fiber. Thin section studies of Bacillus subtilis cells infected with fibered or fiberless 29 suggest that the fibers might enhance the attachment of the virions onto the host cell wall.

  3. Structural basis for the potent inhibition of the HIV integrase-LEDGF/p75 protein-protein interaction.

    Science.gov (United States)

    Ribone, Sergio R; Quevedo, Mario A

    2017-08-01

    Integrase (IN) constitutes one of the key enzymes involved in the lifecycle of the Human Immunodeficiency Virus (HIV), the etiological agent of AIDS. The biological role of IN strongly depends on the recognition and binding of cellular cofactors belonging to the infected host cell. Thus, the inhibition of the protein-protein interaction (PPI) between IN and cellular cofactors has been envisioned as a promising therapeutic target. In the present work we explore a structure-activity relationship for a set of 14 compounds reported as inhibitors of the PPI between IN and the lens epithelium-derived growth factor (LEDGF/p75). Our results demonstrate that the possibility to adopt the bioactive conformation capable of interacting with the hotspots IN-LEDGF/p75 hotspots residues constitutes a critical feature to obtain a potent inhibition. A ligand efficiency (|Lig-Eff|) quantitative descriptor combining both interaction energetics and conformational requirements was developed and correlated with the reported biological activity. Our results contribute to the rational development of IN-LEDGF/p75 interaction inhibitors providing a solid quantitative structure-activity relationship aimed for the screening of new IN-LEDGF/p75 interaction inhibitors. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. A QSAR study of integrase strand transfer inhibitors based on a large set of pyrimidine, pyrimidone, and pyridopyrazine carboxamide derivatives

    Science.gov (United States)

    de Campos, Luana Janaína; de Melo, Eduardo Borges

    2017-08-01

    In the present study, 199 compounds derived from pyrimidine, pyrimidone and pyridopyrazine carboxamides with inhibitory activity against HIV-1 integrase were modeled. Subsequently, a multivariate QSAR study was conducted with 54 molecules employed by Ordered Predictors Selection (OPS) and Partial Least Squares (PLS) for the selection of variables and model construction, respectively. Topological, electrotopological, geometric, and molecular descriptors were used. The selected real model was robust and free from chance correlation; in addition, it demonstrated favorable internal and external statistical quality. Once statistically validated, the training model was used to predict the activity of a second data set (n = 145). The root mean square deviation (RMSD) between observed and predicted values was 0.698. Although it is a value outside of the standards, only 15 (10.34%) of the samples exhibited higher residual values than 1 log unit, a result considered acceptable. Results of Williams and Euclidean applicability domains relative to the prediction showed that the predictions did not occur by extrapolation and that the model is representative of the chemical space of test compounds.

  5. Mutations in nonconserved domains of Ty3 integrase affect multiple stages of the Ty3 life cycle.

    Science.gov (United States)

    Nymark-McMahon, M H; Sandmeyer, S B

    1999-01-01

    Ty3, a retroviruslike element of Saccharomyces cerevisiae, transposes into positions immediately upstream of RNA polymerase III-transcribed genes. The Ty3 integrase (IN) protein is required for integration of the replicated, extrachromosomal Ty3 DNA. In retroviral IN, a conserved core region is sufficient for strand transfer activity. In this study, charged-to-alanine scanning mutagenesis was used to investigate the roles of the nonconserved amino- and carboxyl-terminal regions of Ty3 IN. Each of the 20 IN mutants was defective for transposition, but no mutant was grossly defective for capsid maturation. All mutations affecting steady-state levels of mature IN protein resulted in reduced levels of replicated DNA, even when polymerase activity was not grossly defective as measured by exogenous reverse transcriptase activity assay. Thus, IN could contribute to nonpolymerase functions required for DNA production in vivo or to the stability of the DNA product. Several mutations in the carboxyl-terminal domain resulted in relatively low levels of processed 3' ends of the replicated DNA, suggesting that this domain may be important for binding of IN to the long terminal repeat. Another class of mutants produced wild-type amounts of DNA with correctly processed 3' ends. This class could include mutants affected in nuclear entry and target association. Collectively, these mutations demonstrate that in vivo, within the preintegration complex, IN performs a central role in coordinating multiple late stages of the retrotransposition life cycle.

  6. Design of Formed Milling Cutter for Double-Helix Screw Based on Noninstantaneous Envelope Method

    Directory of Open Access Journals (Sweden)

    Yun Li

    2013-01-01

    Full Text Available The design theory and method of formed milling cutter for double-helix screw of progressing cavity pump are presented. Through analyzing the shape and characteristic parameters of double-helix screw, the helicoids equation and axial curve equation of double-helix screw were established. According to the relative position relations between formed milling cutter and double-helix screw in the machining process, the geometric mapping relationship of screw coordinate system and formed milling cutter coordinate system was established by using the coordinate transformation theory. Based on noninstantaneous envelope method and the meshing conditions between formed milling cutter and double-helix screw, the contact line equations were established by minimum value method. By analyzing the machining errors caused by resharpening the formed milling cutter, the tooth back curve equation was established based on spiral of Archimedes, and the profile equation of formed milling cutter with constant back angle was got. On this basis, the formed milling cutter of processing double-helix screw was designed, and the cutter head and tool post were manufactured, respectively. The measuring results have shown that this method can satisfy the requirements of machining accuracy for double-helix screw. So this is an effective method to get formed milling cutter profile for double-helix screw.

  7. Proposal of a new hydrogen-bonding form to maintain curdlan triple helix.

    Science.gov (United States)

    Miyoshi, Kentaro; Uezu, Kazuya; Sakurai, Kazuo; Shinkai, Seiji

    2004-06-01

    Curdlan and other beta-1,3-D-glucans form right-handed triple helices, and it has been believed that the intermolecular H-bond is present at the center of the helix to maintain the structure. In this H-bond model, three secondary OH groups form an inequilateral hexagonal shape perpendicular to the helix axis. This hexagonal form seems to be characteristic for beta-1,3-D-glucans and is widely accepted. We carried out MOPAC and ab initio calculations for the curdlan helix, and we propose a new intermolecular H-bonding model. In our model, the H-bonds are formed between the O2-atoms on different x-y planes along the curdlan helix, hence the H-bonds are not perpendicular to the helix axis. The new H-bonds are connected along the helix, traversing three curdlan chains to make a left-handed helix. Therefore, the H-bonding array leads to a reverse helix of the main chain. According to our MOPAC calculation, this model is more stable than the previous one. We believe that the continuous H-bonding array is stabilized by cooperative phenomena in the polymeric system.

  8. Triple Helix Systems: An Analytical Framework for Innovation Policy and Practice in the Knowledge Society

    Science.gov (United States)

    Ranga, Marina; Etzkowitz, Henry

    2013-01-01

    This paper introduces the concept of Triple Helix systems as an analytical construct that synthesizes the key features of university--industry--government (Triple Helix) interactions into an "innovation system" format, defined according to systems theory as a set of components, relationships and functions. Among the components of Triple…

  9. Triple helix conformation-specific blinking of Cy3 in DNA.

    Science.gov (United States)

    Kawai, Kiyohiko; Maruyama, Atsushi

    2015-03-21

    We report that Cy3 undergoes triple helix conformation-specific blinking in DNA. Blinking patterns were affected by the stabilization of the Hoogsteen base-pair, suggesting that not only the presence but also the fluctuating behaviour of the triple helix can be monitored by the changes in the Cy3 blinking patterns.

  10. Comparative Study on the Adaptation and Growth Dynamics of the Helix pomatia and Helix aspersa Muller Terrestrial Snails Under Different Feeding Regimes

    Directory of Open Access Journals (Sweden)

    Adrian Toader-Williams

    2010-05-01

    Full Text Available We used Helix pomatia and Helix aspersa species and measure their growth as the snails were approaching the hibernation season. Helix pomatia 2yo shown a decrease in weight while being raised in enclosed parcels of 4sqm the younger Helix pomatia 1yo as well as Helix aspersa Muller demonstrated the ability to adapt relatively fast to the same conditions. We established 5 experimental lots in a Helix pomatia farm, GPS coordinates N46.606040 E23.599950. Control lot contained Taraxacum officinales, Sonchus oleraceus, Equisetum arvense and Atriplex hortensis, wild flora found within the farm. The other lots contained the same plants as the control lot plus different combinations of imported plants from other areals. The H. pomatia 2yo weight decreased in the control lot by a mean of -3.86% while H. aspersa 1yo marked an increase of +16.89% in the same lot during the same period. The lot containing lupinus polyphyllus delivered snails with weight gain of +24.66% for H. pomatia 2yo and an increase of only +1.98% for H. aspersa 1yo. As a contrast, H. pomatia 2yo gained only +7.72% while H. aspersa 1yo gained +28.89%, in the lot containing Lavanda officinalis, Foeniculum vulgare and Hyssopus officinalis among the other plants.

  11. Photo-active collagen systems with controlled triple helix architecture

    CERN Document Server

    Tronci, Giuseppe; Wood, David J

    2013-01-01

    The design of photo-active collagen systems is presented as a basis for establishing biomimetic materials with varied network architecture and programmable macroscopic properties. Following in-house isolation of type I collagen, reaction with vinyl-bearing compounds of varied backbone rigidity, i.e. 4-vinylbenzyl chloride (4VBC) and glycidyl methacrylate (GMA), was carried out. TNBS colorimetric assay, 1H-NMR and ATR-FTIR confirmed covalent and tunable functionalization of collagen lysines. Depending on the type and extent of functionalization, controlled stability and thermal denaturation of triple helices were observed via circular dichroism (CD), whereby the hydrogen-bonding capability of introduced moieties was shown to play a major role. Full gel formation was observed following photo-activation of functionalized collagen solutions. The presence of a covalent network only slightly affected collagen triple helix conformation (as observed by WAXS and ATR-FTIR), confirming the structural organization of fun...

  12. Helix versus sheet formation in a small peptide

    Science.gov (United States)

    Peng, Yong; Hansmann, Ulrich H.

    2003-10-01

    Segments with the amino acid sequence EKAYLRT (glutamine-lysine-alanine-tyrosine-leucine-arginine-threonine) appear in naturally occurring proteins both in α-helices and β-sheets. For this reason, we have used this peptide to study how secondary structure formation in proteins depends on the local environment. Our data rely on multicanonical Monte Carlo simulations where the interactions among all atoms are taken into account. Results in gas phase are compared with that in an implicit solvent. We find that both the solvated molecule and EKAYLRT in gas phase form an α-helix when not interacting with other molecules. However, in the vicinity of a β-strand, the peptide forms a β-strand. Because of this change in secondary structure our peptide may provide a simple model for the α→β transition that is supposedly related to the outbreak of prion diseases and similar illnesses.

  13. A potential smoothing algorithm accurately predicts transmembrane helix packing.

    Science.gov (United States)

    Pappu, R V; Marshall, G R; Ponder, J W

    1999-01-01

    Potential smoothing, a deterministic analog of stochastic simulated annealing, is a powerful paradigm for the solution of conformational search problems that require extensive sampling, and should be a useful tool in computational approaches to structure prediction and refinement. A novel potential smoothing and search (PSS) algorithm has been developed and applied to predict the packing of transmembrane helices. The highlight of this method is the efficient manner in which it circumvents the combinatorial explosion associated with the large number of minima on multidimensional potential energy surfaces in order to converge to the global energy minimum. Here we show how our potential smoothing and search method succeeds in finding the global minimum energy structure for the glycophorin A (GpA) transmembrane helix dimer by optimizing interhelical van der Waals interactions over rigid and semi-rigid helices. Structures obtained from our ab initio predictions are in close agreement with recent experimental data.

  14. [Allergic contact dermatitis to common ivy (Hedera helix L.)].

    Science.gov (United States)

    Ozdemir, C; Schneider, L A; Hinrichs, R; Staib, G; Weber, L; Weiss, J M; Scharffetter-Kochanek, K

    2003-10-01

    Common ivy (Hedera helix L.) is a ubiquitous plant in Europe whose major allergen falcarinol has moderate allergic potential. It is not related to poison ivy (Toxicodendron spp.). There are no cross reactions between the allergens of common ivy (falcarinol) and poison ivy (urushiol). Contact with common ivy or falcarinol may lead to sensitization and then a delayed hypersensitivity reaction. There are only few cases described in the literature. We report on a male hobby gardener with appropriate clinical history and positive patch test. The pathogenic mechanism is a type IV reaction following a sensitization exposure. Gardeners and landscape architects with frequent exposure to common ivy and thus a high risk of sensitization should wear appropriate protective clothing.

  15. RFID Tag Helix Antenna Sensors for Wireless Drug Dosage Monitoring.

    Science.gov (United States)

    Huang, Haiyu; Zhao, Peisen; Chen, Pai-Yen; Ren, Yong; Liu, Xuewu; Ferrari, Mauro; Hu, Ye; Akinwande, Deji

    2014-01-01

    Miniaturized helix antennas are integrated with drug reservoirs to function as RFID wireless tag sensors for real-time drug dosage monitoring. The general design procedure of this type of biomedical antenna sensors is proposed based on electromagnetic theory and finite element simulation. A cost effective fabrication process is utilized to encapsulate the antenna sensor within a biocompatible package layer using PDMS material, and at the same time form a drug storage or drug delivery unit inside the sensor. The in vitro experiment on two prototypes of antenna sensor-drug reservoir assembly have shown the ability to monitor the drug dosage by tracking antenna resonant frequency shift from 2.4-2.5-GHz ISM band with realized sensitivity of 1.27 [Formula: see text] for transdermal drug delivery monitoring and 2.76-[Formula: see text] sensitivity for implanted drug delivery monitoring.

  16. Preparation and evaluation of appertized from snail Helix aspersa M

    Directory of Open Access Journals (Sweden)

    Nelson Loyola López

    2015-01-01

    Full Text Available This study includes the development and evaluation of snails (Helix aspersa M. appertized, collected at a heliciculture breeding center, located in Los Niches sector, Curico, Maule region, South-central of Chile. The test was conducted at the Laboratory of Sciences of the Catholic University of Maule, Nuestra Señora del Carmen Campus, Curico. The main objective of this work was to study the influence of appertized on sensory attributes and commercial durability of snail Helix aspersa M. Additionally, some specific objectives were proposed as follow: to provide this mollusc with a commercial alternative for it consume, to evaluate its organoleptic characteristics and guarantee the product from both the microbiological and nutritional points of view. Three media cover were used (T0: water + NaCl 2%; T1: Water + NaCl 2% + citric acid 0.5% + kilol and T2: extra virgin olive oil + spices + tocopherol. The product was assessed at two different times, after 30 and 90 days of storage. Two sensory evaluations were conducted to measure various organoleptic attributes and acceptability of the appertized by 14 trained panelists. Amino acid, vitamins, cholesterol, acidity, heavy metals, phosphorus and organochlorines analysis were performed. The presence of both total and fecal contaminant microorganisms was determined. Attributes such as color, flavor, aroma, texture and overall acceptability were also measured. Preserves made by T0 and T1 treatments were equally accepted by the panelists. However, preserve from treatment T2 was rejected because of the detection in them of a very dark color, odor and mealy texture. Positive results regarding the content of amino acids, vitamin C and low cholesterol, as well as the absence of pathogenic microorganisms were obtained for the three treatments.

  17. SU(5) & A4

    CERN Document Server

    Urbano, Alfredo

    2009-01-01

    The introduction of a Flavour Symmetry can represent an interesting way in which one can try to find an answer to some intriguing problems in Flavour Physics, like the hierarchy between the fermion masses or the particular values of mixing angles. In the meantime the necessity to set this symmetry in a realistic context grows up; this context should be able to enlarge our incomplete knowledge of fundamental interactions as described in the framework of the Standard Model. Following this direction a merging between A(4) and SU(5) can be possible.

  18. A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD

    Directory of Open Access Journals (Sweden)

    Yu Wei-Hsuan

    2012-05-01

    Full Text Available Abstract Background The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C. The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?” Methods We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. Results This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6

  19. De novo design of protein-protein interactions through modification of inter-molecular helix-helix interface residues.

    Science.gov (United States)

    Yagi, Sota; Akanuma, Satoshi; Yamagishi, Manami; Uchida, Tatsuya; Yamagishi, Akihiko

    2016-05-01

    For de novo design of protein-protein interactions (PPIs), information on the shape and chemical complementarity of their interfaces is generally required. Recent advances in computational PPI design have allowed for de novo design of protein complexes, and several successful examples have been reported. In addition, a simple and easy-to-use approach has also been reported that arranges leucines on a solvent-accessible region of an α-helix and places charged residues around the leucine patch to induce interactions between the two helical peptides. For this study, we adopted this approach to de novo design a new PPI between the helical bundle proteins sulerythrin and LARFH. A non-polar patch was created on an α-helix of LARFH around which arginine residues were introduced to retain its solubility. The strongest interaction found was for the LARFH variant cysLARFH-IV-3L3R and the sulerythrin mutant 6L6D (KD=0.16 μM). This artificial protein complex is maintained by hydrophobic and ionic interactions formed by the inter-molecular helical bundle structure. Therefore, by the simple and easy-to-use approach to create de novo interfaces on the α-helices, we successfully generated an artificial PPI. We also created a second LARFH variant with the non-polar patch surrounded by positively charged residues at each end. Upon mixing this LARFH variant with 6L6D, mesh-like fibrous nanostructures were observed by atomic force microscopy. Our method may, therefore, also be applicable to the de novo design of protein nanostructures. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Advances of screening methods in vitro for HIV-1 integrase inhibitors%HIV-1整合酶抑制剂体外筛选方法研究进展

    Institute of Scientific and Technical Information of China (English)

    张旋; 杨柳萌; 郑永唐

    2013-01-01

    整合酶是HIV基因表达和复制所必需的酶,而且宿主细胞内不存在该酶的类似物.因此,HIV-1整合酶已成为设计、筛选抗HIV药物的理想靶点.迄今为止,Raltegravir仍是唯一上市的HIV整合酶抑制剂,而且临床上也已经出现耐药问题.研发新一代整合酶抑制剂非常必要.高通量、高灵敏度、简单易行的筛选方法是研究开发新一代HIV-1整合酶抑制剂的关键.目前,HIV-1整合酶抑制剂筛选方法有多种,各有优缺点,该文将对文献报道的整合酶抑制剂体外筛选方法的最新进展做一介绍.%Integrase is an essential enzyme for HIV-1 replication and has no functional analogue in host cells. The integrase is an ideal target for designing and screening anti-HIV drugs. Ralte-gravir is the only marked HIV-1 integrase inhibitor so far, and has caused drug resistance. It is necessary to developing new generation of HIV-1 integrase inhibitors. High-throughput, highly sensitive, easy and feasible screening methods are the key to developing new HIV-1 integrase inhibitors. This review introduces the various HIV-1 integrase inhibitors screening methods that were reported recently.

  1. The triple helix frame for Small and Medium-sized Enterprises for innovation and development of offshore wind energy

    DEFF Research Database (Denmark)

    Brink, Tove; Madsen, Svend Ole

    2016-01-01

    from integrating SMEs in a triple helix context. The triple helix approach with government, university and industry participants typically include larger organisations. The research indicates that SMEs could join the triple helix and both contribute and receive benefit from their presence. The findings...

  2. Mutations affecting interaction of integrase with TNPO3 do not prevent HIV-1 cDNA nuclear import

    Directory of Open Access Journals (Sweden)

    Cribier Alexandra

    2011-12-01

    Full Text Available Abstract Background Integration of human immunodeficiency virus type 1 (HIV-1 into a host cell chromosome is an essential step under the control of the viral integrase (IN. Although this enzyme is necessary and sufficient to catalyze the integration reaction in vitro, cellular cofactors are involved in the process in vivo. The chromatin-associated factor LEDGF/p75 interacts with IN and promotes integration to transcription units of the host genome. HIV-1 IN also binds the karyopherin TNPO3, however the significance of this interaction during viral replication remains to be explored. Results Here we present a functional analysis of IN mutants impaired for LEDGF/p75 and TNPO3 interaction. Among them, IN W131A and IN Q168L, that were previously identified to be deficient for LEDGF/p75 interaction, were also partially impaired for TNPO3 binding. We observed that mutations abolishing IN ability to form tetramers resulted in a severe reduction in LEDGF/p75 binding. In sharp contrast, no correlation could be found between the ability of IN to multimerize and TNPO3 interaction. Most of the mutant viruses were essentially impaired for the integration step whereas the amount of 2-LTR circles, reflecting the nuclear import of the viral DNA, was not significantly affected. Conclusion Our functional analysis of HIV-1 IN mutants reveals distinct structural basis for TNPO3 interaction and suggests that the interaction between IN and TNPO3 is not a major determinant of nuclear import but could take place at a nuclear step prior to integration.

  3. A novel high-throughput format assay for HIV-1 integrase strand transfer reaction using magnetic beads

    Institute of Scientific and Technical Information of China (English)

    Hong-qiu HE; Xiao-hui MA; Bin LIU; Wei-zu CHEN; Cun-xin WANG; Shao-hui CHENG

    2008-01-01

    Aim:To develop a novel high-throughput format assay to monitor the integrase (IN) strand transfer (ST) reaction in vitro and apply it to a reaction character study and the identification of antiviral drugs.Methods:The donor DNA duplex,with a sequence identical to the U5 end of HIV-1 long terminal repeats,is labeled at its 5' end with biotin (BIO).The target DNA duplex is labeled at its 3' end with digoxin (DIG).IN mediates the integration of donor DNA into target DNA and results in a 5' BIO and 3' DIG-labeled duplex DNA product.Streptavidin-coated magnetic beads were used to capture the product,and the amount of DIG was measured as the ST reaction product.The assay was optimized in 96-well microplate format for high-throughput screening purpose.Moreover,the assay was applied in a ST reaction character study,and the efficiency of the assay in the identification of antiviral compounds was tested.Results:The end-point values,measured as absorbance at 405 nm was approximately 1.5 for the IN-mediated ST reaction as compared with no more than 0.05 of background readings.The ST reaction char-acter and the half maximal inhibitory concentration (IC50) values of 2 known IN inhibitors obtained in our assay were similar to previously reported results using other assays.The evaluation parameter Z' factor for this assay ranged from 0.6 to 0.9.Conclusion:The assay presented here has been proven to be rapid,sensitive,and specific for the detection of IN ST activity,the reaction character study,as well as for the identification of antiviral drugs targeting IN.

  4. In vitro analysis of the susceptibility of HIV-1 subtype A and CRF01_AE integrases to raltegravir.

    Science.gov (United States)

    Bellecave, Pantxika; Malato, Laurent; Calmels, Christina; Reigadas, Sandrine; Parissi, Vincent; Andreola, Marie-Line; Fleury, Hervé

    2014-08-01

    The antiviral efficacy of raltegravir (RAL) has been proven against human immunodeficiency virus type 1 (HIV-1) subtypes B and C but remained to be determined against other subtypes. Therefore, the enzymatic activities as well as RAL resistance of HIV-1 subtype A and CRF01_AE integrases (INs) were investigated. Previously published subtype A and CRF01_AE IN sequences from RAL-naïve patients were aligned to generate consensus sequences for both IN subtypes. Subtype A and CRF01_AE INs encoded by these consensus sequences as well as the corresponding enzymes harbouring the N155H resistance mutation were expressed and purified. Enzymatic activities of subtype A and CRF01_AE INs were analysed with regard to typical 3'-end processing (3'-P) and strand transfer (ST) activities both in the presence and absence of RAL and were compared with subtype B IN as well as with the corresponding INs harbouring the N155H resistance mutation. Subtypes B, A and CRF01_AE INs showed similar 3'-P and ST activities. In the presence of RAL, the three wild-type INs exhibited ST activity IC50 values (50% inhibitory concentrations) of 86.3 ± 32.5, 158.3 ± 99.0 and 100.0 ± 65.7 nM, respectively. Analysis of 3'-P activity in the presence of RAL revealed IC(50) > 10 μM for all three enzymes. The three INs harbouring the N155H mutation presented in vitro low but similar resistance levels to RAL. In conclusion, INs from HIV-1 subtypes B, A and CRF01_AE showed similar responses to RAL in vitro, suggesting the potency of this antiretroviral drug to treat HIV-1 subtype A- and CRF01_AE-infected patients.

  5. Identification of low molecular weight nuclear complexes containing integrase during the early stages of HIV-1 infection.

    Science.gov (United States)

    Gérard, Annabelle; Soler, Nicolas; Ségéral, Emmanuel; Belshan, Michael; Emiliani, Stéphane

    2013-02-01

    HIV-1 replication requires integration of its reverse transcribed viral cDNA into a host cell chromosome. The DNA cutting and joining reactions associated to this key step are catalyzed by the viral protein integrase (IN). In infected cells, IN binds the viral cDNA, together with viral and cellular proteins, to form large nucleoprotein complexes. However, the dynamics of IN complexes formation is still poorly understood. Here, we characterized IN complexes during the early stages of T-lymphocyte infection. We found that following viral entry into the host cell, IN was rapidly targeted to proteasome-mediated degradation. Interactions between IN and cellular cofactors LEDGF/p75 and TNPO3 were detected as early as 6 h post-infection. Size exclusion chromatography of infected cell extracts revealed distinct IN complexes in vivo. While at 2 h post-infection the majority of IN eluted within a high molecular weight complex competent for integration (IN complex I), IN was also detected in a low molecular weight complex devoid of full-length viral cDNA (IN complex II, ~440 KDa). At 6 h post-infection the relative proportion of IN complex II increased. Inhibition of reverse transcription or integration did not alter the elution profile of IN complex II in infected cells. However, in cells depleted for LEDGF/p75 IN complex II shifted to a lower molecular weight complex (IN complex III, ~150 KDa) containing multimers of IN. Notably, cell fractionation experiments indicated that both IN complex II and III were exclusively nuclear. Finally, IN complex II was not detected in cells infected with a virus harboring a mutated IN defective for LEDGF/p75 interaction and tetramerization. Our findings indicate that, shortly after viral entry, a significant portion of DNA-free IN that is distinct from active pre-integration complexes accumulates in the nucleus.

  6. Substrate recognition and motion mode analyses of PFV integrase in complex with viral DNA via coarse-grained models.

    Directory of Open Access Journals (Sweden)

    Jianping Hu

    Full Text Available HIV-1 integrase (IN is an important target in the development of drugs against the AIDS virus. Drug design based on the structure of IN was markedly hampered due to the lack of three-dimensional structure information of HIV-1 IN-viral DNA complex. The prototype foamy virus (PFV IN has a highly functional and structural homology with HIV-1 IN. Recently, the X-ray crystal complex structure of PFV IN with its cognate viral DNA has been obtained. In this study, both Gaussian network model (GNM and anisotropy network model (ANM have been applied to comparatively investigate the motion modes of PFV DNA-free and DNA-bound IN. The results show that the motion mode of PFV IN has only a slight change after binding with DNA. The motion of this enzyme is in favor of association with DNA, and the binding ability is determined by its intrinsic structural topology. Molecular docking experiments were performed to gain the binding modes of a series of diketo acid (DKA inhibitors with PFV IN obtained from ANM, from which the dependability of PFV IN-DNA used in the drug screen for strand transfer (ST inhibitors was confirmed. It is also found that the functional groups of keto-enol, bis-diketo, tetrazole and azido play a key role in aiding the recognition of viral DNA, and thus finally increase the inhibition capability for the corresponding DKA inhibitor. Our study provides some theoretical information and helps to design anti-AIDS drug based on the structure of IN.

  7. Identification of low molecular weight nuclear complexes containing integrase during the early stages of HIV-1 infection

    Directory of Open Access Journals (Sweden)

    Gérard Annabelle

    2013-02-01

    Full Text Available Abstract Background HIV-1 replication requires integration of its reverse transcribed viral cDNA into a host cell chromosome. The DNA cutting and joining reactions associated to this key step are catalyzed by the viral protein integrase (IN. In infected cells, IN binds the viral cDNA, together with viral and cellular proteins, to form large nucleoprotein complexes. However, the dynamics of IN complexes formation is still poorly understood. Results Here, we characterized IN complexes during the early stages of T-lymphocyte infection. We found that following viral entry into the host cell, IN was rapidly targeted to proteasome-mediated degradation. Interactions between IN and cellular cofactors LEDGF/p75 and TNPO3 were detected as early as 6 h post-infection. Size exclusion chromatography of infected cell extracts revealed distinct IN complexes in vivo. While at 2 h post-infection the majority of IN eluted within a high molecular weight complex competent for integration (IN complex I, IN was also detected in a low molecular weight complex devoid of full-length viral cDNA (IN complex II, ~440 KDa. At 6 h post-infection the relative proportion of IN complex II increased. Inhibition of reverse transcription or integration did not alter the elution profile of IN complex II in infected cells. However, in cells depleted for LEDGF/p75 IN complex II shifted to a lower molecular weight complex (IN complex III, ~150 KDa containing multimers of IN. Notably, cell fractionation experiments indicated that both IN complex II and III were exclusively nuclear. Finally, IN complex II was not detected in cells infected with a virus harboring a mutated IN defective for LEDGF/p75 interaction and tetramerization. Conclusions Our findings indicate that, shortly after viral entry, a significant portion of DNA–free IN that is distinct from active pre-integration complexes accumulates in the nucleus.

  8. Procollagen triple helix assembly: an unconventional chaperone-assisted folding paradigm.

    Science.gov (United States)

    Makareeva, Elena; Leikin, Sergey

    2007-10-10

    Fibers composed of type I collagen triple helices form the organic scaffold of bone and many other tissues, yet the energetically preferred conformation of type I collagen at body temperature is a random coil. In fibers, the triple helix is stabilized by neighbors, but how does it fold? The observations reported here reveal surprising features that may represent a new paradigm for folding of marginally stable proteins. We find that human procollagen triple helix spontaneously folds into its native conformation at 30-34 degrees C but not at higher temperatures, even in an environment emulating Endoplasmic Reticulum (ER). ER-like molecular crowding by nonspecific proteins does not affect triple helix folding or aggregation of unfolded chains. Common ER chaperones may prevent aggregation and misfolding of procollagen C-propeptide in their traditional role of binding unfolded polypeptide chains. However, such binding only further destabilizes the triple helix. We argue that folding of the triple helix requires stabilization by preferential binding of chaperones to its folded, native conformation. Based on the triple helix folding temperature measured here and published binding constants, we deduce that HSP47 is likely to do just that. It takes over 20 HSP47 molecules to stabilize a single triple helix at body temperature. The required 50-200 microM concentration of free HSP47 is not unusual for heat-shock chaperones in ER, but it is 100 times higher than used in reported in vitro experiments, which did not reveal such stabilization.

  9. Experimental investigation of initial steps of helix propagation in model peptides.

    Science.gov (United States)

    Goch, Grazyna; Maciejczyk, Maciej; Oleszczuk, Marta; Stachowiak, Damian; Malicka, Joanna; Bierzyński, Andrzej

    2003-06-10

    It is not certain whether the helix propagation parameters s(n)() (i.e., the equilibrium constants between (n - 1)- and n-residue long alpha-helices) determined from numerous studies of rather long model peptides are applicable for description of the initial steps of the helix formation during the protein folding process. From fluorescence, NMR, and calorimetric studies of a series of model peptides, containing the La(3+)-binding sequence nucleating the helix (Siedlecka, M., Goch, G., Ejchart, A., Sticht, H., and Bierzynski, A. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 903-908), we have determined, at 25 degrees C, the average values of the enthalpy DeltaH(n)() and of the helix growth parameters s(n)() describing the first four steps of helix propagation in polyalanine. The absolute values of the C-cap parameters, describing the contribution of the C-terminal residues to the helix free energy, have also been estimated for alanine (1.2 +/- 0.5) and NH(2) group (1.6 +/- 0.7). The initial four steps of the helix growth in polyalanine can be described by a common propagation parameter s = 1.54 +/- 0.04. The enthalpy DeltaH(n)() is also constant and equals -980 +/- 100 cal mol(-)(1).

  10. Discrete step model of helix-coil kinetics: Distribution of fluctuation times

    Science.gov (United States)

    Poland, Douglas

    1996-07-01

    A method is outlined for the computer simulation of the cooperative kinetics required to construct the distribution function for time intervals between fluctuations in conformational states in macromolecules. Using the helix-coil transition in polyamino acids as an example, we develop a Monte Carlo cellular automata approximation of the kinetics of this system in discrete time. This approximation is tested against a number of exact solutions for homopolymers and is then used to calculate moments of the distribution function for the time intervals between switches in conformational state at a given site (e.g., given a switch from coil to helix at zero time, how long will it take before the state switches back). The maximum-entropy method is used to construct the very broad distribution function from the moments. In heteropolymers the diffusion of helix-coil boundaries is reduced, helix being more localized on strong helix-forming residues. We investigate the effect of a specific sequence of amino acid residues on conformational fluctuations by using the known σ and s values for the naturally occurring amino acids to simulate the kinetics of helix formation (limiting the range of cooperativity to the α-helix) in sperm whale myoglobin, giving the time evolution to the equilibrium probability profile in this system.

  11. Modulation of the oligomerization of myelin proteolipid protein by transmembrane helix interaction motifs.

    Science.gov (United States)

    Ng, Derek P; Deber, Charles M

    2010-08-17

    Proteolipid protein (PLP) is a highly hydrophobic 276-residue integral membrane protein that constitutes more than 50% of the total protein in central nervous system myelin. Previous studies have shown that this protein exists in myelin as an oligomer rather than as a monomer, and mutations in PLP that lead to neurological disorders such as Pelizaeus-Merzbacher disease and spastic paraplegia type 2 have been reported to affect its normal oligomerization. Here we employ peptide-based and in vivo approaches to examine the role of the TM domain in the formation of PLP quaternary structure through homo-oligomeric helix-helix interactions. Focusing on the TM4 alpha-helix (sequence (239)FIAAFVGAAATLVSLLTFMIAATY(262)), the site of several disease-causing point mutations that involve putative small residue helix-helix interaction motifs in the TM4 sequence, we used SDS-PAGE, fluorescence resonance energy transfer, size-exclusion chromatography, and TOXCAT assays in an Escherichia coli membrane to show that the PLP TM4 helix readily assembles into varying oligomeric states. In addition, through targeted studies of the PLP TM4 alpha-helix with point mutations that selectively eliminate these small residue motifs via substitution of Gly, Ala, or Ser residues with Ile residues, we describe a potential mechanism through which disease-causing point mutations can lead to aberrant PLP assembly. The overall results suggest that TM segments in misfolded PLP monomers that expose and/or create surface-exposed helix-helix interaction sites that are normally masked may have consequences for disease.

  12. Statistical thermodynamics of the collagen triple-helix/coil transition. Free energies for amino acid substitutions within the triple-helix.

    Science.gov (United States)

    Doig, Andrew J

    2008-11-27

    Collagen sequences frequently deviate from the most thermally stable (Gly-Pro-Hyp)(n) pattern, with many mutations causing osteogenesis imperfecta (or "brittle bone disease"). The effects of collagen mutations have been studied in short peptides. The analysis of this work is problematic, however, as triple-helices fray from their ends, making the coil/triple-helix equilibrium non-two-state. Here, I develop a statistical thermodynamic model to handle this equilibrium that is applicable to peptides that follow the (G-X-Y)(n) pattern, where Gly is present at every third position and where all three chains are identical. Parameters for substitutions at each position are included, as well as a penalty for initiating triple-helix formation. The model is applied to equilibrium experimental data at 37 degrees C to show that the extension of a triple-helix by a three residue unit stabilizes the triple-helix by 0.76 kcal/mol for Gly-Pro-Hyp and 0.33 kcal/mol for Gly-Pro-Pro. The replacement of Hyp by Arg, Asp, or Trp destabilizes the triple-helix by 1.5, 2.4, and 2.9 kcal/mol, respectively, where the substitution is present once in each chain. The model can thus be used to quantitatively interpret data on collagen peptides, giving free energies that can help rationalize mutations that affect collagen stability, and to design new collagen sequences.

  13. Solvent-Directed Switch of a Left-Handed 10/12-Helix into a Right-Handed 12/10-Helix in Mixed β-Peptides.

    Science.gov (United States)

    Thodupunuri, Prashanth; Katukuri, Sirisha; Ramakrishna, Kallaganti V S; Sharma, Gangavaram V M; Kunwar, Ajit C; Sarma, Akella V S; Hofmann, Hans-Jörg

    2017-02-17

    Present study describes the synthesis and conformational analysis of β-peptides from C-linked carbo-β-amino acids [β-Caa(l)] with a d-lyxo furanoside side chain and β-hGly in 1:1 alternation. NMR and CD investigations on peptides with an (S)-β-Caa(l) monomer at the N-terminus revealed a right-handed 10/12-mixed helix. An unprecedented solvent-directed "switch" both in helical pattern and handedness was observed when the sequence begins with a β-hGly residue instead of a (S)-β-Caa(l) constituent. NMR studies on these peptides in chloroform indicated a left-handed 10/12-helix, while the CD spectrum in methanol inferred a right-handed secondary structure. The NMR data for these peptides in CD3OH showed the presence of a right-handed 12/10-helix. NMR investigations in acetonitrile indicated the coexistence of both helix types. Quantum chemical studies predicted a small energy difference of 0.3 kcal/mol between the two helix types, which may explain the possibility of solvent influence. Examples for a solvent-directed switch of both the H-bonding pattern and the handedness of foldamer helices are rare so far. A comparable solvent effect was not found in the corresponding peptides with (R)-β-Caa(l) residues, where right-handed 12/10-helices are predominating.

  14. Translocation of Biopolymer Chain Through a Nanopore: Coil-Helix Transition

    Institute of Scientific and Technical Information of China (English)

    GU Fang; WANG Hai-Jun; HONG Xiao-Zhong; BA Xin-Wu

    2008-01-01

    @@ The translocation dynamics of a single biopolymer chain through a nanopore in a membrane is investigated by taking the coil-helix transition into account. Based on the changing of the free energy due to the coil-helix transition, the mean first passage time τ is obtained, and then the corresponding numerical simulations are presented under different conditions. It is shown that the coil helix transition can significantly shorten the translocation time of the biopolymer chain. In addition, we also discuss the scaling behaviour for τ with the chain length N and some related problems.

  15. Scalar Casimir effect in the presence of extra dimensions with helix boundary conditions

    Directory of Open Access Journals (Sweden)

    GE Feifei

    2012-12-01

    Full Text Available In this paper,we consider the scalar Casimir effect for parallel plates in the presence of extra dimensions with helix boundary conditions.Using zeta function regularization technique,we get the Casimir pressure explicitly expressed by the modified Bessel function of the second kind.We find the Casimir force is always attractive but for the same magnitude of the helix parameter as the parallel separation,the magnitude of the force between the parallel plates decreases by one order of magnitude.The smaller the helix parameter compared to the plate separation,the more magnitudes the force decreases.

  16. Adsorption of Pb2+, Zn2+ and Ni2+ from Aqueous Solution by Helix aspera Shell

    OpenAIRE

    A. S. Ekop; Eddy, N. O.

    2009-01-01

    The adsorption capacity of Helix aspera shell for Pb2+, Zn2+ and Ni2+ has been studied. This shell has the potential of adsorbing Pb2+, Zn2+ and Ni2+ from aqueous solution. The adsorption potentials of Helix aspera shell is largely influenced by the ionic character of the ions and occurred according to the order Pb2+ > Ni2+ > Zn2+. The adsorption of Pb(II), Zn(II) and Ni(II) ions from aqueous solutions by Helix aspera shell is thermodynamically feasible and is consistent with the models of La...

  17. Accessing commercial cloud resources within the European Helix Nebula cloud marketplace

    Science.gov (United States)

    Cordeiro, C.; De Salvo, A.; Di Girolamo, A.; Field, L.; Giordano, D.; Jones, R.; Villazon, L.

    2015-12-01

    Helix Nebula - the Science Cloud Initiative - is a public-private partnership between Europe's leading scientific research organisations and European IT cloud providers. CERN contributed to this initiative by providing a flagship use case: the workloads from the ATLAS experiment. Aiming to gain experience in managing and monitoring large-scale deployments, as well as in benchmarking the cloud resources, a sizable Monte Carlo production was performed using the Helix Nebula platform. This contribution describes the Helix Nebula initiative and summarizes the experience and the lessons learned from deploying ATLAS experiment application within large cloud setups involving several commercial providers.

  18. Allergic and irritant contact dermatitis from falcarinol and didehydrofalcarinol in common ivy (Hedera helix L.).

    Science.gov (United States)

    Hausen, B M; Bröhan, J; König, W A; Faasch, H; Hahn, H; Bruhn, G

    1987-07-01

    Experimental and chemical investigations revealed that common ivy (Hedera helix susp. helix) contains 3 compounds which are powerful irritants and moderate sensitizers. Only 2 of these constituents, falcarinol and didehydrofalcarinol, are present in the plant during the whole year. Besides Panax ginseng and Schefflera arboricola, this is the third species of the Araliaceae in which these polyacetylenic sensitizers have been found. Falcarinol and didehydrofalcarinol also occur in Hedera helix subsp. canariensis. 4 patients have been patch tested. Even in low concentrations (0.03%), the main allergen falcarinol elicited strong reactions in all of them. One of the authors became sensitized during the investigations.

  19. Thermal helix-coil transition in UV irradiated collagen from rat tail tendon.

    Science.gov (United States)

    Sionkowska, A; Kamińska, A

    1999-05-01

    The thermal helix-coil transition in UV irradiated collagen solution, collagen film and pieces of rat tail tendon (RTT) were compared. Their thermal stability's were determined by differential scanning calorimeter (DSC) and by viscometric measurements. The denaturation temperatures of collagen solution, film and pieces of RTT were different. The helix-coil transition occur near 40 degrees C in collagen solution, near 112 degrees C in collagen film, and near 101 degrees C in pieces of RTT. After UV irradiation the thermal helix-coil transition of collagen samples were changed. These changes depend on the degree of hydratation.

  20. Helix Nebula: sunshine and clouds on the CERN computing horizon

    CERN Multimedia

    Joannah Caborn Wengler

    2012-01-01

    23 petabytes is how much data CERN recorded during 2011, and this number will rise in 2012. In order to respond to the challenge, the IT department is upping its game, amongst other things by participating in the Helix Nebula project, a public-private partnership to create a European cloud-computing platform, as announced in a recent CERN press release.   “We’re not replacing the Grid,” clarifies Bob Jones, responsible for CERN openlab who is also responsible for EC-funded projects in IT, “but looking at three complementary ways of increasing CERN’s computing capacity, so that as demand goes up we can continue to satisfy our users.” “First we are upgrading the electrical and cooling infrastructure of the computer centre in order to increase the availability of critical IT services needed for the Laboratory. This will also provide more floor space in the area called The Barn, allowing for more servers to fit in.”...

  1. Double helix boron-10 powder thermal neutron detector

    Science.gov (United States)

    Wang, Zhehui; Morris, Christopher L.; Bacon, Jeffrey D.

    2015-06-02

    A double-helix Boron-10 powder detector having intrinsic thermal neutron detection efficiency comparable to 36'' long, 2-in diameter, 2-bar Helium-3 detectors, and which can be used to replace such detectors for use in portal monitoring, is described. An embodiment of the detector includes a metallic plate coated with Boron-10 powder for generating alpha and Lithium-7 particles responsive to neutrons impinging thereon supported by insulators affixed to at least two opposing edges; a grounded first wire wound in a helical manner around two opposing insulators; and a second wire having a smaller diameter than that of the first wire, wound in a helical manner around the same insulators and spaced apart from the first wire, the second wire being positively biased. A gas, disposed within a gas-tight container enclosing the plate, insulators and wires, and capable of stopping alpha and Lithium-7 particles and generating electrons produces a signal on the second wire which is detected and subsequently related to the number of neutrons impinging on the plate.

  2. Hydrophobic pulses predict transmembrane helix irregularities and channel transmembrane units

    Directory of Open Access Journals (Sweden)

    Claustres Mireille

    2011-05-01

    Full Text Available Abstract Background Few high-resolution structures of integral membranes proteins are available, as crystallization of such proteins needs yet to overcome too many technical limitations. Nevertheless, prediction of their transmembrane (TM structure by bioinformatics tools provides interesting insights on the topology of these proteins. Methods We describe here how to extract new information from the analysis of hydrophobicity variations or hydrophobic pulses (HPulses in the sequence of integral membrane proteins using the Hydrophobic Pulse Predictor, a new tool we developed for this purpose. To analyze the primary sequence of 70 integral membrane proteins we defined two levels of analysis: G1-HPulses for sliding windows of n = 2 to 6 and G2-HPulses for sliding windows of n = 12 to 16. Results The G2-HPulse analysis of 541 transmembrane helices allowed the definition of the new concept of transmembrane unit (TMU that groups together transmembrane helices and segments with potential adjacent structures. In addition, the G1-HPulse analysis identified helix irregularities that corresponded to kinks, partial helices or unannotated structural events. These irregularities could represent key dynamic elements that are alternatively activated depending on the channel status as illustrated by the crystal structures of the lactose permease in different conformations. Conclusions Our results open a new way in the understanding of transmembrane secondary structures: hydrophobicity through hydrophobic pulses strongly impacts on such embedded structures and is not confined to define the transmembrane status of amino acids.

  3. Helix kinks are equally prevalent in soluble and membrane proteins.

    Science.gov (United States)

    Wilman, Henry R; Shi, Jiye; Deane, Charlotte M

    2014-09-01

    Helix kinks are a common feature of α-helical membrane proteins, but are thought to be rare in soluble proteins. In this study we find that kinks are a feature of long α-helices in both soluble and membrane proteins, rather than just transmembrane α-helices. The apparent rarity of kinks in soluble proteins is due to the relative infrequency of long helices (≥20 residues) in these proteins. We compare length-matched sets of soluble and membrane helices, and find that the frequency of kinks, the role of Proline, the patterns of other amino acid around kinks (allowing for the expected differences in amino acid distributions between the two types of protein), and the effects of hydrogen bonds are the same for the two types of helices. In both types of protein, helices that contain Proline in the second and subsequent turns are very frequently kinked. However, there are a sizeable proportion of kinked helices that do not contain a Proline in either their sequence or sequence homolog. Moreover, we observe that in soluble proteins, kinked helices have a structural preference in that they typically point into the solvent.

  4. The development of bioactive triple helix-forming oligonucleotides.

    Science.gov (United States)

    Seidman, Michael M; Puri, Nitin; Majumdar, Alokes; Cuenoud, Bernard; Miller, Paul S; Alam, Rowshon

    2005-11-01

    We are developing triple helix-forming oligonucleotides (TFOs) as gene targeting reagents in mammalian cells. We have described psoralen-conjugated TFOs containing 2'-O-methyl (2'OMe) and 2'-O-aminoethoxy (AE) ribose substitutions. TFOs with a cluster of 3-4 AE residues, with all other sugars as 2'OMe, were bioactive in a gene knockout assay in mammalian cells. In contrast, TFOs with one or two clustered, or three dispersed, AE residues were inactive. Thermal stability analysis of the triplexes indicated that there were only incremental differences between the active and inactive TFOs. However the active and inactive TFOs could be distinguished by their association kinetics. The bioactive TFOs showed markedly greater on-rates than the inactive TFOs. It appears that the on-rate is a better predictor of TFO bioactivity than thermal stability. Our data are consistent with a model in which a cluster of 3-4 AE residues stabilizes the nucleation event that precedes formation of a complete triplex. It is likely that triplexes in cells are much less stable than triplexes in vitro probably as a result of elution by chromatin-associated translocases and helicases. Consequently the biologic assay will favor TFOs that can bind and rebind genomic targets quickly.

  5. Hydration dynamics of the collagen triple helix by NMR.

    Science.gov (United States)

    Melacini, G; Bonvin, A M; Goodman, M; Boelens, R; Kaptein, R

    2000-07-28

    The hydration of the collagen-like Ac-(Gly-Pro-Hyp)(6)-NH(2) triple-helical peptide in solution was investigated using an integrated set of high-resolution NMR hydration experiments, including different recently developed exchange-network editing methods. This approach was designed to explore the hydration dynamics in the proximity of labile groups, such as the hydroxyproline hydroxyl group, and revealed that the first shell of hydration in collagen-like triple helices is kinetically labile with upper limits for water molecule residence times in the nanosecond to sub-nanosecond range. This result is consistent with a "hopping" hydration model in which solvent molecules are exchanged in and out of solvation sites at a rate that is not directly correlated to the degree of site localization. The hopping model thus reconciles the dynamic view of hydration revealed by NMR with the previously suggested partially ordered semi-clathrate-like cylinder of hydration. In addition, the nanosecond to sub-nanosecond upper limits for water molecule residence times imply that hydration-dehydration events are not likely to be the rate-limiting step for triple helix self-recognition, complementing previous investigations on water dynamics in collagen fibers. This study has also revealed labile proton features expected to facilitate the characterization of the structure and folding of triple helices in collagen peptides.

  6. Helix Electrohydrodynamic Printing of Highly Aligned Serpentine Micro/Nanofibers

    Directory of Open Access Journals (Sweden)

    Yongqing Duan

    2017-09-01

    Full Text Available Micro/nano serpentine structures have widespread applications in flexible/stretchable electronics; however, challenges still exist for low-cost, high-efficiency and controllable manufacturing. Helix electrohydrodynamic printing (HE-printing has been proposed here to realize controllable direct-writing of large area, highly aligned serpentine micro/nanofibers by introducing the rope coiling effect into printing process. By manipulating the flying trajectory and solidification degree of the micro/nano jet, the solidified micro/nanofiber flying in a stabilized helical manner and versatile serpentine structures deposited on a moving collector have been achieved. Systematic experiments and theoretical analysis were conducted to study the transformation behavior and the size changing rules for various deposited microstructures, and highly aligned serpentine microfibers were directly written by controlling the applied voltage, nozzle-to-collector distance and collector velocity. Furthermore, a hyper-stretchable piezoelectric device that can detect stretching, bending and pressure has been successfully fabricated using the printed serpentine micro/nanofibers, demonstrating the potential of HE-printing in stretchable electronics manufacturing.

  7. Double helix boron-10 powder thermal neutron detector

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zhehui; Morris, Christopher L.; Bacon, Jeffrey D.

    2015-06-02

    A double-helix Boron-10 powder detector having intrinsic thermal neutron detection efficiency comparable to 36'' long, 2-in diameter, 2-bar Helium-3 detectors, and which can be used to replace such detectors for use in portal monitoring, is described. An embodiment of the detector includes a metallic plate coated with Boron-10 powder for generating alpha and Lithium-7 particles responsive to neutrons impinging thereon supported by insulators affixed to at least two opposing edges; a grounded first wire wound in a helical manner around two opposing insulators; and a second wire having a smaller diameter than that of the first wire, wound in a helical manner around the same insulators and spaced apart from the first wire, the second wire being positively biased. A gas, disposed within a gas-tight container enclosing the plate, insulators and wires, and capable of stopping alpha and Lithium-7 particles and generating electrons produces a signal on the second wire which is detected and subsequently related to the number of neutrons impinging on the plate.

  8. New nucleic acid triple helix, Poly(AAU)

    Energy Technology Data Exchange (ETDEWEB)

    Broitman, S.L.; Im, D.D.; Fresco, J.R.

    1987-05-01

    A polynucleotide helical structure containing two strands of poly(A) and one of poly(U) has been discovered. The stoichiometry of the complex was determined by continuous variation titrations and isosbestic wavelength analysis. Thermal denaturation profiles were used to examine complex stability over a wide range of conditions. The complex forms only when the poly(A) strands are of molecular weight between 9000-50,000 Daltons (dp approx. 28-150), whereas the size of the poly(U) strand has no effect. This limitation may explain why poly(AAU) was not observed in previous investigations. The complex shows inverse dependence of stability on ionic strength, but is not favored by decreasing pH. This behavior, together with the intermediate poly(A) size requirement suggest that the conformational entropy of the poly(A) strands is a critical determinant of the stability of this complex. The potential of the poly(A) tails of mRNA for formation of this triple helix, and of AAU/T triplet formation to contribute to the binding of unique sequence RNA strands to gene-encoding nucleic acid double helices are noted.

  9. Optical and dielectric properties of double helix DNA thin films

    Energy Technology Data Exchange (ETDEWEB)

    Soenmezoglu, Savas, E-mail: svssonmezoglu@kmu.edu.tr [Department of Physics, Faculty of Kamil Ozdag Science, Karamanoglu Mehmetbey University, 70100, Karaman (Turkey); Ates Soenmezoglu, Ozlem [Department of Biology, Faculty of Kamil Ozdag Science, Karamanoglu Mehmetbey University, 70100, Karaman (Turkey)

    2011-12-01

    In this work, the thin film of wheat DNA was deposited by spin-coating technique onto glass substrate, and the optical and dielectric properties of the double helix DNA thin film were investigated. The optical constants such as refractive index, extinction coefficient, dielectric constant, dissipation factor, relaxation time, and optical conductivity were determined from the measured transmittance spectra in the wavelength range 190-1100 nm. Meanwhile, the dispersion behavior of the refractive index was studied in terms of the single oscillator Wemple-DiDomenico (W-D) model, and the physical parameters of the average oscillator strength, average oscillator wavelength, average oscillator energy, the refractive index dispersion parameter and the dispersion energy were achieved. Furthermore, the optical band gap values were calculated by W-D model and Tauc model, respectively, and the values obtained from W-D model are in agreement with those determined from the Tauc model. The analysis of the optical absorption data indicates that the optical band gap E{sub g} was indirect transitions. These results provide some useful references for the potential application of the DNA thin films in fiber optic, solar cell and optoelectronic devices. Highlights: {yields} The optical constants of DNA in full UV-vis spectrum were determined. {yields} The change in optical and dielectric property demonstrates that this material has potential to be used as a novel technology. {yields} DNA shows promise to be more suitable material than other materials currently being used for photonic devices.

  10. Photo-active collagen systems with controlled triple helix architecture.

    Science.gov (United States)

    Tronci, Giuseppe; Russell, Stephen J; Wood, David J

    2013-08-14

    The design of photo-active collagen systems is presented as a basis for establishing biomimetic materials with varied network architecture and programmable macroscopic properties. Following in-house isolation of type I collagen, reaction with vinyl-bearing compounds of varied backbone rigidity, i.e. 4-vinylbenzyl chloride (4VBC) and glycidyl methacrylate (GMA), was carried out. TNBS colorimetric assay, (1)H-NMR and ATR-FTIR confirmed covalent and tunable functionalization of collagen lysines. Depending on the type and extent of functionalization, controlled stability and thermal denaturation of triple helices were observed via circular dichroism (CD), whereby the hydrogen-bonding capability of introduced moieties was shown to play a major role. Full gel formation was observed following photo-activation of functionalized collagen solutions. The presence of a covalent network only slightly affected collagen triple helix conformation (as observed by WAXS and ATR-FTIR), confirming the structural organization of functionalized collagen precursors. Photo-activated hydrogels demonstrated an increased denaturation temperature (DSC) with respect to native collagen, suggesting that the formation of the covalent network successfully stabilized collagen triple helices. Moreover, biocompatibility and mechanical competence of obtained hydrogels were successfully demonstrated under physiologically-relevant conditions. These results demonstrate that this novel synthetic approach enabled the formation of biocompatible collagen systems with defined network architecture and programmable macroscopic properties, which can only partially be obtained with current synthetic methods.

  11. Assembly of liposomes controlled by triple helix formation.

    Science.gov (United States)

    Jakobsen, Ulla; Vogel, Stefan

    2013-09-18

    Attachment of DNA to the surface of different solid nanoparticles (e.g., gold and silica nanoparticles) is well established, and a number of DNA-modified solid nanoparticle systems have been applied to thermal denaturation analysis of oligonucleotides. We report herein the noncovalent immobilization of oligonucleotides on the surface of soft nanoparticles (i.e., liposomes) and the subsequent controlled assembly by DNA triple helix formation. The noncovalent approach avoids tedious surface chemistry and necessary purification procedures and can simplify and extend the available methodology for the otherwise difficult thermal denaturation analysis of complex triple helical DNA assemblies. The approach is based on lipid modified triplex forming oligonucleotides (TFOs) which control the assembly of liposomes in solution in the presence of single- or double-stranded DNA targets. The thermal denaturation analysis is monitored by ultraviolet spectroscopy at submicromolar concentrations and compared to regular thermal denaturation assays in the absence of liposomes. We report on triplex forming oligonucleotides (TFOs) based on DNA and locked nucleic acid (LNA)/DNA hybrid building blocks and different target sequences (G or C-rich) to explore the applicability of the method for different triple helical assembly modes. We demonstrate advantages and limitations of the approach and show the reversible and reproducible formation of liposome aggregates during thermal denaturation cycles. Nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) show independently from ultraviolet spectroscopy experiments the formation of liposome aggregates.

  12. RFID Tag Helix Antenna Sensors for Wireless Drug Dosage Monitoring

    Science.gov (United States)

    Huang, Haiyu; Zhao, Peisen; Chen, Pai-Yen; Ren, Yong; Liu, Xuewu; Ferrari, Mauro; Hu, Ye; Akinwande, Deji

    2014-01-01

    Miniaturized helix antennas are integrated with drug reservoirs to function as RFID wireless tag sensors for real-time drug dosage monitoring. The general design procedure of this type of biomedical antenna sensors is proposed based on electromagnetic theory and finite element simulation. A cost effective fabrication process is utilized to encapsulate the antenna sensor within a biocompatible package layer using PDMS material, and at the same time form a drug storage or drug delivery unit inside the sensor. The in vitro experiment on two prototypes of antenna sensor-drug reservoir assembly have shown the ability to monitor the drug dosage by tracking antenna resonant frequency shift from 2.4–2.5-GHz ISM band with realized sensitivity of 1.27 \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}$\\mu~{\\rm l}/{\\rm MHz}$\\end{document} for transdermal drug delivery monitoring and 2.76-\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}$\\mu~{\\rm l}/{\\rm MHz}$\\end{document} sensitivity for implanted drug delivery monitoring. PMID:27170865

  13. Dynamic headspace time-extended helix liquid-phase microextraction.

    Science.gov (United States)

    Huang, Shih-Pin; Chen, Pai-Shan; Huang, Shang-Da

    2009-05-15

    Liquid-phase microextraction (LPME) has been proved to be a fast, inexpensive and effective sample pre-treatment technique for the analyses of pesticides and many other compounds. In this investigation, a new headspace microextraction technique, dynamic headspace time-extended helix liquid-phase microextraction (DHS-TEH-LPME), is presented. In this work, use of a solvent cooling system, permits the temperature of the extraction solvent to be lowered. Lowering the temperature of the extraction solvent not only reduces solvent loss but also extends the feasible extraction time, thereby improving extraction efficiency. Use of a larger volume of the solvent not only extends the feasible extraction time but also, after extraction, leaves a larger volume to be directly injected into the gas chromatography (GC) to increase extraction efficiency and instrument signal. The DHS-TEH-LPME technique was used to extract six organochlorine pesticides (OCPs) from 110ml water samples that had been spiked with the analytes at ng/l levels, and stirred for 60min. The proposed method attained enrichments up to 2121 fold. The effects of extraction solvent identity, sample agitation, extraction time, extraction temperature, and salt concentration on extraction performance were also investigated. The method detection limits (MDLs) varied from 0.2 to 25ng/l. The calibration curves were linear for at least 2 orders of magnitude with R(2)>==0.996. Relative recoveries in river water were more than 86%.

  14. The Quality Factor of the Folded Cylindrical Helix

    Directory of Open Access Journals (Sweden)

    S. R. Best

    2009-12-01

    Full Text Available Any electrically small antenna can be impedance matched at any single frequency using a number of well known techniques. Once the small antenna is impedance matched, the primary characteristics of interest are its radiation efficiency, its operating bandwidth and to a lesser extent, its radiation patterns. The bandwidth of the small antenna is often quantified using the antenna's quality factor (Q since fundamental lower bounds for Q are defined in terms of the antenna's occupied volume. The lower bound on Q, also known as the Chu-limit, is defined in terms of the spherical volume occupied by the antenna. However, many small antenna designs are constrained to fit within volumes other than a sphere. To address this issue, Gustafsson et al derived lower bounds for antennas of arbitrary shape with a specific focus on cylindrical and planar shaped antennas. In this paper we consider the quality factor of the folded cylindrical helix, an antenna design that effectively utilizes the available cylindrical volume. We compare its Q to the Gustaffson limit as a function of length-to-diameter ratio, while maintaining a fixed value of ka, and show that it’s Q is at or above Gustafsson’s lower bound for cylindrical shaped antennas.

  15. Knots in the Helix Nebula found in H2

    CERN Document Server

    Matsuura, M; McHunu, B M; Tanaka, I; Wright, N J; Smith, M D; Zijlstra, A A; Viti, S; Wesson, R

    2009-01-01

    We present a deep and wide field-of-view (4'x 7') image of the planetary nebula (PN) NGC 7293 (the Helix Nebula) in the 2.12 micron H2 v=1-0 S(1) line. The excellent seeing (0.4'') at the Subaru Telescope, allows the details of cometary knots to be examined. The knots are found at distances of 2.2'-6.4' from the central star (CS). At the inner edge and in the inner ring (up to 4.5' fromthe CS), the knot often show a `tadpole' shape, an elliptical head with a bright crescent inside and a long tail opposite to the CS. In detail, there are variations in the tadpole shapes, such as narrowing tails, widening tails, meandering tails, or multi-peaks within a tail. In the outer ring (4.5'-6.4' from the CS), the shapes are more fractured, and the tails do not collimate into a single direction. The transition in knot morphology from the inner edge to the outer ring is clearly seen. The number density of knots governs the H2 surface brightness in the inner ring: H2 exists only within the knots. Possible mechanisms which...

  16. The Triple Helix, Quadruple Helix, . . ., and an N-tuple of Helices: Explanatory Models for Analyzing the Knowledge-based Economy?

    CERN Document Server

    Leydesdorff, Loet

    2010-01-01

    Using the Triple Helix model of university-industry-government relations, one can measure the extent to which innovation has become systemic instead of assuming the existence of national (or regional) systems of innovations on a priori grounds. Systemness of innovation patterns, however, can be expected to remain in transition because of integrating and differentiating forces. Integration among the functions of wealth creation, knowledge production, and normative control takes place at the interfaces in organizations, while exchanges on the market, scholarly communication in knowledge production, and political discourse tend to differentiate globally. The neo-institutional and the neo-evolutionary versions of the Triple Helix model enable us to capture this tension reflexively. Empirical studies inform us whether more than three helices are needed for the explanation. The Triple Helix indicator can be extended algorithmically, for example, with local-global as a fourth dimension or, more generally, to an N-tu...

  17. Fluorescence Resonance Energy Transfer (FRET as a method to calculate the dimerization strength of basic Helix-Loop-Helix (bHLH proteins

    Directory of Open Access Journals (Sweden)

    Centonze Victoria E.

    2004-01-01

    Full Text Available Post-translational modifications such as phosphorylation play a vital role in the regulation of protein function. In our study of the basic Helix-loop-Helix (bHLH transcription factor HAND1, we show that HAND1 is phosphorylated during the trophoblast giant cell differentiation on residues residing in Helix I of the bHLH domain. Our hypothesis is that these modifications result in changes in HAND1 dimerization affinities with other bHLH factors. To test this idea, we employed FRET to measure the protein-protein interactions of HAND1 and HAND1 point mutants in HEK293 cells using YFP and CFP fusion proteins and laser scanning confocal microscopy.

  18. The Basic/Helix-Loop-Helix Protein Family in Gossypium: Reference Genes and Their Evolution during Tetraploidization.

    Directory of Open Access Journals (Sweden)

    Qian Yan

    Full Text Available Basic/helix-loop-helix (bHLH proteins comprise one of the largest transcription factor families and play important roles in diverse cellular and molecular processes. Comprehensive analyses of the composition and evolution of the bHLH family in cotton are essential to elucidate their functions and the molecular basis of cotton development. By searching bHLH homologous genes in sequenced diploid cotton genomes (Gossypium raimondii and G. arboreum, a set of cotton bHLH reference genes containing 289 paralogs were identified and named as GobHLH001-289. Based on their phylogenetic relationships, these cotton bHLH proteins were clustered into 27 subfamilies. Compared to those in Arabidopsis and cacao, cotton bHLH proteins generally increased in number, but unevenly in different subfamilies. To further uncover evolutionary changes of bHLH genes during tetraploidization of cotton, all genes of S5a and S5b subfamilies in upland cotton and its diploid progenitors were cloned and compared, and their transcript profiles were determined in upland cotton. A total of 10 genes of S5a and S5b subfamilies (doubled from A- and D-genome progenitors maintained in tetraploid cottons. The major sequence changes in upland cotton included a 15-bp in-frame deletion in GhbHLH130D and a long terminal repeat retrotransposon inserted in GhbHLH062A, which eliminated GhbHLH062A expression in various tissues. The S5a and S5b bHLH genes of A and D genomes (except GobHLH062 showed similar transcription patterns in various tissues including roots, stems, leaves, petals, ovules, and fibers, while the A- and D-genome genes of GobHLH110 and GobHLH130 displayed clearly different transcript profiles during fiber development. In total, this study represented a genome-wide analysis of cotton bHLH family, and revealed significant changes in sequence and expression of these genes in tetraploid cottons, which paved the way for further functional analyses of bHLH genes in the cotton genus.

  19. Short communication: analysis of the integrase gene from HIV type 1-positive patients living in a rural area of West Cameroon.

    Science.gov (United States)

    Turriziani, Ombretta; Montagna, Claudia; Falasca, Francesca; Bucci, Mauro; Russo, Gianluca; Lichtner, Miriam; Sobze, Martin Sanou; Vullo, Vincenzo; Pistello, Mauro; Antonelli, Guido

    2012-12-01

    Major mutations associated with HIV-I integrase inhibitors (INI) resistance are rare in INI-naive patients. However, polymorphisms at positions that may influence the genetic barrier and/or drive the selection of specific INI resistance pathways are common in HIV non-B subtypes. The aim was to evaluate the presence of natural polymorphisms and/or INI resistance mutations in HIV-1 non-B subtype samples obtained from INI-naive patients living in rural west Cameroon. Thirty-three HIV-1 non-B samples were obtained from INI-naive African women and, as controls, 15 samples of HIV-1 subtype B were obtained from antiretroviral-naive Italian patients. The integrase gene was amplified and sequenced using Trugene Core Reagents. Several amino acid positions in B and non-B subtypes were found to be polymorphic. Interestingly, two patients infected with the CRF02_AG subtype had the resistance mutations N155H and E157Q/E and 12% of African samples had an amino acid substitution at position 143. Silent mutations leading to a higher increment of genetic barriers were detected at 140 and 151 positions in non B-subtypes. Although most polymorphisms may have little effect on INI susceptibility, the IN gene variations found in the present study should be taken into consideration as they may facilitate or delay the emergence of variants fully resistant to INIs.

  20. Electric Field-induced Conformational Transition of Bovine Serum Albumin from α -helix to β -sheet

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The irreversible conformational transition of bovine serum albumin (BSA) from α -helix to β -sheet, induced by electric field near the electrode surface, was monitored by circular dichroism (CD) with a long optical path thin layer cell (LOPTLC).

  1. Adsorption of Pb2+, Zn2+ and Ni2+ from Aqueous Solution by Helix aspera Shell

    Directory of Open Access Journals (Sweden)

    A. S. Ekop

    2009-01-01

    Full Text Available The adsorption capacity of Helix aspera shell for Pb2+, Zn2+ and Ni2+ has been studied. This shell has the potential of adsorbing Pb2+, Zn2+ and Ni2+ from aqueous solution. The adsorption potentials of Helix aspera shell is largely influenced by the ionic character of the ions and occurred according to the order Pb2+ > Ni2+ > Zn2+. The adsorption of Pb(II, Zn(II and Ni(II ions from aqueous solutions by Helix aspera shell is thermodynamically feasible and is consistent with the models of Langmuir and Freundlich adsorption isotherms. From the results of the study, the shell of Helix aspera is recommended for use in the removal of Pb2+, Zn2+ and Ni2+ from aqueous solution.

  2. Intramolecular triple helix as a model for regular polyribonucleotide (CAA)(n).

    Science.gov (United States)

    Efimov, Alexander V; Spirin, Alexander S

    2009-10-09

    The regular (CAA)(n) polyribonucleotide, as well as the omega leader sequence containing (CAA)-rich core, have recently been shown to form cooperatively melted and compact structures. In this report, we propose a structural model for the (CAA)(n) sequence in which the polyribonucleotide chain is folded upon itself, so that it forms an intramolecular triple helix. The triple helix is stabilized by hydrogen bonding between bases thus forming coplanar triads, and by stacking interactions between the base triads. A distinctive feature of the proposed triple helix is that it does not contain the canonical double-helix elements. The difference from the known triple helices is that Watson-Crick hydrogen bond pairings do not take place in the interactions between the bases within the base triads.

  3. Gold triple-helix mid-infrared metamaterial by STED-inspired laser lithography.

    Science.gov (United States)

    Kaschke, Johannes; Wegener, Martin

    2015-09-01

    In analogy to wire-grid polarizers for linear polarization, metal-helix metamaterials can act as broadband circular polarizers. This concept has brought circular-polarization capabilities to mid-infrared and terahertz frequencies, which were previously difficult to access. Due to the lack of rotational symmetry, however, single-helix metamaterials exhibit unwanted circular-polarization conversions. Recent theoretical work showed that conversions can be fully eliminated by intertwining N=3 or 4 helices within each unit cell. While direct laser writing in positive-tone photo-resist yielded good results for single-helix metamaterials operating at mid-infrared frequencies, the axial resolution is insufficient for N-helix metamaterials. Here, we use stimulated emission depletion-inspired three-dimensional laser lithography to fabricate such microstructures. We measure all entries of the Jones transmission and reflection matrices and show experimentally that polarization conversions are minimized, in good agreement with theory.

  4. Helix-Sense-Selective Polymerization of N,N-Diphenyl(Meth)acrylamide by Anionic Catalysts

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    In this paper, the helix-sense-selective polymerization of N,N-diphenyl acrylamide (DPAA) and N,N-diphenyl methacrylamide(DPMAA) were studied with living helix prepolymer as anionic initiator, and the chiral optical properties of the obtained polymers were investigated too.It was shown that optically active polymers of DPAA and DPMAA could be obtained under the experimental condition, and exhibited the same screw sense as that of the prepolymer.

  5. The triple helix model and dynamics of innovation: a case study

    OpenAIRE

    Natário, Maria Manuela; Couto, João Pedro Almeida; Almeida, Carlos Fernandes

    2012-01-01

    Copyright © 2012 Emerald Group Publishing Limited. The purpose of this paper is to analyze the dynamics of the triple helix model in less favoured regions, examining the role of three spheres: universities, firms, and government. The paper identifies profiles of behavior in terms of triple helix model performance from the firm's perspective and recognizes key factors for successful innovation dynamics in a less favored region of Portugal.

  6. Macroscopic control of helix orientation in films dried from cholesteric liquid crystalline cellulose nanocrystal suspensions


    OpenAIRE

    2014-01-01

    The intrinsic ability of cellulose nanocrystals (CNCs) to self-organize into films and bulk materials with helical order in a cholesteric liquid crystal is scientifically intriguing and potentially important for the production of renewable multifunctional materials with attractive optical properties. A major obstacle, however, has been the lack of control of helix direction, which results in a defect-rich, mosaic-like domain structure. Herein, a method for guiding the helix during film format...

  7. Development of elvitegravir resistance and linkage of integrase inhibitor mutations with protease and reverse transcriptase resistance mutations.

    Directory of Open Access Journals (Sweden)

    Mark A Winters

    Full Text Available Failure of antiretroviral regimens containing elvitegravir (EVG and raltegravir (RAL can result in the appearance of integrase inhibitor (INI drug-resistance mutations (DRMs. While several INI DRMs have been identified, the evolution of EVG DRMs and the linkage of these DRMs with protease inhibitor (PI and reverse transcriptase inhibitor (RTI DRMs have not been studied at the clonal level. We examined the development of INI DRMs in 10 patients failing EVG-containing regimens over time, and the linkage of INI DRMs with PI and RTI DRMs in these patients plus 6 RAL-treated patients. A one-step RT-nested PCR protocol was used to generate a 2.7 kB amplicon that included the PR, RT, and IN coding region, and standard cloning and sequencing techniques were used to determine DRMs in 1,277 clones (mean 21 clones per time point. Results showed all patients had multiple PI, NRTI, and/or NNRTI DRMs at baseline, but no primary INI DRM. EVG-treated patients developed from 2 to 6 strains with different primary INI DRMs as early as 2 weeks after initiation of treatment, predominantly as single mutations. The prevalence of these strains fluctuated and new strains, and/or strains with new combinations of INI DRMs, developed over time. Final failure samples (weeks 14 to 48 typically showed a dominant strain with multiple mutations or N155H alone. Single N155H or multiple mutations were also observed in RAL-treated patients at virologic failure. All patient strains showed evidence of INI DRM co-located with single or multiple PI and/or RTI DRMs on the same viral strand. Our study shows that EVG treatment can select for a number of distinct INI-resistant strains whose prevalence fluctuates over time. Continued appearance of new INI DRMs after initial INI failure suggests a potent, highly dynamic selection of INI resistant strains that is unaffected by co-location with PI and RTI DRMs.

  8. Mode of inhibition of HIV-1 Integrase by a C-terminal domain-specific monoclonal antibody*

    Directory of Open Access Journals (Sweden)

    Merkel George

    2006-06-01

    Full Text Available Abstract Background To further our understanding of the structure and function of HIV-1 integrase (IN we developed and characterized a library of monoclonal antibodies (mAbs directed against this protein. One of these antibodies, mAb33, which is specific for the C-terminal domain, was found to inhibit HIV-1 IN processing activity in vitro; a corresponding Fv fragment was able to inhibit HIV-1 integration in vivo. Our subsequent studies, using heteronuclear nuclear magnetic resonance spectroscopy, identified six solvent accessible residues on the surface of the C-terminal domain that were immobilized upon binding of the antibody, which were proposed to comprise the epitope. Here we test this hypothesis by measuring the affinity of mAb33 to HIV-1 proteins that contain Ala substitutions in each of these positions. To gain additional insight into the mode of inhibition we also measured the DNA binding capacity and enzymatic activities of the Ala substituted proteins. Results We found that Ala substitution of any one of five of the putative epitope residues, F223, R224, Y226, I267, and I268, caused a decrease in the affinity of the mAb33 for HIV-1 IN, confirming the prediction from NMR data. Although IN derivatives with Ala substitutions in or near the mAb33 epitope exhibited decreased enzymatic activity, none of the epitope substitutions compromised DNA binding to full length HIV-1 IN, as measured by surface plasmon resonance spectroscopy. Two of these derivatives, IN (I276A and IN (I267A/I268A, exhibited both increased DNA binding affinity and uncharacteristic dissociation kinetics; these proteins also exhibited non-specific nuclease activity. Results from these investigations are discussed in the context of current models for how the C-terminal domain interacts with substrate DNA. Conclusion It is unlikely that inhibition of HIV-1 IN activity by mAb33 is caused by direct interaction with residues that are essential for substrate binding. Rather

  9. Small-signal analysis of a rectangular helix structure traveling-wave-tube

    Institute of Scientific and Technical Information of China (English)

    Fu Cheng-Fang; Wei Yan-Yu; Duan Zhao-Yun; Wang Wen-Xiang; Gong Yu-Bin

    2009-01-01

    This paper investigates the properties of traveling wave-beam interaction in a rectangular helix traveling-wave-tube (TWT) for a solid sheet electron beam. The 'hot' dispersion equation is obtained by means of the self-consistent field theory. The small signal analysis, which includes the effects of the beam parameters and slow-wave structure (SWS)parameters, is carried out by theoretical computation. The numerical results show that the bandwidth and the smallsignal gain of the rectangular helix TWT increase as the beam current increases; and the beam voltage not obviously influences the small signal gain. Among different rectangular helix structures, the small-signal gain increases as the width of the rectangular helix SWS increases, however, the bandwidth decreases whether structure parameters a and Lor ψ and L are fixed or not. In addition, a comparison of the small-signal gain of this structure with a conventional round helix is made. The presented analysis will be useful for the design of the TWT with a rectangular helix circuit.

  10. [Study of collagen mimetic peptide's triple-helix structure and its thermostability by circular dichroism].

    Science.gov (United States)

    Zhang, Zhi-Bao; Wang, Jing-Jie; Chen, Hui-Juan; Xiong, Qing-Qing; Liu, Ling-Rong; Zhang, Qi-Qing

    2014-04-01

    In the present study, the authors explore the triple-helix conformation and thermal stability of collagen mimetic peptides (CMPs) as a function of peptide sequence and/or chain length by circular dichroism(CD). Five CMPs were designed and synthetized varying the number of POG triplets or incorporating an integrin alpha2beta1 binding motif Gly-Phe-Hyp-Gly-Glu-Arg (GFOGER). CD spectroscopy from 260 to 190 nm was recorded to confirm the existence of triple-helix conformation at room temperature, while thermal melting and thermal annealing of triple-helix (thermal unfolding and refolding of triple-helix, respectively) was characterized by monitoring ellipticity at 225 nm as a function of temperature. The results demonstrated that all the CMPs adopted triple-helix conformation, and the thermal stability of the CMPs was enhanced with increasing the number of POG triplets. In contrast to natural collagen, the thermal denaturation processes of CMPs were reversible, i. e. the triple-helix unfolded upon heating while refolded upon cooling. Meanwhile, the phenomenon of "hysteresis" was observed by comparing melting and thermal curves. These findings add new insights to the mechanisms of collagen and CMPs assembly, as well as provide an alternative approach to the fabrication of artificial collagen-likes biomaterials.

  11. Assessment of vertical changes during maxillary expansion using quad helix or bonded rapid maxillary expander.

    Science.gov (United States)

    Conroy-Piskai, Cara; Galang-Boquiren, Maria Therese S; Obrez, Ales; Viana, Maria Grace Costa; Oppermann, Nelson; Sanchez, Flavio; Edgren, Bradford; Kusnoto, Budi

    2016-11-01

    To determine if there is a significantly different effect on vertical changes during phase I palatal expansion treatment using a quad helix and a bonded rapid maxillary expander in growing skeletal Class I and Class II patients. This retrospective study looked at 2 treatment groups, a quad helix group and a bonded rapid maxillary expander group, before treatment (T1) and at the completion of phase I treatment (T2). Each treatment group was compared to an untreated predicted growth model. Lateral cephalograms at T1 and T2 were traced and analyzed for changes in vertical dimension. No differences were found between the treatment groups at T1, but significant differences at T2 were found for convexity, lower facial height, total facial height, facial axis, and Frankfort Mandibular Plane Angle (FMA) variables. A comparison of treatment groups at T2 to their respective untreated predicted growth models found a significant difference for the lower facial height variable in the quad helix group and for the upper first molar to palatal plane (U6-PP) variable in the bonded expander group. Overall, both the quad helix expander and the bonded rapid maxillary expander showed minimal vertical changes during palatal expansion treatment. The differences at T2 suggested that the quad helix expander had more control over skeletal vertical measurements. When comparing treatment results to untreated predicted growth values, the quad helix expander appeared to better maintain lower facial height and the bonded rapid maxillary expander appeared to better maintain the maxillary first molar vertical height.

  12. Emergence of the Persistent Spin Helix in Semiconductor Quantum Wells

    Energy Technology Data Exchange (ETDEWEB)

    Koralek, Jake; Weber, Chris; Orenstein, Joe; Bernevig, Andrei; Zhang, Shoucheng; Mack, Shawn; Awschalom, David

    2011-08-24

    According to Noether's theorem, for every symmetry in nature there is a corresponding conservation law. For example, invariance with respect to spatial translation corresponds to conservation of momentum. In another well-known example, invariance with respect to rotation of the electron's spin, or SU(2) symmetry, leads to conservation of spin polarization. For electrons in a solid, this symmetry is ordinarily broken by spin-orbit (SO) coupling, allowing spin angular momentum to flow to orbital angular momentum. However, it has recently been predicted that SU(2) can be recovered in a two-dimensional electron gas (2DEG), despite the presence of SO coupling. The corresponding conserved quantities include the amplitude and phase of a helical spin density wave termed the 'persistent spin helix' (PSH). SU(2) is restored, in principle, when the strength of two dominant SO interactions, the Rashba ({alpha}) and linear Dresselhaus ({beta}{sub 1}), are equal. This symmetry is predicted to be robust against all forms of spin-independent scattering, including electron-electron interactions, but is broken by the cubic Dresselhaus term ({beta}{sub 3}) and spin-dependent scattering. When these terms are negligible, the distance over which spin information can propagate is predicted to diverge as {alpha} {yields} {beta}{sub 1}. Here we observe experimentally the emergence of the PSH in GaAs quantum wells (QW's) by independently tuning {alpha} and {beta}{sub 1}. Using transient spin-grating spectroscopy (TSG), we find a spin-lifetime enhancement of two orders of magnitude near the symmetry point. Excellent quantitative agreement with theory across a wide range of sample parameters allows us to obtain an absolute measure of all relevant SO terms, identifying {beta}{sub 3} as the main SU(2) violating term in our samples. The tunable suppression of spin-relaxation demonstrated in this work is well-suited for application to spintronics.

  13. Natural polymorphisms of HIV-1 CRF01_AE integrase coding region in ARV-naïve individuals in Cambodia, Thailand and Vietnam: an ANRS AC12 working group study.

    Science.gov (United States)

    Nouhin, Janin; Donchai, Tawee; Hoang, Khanh Thu Huynh; Ken, Sreymom; Kamkorn, Jiraporn; Tran, Ton; Ayouba, Ahidjo; Peeters, Martine; Chaix, Marie-Laure; Lien, Truong Xuan; Nerrienet, Eric; Ngo-Giang-Huong, Nicole

    2011-01-01

    The HIV integrase enzyme is essential for the HIV life cycle as it mediates integration of HIV-1 proviral DNA into the infected cell's genome. Recently, the development of drugs capable of inhibiting integrase has provided major new options for HIV-infected, treatment-experienced patients with multidrug resistant virus, as well treatment-naïve patients. More than 40 amino acid substitutions within integrase have been described as associated mostly with resistance of HIV B-subtypes to currently available integrase inhibitors (INIs). We have analyzed the natural polymorphisms of the integrase coding region in 87 antiretroviral-naïve subjects (32 from Cambodia, 37 from Thailand and 18 from Vietnam) infected with CRF01_AE virus, the predominant HIV-1 strain circulating in Southeast Asia. The 864bp integrase coding region was sequenced using the ANRS consensus sequencing technique from plasma samples, and amino acid results were interpreted for drug resistance according to the ANRS (Updated July 2009, version 18) and Stanford algorithms (Version November 6, 2009). Alignment of the 87 amino acid sequences against the 2004 Los Alamos HIV-1 clade B consensus sequence showed that overall, 119 of 288 (41.3%) amino acid positions presented at least one polymorphism each. Substitutions found in >60% of study subjects occurred at: K14, A21, V31, S39, I72, T112, T124, T125, G134, I135, K136, D167, V201, L234 and S283. Also, new amino acid substitutions of as yet unknown significance were identified: E152K/H, S153F/L, N155I and E157G. None of the known integrase resistance mutations were observed, except E157Q found in one Cambodian subject (1.1%, CI 95% 0.02-6.3%). The clinical impact of this substitution on resistance of B and nonB-viruses to the licensed INI raltegravir is unclear. If this substitution is confirmed to compromise the virologic response to raltegravir, further studies will be needed to better assess the prevalence of this substitution among CRF01_AE virus.

  14. The C-Terminal RpoN Domain of sigma54 Forms an unpredictedHelix-Turn-Helix Motif Similar to domains of sigma70

    Energy Technology Data Exchange (ETDEWEB)

    Doucleff, Michaeleen; Malak, Lawrence T.; Pelton, Jeffrey G.; Wemmer, David E.

    2005-11-01

    The ''{delta}'' subunit of prokaryotic RNA-polymerase allows gene-specific transcription initiation. Two {sigma} families have been identified, {sigma}{sup 70} and {sigma}{sup 54}, which use distinct mechanisms to initiate transcription and share no detectable sequence homology. Although the {sigma}{sup 70}-type factors have been well characterized structurally by x-ray crystallography, no high-resolution structural information is available for the {sigma}{sup 54}-type factors. Here we present the NMR derived structure of the C-terminal domain of {sigma}{sup 54} from Aquifex aeolicus. This domain (Thr323 to Gly389), which contains the highly conserved RpoN box sequence, consists of a poorly structured N-terminal tail followed by a three-helix bundle, which is surprisingly similar to domains of the {sigma}{sup 70}-type proteins. Residues of the RpoN box, which have previously been shown to be critical for DNA binding, form the second helix of an unpredicted helix-turn-helix motif. This structure's homology with other DNA binding proteins, combined with previous biochemical data, suggest how the C-terminal domain of {sigma}{sup 54} binds to DNA.

  15. Heterogeneity of myotubes generated by the MyoD and E12 basic helix-loop-helix transcription factors in otherwise non-differentiation growth conditions.

    Science.gov (United States)

    Grubišić, Vladimir; Gottipati, Manoj K; Stout, Randy F; Grammer, J Robert; Parpura, Vladimir

    2014-02-01

    We used a synthetic biology approach to produce myotubes from mammalian C2C12 myoblasts in non-differentiation growth conditions using the expression of basic helix-loop-helix transcription factors, MyoD and E12, in various combinations and configurations. Our approach not only recapitulated the basics of muscle development and physiology, as the obtained myotubes showed qualities similar to those seen in striated muscle fibers in vivo, but also allowed for the synthesis of populations of myotubes which assumed distinct morphology, myofibrillar development and Ca(2+) dynamics. This fashioned class of biomaterials is suitable for the building blocks of soft actuators in micro-scale biomimetic robotics. This production line strategy can be embraced in reparative medicine as synthetic human myotubes with predetermined morphological/functional properties could be obtained using this very approach. This methodology can be adopted beyond striated muscle for the engineering of other tissue components/cells whose differentiation is governed by the principles of basic helix-loop-helix transcription factors, as in the case, for example, of neural or immune cell types.

  16. A routine for measuring synergy in university-industry-government relations: mutual information as a Triple-Helix and Quadruple-Helix indicator

    NARCIS (Netherlands)

    Leydesdorff, L.; Park, H.W.; Lengyel, B.

    2014-01-01

    Mutual information in three (or more) dimensions can be considered as a Triple-Helix indicator of possible synergy in university-industry-government relations. An open-source routine th4.exe makes the computation of this indicator interactively available at the internet, and thus applicable to large

  17. CFD analysis and flow model reduction for surfactant production in helix reactor = CFD analiza i redukcija modela strujanja za proizvodnju surfaktanta u helix reaktoru

    NARCIS (Netherlands)

    Nikačević, N.M.; Thielen, L.; Twerda, A.; Hof, P.M.J. van den

    2015-01-01

    Flow pattern analysis in a spiral Helix reactor is conducted, for the application in commercial surfactant production. Step change response curves (SCR) were obtained from numerical tracer experiments by three-dimensional computational fluid dynamics (CFD) simulations. Non-reactive flow is simulated

  18. A routine for measuring synergy in university-industry-government relations: mutual information as a Triple-Helix and Quadruple-Helix indicator

    NARCIS (Netherlands)

    Leydesdorff, L.; Park, H.W.; Lengyel, B.

    2014-01-01

    Mutual information in three (or more) dimensions can be considered as a Triple-Helix indicator of possible synergy in university-industry-government relations. An open-source routine th4.exe makes the computation of this indicator interactively available at the internet, and thus applicable to large

  19. The triple helix, quadruple helix, ..., and an N-tuple of helices: explanatory models for analyzing the knowledge-based economy?

    NARCIS (Netherlands)

    Leydesdorff, L.

    2012-01-01

    Using the Triple Helix model of university-industry-government relations, one can measure the extent to which innovation has become systemic instead of assuming the existence of national (or regional) systems of innovations on a priori grounds. Systemness of innovation patterns, however, can be expe

  20. CFD analysis and flow model reduction for surfactant production in helix reactor = CFD analiza i redukcija modela strujanja za proizvodnju surfaktanta u helix reaktoru

    NARCIS (Netherlands)

    Nikačević, N.M.; Thielen, L.; Twerda, A.; Hof, P.M.J. van den

    2015-01-01

    Flow pattern analysis in a spiral Helix reactor is conducted, for the application in commercial surfactant production. Step change response curves (SCR) were obtained from numerical tracer experiments by three-dimensional computational fluid dynamics (CFD) simulations. Non-reactive flow is

  1. Osteogenesis Imperfecta Model Peptides: Incorporation of Residues Replacing Gly within a Triple Helix Achieved by Renucleation and Local Flexibility

    OpenAIRE

    Xiao, Jianxi; Madhan, Balaraman; Li, Yingjie; Brodsky, Barbara; Baum, Jean

    2011-01-01

    Missense mutations, which replace one Gly with a larger residue in the repeating sequence of the type I collagen triple helix, lead to the hereditary bone disorder osteogenesis imperfecta (OI). Previous studies suggest that these mutations may interfere with triple-helix folding. NMR was used to investigate triple-helix formation in a series of model peptides where the residue replacing Gly, as well as the local sequence environment, was varied. NMR measurement of translational diffusion coef...

  2. Synthesis and anti-HIV activity of some [Nucleoside Reverse Transcriptase Inhibitor]-C5'-linker-[Integrase Inhibitor] heterodimers as inhibitors of HIV replication.

    Science.gov (United States)

    Sugeac, Elena; Fossey, Christine; Ladurée, Daniel; Schmidt, Sylvie; Laumond, Geraldine; Aubertin, Anne-Marie

    2004-12-01

    Selected for their expected ability to inhibit HIV replication, a series of eight heterodimers containing a Nucleoside Reverse Transcriptase Inhibitor (NRTI) and an Integrase Inhibitor (INI), bound by a linker, were designed and synthesized. For the NRTIs, d4U, d2U and d4T were chosen. For the INIs, 4-[1-(4-fluorobenzyl)-1H-pyrrol-2-yl]-2,4-dioxobutyric acid (6) and 4-(3,5-dibenzyloxyphenyl)-2,4-dioxobutyric acid (9) (belonging to the beta-diketo acids class) were chosen. The conjugation of the two different inhibitors (NRTI and INI) was performed using an amino acid (glycine or beta-alanine) as a cleavable linker.

  3. Genome engineering and direct cloning of antibiotic gene clusters via phage ϕBT1 integrase-mediated site-specific recombination in Streptomyces.

    Science.gov (United States)

    Du, Deyao; Wang, Lu; Tian, Yuqing; Liu, Hao; Tan, Huarong; Niu, Guoqing

    2015-03-04

    Several strategies have been used to clone large DNA fragments directly from bacterial genome. Most of these approaches are based on different site-specific recombination systems consisting of a specialized recombinase and its target sites. In this study, a novel strategy based on phage ϕBT1 integrase-mediated site-specific recombination was developed, and used for simultaneous Streptomyces genome engineering and cloning of antibiotic gene clusters. This method has been proved successful for the cloning of actinorhodin gene cluster from Streptomyces coelicolor M145, napsamycin gene cluster and daptomycin gene cluster from Streptomyces roseosporus NRRL 15998 at a frequency higher than 80%. Furthermore, the system could be used to increase the titer of antibiotics as we demonstrated with actinorhodin and daptomycin, and it will be broadly applicable in many Streptomyces.

  4. Effect of sterically demanding substituents on the conformational stability of the collagen triple helix.

    Science.gov (United States)

    Erdmann, Roman S; Wennemers, Helma

    2012-10-17

    The effect of sterically demanding groups at proline residues on the conformational stability of the collagen triple helix was examined. The thermal stabilities (T(m) and ΔG) of eight different triple helices derived from collagen model peptides with (4R)- or (4S)-configured amidoprolines bearing either methyl or bulkier tert-butyl groups in the Xaa or Yaa position were determined and served as a relative measure for the conformational stability of the corresponding collagen triple helices. The results show that sterically demanding substituents are tolerated in the collagen triple helix when they are attached to (4R)-configured amidoprolines in the Xaa position or to (4S)-configured amidoprolines in the Yaa position. Structural studies in which the preferred conformation of (4R)- or (4S)-configured amidoproline were overlaid with the Pro and Hyp residues within a crystal structure of collagen revealed that the sterically demanding groups point to the outside of these two triple helices and thereby do not interfere with the formation of the triple helix. In all of the other examined collagen derivatives with lower stability of the triple helices, the acetyl or pivaloyl residues point toward the inside of the triple helix and clash with a residue of the neighboring strand. The results also revealed that unfavorable steric dispositions affect the conformational stability of the collagen triple helix more than unfavorable ring puckers of the proline residues. The results are useful for the design of functionalized collagen based materials.

  5. Contribution of dipole-dipole interactions to the stability of the collagen triple helix.

    Science.gov (United States)

    Improta, Roberto; Berisio, Rita; Vitagliano, Luigi

    2008-05-01

    Unveiling sequence-stability and structure-stability relationships is a major goal of protein chemistry and structural biology. Despite the enormous efforts devoted, answers to these issues remain elusive. In principle, collagen represents an ideal system for such investigations due to its simplified sequence and regular structure. However, the definition of the molecular basis of collagen triple helix stability has hitherto proved to be a difficult task. Particularly puzzling is the decoding of the mechanism of triple helix stabilization/destabilization induced by imino acids. Although the propensity-based model, which correlates the propensities of the individual imino acids with the structural requirements of the triple helix, is able to explicate most of the experimental data, it is unable to predict the rather high stability of peptides embedding Gly-Hyp-Hyp triplets. Starting from the available X-ray structures of this polypeptide, we carried out an extensive quantum chemistry analysis of the mutual interactions established by hydroxyproline residues located at the X and Y positions of the Gly-X-Y motif. Our data clearly indicate that the opposing rings of these residues establish significant van der Waals and dipole-dipole interactions that play an important role in triple helix stabilization. These findings suggest that triple helix stabilization can be achieved by distinct structural mechanisms. The interplay of these subtle but recurrent effects dictates the overall stability of this widespread structural motif.

  6. Position and motions of the S4 helix during opening of the Shaker potassium channel.

    Science.gov (United States)

    Phillips, L Revell; Swartz, Kenton J

    2010-12-01

    The four voltage sensors in voltage-gated potassium (Kv) channels activate upon membrane depolarization and open the pore. The location and motion of the voltage-sensing S4 helix during the early activation steps and the final opening transition are unresolved. We studied Zn(2+) bridges between two introduced His residues in Shaker Kv channels: one in the R1 position at the outer end of the S4 helix (R362H), and another in the S5 helix of the pore domain (A419H or F416H). Zn(2+) bridges readily form between R362H and A419H in open channels after the S4 helix has undergone its final motion. In contrast, a distinct bridge forms between R362H and F416H after early S4 activation, but before the final S4 motion. Both bridges form rapidly, providing constraints on the average position of S4 relative to the pore. These results demonstrate that the outer ends of S4 and S5 remain in close proximity during the final opening transition, with the S4 helix translating a significant distance normal to the membrane plane.

  7. Construction of transformed, cultured silkworm cells and transgenic silkworm using the site-specific integrase system from phage φC31.

    Science.gov (United States)

    Yin, Yajuan; Cao, Guangli; Xue, Renyu; Gong, Chengliang

    2014-10-01

    The Streptomyces bacteriophage, φC31, uses a site-specific integrase enzyme to perform efficient recombination. The recombination system uses specific sequences to integrate exogenous DNA from the phage into a host. The sequences are known as the attP site in the phage and the attB site in the host. The system can be used as a genetic manipulation tool. In this study it has been applied to the transformation of cultured BmN cells and the construction of transgenic Bombyx mori individuals. A plasmid, pSK-attB/Pie1-EGFP/Zeo-PASV40, containing a cassette designed to express a egfp-zeocin fusion gene, was co-transfected into cultured BmN cells with a helper plasmid, pSK-Pie1/NLS-Int/NSL. Expression of the egfp-zeocin fusion gene was driven by an ie-1 promoter, downstream of a φC31 attB site. The helper plasmid encoded the φC31 integrase enzyme, which was flanked by two nuclear localization signals. Expression of the egfp-zeocin fusion gene could be observed in transformed cells. The two plasmids were also transferred into silkworm eggs to obtain transgenic silkworms. Successful integration of the fusion gene was indicated by the detection of green fluorescence, which was emitted by the silkworms. Nucleotide sequence analysis demonstrated that the attB site had been cut, to allow recombination between the attB and endogenous pseudo attP sites in the cultured silkworm cells and silkworm individuals.

  8. Inorganic and organic fertilizers impact the abundance and proportion of antibiotic resistance and integron-integrase genes in agricultural grassland soil.

    Science.gov (United States)

    Nõlvak, Hiie; Truu, Marika; Kanger, Kärt; Tampere, Mailiis; Espenberg, Mikk; Loit, Evelin; Raave, Henn; Truu, Jaak

    2016-08-15

    Soil fertilization with animal manure or its digestate may facilitate an important antibiotic resistance dissemination route from anthropogenic sources to the environment. This study examines the effect of mineral fertilizer (NH4NO3), cattle slurry and cattle slurry digestate amendment on the abundance and proportion dynamics of five antibiotic resistance genes (ARGs) and two classes of integron-integrase genes (intI1 and intI2) in agricultural grassland soil. Fertilization was performed thrice throughout one vegetation period. The targeted ARGs (sul1, tetA, blaCTX-M, blaOXA2 and qnrS) encode resistance to several major antibiotic classes used in veterinary medicine such as sulfonamides, tetracycline, cephalosporins, penicillin and fluoroquinolones, respectively. The non-fertilized grassland soil contained a stable background of tetA, blaCTX-M and sul1 genes. The type of applied fertilizer significantly affected ARGs and integron-integrase genes abundances and proportions in the bacterial community (porganic fertilizer's application event, but this increase was followed by a stage of decrease, suggesting that microbes possessing these genes were predominantly entrained into soil via cattle slurry or its digestate application and had somewhat limited survival potential in a soil environment. However, the abundance of these three target genes did not decrease to a background level by the end of the study period. TetA was most abundant in mineral fertilizer treated soil and blaCTX-M in cattle slurry digestate amended soil. Despite significantly different abundances, the abundance dynamics of bacteria possessing these genes were similar (p<0.05 in all cases) in different treatments and resembled the dynamics of the whole bacterial community abundance in each soil treatment.

  9. Genetic variation of the HIV-1 integrase region in newly diagnosed anti-retroviral drug-naïve patients with HIV/AIDS in Korea.

    Science.gov (United States)

    Kim, J-Y; Kim, E-J; Choi, J-Y; Kwon, O-K; Kim, G J; Choi, S Y; Kim, S S

    2011-08-01

    The survival time of HIV/AIDS patients in Korea has increased since HAART (highly active anti-retroviral therapy) was introduced. However, the occurrence of drug-resistant strains requires new anti-retroviral drugs, one of which, an integrase inhibitor (INI), was approved by the US Food and Drug Administration (FDA) in 2007. INIs have been used for therapy in many countries and are about to be employed in Korea. Therefore, it is important to identify basic mutant variants prior to the introduction of INIs in order to estimate their efficacy. To monitor potential drug-resistant INI mutations in Korean HIV/AIDS patients, the polymorphism of the int gene was investigated together with the pol gene using a genotypic assay for 75 randomly selected Korean HIV-1 patients newly diagnosed in 2007. The drug-resistant mutation sequences were analysed using the Stanford HIV DB and the International AIDS Society resistance testing-USA panel (IAS-USA). Seventy strains of Korean subtype B were compared with foreign subtype-B strains, and there were no significantly different variants of the int gene region in the study population. Major mutation sites in the integrase (E92Q, F121Y, G140A/S, Y143C/R, Q148H/R/K and N155H) were not detected, and only a few minor mutation sites (L74M, V151I, E157Q, V165I, I203M, S230N and D232N) were identified in 21 strains (28%). Resistance due to mutations in the pol gene was observed in a single strain (1.3%) resistant to protease inhibitors (PIs) and in four strains (5.3%) resistant to reverse transcriptase inhibitors (RTIs). In summary, this demonstrates that INIs will be susceptible to drug naïve HIV/AIDS patients in Korea.

  10. Removal of bacterial cells, antibiotic resistance genes and integrase genes by on-site hospital wastewater treatment plants: surveillance of treated hospital effluent quality

    KAUST Repository

    Timraz, Kenda

    2016-12-15

    This study aims to evaluate the removal efficiency of microbial contaminants, including total cell counts, antibiotic-resistant bacteria (ARB), antibiotic resistance genes (ARGs, e.g. tetO, tetZ, sul1 and sul2) and integrase genes (e.g. intl1 and intl2), by wastewater treatment plants (WWTPs) operated on-site of two hospitals (i.e., SH WWTP and IH WWTP). Both SH and IH WWTPs utilize the conventional activated sludge process but differences in the removal efficiencies were observed. Over the 2 week sampling period, IH WWTP outperformed SH WWTP, and achieved an approximate 0.388 to 2.49-log log removal values (LRVs) for total cell counts compared to the 0.010 to 0.162-log removal in SH WWTP. Although ARB were present in the hospital influent, the treatment process of both hospitals effectively removed ARB from most of the effluent samples. In instances where ARB were recovered in the effluent, none of the viable isolates were identified to be opportunistic pathogenic species based on 16S rRNA gene sequencing. However, sul1 and intl1 genes remained detectable at up to 105 copies per mL or 8 x 10(-1) copies per 16S rRNA gene in the treated effluent, with an LRV of less than 1.2. When the treated effluent is discharged from hospital WWTPs into the public sewer for further treatment as per requirement in many countries, the detected amount of ARGs and integrase genes in the hospital effluent can become a potential source of horizontal gene dissemination in the municipal WWTP. Proper on-site wastewater treatment and surveillance of the effluent quality for emerging contaminants are therefore highly recommended.

  11. N-terminal helix-cap in α-helix 2 modulates β-state misfolding in rabbit and hamster prion proteins.

    Science.gov (United States)

    Sweeting, Braden; Brown, Eric; Khan, M Qasim; Chakrabartty, Avijit; Pai, Emil F

    2013-01-01

    Susceptibility of a particular species to prion disease is affected by small differences in the sequence of PrP and correlates with the propensity of its PrP to assume the β-state. A helix-cap motif in the β2-α2-loop of native α-helical rabbit PrP, a resistant species, contains sequence differences that influence intra- and interspecies transmission. To determine the effect the helix-cap motif on β-state refolding propensity, we mutated S170N, S174N, and S170N/S174N of the rabbit PrP helix-cap to resemble that of hamster PrP and conversely, N170S, N174S, and N170S/N174S of hamster PrP to resemble the helix-cap of rabbit PrP. High-resolution crystal structures (1.45-1.6 Å) revealed that these mutations ablate hydrogen-bonding interactions within the helix-cap motif in rabbit PrP(C). They also alter the β-state-misfolding propensity of PrP; the serine mutations in hamster PrP decrease the propensity up to 35%, whereas the asparagine mutations in rabbit PrP increase it up to 42%. Rapid dilution of rabbit and hamster into β-state buffer conditions causes quick conversion to β-state monomers. Kinetic monitoring using size-exclusion chromatography showed that the monomer population decreases exponentially mirrored by an increase in an octameric species. The monomer-octamer transition rates are faster for hamster than for rabbit PrP. The N170S/N174S mutant of hamster PrP has a smaller octamer component at the endpoint compared to the wild-type, whereas the kinetics of octamer formation in mutant and wild-type rabbit PrP are comparable. These findings demonstrate that the sequence of the β2-α2 helix-cap affects refolding to the β-state and subsequently, may influence susceptibility to prion disease.

  12. Exactly Solvable Model for Helix-Coil-Sheet Transitions in Protein Systems

    CERN Document Server

    Schreck, John S

    2010-01-01

    In view of the important role helix-sheet transitions play in protein aggregation, we introduce a simple model to study secondary structural transitions of helix-coil-sheet systems using a Potts model starting with an effective Hamiltonian. This energy function depends on four parameters that approximately describe entropic and enthalpic contributions to the stability of a polypeptide in helical and sheet conformations. The sheet structures involve long-range interactions between residues which are far in sequence, but are in contact in real space. Such contacts are included in the Hamiltonian. Using standard statistical mechanical techniques, the partition function is solved exactly using transfer matrices. Based on this model, we study thermodynamic properties of polypeptides, including phase transitions between helix, sheet, and coil structures.

  13. The auto-inhibitory role of the EPAC hinge helix as mapped by NMR.

    Directory of Open Access Journals (Sweden)

    Rajeevan Selvaratnam

    Full Text Available The cyclic-AMP binding domain (CBD is the central regulatory unit of exchange proteins activated by cAMP (EPAC. The CBD maintains EPAC in a state of auto-inhibition in the absence of the allosteric effector, cAMP. When cAMP binds to the CBD such auto-inhibition is released, leading to EPAC activation. It has been shown that a key feature of such cAMP-dependent activation process is the partial destabilization of a structurally conserved hinge helix at the C-terminus of the CBD. However, the role of this helix in auto-inhibition is currently not fully understood. Here we utilize a series of progressive deletion mutants that mimic the hinge helix destabilization caused by cAMP to show that such helix is also a pivotal auto-inhibitory element of apo-EPAC. The effect of the deletion mutations on the auto-inhibitory apo/inactive vs. apo/active equilibrium was evaluated using recently developed NMR chemical shift projection and covariance analysis methods. Our results show that, even in the absence of cAMP, the C-terminal region of the hinge helix is tightly coupled to other conserved allosteric structural elements of the CBD and perturbations that destabilize the hinge helix shift the auto-inhibitory equilibrium toward the apo/active conformations. These findings explain the apparently counterintuitive observation that cAMP binds more tightly to shorter than longer EPAC constructs. These results are relevant for CBDs in general and rationalize why substrates sensitize CBD-containing systems to cAMP. Furthermore, the NMR analyses presented here are expected to be generally useful to quantitatively evaluate how mutations affect conformational equilibria.

  14. The auto-inhibitory role of the EPAC hinge helix as mapped by NMR.

    Science.gov (United States)

    Selvaratnam, Rajeevan; Mazhab-Jafari, Mohammad T; Das, Rahul; Melacini, Giuseppe

    2012-01-01

    The cyclic-AMP binding domain (CBD) is the central regulatory unit of exchange proteins activated by cAMP (EPAC). The CBD maintains EPAC in a state of auto-inhibition in the absence of the allosteric effector, cAMP. When cAMP binds to the CBD such auto-inhibition is released, leading to EPAC activation. It has been shown that a key feature of such cAMP-dependent activation process is the partial destabilization of a structurally conserved hinge helix at the C-terminus of the CBD. However, the role of this helix in auto-inhibition is currently not fully understood. Here we utilize a series of progressive deletion mutants that mimic the hinge helix destabilization caused by cAMP to show that such helix is also a pivotal auto-inhibitory element of apo-EPAC. The effect of the deletion mutations on the auto-inhibitory apo/inactive vs. apo/active equilibrium was evaluated using recently developed NMR chemical shift projection and covariance analysis methods. Our results show that, even in the absence of cAMP, the C-terminal region of the hinge helix is tightly coupled to other conserved allosteric structural elements of the CBD and perturbations that destabilize the hinge helix shift the auto-inhibitory equilibrium toward the apo/active conformations. These findings explain the apparently counterintuitive observation that cAMP binds more tightly to shorter than longer EPAC constructs. These results are relevant for CBDs in general and rationalize why substrates sensitize CBD-containing systems to cAMP. Furthermore, the NMR analyses presented here are expected to be generally useful to quantitatively evaluate how mutations affect conformational equilibria.

  15. Helix A Stabilization Precedes Amino-terminal Lobe Activation upon Calcium Binding to Calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Baowei [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Lowry, David [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Mayer, M. Uljana [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Squier, Thomas C. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2008-08-09

    The structural coupling between opposing domains of CaM was investigated using the conformationally sensitive biarsenical probe 4,5-bis(1,3,2-dithioarsolan-2-yl)-resorufin (ReAsH), which upon binding to an engineered tetracysteine binding motif near the end of helix A (Thr-5 to Phe-19) becomes highly fluorescent. Changes in conformation and dynamics are reflective of the native CaM structure, as there is no change in the 1H-15N HSQC NMR spectrum in comparison to wild-type CaM. We find evidence of a conformational intermediate associated with CaM activation, where calcium occupancy of sites in the amino-terminal and carboxyl-terminal lobes of CaM differentially affect the fluorescence intensity of bound ReAsH. Insight into the structure of the conformational intermediate is possible from a consideration of calcium-dependent changes in rates of ReAsH binding and helix A mobility, which respectively distinguish secondary structural changes associated with helix A stabilization from the tertiary structural reorganization of the amino-terminal lobe of CaM necessary for high-affinity binding to target proteins. Helix A stabilization is associated with calcium occupancy of sites in the carboxyl-terminal lobe (Kd = 0.36 ± 0.04 μM), which results in a reduction in the rate of ReAsH binding from 4900 M-1 sec-1 to 370 M-1 sec-1. In comparison, tertiary structural changes involving helix A and other structural elements in the amino-terminal lobe requires calcium-occupancy of amino-terminal sites (Kd = 18 ± 3 μM). Observed secondary and tertiary structural changes involving helix A in response to the sequential calcium occupancy of carboxyl- and amino-terminal lobe calcium binding sites suggest an important involvement of helix A in mediating the structural coupling between the opposing domains of CaM. These results are discussed in terms of a model in which carboxyl-terminal lobe calcium activation induces

  16. The signaling helix: a common functional theme in diverse signaling proteins

    Directory of Open Access Journals (Sweden)

    Aravind L

    2006-09-01

    Full Text Available Abstract Background The mechanism by which the signals are transmitted between receptor and effector domains in multi-domain signaling proteins is poorly understood. Results Using sensitive sequence analysis methods we identify a conserved helical segment of around 40 residues in a wide range of signaling proteins, including numerous sensor histidine kinases such as Sln1p, and receptor guanylyl cyclases such as the atrial natriuretic peptide receptor and nitric oxide receptors. We term this helical segment the signaling (S-helix and present evidence that it forms a novel parallel coiled-coil element, distinct from previously known helical segments in signaling proteins, such as the Dimerization-Histidine phosphotransfer module of histidine kinases, the intra-cellular domains of the chemotaxis receptors, inter-GAF domain helical linkers and the α-helical HAMP module. Analysis of domain architectures allowed us to reconstruct the domain-neighborhood graph for the S-helix, which showed that the S-helix almost always occurs between two signaling domains. Several striking patterns in the domain neighborhood of the S-helix also became evident from the graph. It most often separates diverse N-terminal sensory domains from various C-terminal catalytic signaling domains such as histidine kinases, cNMP cyclase, PP2C phosphatases, NtrC-like AAA+ ATPases and diguanylate cyclases. It might also occur between two sensory domains such as PAS domains and occasionally between a DNA-binding HTH domain and a sensory domain. The sequence conservation pattern of the S-helix revealed the presence of a unique constellation of polar residues in the dimer-interface positions within the central heptad of the coiled-coil formed by the S-helix. Conclusion Combining these observations with previously reported mutagenesis studies on different S-helix-containing proteins we suggest that it functions as a switch that prevents constitutive activation of linked downstream

  17. An octopaminergic system in the CNS of the snails, Lymnaea stagnalis and Helix pomatia

    OpenAIRE

    1998-01-01

    Octopamine (OA) levels in each ganglion of the terrestrial snail, Helix pomatia, and the pond snail, Lymnaea stagnalis, were measured by using the HPLC technique. In both species an inhomogeneous distribution of OA was found in the central nervous system. The buccal ganglia contained a concentration of OA (12.6 pmol mg-1 and 18.8 pmol mg-1) that was two to three times higher than the pedal (4.93 pmol mg-1 and 9.2 pmol mg-1) or cerebral (4.46 pmol mg-1 and 4.9 pmol mg-1) ganglia of Helix and L...

  18. Discovery of HIV fusion inhibitors targeting gp41 using a comprehensive α-helix mimetic library

    Science.gov (United States)

    Whitby, Landon R.; Boyle, Kristopher E.; Cai, Lifeng; Yu, Xiaoqian; Gochin, Miriam; Boger, Dale L.

    2012-01-01

    The evaluation of a comprehensive α-helix mimetic library for binding the gp41 NHR hydrophobic pocket recognizing an intramolecular CHR α-helix provided a detailed depiction of structural features required for binding and led to the discovery of small molecule inhibitors (Ki 0.6–1.3 µM) that not only match or exceed the potency of those disclosed over the past decade, but that also exhibit effective activity in a cell–cell fusion assay (IC50 5–8 µM). PMID:22424973

  19. Radio-Frequency Characteristics of a Printed Rectangular Helix Slow-Wave Structure

    Institute of Scientific and Technical Information of China (English)

    FU Cheng-Fang; WEI Yan-Yu; WANG Wen-Xiang; GONG Yu-Bin

    2008-01-01

    A new type of printed rectangular he/ix slow-wave structure (SWS) is investigated using the field-matching method and the electromagnetic integral equations at the boundaries. The radio-frequency characteristics including the dispersion equation and the coupling impedance for transverse antisymmetric (odd) modes of this structure are analysed. The numerical results agree well with the results obtained by the EM simulation software HFSS. It is shown that the dispersion of the rectangular helix circuit is weakened, the phase velocity is reduced after filling the dielectric materials in the rectangular helix SWS. As a planar slow-wave structure, this structure has potential applications in compact TWTs.

  20. Rotational symmetry and the transformation of innovation systems in a Triple Helix of university-industry-government relations

    NARCIS (Netherlands)

    Ivanova, I.A.; Leydesdorff, L.

    2014-01-01

    Using a mathematical model, we show that a Triple Helix (TH) system contains self-interaction, and therefore self-organization of innovations can be expected in waves, whereas a Double Helix (DH) remains determined by its linear constituents. (The mathematical model is fully elaborated in the Append

  1. Rotational symmetry and the transformation of innovation systems in a Triple Helix of university-industry-government relations

    NARCIS (Netherlands)

    Ivanova, I.A.; Leydesdorff, L.

    2014-01-01

    Using a mathematical model, we show that a Triple Helix (TH) system contains self-interaction, and therefore self-organization of innovations can be expected in waves, whereas a Double Helix (DH) remains determined by its linear constituents. (The mathematical model is fully elaborated in the Append

  2. A secondary structural transition in the C-helix promotes gating of cyclic nucleotide-regulated ion channels.

    Science.gov (United States)

    Puljung, Michael C; Zagotta, William N

    2013-05-03

    Cyclic nucleotide-regulated ion channels bind second messengers like cAMP to a C-terminal domain, consisting of a β-roll, followed by two α-helices (B- and C-helices). We monitored the cAMP-dependent changes in the structure of the C-helix of a C-terminal fragment of HCN2 channels using transition metal ion FRET between fluorophores on the C-helix and metal ions bound between histidine pairs on the same helix. cAMP induced a change in the dimensions of the C-helix and an increase in the metal binding affinity of the histidine pair. cAMP also caused an increase in the distance between a fluorophore on the C-helix and metal ions bound to the B-helix. Stabilizing the C-helix of intact CNGA1 channels by metal binding to a pair of histidines promoted channel opening. These data suggest that ordering of the C-helix is part of the gating conformational change in cyclic nucleotide-regulated channels.

  3. Progress of φC31 integrase system in site-specific integration%φC31整合酶系统介导的位点特异性整合研究进展

    Institute of Scientific and Technical Information of China (English)

    马晴雯

    2011-01-01

    来源于链霉菌(Streptomyces)噬菌体φC31的整合酶可介导链霉菌附着位点(attB)和噬菌体附着位点(attP)之间的同源重组,这种重组亦可在多种动植物细胞内进行;而且,该整合酶还可介导含attB位点的载体以位点特异性方式整合于多种真核生物基因组内的假attP位点,并使转基因持续高效表达.因此,φC31整合酶在基因修饰、基因治疗及转基因动物研制等方面得到了广泛的应用.文章就近年来φC31整合酶整合规律、提高效率方面的改进及安全性等相关领域的研究进展进行了综述.%Integrase of phage φC31 catalyses the homologous recombination between Streptomyces attachment site attB and the phage attachment site attP.Meanwhile, this integrase can mediate integration of attB-containing donor plasmids into the pseudo attP sites in eukaryotic genomes by a site-specific manner and resulting long-term and robust expression of integrated genes.Nowadays, φC31 integrase system is becoming a potential tool for genome modification, gene therapy and transgenic research.Recent progress of φC31 integrase system in integration mode in mammalian genomes, efficiency improvement and researches concemed on transgenic safety were summarized in this review.

  4. Symmetrical-geometry constructions defining helicoidal biostructures. The case of alpha-helix

    CERN Document Server

    Samoylovich, Mikhail

    2016-01-01

    The chain of algebraic geometry constructions permits to transfer from the minimal surface with zero instability index, and from the lattice over the ring of cyclotomic integers to the tetra-block helix. The tetra-block is the 7-vertex joining of four tetrahedra sharing common faces; it is considered as a building unit for structures approximated by the chains of regular tetrahedra. The minimality condition of the 7 - vertex tetrablock as a building unit is the consequence of its unique mapping by the Klein's quartic (which is characterized by the minimal hyperbolic Schwartz triangle) into the minimal finite projective geometry. The topological stability of this helix provided by the pitch to radius ratio H/R of 2{\\pi}/({\\tau}+1) ({\\tau} is the golden section) and by the local rotation axis order of 40/11=40exp(-H/R). These parameters determine the helix of C{\\alpha} atoms inside the alpha - helix with the accuracy of up to 2%. They explain also the bonding relationship i -- i+4 between the i-th amide group a...

  5. Rendezvous of the "Third Kind": Triple Helix Origins and Future Possibilities

    Science.gov (United States)

    Etzkowitz, Henry

    2015-01-01

    The Triple Helix, representing university-industry-government interactions, was rooted in a 1993 International Workshop on University-Industry Relations at UNAM's Centro Para la Innovacion Technologica in Mexico City. Impelled by Mexican reality, where university-industry interactions and the institutions themselves operated within a governmental…

  6. Alpha-helix <-> random coil phase transition: analysis of ab initio theory predictions

    DEFF Research Database (Denmark)

    Solov'yov, Ilia; Yakubovich, Alexander V.; Solov'yov, Andrey V.;

    2008-01-01

    In the present paper we present results of calculations obtained with the use of the theoretical method described in our preceding paper [Eur. Phys. J. D, DOI 10.1140/epjd/e2007-00328-9] and perform detail analysis of -helix random coil transition in alanine polypeptides of different length. We...

  7. Unpredictable responses of garden snail (Helix aspersa) populations to climate change

    NARCIS (Netherlands)

    Bezemer, T.M.; Knight, K.J.

    2001-01-01

    We studied the impact of climate change on the population dynamics of the garden snail (Helix aspersa) in the Ecotron controlled environment facility. The experimental series ran for three plant generations, allowing the snails to reproduce. We investigated the isolated and combined effects of eleva

  8. [Stages of organogenesis and cytodifferentiation of the albumen gland in the snail Helix aspersa Muller].

    Science.gov (United States)

    Courtot, A M; Gomot, L

    1982-01-01

    The differentiation of the albumen gland of the pulmonate stylommatophora Helix aspersa has been divided into five stages. An ultrastructural study showed, the differentiation of undifferentiated epithelial cells into two cell types: ciliated cells and secretory cells. The glandular differentiation of epithelial cells was characterized by the development of ergastoplasma and the Golgi apparatus which were both involved in protein and galactogen synthesis.

  9. Restrictions on TWT Helix Voltage Ripple for Acceptable Notch Filter Performance

    Energy Technology Data Exchange (ETDEWEB)

    Hyslop, B.

    1984-12-01

    An ac ripple on the helix voltage of the 1-2 GHz TWT's creates FM sidebands that cause amplitude and phase modulation of the microwave TWT output signal. A limit of 16 volts peak-to-peak is required for acceptable superconducting notch filter performance.

  10. Desain Antena Helix Quadrifilar pada Frekuensi 2,4 GHz Untuk Perangkat Ground Station Satelit Nano

    Directory of Open Access Journals (Sweden)

    Vivin Violita

    2013-09-01

    Full Text Available Pada penelitian ini akan dibuat desain antena helix quadrifilar untuk ground station satelit nano yang bekerja pada frekuensi S-band 2,4 GHz. Antena ini membutuhkan arus yang berbeda fase 900 untuk mengeksitasi pencatuannya. Untuk menghasilkan arus tersebut tanpa menambah perangkat pencatu tambahan, maka antena ini menggunakan metode self-phased. Pada metode self-phased, digunakan lilitan kawat yang berbeda dimensi. Antena ini terdiri dari dua lilitan kawat tembaga yang memiliki dimensi berbeda, yang kemudian disebut smaller loop dan larger loop. Perbedaan dimensi ini akan menyebabkan resistansi smaller loop bersifat kapasitif dan resistansi larger loop bersifat induktif. Reflektor parabola ditambahkan pada antena helix quadrifilar untuk meningkatkan gain dan direktivitas. Hasil simulasi serta implementasi menunjukkan bahwa antena helix quadrifilar telah memenuhi kriteria desain . Antena ini menggunakan metode pencatuan self-phased. Pola radiasi yang dihasilkan merupakan directional. Nilai return loss dari hasil pengukuran bernilai -21.45 dB dengan VSWR 1.17. Bandwidth yang didapatkan adalah 18,53% dari frekuensi tengah 2.4 GHz atau sebesar 444.8 MHz. Impedansi hasil pengukuran sebesar 57.68 Ω. Gain antena helix quadrifilar dengan reflektor parabola adalah 20,61 dB.

  11. Predicting transmembrane helix packing arrangements using residue contacts and a force-directed algorithm.

    Science.gov (United States)

    Nugent, Timothy; Jones, David T

    2010-03-19

    Alpha-helical transmembrane proteins constitute roughly 30% of a typical genome and are involved in a wide variety of important biological processes including cell signalling, transport of membrane-impermeable molecules and cell recognition. Despite significant efforts to predict transmembrane protein topology, comparatively little attention has been directed toward developing a method to pack the helices together. Here, we present a novel approach to predict lipid exposure, residue contacts, helix-helix interactions and finally the optimal helical packing arrangement of transmembrane proteins. Using molecular dynamics data, we have trained and cross-validated a support vector machine (SVM) classifier to predict per residue lipid exposure with 69% accuracy. This information is combined with additional features to train a second SVM to predict residue contacts which are then used to determine helix-helix interaction with up to 65% accuracy under stringent cross-validation on a non-redundant test set. Our method is also able to discriminate native from decoy helical packing arrangements with up to 70% accuracy. Finally, we employ a force-directed algorithm to construct the optimal helical packing arrangement which demonstrates success for proteins containing up to 13 transmembrane helices. This software is freely available as source code from http://bioinf.cs.ucl.ac.uk/memsat/mempack/.

  12. Predicting transmembrane helix packing arrangements using residue contacts and a force-directed algorithm.

    Directory of Open Access Journals (Sweden)

    Timothy Nugent

    2010-03-01

    Full Text Available Alpha-helical transmembrane proteins constitute roughly 30% of a typical genome and are involved in a wide variety of important biological processes including cell signalling, transport of membrane-impermeable molecules and cell recognition. Despite significant efforts to predict transmembrane protein topology, comparatively little attention has been directed toward developing a method to pack the helices together. Here, we present a novel approach to predict lipid exposure, residue contacts, helix-helix interactions and finally the optimal helical packing arrangement of transmembrane proteins. Using molecular dynamics data, we have trained and cross-validated a support vector machine (SVM classifier to predict per residue lipid exposure with 69% accuracy. This information is combined with additional features to train a second SVM to predict residue contacts which are then used to determine helix-helix interaction with up to 65% accuracy under stringent cross-validation on a non-redundant test set. Our method is also able to discriminate native from decoy helical packing arrangements with up to 70% accuracy. Finally, we employ a force-directed algorithm to construct the optimal helical packing arrangement which demonstrates success for proteins containing up to 13 transmembrane helices. This software is freely available as source code from http://bioinf.cs.ucl.ac.uk/memsat/mempack/.

  13. Monosaccharide templates for de novo designed 4-alpha-helix bundle proteins: template effects in carboproteins

    DEFF Research Database (Denmark)

    Brask, Jesper; Dideriksen, J.M.; Nielsen, John;

    2003-01-01

    De novo design and total chemical synthesis of proteins provide powerful approaches to critically test our understanding of protein folding, structure, and stability. The 4-alpha-helix bundle is a frequently studied structure in which four amphiphilic alpha-helical peptide strands form a hydropho......)) and melting points in chemical and thermal denaturation experiments....

  14. A triple Helix Approach to the Future Innovation Flagship of Europe:

    DEFF Research Database (Denmark)

    Jofre, Sergio; Andersen, Per Dannemand

    2009-01-01

    , are transforming the profile of the triple helix relationships. This transformation is bringing the American and the Japanese innovation system to an unprecedented level of commonality and the EU to a yet uncertain stage of transition characterized by the conflict between national and supranational priorities...

  15. On the Helix Propensity in Generalized Born Solvent Descriptions of Modeling the Dark Proteome

    Science.gov (United States)

    2017-01-10

    and their probability density of states on a conformational landscape . Particularly missing among the reported studies are comparative assessments of...multidimensional energy landscape . This work explores the application of parallel tempering methods with implicit solvent models as a computational...turn-helix fold upon molecular association with the Ebola protein NP. An assessment is provided of the accuracy of two generalized Born solvent

  16. pH-jump induced α-helix folding of poly-L-glutamic acid

    Energy Technology Data Exchange (ETDEWEB)

    Donten, Mateusz L. [Institute of Physical Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich (Switzerland); Hamm, Peter, E-mail: phamm@pci.uzh.ch [Institute of Physical Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich (Switzerland)

    2013-08-30

    Highlights: ► pH-jump as truly biomimetic tool to initiate non-equilibrium dynamics of biomolecules. ► Design criteria to widen the applicability of pH-jumps are developed. ► Folding of poly-L-Glu in dependence of starting pH, pH jump size and helix length. ► Length dependence provides strong evidence for a nucleation–propagation scenario. - Abstract: pH jumps are a truly biomimetic technique to initiate non-equilibrium dynamics of biomolecules. In this work, the pH jump induced α-helix folding of poly-L-glutamic acid is investigated upon proton release from o-nitrobenzaldehyde. The aim of this work is twofold: On the one hand, design criteria of pH jump experiments are discussed, on the other hand, the folding mechanism of poly-L-glutamic acid is clarified by probing the IR response of the amide I band. Its folding kinetics is studied in dependence of the starting pD, the size of the pD jump and the length of the helix. While no dependence on the first two parameters could be detected, the folding time varies from 0.6 μs to 1.8 μs for helix lengths of 20 residue to 440 residue, respectively. It converges to a long-length limit at about 50 residue, a result which is attributed to a nucleation–propagation mechanism.

  17. The triple-helix model of smart cities: a neo-evolutionary perspective

    NARCIS (Netherlands)

    Leydesdorff, L.; Deakin, M.

    2011-01-01

    This paper sets out to demonstrate how the triple-helix model enables us to study the knowledge base of an urban economy in terms of its civil society's support for the evolution of the city as a key component of an innovation system. It argues that cities can be considered as densities in networks

  18. CFD analysis and flow model reduction for surfactant production in helix reactor

    NARCIS (Netherlands)

    Nikačević, N.M.; Thielen, L.; Twerda, A.; Hof, P.M.J. van den

    2014-01-01

    Flow pattern analysis in a spiral Helix reactor is conducted, for the application in the commercial surfactant production. Step change response curves (SCR) were obtained from numerical tracer experiments by three-dimensional computational fluid dynamics (CFD) simulations. Non-reactive flow is simul

  19. Characterization by immunocytochemistry of ionic channels in Helix aspersa suboesophageal brain ganglia neurons.

    Science.gov (United States)

    Azanza, M J; Pérez-Castejón, C; Pes, N; Pérez-Bruzón, R N; Aisa, J; Junquera, C; Maestú, C; Lahoz, M; Martínez-Ciriano, C; Vera-Gil, A; Del Moral, A

    2008-04-01

    The aim of this work was to characterize several ionic channels in nervous cells of the suboesophageal visceral, left and right parietal, and left and right pleural brain ganglia complex of the snail Helix aspersa by immunocytochemistry. We have studied the immunostaining reaction for a wide panel of eleven polyclonal antibodies raised against mammal antigens as follows: voltage-gated-Na+ channel; voltage-gated-delayed-rectifier-K+ channel; SK2-small-conductance-Ca2+-dependent-K+ channel apamin sensitive; SK3 potassium channel; charybdotoxin-sensitive voltage-dependent potassium channel; BKCa-maxi-conductance-Ca2+-dependent-K+ channel; hyperpolarization-activated cyclic nucleotide-gated potassium channel 4; G-protein-activated inwardly rectifying potassium channel GIRK2 and voltage-gated-calcium of L, N and P/Q type channels. Our results show positive reaction in neurons, but neither in glia cells nor in processes in the Helix suboesophageal ganglia. Our results suggest the occurrence of molecules in Helix neurons sharing antigenic determinants with mammal ionic channels. The reaction density and distribution of immunoreactive staining within neurons is specific for each one of the antisera tested. The studies of co-localization of immunoreaction, on alternate serial sections of the anterior right parietal ganglion, have shown for several recognized mapped neurons that they can simultaneously be expressed among two and seven different ionic protein channels. These results are considered a key structural support for the interpretation of Helix aspersa neuron electrophysiological activity.

  20. A Role-Based Approach to Adult Development: The Triple-Helix Model.

    Science.gov (United States)

    Juhasz, Anne McCreary

    1989-01-01

    Presents triple-helix model of adult development which incorporates three major roles: family, work, and self, each powered by drive for self-esteem. Asserts that this approach accommodates wide range of possible patterns and varied timing of life events relative to career options, family and relationship choices, and emphasis on self-development.…

  1. The triple-helix model of smart cities: a neo-evolutionary perspective

    NARCIS (Netherlands)

    Leydesdorff, L.; Deakin, M.

    2011-01-01

    This paper sets out to demonstrate how the triple-helix model enables us to study the knowledge base of an urban economy in terms of its civil society's support for the evolution of the city as a key component of an innovation system. It argues that cities can be considered as densities in networks

  2. The University in the Knowledge Economy: The Triple Helix Model and Its Implications

    Science.gov (United States)

    Zheng, Peijun; Harris, Michael

    2007-01-01

    In the context of the global knowledge economy, the three major players--university, industry, and government--are becoming increasingly interdependent. As more intensified interactions and relationships of increasing complexity among the institutions evolve, the Triple Helix model attempts to describe not only interactions among university,…

  3. Open Innovation, Triple Helix and Regional Innovation Systems: Exploring CATAPULT Centres in the UK

    Science.gov (United States)

    Kerry, Christopher; Danson, Michael

    2016-01-01

    Through the lens of UK CATAPULT Centres this conceptual paper presents an examination of the links between open innovation, the Triple Helix model and regional innovation systems. Highlighting the importance of boundary-spanning intermediaries, the combined role of these concepts is explored in detail. A conceptual model is then proposed which…

  4. Reversible Helix Sense Inversion in Surface-Grafted Poly(β-phenethyl-L-aspartate) Films

    NARCIS (Netherlands)

    Luijten, Jeroen; Vorenkamp, Eltjo J.; Schouten, Arend J.

    2007-01-01

    The reversible manipulation of the helix screw sense in surface-grafted poly(β-phenethyl-L-aspartate) (PPELA) films by means of external stimuli was investigated. Ringopening polymerization of β-phenethyl-L-aspartate N-carboxyanhydride initiated from primary amino-functionalized silicon and quartz s

  5. Reversible helix sense inversion in surface-grafted poly(beta-phenethyl-L-aspartate) films

    NARCIS (Netherlands)

    Luijten, Jeroen; Vorenkamp, Eltjo J.; Schouten, Arend J.

    2007-01-01

    The reversible manipulation of the helix screw sense in surface-grafted poly(beta-phenethyl-L-aspartate) (PPELA) films by means of external stimuli was investigated. Ringopening polymerization of beta-phenethyl-L-aspartate N-carboxyanhydride initiated from primary amino-functionalized silicon and qu

  6. Nano-mechanical characterization of tension-sensitive helix bundles in talin rod.

    Science.gov (United States)

    Maki, Koichiro; Nakao, Nobuhiko; Adachi, Taiji

    2017-03-04

    Tension-induced exposure of a cryptic signaling binding site is one of the most fundamental mechanisms in molecular mechanotransduction. Helix bundles in rod domains of talin, a tension-sensing protein at focal adhesions, unfurl under tension to expose cryptic vinculin binding sites. Although the difference in their mechanical stabilities would determine which helix bundle is tension-sensitive, their respective mechanical behaviors under tension have not been characterized. In this study, we evaluated the mechanical behaviors of residues 486-654 and 754-889 of talin, which form helix bundles with low and high tension-sensitivity, by employing AFM nano-tensile testing. As a result, residues 754-889 exhibited lower unfolding energy for complete unfolding than residues 486-654. In addition, we found that residues 754-889 transition into intermediate conformations under lower tension than residues 486-654. Furthermore, residues 754-889 showed shorter persistence length in the intermediate conformation than residues 486-654, suggesting that residues 754-889 under tension exhibit separated α-helices, while residues 486-654 assume a compact conformation with inter-helix interactions. Therefore, we suggest that residues 754-889 of talin work as a tension-sensitive domain to recruit vinculin at the early stage of focal adhesion development, while residues 486-654 contribute to rather robust tension-sensitivity by recruiting vinculin under high tension.

  7. A note on the pollen representation of ivy (Hedera helix L.)

    NARCIS (Netherlands)

    Bottema, S

    2001-01-01

    Pollen productivity and dispersal of ivy (Hedera helix) is discussed on the basis of a simple surface sample study in a situation that has been followed for about 30 yr. The pollen production of a special ivy plant was estimated for the flowering season of 1998. Hedera pollen dispersal was followed

  8. Synthetic Aperture Focusing for a Single Element Transducer undergoing Helix Motion

    DEFF Research Database (Denmark)

    Andresen, Henrik; Nikolov, Svetoslav Ivanov; Jensen, Jørgen Arendt

    2011-01-01

    This paper describes the application of 3D synthetic aperture focusing (SAF) to a single element trans-rectal ultrasound transducer. The transducer samples a 3D volume by simultaneous rotation and translation giving a helix motion. Two different 3D SAF methods are investigated, a direct and a two...

  9. The close-packed triple helix as a possible new structural motif for collagen

    CERN Document Server

    Bohr, Jakob

    2010-01-01

    The one-dimensional problem of selecting the triple helix with the highest volume fraction is solved and hence the condition for a helix to be close-packed is obtained. The close-packed triple helix is shown to have a pitch angle of $v_{CP} =43.3 ^\\circ$. Contrary to the conventional notion, we suggest that close packing form the underlying principle behind the structure of collagen, and the implications of this suggestion are considered. Further, it is shown that the unique zero-twist structure with no strain-twist coupling is practically identical to the close-packed triple helix. Some of the difficulties for the current understanding of the structure of collagen are reviewed: The ambiguity in assigning crystal structures for collagen-like peptides, and the failure to satisfactorily calculate circular dichroism spectra. Further, the proposed new geometrical structure for collagen is better packed than both the 10/3 and the 7/2 structure. A feature of the suggested collagen structure is the existence of a ce...

  10. Infrared spectroscopic studies of cells and tissues: triple helix proteins as a potential biomarker for tumors.

    Science.gov (United States)

    Stelling, Allison L; Toher, Deirdre; Uckermann, Ortrud; Tavkin, Jelena; Leipnitz, Elke; Schweizer, Julia; Cramm, Holger; Steiner, Gerald; Geiger, Kathrin D; Kirsch, Matthias

    2013-01-01

    In this work, the infrared (IR) spectra of living neural cells in suspension, native brain tissue, and native brain tumor tissue were investigated. Methods were developed to overcome the strong IR signal of liquid water so that the signal from the cellular biochemicals could be seen. Measurements could be performed during surgeries, within minutes after resection. Comparison between normal tissue, different cell lineages in suspension, and tumors allowed preliminary assignments of IR bands to be made. The most dramatic difference between tissues and cells was found to be in weaker IR absorbances usually assigned to the triple helix of collagens. Triple helix domains are common in larger structural proteins, and are typically found in the extracellular matrix (ECM) of tissues. An algorithm to correct offsets and calculate the band heights and positions of these bands was developed, so the variance between identical measurements could be assessed. The initial results indicate the triple helix signal is surprisingly consistent between different individuals, and is altered in tumor tissues. Taken together, these preliminary investigations indicate this triple helix signal may be a reliable biomarker for a tumor-like microenvironment. Thus, this signal has potential to aid in the intra-operational delineation of brain tumor borders.

  11. A peptide study of the relationship between the collagen triple-helix and amyloid.

    Science.gov (United States)

    Parmar, Avanish S; Nunes, Ana Monica; Baum, Jean; Brodsky, Barbara

    2012-10-01

    Type XXV collagen, or collagen-like amyloidogenic component, is a component of amyloid plaques, and recent studies suggest this collagen affects amyloid fibril elongation and has a genetic association with Alzheimer's disease. The relationship between the collagen triple helix and amyloid fibrils was investigated by studying peptide models, including a very stable triple helical peptide (Pro-Hyp-Gly)₁₀ , an amyloidogenic peptide GNNQQNY, and a hybrid peptide where the GNNQQNY sequence was incorporated between (GPO)(n) domains. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopy showed the GNNQQNY peptide formed a random coil structure, whereas the hybrid peptide contained a central disordered GNNQQNY region transitioning to triple-helical ends. Light scattering confirmed the GNNQQNY peptide had a high propensity to form amyloid fibrils, whereas amyloidogenesis was delayed in the hybrid peptide. NMR data suggested the triple-helix constraints on the GNNQQNY sequence within the hybrid peptide may disfavor the conformational change necessary for aggregation. Independent addition of a triple-helical peptide to the GNNQQNY peptide under aggregating conditions delayed nucleation and amyloid fibril growth. The inhibition of amyloid nucleation depended on the Gly-Xaa-Yaa sequence and required the triple-helix conformation. The inhibitory effect of the collagen triple-helix on an amyloidogenic sequence, when in the same molecule or when added separately, suggests Type XXV collagen, and possibly other collagens, may play a role in regulating amyloid fibril formation.

  12. Inside the Triple Helix: An Integrative Conceptual Framework of the Academic Researcher's Activities, a Systematic Review

    Science.gov (United States)

    Halilem, Norrin

    2010-01-01

    In the Triple Helix of University-Industry-Government relations, the academic researcher plays a predominant role as he participates in research, which provides opportunities for innovation; in teaching, which develops highly qualified personnel; and in entrepreneurialism, which represents the transformation of knowledge in a more usable form, and…

  13. Structural insights into the stabilization of MALAT1 noncoding RNA by a bipartite triple helix.

    Science.gov (United States)

    Brown, Jessica A; Bulkley, David; Wang, Jimin; Valenstein, Max L; Yario, Therese A; Steitz, Thomas A; Steitz, Joan A

    2014-07-01

    Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a highly abundant nuclear long noncoding RNA that promotes malignancy. A 3'-stem-loop structure is predicted to confer stability by engaging a downstream A-rich tract in a triple helix, similar to the expression and nuclear retention element (ENE) from the KSHV polyadenylated nuclear RNA. The 3.1-Å-resolution crystal structure of the human MALAT1 ENE and A-rich tract reveals a bipartite triple helix containing stacks of five and four U•A-U triples separated by a C+•G-C triplet and C-G doublet, extended by two A-minor interactions. In vivo decay assays indicate that this blunt-ended triple helix, with the 3' nucleotide in a U•A-U triple, inhibits rapid nuclear RNA decay. Interruption of the triple helix by the C-G doublet induces a 'helical reset' that explains why triple-helical stacks longer than six do not occur in nature.

  14. Infrared spectroscopic studies of cells and tissues: triple helix proteins as a potential biomarker for tumors.

    Directory of Open Access Journals (Sweden)

    Allison L Stelling

    Full Text Available In this work, the infrared (IR spectra of living neural cells in suspension, native brain tissue, and native brain tumor tissue were investigated. Methods were developed to overcome the strong IR signal of liquid water so that the signal from the cellular biochemicals could be seen. Measurements could be performed during surgeries, within minutes after resection. Comparison between normal tissue, different cell lineages in suspension, and tumors allowed preliminary assignments of IR bands to be made. The most dramatic difference between tissues and cells was found to be in weaker IR absorbances usually assigned to the triple helix of collagens. Triple helix domains are common in larger structural proteins, and are typically found in the extracellular matrix (ECM of tissues. An algorithm to correct offsets and calculate the band heights and positions of these bands was developed, so the variance between identical measurements could be assessed. The initial results indicate the triple helix signal is surprisingly consistent between different individuals, and is altered in tumor tissues. Taken together, these preliminary investigations indicate this triple helix signal may be a reliable biomarker for a tumor-like microenvironment. Thus, this signal has potential to aid in the intra-operational delineation of brain tumor borders.

  15. Rendezvous of the "Third Kind": Triple Helix Origins and Future Possibilities

    Science.gov (United States)

    Etzkowitz, Henry

    2015-01-01

    The Triple Helix, representing university-industry-government interactions, was rooted in a 1993 International Workshop on University-Industry Relations at UNAM's Centro Para la Innovacion Technologica in Mexico City. Impelled by Mexican reality, where university-industry interactions and the institutions themselves operated within a governmental…

  16. Efficient Fatigue Analysis of Helix Elements in Umbilicals and Flexible Risers: Theory and Applications

    Directory of Open Access Journals (Sweden)

    Geir Skeie

    2012-01-01

    Full Text Available Fatigue analysis of structural components such as helix tensile armors and steel tubes is a critical design issue for dynamic umbilicals and flexible pipes. The basis for assessment of fatigue damage of such elements is the long-term stress cycle distribution at critical locations on the helix elements caused by long-term environmental loading on the system. The long-term stress cycle distribution will hence require global dynamic time domain analysis followed by a detailed cross-sectional analysis in a large number of irregular sea states. An overall computational consistent and efficient fatigue analysis scheme is outlined with due regard of the cross-sectional analysis technique required for fatigue stress calculation with particular attention to the helix elements. The global cross-section is exposed to pure bending, tensile, torsion, and pressure loading. The state of the different cross-section elements is based on the global response. Special emphasis is placed on assessment of friction stresses caused by the stick-slip behavior of helix elements in bending that are of special importance for fatigue life assessments. The described cross-sectional analysis techniques are based on an extensive literature survey and are hence considered to represent industry consensus. The performance of the described calculation scheme is illustrated by case studies.

  17. Effect of four-alpha-helix bundle cavity size on volatile anesthetic binding energetics.

    Science.gov (United States)

    Manderson, Gavin A; Michalsky, Stuart J; Johansson, Jonas S

    2003-09-30

    Currently, it is thought that inhalational anesthetics cause anesthesia by binding to ligand-gated ion channels. This is being investigated using four-alpha-helix bundles, small water-soluble analogues of the transmembrane domains of the "natural" receptor proteins. The study presented here specifically investigates how multiple alanine-to-valine substitutions (which each decrease the volume of the internal binding cavity by 38 A(3)) affect structure, stability, and anesthetic binding affinity of the four-alpha-helix bundles. Structure remains essentially unchanged when up to four alanine residues are changed to valine. However, stability increases as the number of these substitutions is increased. Anesthetic binding affinities are also affected. Halothane binds to the four-alpha-helix bundle variants with 0, 1, and 2 substitutions with equivalent affinities but binds to the variants with 3 and 4 more tightly. The same order of binding affinities was observed for chloroform, although for a particular variant, chloroform was bound less tightly. The observed differences in binding affinities may be explained in terms of a modulation of van der Waals and hydrophobic interactions between ligand and receptor. These, in turn, could result from increased four-alpha-helix bundle binding cavity hydrophobicity, a decrease in cavity size, or improved ligand/receptor shape complementarity.

  18. Modelling packing interactions in parallel helix bundles: pentameric bundles of nicotinic receptor M2 helices.

    Science.gov (United States)

    Sankararamakrishnan, R; Sansom, M S

    1995-11-01

    The transbilayer pore of the nicotinic acetylcholine receptor (nAChR) is formed by a pentameric bundle of M2 helices. Models of pentameric bundles of M2 helices have been generated using simulated annealing via restrained molecular dynamics. The influence of: (a) the initial C alpha template; and (b) screening of sidechain electrostatic interactions on the geometry of the resultant M2 helix bundles is explored. Parallel M2 helices, in the absence of sidechain electrostatic interactions, pack in accordance with simple ridges-in-grooves considerations. This results in a helix crossing angle of ca. +12 degrees, corresponding to a left-handed coiled coil structure for the bundle as a whole. Tilting of M2 helices away from the central pore axis at their C-termini and/or inclusion of sidechain electrostatic interactions may perturb such ridges-in-grooves packing. In the most extreme cases right-handed coiled coils are formed. An interplay between inter-helix H-bonding and helix bundle geometry is revealed. The effects of changes in electrostatic screening on the dimensions of the pore mouth are described and the significance of these changes in the context of models for the nAChR pore domain is discussed.

  19. Identification of a site critical for kinase regulation on the central processing unit (CPU) helix of the aspartate receptor.

    Science.gov (United States)

    Trammell, M A; Falke, J J

    1999-01-05

    Ligand binding to the homodimeric aspartate receptor of Escherichia coli and Salmonella typhimurium generates a transmembrane signal that regulates the activity of a cytoplasmic histidine kinase, thereby controlling cellular chemotaxis. This receptor also senses intracellular pH and ambient temperature and is covalently modified by an adaptation system. A specific helix in the cytoplasmic domain of the receptor, helix alpha6, has been previously implicated in the processing of these multiple input signals. While the solvent-exposed face of helix alpha6 possesses adaptive methylation sites known to play a role in kinase regulation, the functional significance of its buried face is less clear. This buried region lies at the subunit interface where helix alpha6 packs against its symmetric partner, helix alpha6'. To test the role of the helix alpha6-helix alpha6' interface in kinase regulation, the present study introduces a series of 13 side-chain substitutions at the Gly 278 position on the buried face of helix alpha6. The substitutions are observed to dramatically alter receptor function in vivo and in vitro, yielding effects ranging from kinase superactivation (11 examples) to complete kinase inhibition (one example). Moreover, four hydrophobic, branched side chains (Val, Ile, Phe, and Trp) lock the kinase in the superactivated state regardless of whether the receptor is occupied by ligand. The observation that most side-chain substitutions at position 278 yield kinase superactivation, combined with evidence that such facile superactivation is rare at other receptor positions, identifies the buried Gly 278 residue as a regulatory hotspot where helix packing is tightly coupled to kinase regulation. Together, helix alpha6 and its packing interactions function as a simple central processing unit (CPU) that senses multiple input signals, integrates these signals, and transmits the output to the signaling subdomain where the histidine kinase is bound. Analogous CPU

  20. Structural determinants of salmon calcitonin bioactivity: the role of the Leu-based amphipathic alpha-helix.

    Science.gov (United States)

    Andreotti, Giuseppina; Méndez, Blanca López; Amodeo, Pietro; Morelli, Maria A Castiglione; Nakamuta, Hiromichi; Motta, Andrea

    2006-08-25

    Salmon calcitonin (sCT) forms an amphipathic helix in the region 9-19, with the C-terminal decapeptide interacting with the helix (Amodeo, P., Motta, A., Strazzullo, G., Castiglione Morelli, M. A. (1999) J. Biomol. NMR 13, 161-174). To uncover the structural requirements for the hormone bioactivity, we investigated several sCT analogs. They were designed so as to alter the length of the central helix by removal and/or replacement of flanking residues and by selectively mutating or deleting residues inside the helix. The helix content was assessed by circular dichroism and NMR spectroscopies; the receptor binding affinity in human breast cancer cell line T 47D and the in vivo hypocalcemic activity were also evaluated. In particular, by NMR spectroscopy and molecular dynamics calculations we studied Leu(23),Ala(24)-sCT in which Pro(23) and Arg(24) were replaced by helix inducing residues. Compared with sCT, it assumes a longer amphipathic alpha-helix, with decreased binding affinity and one-fifth of the hypocalcemic activity, therefore supporting the idea of a relationship between a definite helix length and bioactivity. From the analysis of other sCT mutants, we inferred that the correct helix length is located in the 9-19 region and requires long range interactions and the presence of specific regions of residues within the sequence for high binding affinity and hypocalcemic activity. Taken together, the structural and biological data identify well defined structural parameters of the helix for sCT bioactivity.

  1. Democracy and environment as references for quadruple and quintuple helix innovation systems

    Science.gov (United States)

    Carayannis, Elias G.; Campbell, David F. J.; Orr, Barron J.

    2015-04-01

    The perspective of democracy and the ecological context define key references for knowledge production and innovation in innovation systems. Particularly under conditions of environmental change where enhancing the potential for adaptation is critical, this requires a closer look at ecological responsibility and sensitivity in the different innovation models and governance regimes. The "Quintuple Helix" innovation model is an approach that stresses the necessary socio-ecological transition of society and economy by adding an environment helix to an innovation system already made up of three (university-industry-government) or four (civil society relations) helices in a way that supports adaptation by incorporating global warming as both a challenge to and a driver of innovation. There is the proposition that knowledge production and innovation co-evolve with democracy (Carayannis and Campbell, 2014). In the Triple Helix model (Etzkowitz and Leydesdorff, 2000) the existence of a democracy does not appear to be necessary for knowledge production and innovation. However, the Quadruple Helix (Carayannis and Campbell, 2009, 2010 and 2014) is defined and represented by additional key attributes and components: "media-based and culture-based public", "civil society" and "arts, artistic research and arts-based innovation" (Bast, Carayannis and Campbell, 2015). Implications of this are that the fourth helix in the Quadruple Helix innovation systems brings in and represents the perspective of "dimension of democracy" or the "context of democracy" for knowledge in general and knowledge production and innovation in more particular. Within theories of democracy there is a competition between narrow and broader concepts of democracy (Campbell, 2013). This is particularly true when democracy is to be understood to transcend more substantially the narrow understanding of being primarily based on or being primarily rooted in government institutions (within a Triple Helix

  2. Tah1 helix-swap dimerization prevents mixed Hsp90 co-chaperone complexes

    Energy Technology Data Exchange (ETDEWEB)

    Morgan, Rhodri M. L.; Pal, Mohinder; Roe, S. Mark; Pearl, Laurence H., E-mail: laurence.pearl@sussex.ac.uk; Prodromou, Chrisostomos, E-mail: laurence.pearl@sussex.ac.uk [University of Sussex, Falmer, Brighton BN1 9RQ (United Kingdom)

    2015-05-01

    A helix swap involving the fifth helix between two adjacently bound Tah1 molecules restores the normal binding environment of the conserved MEEVD peptide of Hsp90. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes with Hsp90 and Tah1. Specific co-chaperone adaptors facilitate the recruitment of client proteins to the Hsp90 system. Tah1 binds the C-terminal conserved MEEVD motif of Hsp90, thus linking an eclectic set of client proteins to the R2TP complex for their assembly and regulation by Hsp90. Rather than the normal complement of seven α-helices seen in other tetratricopeptide repeat (TPR) domains, Tah1 unusually consists of the first five only. Consequently, the methionine of the MEEVD peptide remains exposed to solvent when bound by Tah1. In solution Tah1 appears to be predominantly monomeric, and recent structures have failed to explain how Tah1 appears to prevent the formation of mixed TPR domain-containing complexes such as Cpr6–(Hsp90){sub 2}–Tah1. To understand this further, the crystal structure of Tah1 in complex with the MEEVD peptide of Hsp90 was determined, which shows a helix swap involving the fifth α-helix between two adjacently bound Tah1 molecules. Dimerization of Tah1 restores the normal binding environment of the bound Hsp90 methionine residue by reconstituting a TPR binding site similar to that in seven-helix-containing TPR domain proteins. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes.

  3. From Family Based to Industrial Based Production: Local Economic Development Initiatives and the HELIX Model

    Directory of Open Access Journals (Sweden)

    Bartjan W Pennink

    2013-01-01

    Full Text Available To build a strong local economy, good practice tells us that each community should undertake a collaborative, strategically planned process to understand and then act upon its own strengths, weaknesses, opportunities and threats. From this perspective we start with the local communities but how is this related to the perspective from the Helix model in which three actors are explicitly introduced: the Government, the Industry and the Universities? The purpose of local economic development (LED is to build up the economic capacity of a local area to improve its economic future and the quality of life for all. To support  the Local Economic Development in remote areas,   a program  has been developed based on the LED frame work of the world bank. This approach and  the experiences over  the past years with this program are  described in the first part.  In the second part of the paper, We analyse work done with that program with the help of the social capital concept and the triple helix model.  In all cases it is important to pay attention to who is taken the initiative after the first move (and it is not always the governance as actor and for the triple helix we suggest  that the concepts of (national Government, Industry and University need a translation to Local Governance Agency, Cooperation or other ways of cooperation of local communities and Local Universities. Although a push from outside might help  a local region in development the endogenous factors are  also needed. Keywords: Triple Helix model, Local Economic Development, Local Actors, Double Triangle within the Helix Model

  4. An important base triple anchors the substrate helix recognition surface within the Tetrahymena ribozyme active site.

    Science.gov (United States)

    Szewczak, A A; Ortoleva-Donnelly, L; Zivarts, M V; Oyelere, A K; Kazantsev, A V; Strobel, S A

    1999-09-28

    Key to understanding the structural biology of catalytic RNA is determining the underlying networks of interactions that stabilize RNA folding, substrate binding, and catalysis. Here we demonstrate the existence and functional importance of a Hoogsteen base triple (U300.A97-U277), which anchors the substrate helix recognition surface within the Tetrahymena group I ribozyme active site. Nucleotide analog interference suppression analysis of the interacting functional groups shows that the U300.A97-U277 triple forms part of a network of hydrogen bonds that connect the P3 helix, the J8/7 strand, and the P1 substrate helix. Product binding and substrate cleavage kinetics experiments performed on mutant ribozymes that lack this base triple (C A-U, U G-C) or replace it with the isomorphous C(+).G-C triple show that the A97 Hoogsteen triple contributes to the stabilization of both substrate helix docking and the conformation of the ribozyme's active site. The U300. A97-U277 base triple is not formed in the recently reported crystallographic model of a portion of the group I intron, despite the presence of J8/7 and P3 in the RNA construct [Golden, B. L., Gooding, A. R., Podell, E. R. & Cech, T. R. (1998) Science 282, 259-264]. This, along with other biochemical evidence, suggests that the active site in the crystallized form of the ribozyme is not fully preorganized and that substantial rearrangement may be required for substrate helix docking and catalysis.

  5. Consensus HIV-1 FSU-A integrase gene variants electroporated into mice induce polyfunctional antigen-specific CD4+ and CD8+ T cells.

    Directory of Open Access Journals (Sweden)

    Olga Krotova

    Full Text Available Our objective is to create gene immunogens targeted against drug-resistant HIV-1, focusing on HIV-1 enzymes as critical components in viral replication and drug resistance. Consensus-based gene vaccines are specifically fit for variable pathogens such as HIV-1 and have many advantages over viral genes and their expression-optimized variants. With this in mind, we designed the consensus integrase (IN of the HIV-1 clade A strain predominant in the territory of the former Soviet Union and its inactivated derivative with and without mutations conferring resistance to elvitegravir. Humanized IN gene was synthesized; and inactivated derivatives (with 64D in the active site mutated to V with and without elvitegravir-resistance mutations were generated by site-mutagenesis. Activity tests of IN variants expressed in E coli showed the consensus IN to be active, while both D64V-variants were devoid of specific activities. IN genes cloned in the DNA-immunization vector pVax1 (pVaxIN plasmids were highly expressed in human and murine cell lines (>0.7 ng/cell. Injection of BALB/c mice with pVaxIN plasmids followed by electroporation generated potent IFN-γ and IL-2 responses registered in PBMC by day 15 and in splenocytes by day 23 after immunization. Multiparametric FACS demonstrated that CD8+ and CD4+ T cells of gene-immunized mice stimulated with IN-derived peptides secreted IFN-γ, IL-2, and TNF-α. The multi-cytokine responses of CD8+ and CD4+ T-cells correlated with the loss of in vivo activity of the luciferase reporter gene co-delivered with pVaxIN plasmids. This indicated the capacity of IN-specific CD4+ and CD8+ T-cells to clear IN/reporter co-expressing cells from the injection sites. Thus, the synthetic HIV-1 clade A integrase genes acted as potent immunogens generating polyfunctional Th1-type CD4+ and CD8+ T cells. Generation of such response is highly desirable for an effective HIV-1 vaccine as it offers a possibility to attack virus

  6. Contribution of the C-terminal region within the catalytic core domain of HIV-1 integrase to yeast lethality, chromatin binding and viral replication

    Directory of Open Access Journals (Sweden)

    Belhumeur Pierre

    2008-11-01

    Full Text Available Abstract Background HIV-1 integrase (IN is a key viral enzymatic molecule required for the integration of the viral cDNA into the genome. Additionally, HIV-1 IN has been shown to play important roles in several other steps during the viral life cycle, including reverse transcription, nuclear import and chromatin targeting. Interestingly, previous studies have demonstrated that the expression of HIV-1 IN induces the lethal phenotype in some strains of Saccharomyces cerevisiae. In this study, we performed mutagenic analyses of the C-terminal region of the catalytic core domain of HIV-1 IN in order to delineate the critical amino acid(s and/or motif(s required for the induction of the lethal phenotype in the yeast strain HP16, and to further elucidate the molecular mechanism which causes this phenotype. Results Our study identified three HIV-1 IN mutants, V165A, A179P and KR186,7AA, located in the C-terminal region of the catalytic core domain of IN that do not induce the lethal phenotype in yeast. Chromatin binding assays in yeast and mammalian cells demonstrated that these IN mutants were impaired for the ability to bind chromatin. Additionally, we determined that while these IN mutants failed to interact with LEDGF/p75, they retained the ability to bind Integrase interactor 1. Furthermore, we observed that VSV-G-pseudotyped HIV-1 containing these IN mutants was unable to replicate in the C8166 T cell line and this defect was partially rescued by complementation with the catalytically inactive D64E IN mutant. Conclusion Overall, this study demonstrates that three mutations located in the C-terminal region of the catalytic core domain of HIV-1 IN inhibit the IN-induced lethal phenotype in yeast by inhibiting the binding of IN to the host chromatin. These results demonstrate that the C-terminal region of the catalytic core domain of HIV-1 IN is important for binding to host chromatin and is crucial for both viral replication and the promotion of

  7. Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS

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    Ciervo Alessandra

    2007-10-01

    Full Text Available Abstract Background Treatment of feline immunodeficiency virus (FIV infection has been hampered by the absence of a specific combination antiretroviral treatment (ART. Integrase strand transfer inhibitors (INSTIs are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs. Methods Phylogenetic analysis of lentiviral integrase (IN sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD. Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR. Results The calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810. Conclusion We report a drug class (other than nucleosidic reverse transcriptase inhibitors that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound

  8. HIV-1 integrase inhibitory activity of endophytic fungi from five species of medicinal Dendrobium%五种药用石斛内生真菌抑制HIV-1整合酶活性研究

    Institute of Scientific and Technical Information of China (English)

    祁婧; 张大为; 陈娟; 康永; 郭顺星

    2013-01-01

    目的 评价5种药用石斛内生真菌发酵产物抑制HIV-1整合酶的活性.方法 将分离自石斛的202株内生真菌提取物共404个采用高通量ELISA法评价其抑制HIV-l整合酶活性;对抑制活性大于100%的样品进行量效关系考察并进行体外抑制肿瘤细胞活性筛选.结果 筛选得到19个对HIV-l整合酶抑制活性大于80%的样品,其中样品5119F、5297F、5097F、5140J和5211F的抑制率分别达到117.96%、113.53%、108.62%、103.74%和109.02%,其对应的IC50值分别为0.02024、0.003125、0.00862、0.01007 和 0.01192 mg/ml.结论石斛属药用植物内生真菌是一个潜在的、丰富的用于筛选HIV-1整合酶抑制剂的资源库,值得进一步研究和开发.%Objective To evaluate the HIV integrase inhibitory activity of endophvtic fungi in five species of medicinal Dendrobium.Methods The HIV-1 integrase inhibitory activity was detected with high-throughput ELISA.The samples that displayed more than 100% inhibitory effect for HIV-1 integrase were selected for dose-response experiments and anticancer activity screening.Results Among all the tested samples,19 samples inhibited the HIV-1 integrase activity by more than 80%.Specially,the samples 5119F,5297F,5097F,5140J and 5211F inhibited the enzyme activity by 117.96%,113.53%,108.62%,103.74% and 109.02% respectively.The IC50 of the five samples was 0.02024,0.003125,0.00862,0.01007 and 0.01192 mg/ml respectively.Conclusion Endophytic fungi of medicinal Dendrobium are potential and abundant reservoir for the screening of HIV-1 integrase inhibitors and are worthy of further study.

  9. Structural characterizations of fusion peptide analogs of influenza virus hemagglutinin. Implication of the necessity of a helix-hinge-helix motif in fusion activity.

    Science.gov (United States)

    Hsu, Chun-Hua; Wu, Shih-Hsiung; Chang, Ding-Kwo; Chen, Chinpan

    2002-06-21

    Infection by enveloped viruses initially involves membrane fusion between viral and host cell membranes. The fusion peptide plays a crucial role in triggering this reaction. To clarify how the fusion peptide exerts this specific function, we carried out biophysical studies of three fusion peptide analogs of influenza virus hemagglutinin HA2, namely E5, G13L, and L17A. E5 exhibits an activity similar to the native fusion peptide, whereas G13L and L17A, which are two point mutants of the E5 analog, possess much less fusion activity. Our CD data showed that the conformations of these three analogs in SDS micelles are pH-dependent, with higher alpha-helical contents at acidic pH. Tryptophan fluorescence emission experiments indicated that these three analogs insert deeper into lipid bilayers at acidic pH. The three-dimensional structure of the E5 analog in SDS micelles at pH 4.0 revealed that two segments, Leu(2)-Glu(11) and Trp(14)-Ile(18), form amphipathic helical conformations, with Gly(12)-Gly(13) forming a hinge. The hydrophobic residues in the N- and C-terminal helices form a hydrophobic cluster. At neutral pH, however, the C-terminal helix of Trp(14)-Ile(18) reduces dramatically, and the hydrophobic core observed at acidic pH is severely disrupted. We suggest that the disruption of the C-terminal helix renders the E5 analog fusion-inactive at neutral pH. Furthermore, the decrease of the hinge and the reduction of fusion activity in G13L reveal the importance of the hinge in fusion activity. Also, the decrease in the C-terminal helix and the reduction of fusion activity in L17A demonstrates the importance of the C-terminal helix in fusion activity. Based on these biophysical studies, we propose a model that illustrates the structural change of the HA2 fusion peptide analog and explains how the analog interacts with the lipid bilayer at different pH values.

  10. Location of glycine mutations within a bacterial collagen protein affects degree of disruption of triple-helix folding and conformation.

    Science.gov (United States)

    Cheng, Haiming; Rashid, Shayan; Yu, Zhuoxin; Yoshizumi, Ayumi; Hwang, Eileen; Brodsky, Barbara

    2011-01-21

    The hereditary bone disorder osteogenesis imperfecta is often caused by missense mutations in type I collagen that change one Gly residue to a larger residue and that break the typical (Gly-Xaa-Yaa)(n) sequence pattern. Site-directed mutagenesis in a recombinant bacterial collagen system was used to explore the effects of the Gly mutation position and of the identity of the residue replacing Gly in a homogeneous collagen molecular population. Homotrimeric bacterial collagen proteins with a Gly-to-Arg or Gly-to-Ser replacement formed stable triple-helix molecules with a reproducible 2 °C decrease in stability. All Gly replacements led to a significant delay in triple-helix folding, but a more dramatic delay was observed when the mutation was located near the N terminus of the triple-helix domain. This highly disruptive mutation, close to the globular N-terminal trimerization domain where folding is initiated, is likely to interfere with triple-helix nucleation. A positional effect of mutations was also suggested by trypsin sensitivity for a Gly-to-Arg replacement close to the triple-helix N terminus but not for the same replacement near the center of the molecule. The significant impact of the location of a mutation on triple-helix folding and conformation could relate to the severe consequences of mutations located near the C terminus of type I and type III collagens, where trimerization occurs and triple-helix folding is initiated.

  11. Triple helix stabilization by covalently linked DNA-bisbenzimidazole conjugate synthesized by maleimide-thiol coupling chemistry.

    Science.gov (United States)

    Jain, Akash K; Awasthi, Satish Kumar; Tandon, Vibha

    2006-09-15

    Tethering of BBZPNH2, an analogue of the Hoechst 33258, with a 14 nucleotide long DNA sequence with the help of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), a heterobifunctional crosslinking reagent, using DMF/ water as solvent yields a conjugate which effectively stabilizes the triple helix. The above conjugate was hybridized with 26 bp long double stranded (ds) DNA having 14 bp long polypurine-polypyrimidine stretch to form a pyrimidine motif triple helix. The above conjugate increases the thermal stability of both the transitions, that is, triple helix to double helix by 12 degrees C and double helix to single strand transition by 16 degrees C for the triple helix formed with conjugated TFO over the triple helix made from non-conjugated TFO. Fluorescence and circular dichroism spectra recorded at different temperatures confirm the presence of minor groove binding bisbenzimidazole in the AT-rich minor groove of dsDNA even after the major groove bound TFO separates out.

  12. Osteogenesis imperfecta model peptides: incorporation of residues replacing Gly within a triple helix achieved by renucleation and local flexibility.

    Science.gov (United States)

    Xiao, Jianxi; Madhan, Balaraman; Li, Yingjie; Brodsky, Barbara; Baum, Jean

    2011-07-20

    Missense mutations, which replace one Gly with a larger residue in the repeating sequence of the type I collagen triple helix, lead to the hereditary bone disorder osteogenesis imperfecta (OI). Previous studies suggest that these mutations may interfere with triple-helix folding. NMR was used to investigate triple-helix formation in a series of model peptides where the residue replacing Gly, as well as the local sequence environment, was varied. NMR measurement of translational diffusion coefficients allowed the identification of partially folded species. When Gly was replaced by Ala, the Ala residue was incorporated into a fully folded triple helix, whereas replacement of Gly by Ser or Arg resulted in the presence of some partially folded species, suggesting a folding barrier. Increasing the triple-helix stability of the sequence N-terminal to a Gly-to-Ser replacement allowed complete triple-helix folding, whereas with the substitution of Arg, with its large side chain, the peptide achieved full folding only after flexible residues were introduced N-terminal to the mutation site. These studies shed light on the factors important for accommodation of Gly mutations within the triple helix and may relate to the varying severity of OI.

  13. L'autoroute A4

    Institute of Scientific and Technical Information of China (English)

    DominiqueMaitrot; 文芳

    2005-01-01

    Lorsque je dois me rendre à Metz, grande ville de la Moselle, rivale de Nancy pour le titre de capitale de la Lorraine, je prends l'autoroute A4. Epernay, ma petite ville, n'est pas direetement reliée à l'autoroute:

  14. Stereoelectronic and steric effects in the collagen triple helix: toward a code for strand association.

    Science.gov (United States)

    Hodges, Jonathan A; Raines, Ronald T

    2005-11-16

    Collagen is the most abundant protein in animals. The protein consists of a helix of three strands, each with sequence X-Y-Gly. Natural collagen is most stable when X is (2S)-proline (Pro) and Y is (2S,4R)-4-hydroxyproline (4R-Hyp). We had shown previously that triple helices in which X is (2S,4S)-4-fluoroproline (4S-Flp) or Y is (2S,4R)-4-fluoroproline (4R-Flp) display hyperstability. This hyperstability arises from stereoelectronic effects that preorganize the main-chain dihedral angles in the conformation found in the triple helix. Here, we report the synthesis of strands containing both 4S-Flp in the X-position and 4R-Flp in the Y-position. We find that these strands do not form a stable triple helix, presumably because of an unfavorable steric interaction between fluoro groups on adjacent strands. Density functional theory calculations indicate that (2S,3S)-3-fluoroproline (3S-Flp), like 4S-Flp, should preorganize the main chain properly for triple-helix formation but without a steric conflict. Synthetic strands containing 3S-Flp in the X-position and 4R-Flp in the Y-position do form a triple helix. This helix is, however, less stable than one with Pro in the X-position, presumably because of an unfavorable inductive effect that diminishes the strength of the interstrand 3S-FlpC=O...H-NGly hydrogen bond. Thus, other forces can counter the benefits derived from the proper preorganization. Although (Pro-Pro-Gly)7 and (4S-Flp-4R-Flp-Gly)7 do not form stable homotrimeric helices, mixtures of these two peptides form stable heterotrimeric helices containing one (Pro-Pro-Gly)7 strand and two (4S-Flp-4R-Flp-Gly)7 strands. This stoichiometry can be understood by considering the cross sections of the two possible heterotrimeric helices. This unexpected finding portends the development of a "code" for the self-assembly of determinate triple helices from two or three strands.

  15. Virtual-screening targeting Human Immunodeficiency Virus type 1 integrase-lens epithelium-derived growth factor/p75 interaction for drug development.

    Science.gov (United States)

    Gu, Wan-Gang; Liu, Bai-Nan; Yuan, Jun-Fa

    2015-02-01

    Three integrase (IN) inhibitors have been approved by FDA for clinical treatment of Human Immunodeficiency Virus (HIV) infection. This stimulates more researchers to focus their studies on this target for anti-HIV drug development. Three steps regarding of IN activity have been validated for inhibitor discovery: strand transfer, 3'-terminal processing, and IN-lens epithelium-derived growth factor (LEDGF)/p75 interaction. Among them, IN-LEDGF/p75 interaction is a new target validated in recent years. Emergence of drug-resistant virus strains makes this target appealing to pharmacologists. Compared with the traditional screening methods such as AlphaScreen and cell-based screening developed for IN inhibitor discovery, virtual screening is a powerful technique in modern drug discovery. Here we summarized the recent advances of virtual-screening targeting IN-LEDFG/p75 interaction. The combined application of virtual screening and experiments in drug discovery against IN-LEDFG/p75 interaction sheds light on anti-HIV research and drug discovery.

  16. A novel co-crystal structure affords the design of gain-of-function lentiviral integrase mutants in the presence of modified PSIP1/LEDGF/p75.

    Directory of Open Access Journals (Sweden)

    Stephen Hare

    2009-01-01

    Full Text Available Lens epithelium derived growth factor (LEDGF, also known as PC4 and SFRS1 interacting protein 1 (PSIP1 and transcriptional co-activator p75, is the cellular binding partner of lentiviral integrase (IN proteins. LEDGF accounts for the characteristic propensity of Lentivirus to integrate within active transcription units and is required for efficient viral replication. We now present a crystal structure containing the N-terminal and catalytic core domains (NTD and CCD of HIV-2 IN in complex with the IN binding domain (IBD of LEDGF. The structure extends the known IN-LEDGF interface, elucidating primarily charge-charge interactions between the NTD of IN and the IBD. A constellation of acidic residues on the NTD is characteristic of lentiviral INs, and mutations of the positively charged residues on the IBD severely affect interaction with all lentiviral INs tested. We show that the novel NTD-IBD contacts are critical for stimulation of concerted lentiviral DNA integration by LEDGF in vitro and for its function during the early steps of HIV-1 replication. Furthermore, the new structural details enabled us to engineer a mutant of HIV-1 IN that primarily functions only when presented with a complementary LEDGF mutant. These findings provide structural basis for the high affinity lentiviral IN-LEDGF interaction and pave the way for development of LEDGF-based targeting technologies for gene therapy.

  17. From ligand to complexes. Part 2. Remarks on human immunodeficiency virus type 1 integrase inhibition by beta-diketo acid metal complexes.

    Science.gov (United States)

    Bacchi, Alessia; Biemmi, Mariano; Carcelli, Mauro; Carta, Fabrizio; Compari, Carlotta; Fisicaro, Emilia; Rogolino, Dominga; Sechi, Mario; Sippel, Martin; Sotriffer, Christoph A; Sanchez, Tino W; Neamati, Nouri

    2008-11-27

    Previously, we synthesized a series of beta-diketo acid metal complexes as novel HIV-1 integrase (IN) inhibitors (J. Med. Chem. 2006, 46, 4248-4260). Herein, a further extension of this study is reported. First, detailed docking studies were performed in order to investigate the mode of binding in the active site of the free ligands and of their metal complexes. Second, a series of potentiometric measurements were conducted for two diketo acids chosen as model ligands, with Mn(2+) and Ca(2+), in order to outline a speciation model. Third, we designed and synthesized a new set of complexes with different stoichiometries and tested them in an in vitro assay specific for IN. Finally, we obtained the first X-ray structure of a metal complex with HIV-1 IN inhibition activity. Analysis of these results supports the hypothesis that the diketo acids could act as complexes and form complexes with the metal ions on the active site of the enzyme.

  18. The Microbiota and Abundance of the Class 1 Integron-Integrase Gene in Tropical Sewage Treatment Plant Influent and Activated Sludge.

    Directory of Open Access Journals (Sweden)

    Magna C Paiva

    Full Text Available Bacteria are assumed to efficiently remove organic pollutants from sewage in sewage treatment plants, where antibiotic-resistance genes can move between species via mobile genetic elements known as integrons. Nevertheless, few studies have addressed bacterial diversity and class 1 integron abundance in tropical sewage. Here, we describe the extant microbiota, using V6 tag sequencing, and quantify the class 1 integron-integrase gene (intI1 in raw sewage (RS and activated sludge (AS. The analysis of 1,174,486 quality-filtered reads obtained from RS and AS samples revealed complex and distinct bacterial diversity in these samples. The RS sample, with 3,074 operational taxonomic units, exhibited the highest alpha-diversity indices. Among the 25 phyla, Proteobacteria, Bacteroidetes and Firmicutes represented 85% (AS and 92% (RS of all reads. Increased relative abundance of Micrococcales, Myxococcales, and Sphingobacteriales and reduced pathogen abundance were noted in AS. At the genus level, differences were observed for the dominant genera Simplicispira and Diaphorobacter (AS as well as for Enhydrobacter (RS. The activated sludge process decreased (55% the amount of bacteria harboring the intI1 gene in the RS sample. Altogether, our results emphasize the importance of biological treatment for diminishing pathogenic bacteria and those bearing the intI1 gene that arrive at a sewage treatment plant.

  19. Hierarchical self-assembly of designed 2x2-alpha-helix bundle proteins on Au(111) surfaces

    DEFF Research Database (Denmark)

    Wackerbarth, Hainer; Tofteng, A.P.; Jensen, K.J.

    2006-01-01

    Self-assembled monolayers of biomolecules on atomically planar surfaces offer the prospect of complex combinations of controlled properties, e. g., for bioelectronics. We have prepared a novel hemi-4-alpha-helix bundle protein by attaching two alpha-helical peptides to a cyclo-dithiothreitol (cyclo...... proteins retained. The surface properties of the DTT and 2 x 2- R-helix bundle protein adlayer were characterized by interfacial capacitance and impedance techniques. Reductive desorption was used to determine the coverage of the adlayers, giving values of 65 and 16 mu C cm(-2) for DTT and 2 x 2-helix...

  20. Characterization of Mycobacterium tuberculosis EsxA membrane insertion: roles of N- and C-terminal flexible arms and central helix-turn-helix motif.

    Science.gov (United States)

    Ma, Yue; Keil, Verena; Sun, Jianjun

    2015-03-13

    EsxA (ESAT-6), an important virulence factor of Mycobacterium tuberculosis, plays an essential role in phagosome rupture and bacterial cytosolic translocation within host macrophages. Our previous study showed that EsxA exhibits a unique membrane-interacting activity that is not found in its ortholog from nonpathogenic Mycobacterium smegmatis. However, the molecular mechanism of EsxA membrane insertion remains unknown. In this study, we generated truncated EsxA proteins with deletions of the N- and/or C-terminal flexible arm. Using a fluorescence-based liposome leakage assay, we found that both the N- and C-terminal arms were required for membrane disruption. Moreover, we found that, upon acidification, EsxA converted into a more organized structure with increased α-helical content, which was evidenced by CD analysis and intrinsic tryptophan fluorescence. Finally, using an environmentally sensitive fluorescent dye, we obtained direct evidence that the central helix-turn-helix motif of EsxA inserted into the membranes and formed a membrane-spanning pore. A model of EsxA membrane insertion is proposed and discussed.

  1. Evidence supporting the existence of a NUPR1-like family of helix-loop-helix chromatin proteins related to, yet distinct from, AT hook-containing HMG proteins.

    Science.gov (United States)

    Urrutia, Raul; Velez, Gabriel; Lin, Marisa; Lomberk, Gwen; Neira, Jose Luis; Iovanna, Juan

    2014-08-01

    NUPR1, a small chromatin protein, plays a critical role in cancer development, progression, and resistance to therapy. Here, using a combination of structural bioinformatics and molecular modeling methods, we report several novel findings that enhance our understanding of the biochemical function of this protein. We find that NUPR1 has been conserved throughout evolution, and over time it has undergone duplications and transpositions to form other transcriptional regulators. Using threading, homology-based molecular modeling, molecular mechanics calculations, and molecular dynamics simulations, we generated structural models for four of these proteins: NUPR1a, NUPR1b, NUPR2, and the NUPR-like domain of GTF2-I. Comparative analyses of these models combined with extensive linear motif identification reveal that these four proteins, though similar in their propensities for folding, differ in size, surface changes, and sites amenable for posttranslational modification. Lastly, taking NUPR1a as the paradigm for this family, we built models of a NUPR-DNA complex. Additional structural comparisons revealed that NUPR1 defines a new family of small-groove-binding proteins that share structural features with, yet are distinct from, helix-loop-helix AT-hook-containing HMG proteins. These models and inferences should lead to a better understanding of the function of this group of chromatin proteins, which play a critical role in the development of human malignant diseases.

  2. Mutations within or upstream of the basic helix-loop-helix domain of the TWIST gene are specific to Saethre-Chotzen syndrome.

    Science.gov (United States)

    El Ghouzzi, V; Lajeunie, E; Le Merrer, M; Cormier-Daire, V; Renier, D; Munnich, A; Bonaventure, J

    1999-01-01

    Saethre-Chotzen syndrome (ACS III) is an autosomal dominant craniosynostosis syndrome recently ascribed to mutations in the TWIST gene, a basic helix-loop-helix (b-HLH) transcription factor regulating head mesenchyme cell development during cranial neural tube formation in mouse. Studying a series of 22 unrelated ACS III patients, we have found TWIST mutations in 16/22 cases. Interestingly, these mutations consistently involved the b-HLH domain of the protein. Indeed, mutant genotypes included frameshift deletions/insertions, nonsense and missense mutations, either truncating or disrupting the b-HLH motif of the protein. This observation gives additional support to the view that most ACS III cases result from loss-of-function mutations at the TWIST locus. The P250R recurrent FGFR 3 mutation was found in 2/22 cases presenting mild clinical manifestations of the disease but 4/22 cases failed to harbour TWIST or FGFR 3 mutations. Clinical re-examination of patients carrying TWIST mutations failed to reveal correlations between the mutant genotype and severity of the phenotype. Finally, since no TWIST mutations were detected in 40 cases of isolated coronal craniosynostosis, the present study suggests that TWIST mutations are specific to Saethre-Chotzen syndrome.

  3. TdIF1 recognizes a specific DNA sequence through its Helix-Turn-Helix and AT-hook motifs to regulate gene transcription.

    Directory of Open Access Journals (Sweden)

    Takashi Kubota

    Full Text Available TdIF1 was originally identified as a protein that directly binds to DNA polymerase TdT. TdIF1 is also thought to function in transcription regulation, because it binds directly to the transcriptional factor TReP-132, and to histone deacetylases HDAC1 and HDAC2. Here we show that TdIF1 recognizes a specific DNA sequence and regulates gene transcription. By constructing TdIF1 mutants, we identify amino acid residues essential for its interaction with DNA. An in vitro DNA selection assay, SELEX, reveals that TdIF1 preferentially binds to the sequence 5'-GNTGCATG-3' following an AT-tract, through its Helix-Turn-Helix and AT-hook motifs. We show that four repeats of this recognition sequence allow TdIF1 to regulate gene transcription in a plasmid-based luciferase reporter assay. We demonstrate that TdIF1 associates with the RAB20 promoter, and RAB20 gene transcription is reduced in TdIF1-knocked-down cells, suggesting that TdIF1 stimulates RAB20 gene transcription.

  4. Molecular characterization of cold-responsive basic helix-loop-helix transcription factors MabHLHs that interact with MaICE1 in banana fruit.

    Science.gov (United States)

    Peng, Huan-Huan; Shan, Wei; Kuang, Jian-Fei; Lu, Wang-Jin; Chen, Jian-Ye

    2013-11-01

    Basic helix-loop-helix (bHLH) transcription factors (TFs) are ubiquitously involved in the response of higher plants to various abiotic stresses. However, little is known about bHLH TFs involved in the cold stress response in economically important fruits. Here, five novel full-length bHLH genes, designated as MabHLH1-MabHLH5, were isolated and characterized from banana fruit. Gene expression profiles revealed that MabHLH1/2/4 were induced by cold stress and methyl jasmonate (MeJA) treatment. Transient assays in tobacco BY2 protoplasts showed that MabHLH1/2/4 promoters were activated by cold stress and MeJA treatments. Moreover, protein-protein interaction analysis demonstrated that MabHLH1/2/4 not only physically interacted with each other to form hetero-dimers in the nucleus, but also interacted with an important upstream component of cold signaling MaICE1, with different interaction domains at their N-terminus. These results indicate that banana fruit cold-responsive MabHLHs may form a big protein complex in the nucleus with MaICE1. Taken together, our findings advance our understanding of the possible involvement of bHLH TFs in the regulatory network of ICE-CBF cold signaling pathway.

  5. Elevated endogenous expression of the dominant negative basic helix-loop-helix protein ID1 correlates with significant centrosome abnormalities in human tumor cells

    Directory of Open Access Journals (Sweden)

    Gutmann Anja

    2010-01-01

    Full Text Available Abstract Background ID proteins are dominant negative inhibitors of basic helix-loop-helix transcription factors that have multiple functions during development and cellular differentiation. Ectopic (over-expression of ID1 extends the lifespan of primary human epithelial cells. High expression levels of ID1 have been detected in multiple human malignancies, and in some have been correlated with unfavorable clinical prognosis. ID1 protein is localized at the centrosomes and forced (over-expression of ID1 results in errors during centrosome duplication. Results Here we analyzed the steady state expression levels of the four ID-proteins in 18 tumor cell lines and assessed the number of centrosome abnormalities. While expression of ID1, ID2, and ID3 was detected, we failed to detect protein expression of ID4. Expression of ID1 correlated with increased supernumerary centrosomes in most cell lines analyzed. Conclusions This is the first report that shows that not only ectopic expression in tissue culture but endogenous levels of ID1 modulate centrosome numbers. Thus, our findings support the hypothesis that ID1 interferes with centrosome homeostasis, most likely contributing to genomic instability and associated tumor aggressiveness.

  6. Modeling of helix molecules formation on a surface of nanotube and inside it

    Directory of Open Access Journals (Sweden)

    Belolipetskaya Anna

    2017-01-01

    Full Text Available “On-surface” synthesis of large molecules and assemblies is a great challenge in molecular electronics, biomedical devices, sensors, energy harnessing and catalysis. Usually, plane surfaces are used for these purposes. We study a possibility of macro molecules formation on the surface of nanotube and inside it. It can be used for synthesis of helix molecules, particularly, organic and biological. Nanotubes of different structures were considered. Point-like approximation is used for atoms. The Lennard-Jones 6-12 potential is taken as the interaction potential. Possibilities of helix molecules and ring-like molecules formations were shown. A hypothesis of such processes influence on the organic molecules formation during the early Earth history is suggested and discussed.

  7. Helix Nebula and CERN: A Symbiotic approach to exploiting commercial clouds

    CERN Multimedia

    Barreiro Megino, Fernando Harald; Kucharczyk, Katarzyna; Medrano Llamas, Ramón; van der Ster, Daniel

    2013-01-01

    The recent paradigm shift toward cloud computing in IT, and general interest in "Big Data" in particular, have demonstrated that the computing requirements of HEP are no longer globally unique. Indeed, the CERN IT department and LHC experiments have already made significant R&D; investments in delivering and exploiting cloud computing resources. While a number of technical evaluations of interesting commercial offerings from global IT enterprises have been performed by various physics labs, further technical, security, sociological, and legal issues need to be address before their large-scale adoption by the research community can be envisaged. Helix Nebula - the Science Cloud is an initiative that explores these questions by joining the forces of three European research institutes (CERN, ESA and EMBL) with leading European commercial IT enterprises. The goals of Helix Nebula are to establish a cloud platform federating multiple commercial cloud providers, along with new business models, which can sustain...

  8. The Effect of a Helix-Coil Transition on the Extension Elasticity

    Science.gov (United States)

    Buhot, Arnaud; Halperin, Avi

    2000-03-01

    The secondary structure of a polymer affects its deformation behavior in accordance with the Le Chatelier principle. An important example of such secondary structure is the alpha helix encountered in polypeptides. Similar structure was recently proposed for PEO in aqueous media. Our discussion concerns the coupling of the cooperative helix-coil transition and the extension elasticity. In particular, we analyze the extension of a long single chain by use of optical tweezers or AFM. We consider chains that exist in the coil-state when unperturbed. The transition nevertheless occurs because the extension favors the low entropy helical state. As a result, the corresponding force law exhibits a plateau. The analysis of this situation involves two ingredients: (I) the stretching free energy penalty for a rod-coil mutiblock copolymer (II) the entropy associated with the possible placements of the rod and coil blocks.

  9. Helix Nebula and CERN: A Symbiotic approach to exploiting commercial clouds

    CERN Document Server

    Barreiro Megino, Fernando Harald; Kucharczyk, Katarzyna; Medrano Llamas, Ramón; van der Ster, Daniel

    2014-01-01

    The recent paradigm shift toward cloud computing in IT, and general interest in "Big Data" in particular, have demonstrated that the computing requirements of HEP are no longer globally unique. Indeed, the CERN IT department and LHC experiments have already made significant R&D investments in delivering and exploiting cloud computing resources. While a number of technical evaluations of interesting commercial offerings from global IT enterprises have been performed by various physics labs, further technical, security, sociological, and legal issues need to be address before their large-scale adoption by the research community can be envisaged. Helix Nebula - the Science Cloud is an initiative that explores these questions by joining the forces of three European research institutes (CERN, ESA and EMBL) with leading European commercial IT enterprises. The goals of Helix Nebula are to establish a cloud platform federating multiple commercial cloud providers, along with new business models, which can sustain ...

  10. Conformational Heterogeneity of Bax Helix 9 Dimer for Apoptotic Pore Formation

    Science.gov (United States)

    Liao, Chenyi; Zhang, Zhi; Kale, Justin; Andrews, David W.; Lin, Jialing; Li, Jianing

    2016-07-01

    Helix α9 of Bax protein can dimerize in the mitochondrial outer membrane (MOM) and lead to apoptotic pores. However, it remains unclear how different conformations of the dimer contribute to the pore formation on the molecular level. Thus we have investigated various conformational states of the α9 dimer in a MOM model — using computer simulations supplemented with site-specific mutagenesis and crosslinking of the α9 helices. Our data not only confirmed the critical membrane environment for the α9 stability and dimerization, but also revealed the distinct lipid-binding preference of the dimer in different conformational states. In our proposed pathway, a crucial iso-parallel dimer that mediates the conformational transition was discovered computationally and validated experimentally. The corroborating evidence from simulations and experiments suggests that, helix α9 assists Bax activation via the dimer heterogeneity and interactions with specific MOM lipids, which eventually facilitate proteolipidic pore formation in apoptosis regulation.

  11. Genomics into Healthcare: the 5th Pan Arab Human Genetics Conference and 2013 Golden Helix Symposium.

    Science.gov (United States)

    Fortina, Paolo; Al Khaja, Najib; Al Ali, Mahmoud Taleb; Hamzeh, Abdul Rezzak; Nair, Pratibha; Innocenti, Federico; Patrinos, George P; Kricka, Larry J

    2014-05-01

    The joint 5th Pan Arab Human Genetics conference and 2013 Golden Helix Symposium, "Genomics into Healthcare" was coorganized by the Center for Arab Genomic Studies (http://www.cags.org.ae) in collaboration with the Golden Helix Foundation (http://www.goldenhelix.org) in Dubai, United Arab Emirates from 17 to 19 November, 2013. The meeting was attended by over 900 participants, doctors and biomedical students from over 50 countries and was organized into a series of nine themed sessions that covered cancer genomics and epigenetics, genomic and epigenetic studies, genomics of blood and metabolic disorders, cytogenetic diagnosis and molecular profiling, next-generation sequencing, consanguinity and hereditary diseases, clinical genomics, clinical applications of pharmacogenomics, and genomics in public health.

  12. One-dimensional nonlinear theory for rectangular helix traveling-wave tube

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Chengfang, E-mail: fchffchf@126.com; Zhao, Bo; Yang, Yudong; Ju, Yongfeng [Faculty of Electronic Information Engineering, Huaiyin Institute of Technology, Huai' an 223003 (China); Wei, Yanyu [School of Physical Electronics, University of Electronic and Technology of China, Chengdu 610054 (China)

    2016-08-15

    A 1-D nonlinear theory of a rectangular helix traveling-wave tube (TWT) interacting with a ribbon beam is presented in this paper. The RF field is modeled by a transmission line equivalent circuit, the ribbon beam is divided into a sequence of thin rectangular electron discs with the same cross section as the beam, and the charges are assumed to be uniformly distributed over these discs. Then a method of computing the space-charge field by solving Green's Function in the Cartesian Coordinate-system is fully described. Nonlinear partial differential equations for field amplitudes and Lorentz force equations for particles are solved numerically using the fourth-order Runge-Kutta technique. The tube's gain, output power, and efficiency of the above TWT are computed. The results show that increasing the cross section of the ribbon beam will improve a rectangular helix TWT's efficiency and reduce the saturated length.

  13. Effects of Boundary Condition and Helix Angle On Meshing Performance of TI Worm Gearing

    Institute of Scientific and Technical Information of China (English)

    SUN Yue-hai; DUAN Lu-qian; WANG Shu-ren; ZHANG Ce

    2006-01-01

    To exactly describe the contact state and contact area oftooth surface oftoroidalinvolute(TI) worm gearing.the authors introduced boundary condition into contact line analysis.With helix angle chosen as parameter,the criterion for the existence of meshing boundary line on the surface of TI worm gearing is derived.Results show that there can be four situations for meshing boundary line on the tooth surface of gear.namely,inexistence of meshing boundary line.a unique line,two lines,and two coincident lines.If the helix angle is equal to or slightly smaller than the bigger angle,which makes two meshing boundary lines superpose,a preferable meshing performance is obtained.Computer simulation proves the validity Of the above conclusion.

  14. Local equilibria and state transfer of charged classical particles on a helix in an electric field

    CERN Document Server

    Plettenberg, J; Zampetaki, A V; Schmelcher, P

    2016-01-01

    We explore the effects of a homogeneous external electric field on the static properties and dynamical behavior of two charged particles confined to a helix. In contrast to the field-free setup which provides a separation of the center-of-mass and relative motion, the existence of an external force perpendicular to the helix axis couples the center-of-mass to the relative degree of freedom leading to equilibria with a localized center of mass. By tuning the external field various fixed points are created and/or annihilated through different bifurcation scenarios. We provide a detailed analysis of these bifurcations based on which we demonstrate a robust state transfer between essentially arbitrary equilibrium configurations of the two charges that can be induced by making the external force time-dependent.

  15. Water-mediated conformational transitions in nicotinic receptor M2 helix bundles: a molecular dynamics study.

    Science.gov (United States)

    Sankararamakrishnan, R; Sansom, M S

    1995-12-27

    The ion channel of the nicotinic acetylcholine receptor is a water-filled pore formed by five M2 helix segments, one from each subunit. Molecular dynamics simulations on bundles of five M2 alpha 7 helices surrounding a central column of water and with caps of water molecules at either end of the pore have been used to explore the effects of intrapore water on helix packing. Interactions of water molecules with the N-terminal polar sidechains lead to a conformational transition from right- to left-handed supercoils during these stimulations. These studies reveal that the pore formed by the bundle of M2 helices is flexible. A structural role is proposed for water molecules in determining the geometry of bundles of isolated pore-forming helices.

  16. One-dimensional nonlinear theory for rectangular helix traveling-wave tube

    Science.gov (United States)

    Fu, Chengfang; Wei, Yanyu; Zhao, Bo; Yang, Yudong; Ju, Yongfeng

    2016-08-01

    A 1-D nonlinear theory of a rectangular helix traveling-wave tube (TWT) interacting with a ribbon beam is presented in this paper. The RF field is modeled by a transmission line equivalent circuit, the ribbon beam is divided into a sequence of thin rectangular electron discs with the same cross section as the beam, and the charges are assumed to be uniformly distributed over these discs. Then a method of computing the space-charge field by solving Green's Function in the Cartesian Coordinate-system is fully described. Nonlinear partial differential equations for field amplitudes and Lorentz force equations for particles are solved numerically using the fourth-order Runge-Kutta technique. The tube's gain, output power, and efficiency of the above TWT are computed. The results show that increasing the cross section of the ribbon beam will improve a rectangular helix TWT's efficiency and reduce the saturated length.

  17. Globular structures of a helix-coil copolymer: Self-consistent treatment

    Science.gov (United States)

    Nowak, C.; Rostiashvili, V. G.; Vilgis, T. A.

    2007-01-01

    A self-consistent-field theory was developed in the grand canonical ensemble formulation to study transitions in a helix-coil multiblock globule. Helical and coil parts are treated as stiff rods and self-avoiding walks of variable lengths correspondingly. The resulting field theory takes, in addition to the conventional Zimm-Bragg, [J. Chem. Phys. 31, 526 (1959)] parameters, also three-dimensional interaction terms into account. The appropriate differential equations which determine the self-consistent fields were solved numerically with finite element method. Three different phase states are found: open chain, amorphous globule, and nematic liquid-crystalline (LC) globule. The LC-globule formation is driven by the interplay between the hydrophobic helical segment attraction and the anisotropic globule surface energy of an entropic nature. The full phase diagram of the helix-coil copolymer was calculated and thoroughly discussed. The suggested theory shows a clear interplay between secondary and tertiary structures in globular homopolypeptides.

  18. The Triple Helix Model and the Meta-Stabilization of Urban Technologies in Smart Cities

    CERN Document Server

    Leydesdorff, Loet

    2010-01-01

    The Triple Helix model of university-industry-government relations can be generalized from a neo-institutional model of networks to a neo-evolutionary model of how three selection environments operate upon one another. The neo-evolutionary model enables us to appreciate both organizational integration in university-industry-government relations and differentiation among functions like the generation of intellectual capital, creation of wealth, and their attending legislation. The specification of innovation systems in terms of nations, sectors, cities, and regions can then be formulated as empirical questions: is synergy generated among functions in networks of relations? This Triple Helix model enables us to study the knowledge base of an urban economy in terms of a trade-off between locally stabilized and (potentially locked-in) trajectories versus the techno-economic and cultural development regimes which work with one more degree of freedom at the global level. The meta-stabilizing potentials of urban tec...

  19. Beyond the nearest-neighbor Zimm-Bragg model for helix-coil transition in peptides.

    Science.gov (United States)

    Murza, Adrian; Kubelka, Jan

    2009-02-01

    The nearest-neighbor (micro = 1) variant of the Zimm and Bragg (ZB) model has been extensively used to describe the helix-coil transition in biopolymers. In this work, we investigate the helix-coil transition for a 21-residue alanine peptide (AP) with the ZB model up to fourth nearest neighbor (micro = 1, 2, 3, and 4). We use a matrix approach that takes into account combinations of any number of helical stretches of any length and therefore gives the exact statistical weight of the chain within the assumptions of the ZB model. The parameters of the model are determined by fitting the temperature-dependent circular dichroism and Fourier transform infrared experimental spectra of the AP. All variants of the model fit the experimental data, thus giving similar results in terms of the macroscopic observables, such as temperature-dependent fractional helicity. However, the resulting microscopic parameters, such as distributions of the individual residue helical probabilities and free energy surfaces, vary significantly depending on the variant of the model. Overall, the mean residue enthalpy and entropy (in the absolute value) both increase with micro, but combined yield essentially the same "effective" value of the ZB propagation parameters for all micro. Greater helical probabilities for individual residues are predicted for larger micro, in particular, near the center of the sequence. The ZB nucleation parameters increase with increasing micro, which results in a lower free energy barrier to helix nucleation and lower apparent "cooperativity" of the transition. The significance of the long-range interactions for the predictions of ZB model for helix-coil transition, the calculated model parameters and the limitations of the model are discussed.

  20. Conservation of Hydrophobic and Hydrophilic Residues in Four-Helix Bundle

    Institute of Scientific and Technical Information of China (English)

    秦猛; 王骏; 王炜

    2003-01-01

    The conservation of the hydrophobic and the hydrophilic residue sites obtained from 1000 designed sequences with the Z-score method for a four-helix bundle has been studied. The folding dynamic and thermodynamic features of the designed sequences and their different mutations are also studied. It is found that this conservation is related to the stability and the fast folding of the model proteins. Our results are consistent with the experimental results.

  1. BILATERAL PERICHONDRITIS OF PINNA FOLLOWING PIERCING OF HELIX – A CASE REPORT

    Directory of Open Access Journals (Sweden)

    Rajamani

    2014-01-01

    Full Text Available Piercing of Helix of pinna is a social custom of certain communities in Northern Tamil Nadu. Such a “high helical” piercing is well known to cause Perichondritis of Pinna. Most of the times such a Perichondritis is unilateral. Here we present a case of Bilateral Perichondritis of pinna, following ear piercing, first time from India subcontinent. We also highlight some practical points on management of such cases.

  2. Systematische Untersuchungen zum Helix-Knäuel-Übergang in meta-Phenylenethinylen Polymeren

    OpenAIRE

    Kaiser, Christian

    2010-01-01

    The Sonogashira-Polycondensation, based on chiral AB’-monomers, can be used to construct different substituted and chiral, defect-free structures of PmPE`s. The optical properties of the polymers can be investigated by the methods of absorption-, fluorescence- or CD-spectroscopy. For synthesized PmPE`s the helix-coil-transition from a helical ordered state to an extended random conformation is characteristic, originating from solvophobic interactions with the solvent. Thereby, a different beh...

  3. Triple helix DNA alters nucleosomal histone-DNA interactions and acts as a nucleosome barrier.

    OpenAIRE

    Westin, L; Blomquist, P; Milligan, J F; Wrange, O

    1995-01-01

    Oligonucleotides which form triple helical complexes on double-stranded DNA have been previously reported to selectively inhibit transcription both in vitro and in vivo by physically blocking RNA polymerase or transcription factor access to the DNA template. Here we show that a 16mer oligonucleotide, which forms triple helix DNA by binding to a 16 bp homopurine segment, alters the formation of histone-DNA contacts during in vitro nucleosome reconstitution. This effect was DNA sequence-specifi...

  4. Convention of Optical Vortices in Two-Helix Long-Period Fiber Gratings

    Institute of Scientific and Technical Information of China (English)

    Xiao-Qiang Zhang; Rui-Shan Chen; Yong Zhou; Hai Ming; An-Ting Wang

    2016-01-01

    An effective method to fabricate two-helix long-period fiber gratings (TH-LFGs) is presented.Based on the coupling mode theory,the conversion of optical vortices (OVs) in TH-LFGs are analyzed in detail.The conversions of OVs with different topological charges:0 → ±2 and 1 → 3 are simulated as three examples and the conversion efficiency higher than 98% can be realized.

  5. Mechanism of stabilization of a bacterial collagen triple helix in the absence of hydroxyproline.

    Science.gov (United States)

    Mohs, Angela; Silva, Teresita; Yoshida, Takeshi; Amin, Ravish; Lukomski, Slawomir; Inouye, Masayori; Brodsky, Barbara

    2007-10-12

    The Streptococcus pyogenes cell-surface protein Scl2 contains a globular N-terminal domain and a collagen-like domain, (Gly-Xaa-X'aa)(79), which forms a triple helix with a thermal stability close to that seen for mammalian collagens. Hyp is a major contributor to triple-helix stability in animal collagens, but is not present in bacteria, which lack prolyl hydroxylase. To explore the basis of bacterial collagen triple-helix stability in the absence of Hyp, biophysical studies were carried out on recombinant Scl2 protein, the isolated collagen-like domain from Scl2, and a set of peptides modeling the Scl2 highly charged repetitive (Gly-Xaa-X'aa)(n) sequences. At pH 7, CD spectroscopy, dynamic light scattering, and differential scanning calorimetry of the Scl2 protein all showed a very sharp thermal transition near 36 degrees C, indicating a highly cooperative unfolding of both the globular and triple-helix domains. The collagen-like domain isolated by trypsin digestion showed a sharp transition at the same temperature, with an enthalpy of 12.5 kJ/mol of tripeptide. At low pH, Scl2 and its isolated collagen-like domain showed substantial destabilization from the neutral pH value, with two thermal transitions at 24 and 27 degrees C. A similar destabilization at low pH was seen for Scl2 charged model peptides, and the degree of destabilization was consistent with the strong pH dependence arising from the GKD tripeptide unit. The Scl2 protein contained twice as much charge as human fibril-forming collagens, and the degree of electrostatic stabilization observed for Scl2 was similar to the contribution Hyp makes to the stability of mammalian collagens. The high enthalpic contribution to the stability of the Scl2 collagenous domain supports the presence of a hydration network in the absence of Hyp.

  6. Triple Helix model of relations among university, industry and governments: a critical analysis

    OpenAIRE

    González de la Fe, Teresa

    2009-01-01

    In this essay, I attend to the Triple Helix Model of rela tions among university, industry and government, broadly used in innovation studies. The model was proposed by Henry Etzkowitz and Löet Leyersdorf and it has been very succesful as a heuristic for analysis of innovation process and as a normative guide for innovation policies. Moreover, its theoretical foundations are suited with perspectives from evolutive and institutionalist theories of economic science, at the same time that it tak...

  7. Coupled-mode analysis for single-helix chiral fiber gratings with small core-offset

    Institute of Scientific and Technical Information of China (English)

    Li Yang; Linlin Xue; Jue Su; Jingren Qian

    2011-01-01

    Using conventional coupled-mode theory,a set of coupled-mode equations are formulated for single-helix chiral fiber long-period gratings.A helical-core fiber is analyzed as an example.The analysis is simple in mathematical form and intuitive in physical concept.Based on the analysis,the polarization independence of mode coupling in special fiber gratings is revealed.The transmission characteristics of helical-core fibers are also simulated and discussed.

  8. Isolation and chemical analysis of nanoparticles from English ivy (Hedera helix L.)

    OpenAIRE

    Lenaghan, Scott C.; Burris, Jason N.; Chourey, Karuna; Huang, Yujian; Xia, Lijin; Lady, Belinda; Sharma, Ritin; Pan, Chongle; LeJeune, Zorabel; Foister, Shane; Hettich, Robert L.; Stewart, C Neal; Zhang, Mingjun

    2013-01-01

    Bio-inspiration for novel adhesive development has drawn increasing interest in recent years with the discovery of the nanoscale morphology of the gecko footpad and mussel adhesive proteins. Similar to these animal systems, it was discovered that English ivy (Hedera helix L.) secretes a high strength adhesive containing uniform nanoparticles. Recent studies have demonstrated that the ivy nanoparticles not only contribute to the high strength of this adhesive, but also have ultraviolet (UV) pr...

  9. Herschel imaging of the dust in the Helix Nebula (NGC 7293)

    CERN Document Server

    Van de Steene, G C; Exter, K M; Barlow, M J; Cernicharo, J; Etxaluze, M; Gear, W K; Goicoechea, J R; Gomez, H L; Groenewegen, M A T; Hargrave, P C; Ivison, R J; Leeks, S J; Lim, T L; Matsuura, M; Olofsson, G; Polehampton, E T; Swinyard, B M; Ueta, T; Van Winckel, H; Waelkens, C; Wesson, R

    2014-01-01

    In our series of papers presenting the Herschel imaging of evolved planetary nebulae, we present images of the dust distribution in the Helix nebula (NGC 7293). Images at 70, 160, 250, 350, and 500 micron were obtained with the PACS and SPIRE instruments on board the Herschel satellite. The broadband maps show the dust distribution over the main Helix nebula to be clumpy and predominantly present in the barrel wall. We determined the spectral energy distribution of the main nebula in a consistent way using Herschel, IRAS, and Planck flux values. The emissivity index of 0.99 +/- 0.09, in combination with the carbon rich molecular chemistry of the nebula, indicates that the dust consists mainly of amorphous carbon. The dust excess emission from the central star disk is detected at 70 micron and the flux measurement agree with previous measurement. We present the temperature and dust column density maps. The total dust mass across the Helix nebula (without its halo) is determined to be 0.0035 solar mass at a dis...

  10. Terrestrial gastropods (Helix spp) as sentinels of primary DNA damage for biomonitoring purposes: a validation study.

    Science.gov (United States)

    Angeletti, Dario; Sebbio, Claudia; Carere, Claudio; Cimmaruta, Roberta; Nascetti, Giuseppe; Pepe, Gaetano; Mosesso, Pasquale

    2013-04-01

    We validated the alkaline comet assay in two species of land snail (Helix aspersa and Helix vermiculata) to test their suitability as sentinels for primary DNA damage in polluted environments. The study was conducted under the framework of a biomonitoring program for a power station in Central Italy that had recently been converted from oil to coal-fired plant. After optimizing test conditions, the comet assay was used to measure the % Tail DNA induced by in vitro exposure of hemocytes to different concentrations of a reactive oxygen species (H2 O2 ). The treatment induced significant increases in this parameter with a concentration effect, indicating the effectiveness of the assay in snail hemocytes. After evaluating possible differences between the two species, we sampled them in three field sites at different distances from the power station, and in two reference sites assumed to have low or no levels of pollution. No species differences emerged. Percent Tail DNA values in snails from the sites near the power station were higher than those from control sites. An inverse correlation emerged between % Tail DNA and distance from the power station, suggesting that the primary DNA damage decreased as distance increased away from the pollution source. Detection of a gradient of heavy metal concentration in snail tissues suggests that these pollutants are a potential cause of the observed pattern. The comet assay appears to be a suitable assay and Helix spp. populations suitable sentinels to detect the genotoxic impact of pollutants.

  11. Structural dynamics of a single-stranded RNA–helix junction using NMR

    Science.gov (United States)

    Eichhorn, Catherine D.; Al-Hashimi, Hashim M.

    2014-01-01

    Many regulatory RNAs contain long single strands (ssRNA) that adjoin secondary structural elements. Here, we use NMR spectroscopy to study the dynamic properties of a 12-nucleotide (nt) ssRNA tail derived from the prequeuosine riboswitch linked to the 3′ end of a 48-nt hairpin. Analysis of chemical shifts, NOE connectivity, 13C spin relaxation, and residual dipolar coupling data suggests that the first two residues (A25 and U26) in the ssRNA tail stack onto the adjacent helix and assume an ordered conformation. The following U26-A27 step marks the beginning of an A6-tract and forms an acute pivot point for substantial motions within the tail, which increase toward the terminal end. Despite substantial internal motions, the ssRNA tail adopts, on average, an A-form helical conformation that is coaxial with the helix. Our results reveal a surprising degree of structural and dynamic complexity at the ssRNA–helix junction, which involves a fine balance between order and disorder that may facilitate efficient pseudoknot formation on ligand recognition. PMID:24742933

  12. Does alpha-helix folding necessarily provide an energy source for the protein-lipid binding?

    Science.gov (United States)

    Gursky, Olga

    2007-01-01

    Lipid-induced alpha-helix folding, which occurs in many lipid surface-binding proteins and peptides such as apolipoproteins and synucleins, has been proposed to provide an energy source for protein-lipid interactions. We propose that in a system comprised of a phospholipid surface and a small polypeptide that is unfolded in solution and binds reversibly to lipid surface, helical folding involves expenditure of free energy as compared to a similar polypeptide that is alpha-helical in solution. This is a consequence of the entropic cost of helix folding that is illustrated in a simple thermodynamic model and exemplifies the general "key-into-lock" paradigm of protein-ligand binding. Even though this simple model does not explicitly address the protein-induced lipid re-arrangement and may not directly apply to large proteins that undergo significant tertiary structural changes upon lipid binding, it suggests that the notion of helix folding as an energy source for lipid binding should be treated with caution.

  13. Statistical mechanical model for a closed loop plectoneme with weak helix specific forces.

    Science.gov (United States)

    Lee, Dominic J O'

    2017-04-12

    We develop a statistical mechanical framework, based on a variational approximation, to describe closed loop plectonemes. This framework incorporates weak helix structure dependent forces into the determination of the free energy and average structure of a plectoneme. Notably, due to their chiral nature, helix structure dependent forces break the symmetry between left and right handed supercoiling. The theoretical approach, presented here, also provides a systematic way of enforcing the topological constraint of closed loop supercoiling in the variational approximation. At large plectoneme lengths, by considering correlation functions in an expansion in terms of the spatial mean twist density about its thermally averaged value, it can be argued that topological constraint may be approximated by replacing twist and writhe by their thermal averages. A Lagrange multiplier, containing the sum of average twist and writhe, can be added to the free energy to conveniently inforce this result. The average writhe can be calculated through the thermal average of the Gauss' integral in the variational approximation. Furthermore, this approach allows for a possible way to calculate finite size corrections due to the topological constraint. Using interaction energy terms from the mean-field Kornyshev-Leikin theory, for parameter values that correspond to weak helix dependent forces, we calculate the free energy, fluctuation magnitudes and mean geometric parameters for the plectoneme. We see a slight asymmetry, where interestingly, left handed supercoils have a looser structure than right handed ones, although with a lower free energy, unlike what the previous ground state calculations would suggest.

  14. Forster Resonance Energy Transfer and Laser Fluorescent Analysis of Defects in DNA Double Helix

    CERN Document Server

    Bregadze, Vasil G; Giorgadze, Tamar G; Jaliashvili, Zaza V; Chkhaberidze, Jemal G; Monaselidze, Jamlet R; Khuskivadze, Temur B

    2013-01-01

    Real time laser induced fluorescence spectroscopy usage for microanalysis of DNA double helix defects is shown. The method is based on Forster resonance energy transfer (FRET) in intercalator-donor pair (acridine orange as a donor and ethidium bromide as an acceptor). Transition metal ions such as Cu(II), Cu(I), Ag(I), silver nanoparticles (AgNPs), photo- and thermo effects were used to cause double helix defects in DNA. FRET radii were experimentally estimated in background electrolyte solution (0.01 M NaNO3) and proved to be 3.9 +- 0.3 nm and the data are in satisfactory agreement with the theoretically calculated value Ro = 3.5 +- 0.3 nm. Concentration of DNA sites, exposed to Cu(II), Cu(I), Ag(I) ions, AgNPs impact as well as laser irradiation ({\\lambda} = 457 nm) and temperature, which are applicable for intercalation, were estimated in relative units. FRET method allows to estimate the concentration of double helix areas with high quality stability applicable for intercalation in DNA after it was subjec...

  15. Structure of the Membrane Anchor of Pestivirus Glycoprotein Erns, a Long Tilted Amphipathic Helix

    Science.gov (United States)

    Aberle, Daniel; Muhle-Goll, Claudia; Bürck, Jochen; Wolf, Moritz; Reißer, Sabine; Luy, Burkhard; Wenzel, Wolfgang; Ulrich, Anne S.; Meyers, Gregor

    2014-01-01

    Erns is an essential virion glycoprotein with RNase activity that suppresses host cellular innate immune responses upon being partially secreted from the infected cells. Its unusual C-terminus plays multiple roles, as the amphiphilic helix acts as a membrane anchor, as a signal peptidase cleavage site, and as a retention/secretion signal. We analyzed the structure and membrane binding properties of this sequence to gain a better understanding of the underlying mechanisms. CD spectroscopy in different setups, as well as Monte Carlo and molecular dynamics simulations confirmed the helical folding and showed that the helix is accommodated in the amphiphilic region of the lipid bilayer with a slight tilt rather than lying parallel to the surface. This model was confirmed by NMR analyses that also identified a central stretch of 15 residues within the helix that is fully shielded from the aqueous layer, which is C-terminally followed by a putative hairpin structure. These findings explain the strong membrane binding of the protein and provide clues to establishing the Erns membrane contact, processing and secretion. PMID:24586172

  16. Different effects of 4-hydroxyproline and 4-fluoroproline on the stability of collagen triple helix.

    Science.gov (United States)

    Nishi, Yoshinori; Uchiyama, Susumu; Doi, Masamitsu; Nishiuchi, Yuji; Nakazawa, Takashi; Ohkubo, Tadayasu; Kobayashi, Yuji

    2005-04-26

    Differential scanning calorimetry (DSC) analyses of a series of collagen model peptides suggest that 4-hydroxyproline (Hyp) and 4-fluoroproline (fPro) have different effects on the stability of the collagen triple helices according to the sequence of amino acids and stereochemistry at the 4 positions of these imino acids. The thermodynamic parameters indicate that the enhanced stabilities are classified into two different types: the enthalpy term is primarily responsible for the enhanced stability of the triple helix of (Pro-Hyp(R)-Gly)(10), whereas the entropy term dominates the enhanced stability of (Pro-fPro(R)-Gly)(10). The difference between the molecular volumes observed in solution and intrinsic molecular volumes calculated from the crystal structure indicates the different hydration states of these peptides. (Pro-Hyp(R)-Gly)(10) is highly hydrated compared to (Pro-Pro-Gly)(10), which contributes to the larger enthalpy. In contrast, the volume of (Pro-fPro(R)-Gly)(10) shows a smaller degree of hydration than that of (Pro-Pro-Gly)(10). The entropic cost of forming the triple helix of the fPro-containing peptides is compensated by a decrease in an ordered structure of water molecules surrounding the peptide molecule, although the contribution of enthalpy originating from the hydration is reduced. These arguments about the different contribution of entropic and enthalpic terms were successfully applied to interpret the stability of the triple helix of (fPro(S)-Pro-Gly)(10) as well.

  17. Role of side chains in collagen triple helix stabilization and partner recognition.

    Science.gov (United States)

    Berisio, Rita; De Simone, Alfonso; Ruggiero, Alessia; Improta, Roberto; Vitagliano, Luigi

    2009-03-01

    Collagen is a widespread protein family involved in a variety of biological processes. The complexity of collagen and its fibrous nature prevent detailed investigations on the full-length protein. Reductionist approaches conducted by dissecting the protein complexity through the use of model peptides have proved to be quite effective. There are, however, several issues regarding structure-stability relationships, aggregation in higher-order assemblies, and partner recognition that are still extensively investigated. In this review, we discuss the role that side chains play in triple helix stabilization and in partner recognition. On the basis of recent literature data, we show that collagen triple helix stability is the result of the interplay of different factors. As a general trend, interactions established by amino/imino acid side chains within the triple helix scaffold effectively modulate the intrinsic residue propensity for this common structural motif. The use of peptide models has also highlighted the role that side chains play in collagen self-association and in its interactions with receptors. Valuable examples in these fields are illustrated. Finally, future actions required to obtain more detailed information on the structure and the function of this complex protein are also delineated.

  18. A triple helix within a pseudoknot is a conserved and essential element of telomerase RNA.

    Science.gov (United States)

    Shefer, Kinneret; Brown, Yogev; Gorkovoy, Valentin; Nussbaum, Tamar; Ulyanov, Nikolai B; Tzfati, Yehuda

    2007-03-01

    Telomerase copies a short template within its integral telomerase RNA onto eukaryotic chromosome ends, compensating for incomplete replication and degradation. Telomerase action extends the proliferative potential of cells, and thus it is implicated in cancer and aging. Nontemplate regions of telomerase RNA are also crucial for telomerase function. However, they are highly divergent in sequence among species, and their roles are largely unclear. Using in silico three-dimensional modeling, constrained by mutational analysis, we propose a three-dimensional model for a pseudoknot in telomerase RNA of the budding yeast Kluyveromyces lactis. Interestingly, this structure includes a U-A.U major-groove triple helix. We confirmed the triple-helix formation in vitro using oligoribonucleotides and showed that it is essential for telomerase function in vivo. While triplex-disrupting mutations abolished telomerase function, triple compensatory mutations that formed pH-dependent G-C.C(+) triples restored the pseudoknot structure in a pH-dependent manner and partly restored telomerase function in vivo. In addition, we identified a novel type of triple helix that is formed by G-C.U triples, which also partly restored the pseudoknot structure and function. We propose that this unusual structure, so far found only in telomerase RNA, provides an essential and conserved telomerase-specific function.

  19. Crystal structure of the collagen triple helix model [(Pro-Pro-Gly)(10)](3).

    Science.gov (United States)

    Berisio, Rita; Vitagliano, Luigi; Mazzarella, Lelio; Zagari, Adriana

    2002-02-01

    The first report of the full-length structure of the collagen-like polypeptide [(Pro-Pro-Gly)(10)](3) is given. This structure was obtained from crystals grown in a microgravity environment, which diffracted up to 1.3 A, using synchrotron radiation. The final model, which was refined to an R(factor) of 0.18, is the highest-resolution description of a collagen triple helix reported to date. This structure provides clues regarding a series of aspects related to collagen triple helix structure and assembly. The strict dependence of proline puckering on the position inside the Pro-Pro-Gly triplets and the correlation between backbone and side chain dihedral angles support the propensity-based mechanism of triple helix stabilization/destabilization induced by hydroxyproline. Furthermore, the analysis of [(Pro-Pro-Gly)(10)](3) packing, which is governed by electrostatic interactions, suggests that charges may act as locking features in the axial organization of triple helices in the collagen fibrils.

  20. Left-handed polyproline-II helix revisited: proteins causing proteopathies.

    Science.gov (United States)

    Adzhubei, Alexei A; Anashkina, Anastasia A; Makarov, Alexander A

    2016-09-28

    Left-handed polyproline-II type helix is a regular conformation of polypeptide chain not only of fibrous, but also of folded and natively unfolded proteins and peptides. It is the only class of regular secondary structure substantially represented in non-fibrous proteins and peptides on a par with right-handed alpha-helix and beta-structure. In this study, we have shown that polyproline-II helix is abundant in several peptides and proteins involved in proteopathies, the amyloid-beta peptides, protein tau and prion protein. Polyproline-II helices form two interaction sites in the amyloid-beta peptides, which are pivotal for pathogenesis of Alzheimer's disease (AD). It also with high probability is the structure of the majority of tau phosphorylation sites, important for tau hyperphosphorylation and formation of neurofibrillary tangles, a hallmark of AD. Polyproline-II helices form large parts of the structure of the folded domain of prion protein. They can undergo conversion to beta-structure as a result of relatively small change of one torsional angle of polypeptide chain. We hypothesize that in prions and amyloids, in general polyproline-II helices can serve as structural elements of the normal structure as well as dormant nuclei of structure conversion, and thus play important role in structure changes leading to the formation of fibrils.