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Sample records for nucleotide binding pocket

  1. Rasp21 sequences opposite the nucleotide binding pocket are required for GRF-mediated nucleotide release

    DEFF Research Database (Denmark)

    Leonardsen, L; DeClue, J E; Lybaek, H

    1996-01-01

    The substrate requirements for the catalytic activity of the mouse Cdc25 homolog Guanine nucleotide Release Factor, GRF, were determined using the catalytic domain of GRF expressed in insect cells and E. coli expressed H-Ras mutants. We found a requirement for the loop 7 residues in Ras (amino ac...... and the human Ras like proteins RhoA, Rap1A, Rac1 and G25K revealed a strict Ras specificity; of these only S. pombe Ras was GRF sensitive....

  2. Structure of the HIV-1 reverse transcriptase Q151M mutant: insights into the inhibitor resistance of HIV-1 reverse transcriptase and the structure of the nucleotide-binding pocket of Hepatitis B virus polymerase

    International Nuclear Information System (INIS)

    Nakamura, Akiyoshi; Tamura, Noriko; Yasutake, Yoshiaki

    2015-01-01

    The structure of the HIV-1 reverse transcriptase Q151M mutant was determined at a resolution of 2.6 Å in space group P321. Hepatitis B virus polymerase (HBV Pol) is an important target for anti-HBV drug development; however, its low solubility and stability in vitro has hindered detailed structural studies. Certain nucleotide reverse transcriptase (RT) inhibitors (NRTIs) such as tenofovir and lamivudine can inhibit both HBV Pol and Human immunodeficiency virus 1 (HIV-1) RT, leading to speculation on structural and mechanistic analogies between the deoxynucleotide triphosphate (dNTP)-binding sites of these enzymes. The Q151M mutation in HIV-1 RT, located at the dNTP-binding site, confers resistance to various NRTIs, while maintaining sensitivity to tenofovir and lamivudine. The residue corresponding to Gln151 is strictly conserved as a methionine in HBV Pol. Therefore, the structure of the dNTP-binding pocket of the HIV-1 RT Q151M mutant may reflect that of HBV Pol. Here, the crystal structure of HIV-1 RT Q151M, determined at 2.6 Å resolution, in a new crystal form with space group P321 is presented. Although the structure of HIV-1 RT Q151M superimposes well onto that of HIV-1 RT in a closed conformation, a slight movement of the β-strands (β2–β3) that partially create the dNTP-binding pocket was observed. This movement might be caused by the introduction of the bulky thioether group of Met151. The structure also highlighted the possibility that the hydrogen-bonding network among amino acids and NRTIs is rearranged by the Q151M mutation, leading to a difference in the affinity of NRTIs for HIV-1 RT and HBV Pol

  3. Structure of the HIV-1 reverse transcriptase Q151M mutant: insights into the inhibitor resistance of HIV-1 reverse transcriptase and the structure of the nucleotide-binding pocket of Hepatitis B virus polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, Akiyoshi; Tamura, Noriko; Yasutake, Yoshiaki, E-mail: y-yasutake@aist.go.jp [National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo, Hokkaido 062-8517 (Japan)

    2015-10-23

    The structure of the HIV-1 reverse transcriptase Q151M mutant was determined at a resolution of 2.6 Å in space group P321. Hepatitis B virus polymerase (HBV Pol) is an important target for anti-HBV drug development; however, its low solubility and stability in vitro has hindered detailed structural studies. Certain nucleotide reverse transcriptase (RT) inhibitors (NRTIs) such as tenofovir and lamivudine can inhibit both HBV Pol and Human immunodeficiency virus 1 (HIV-1) RT, leading to speculation on structural and mechanistic analogies between the deoxynucleotide triphosphate (dNTP)-binding sites of these enzymes. The Q151M mutation in HIV-1 RT, located at the dNTP-binding site, confers resistance to various NRTIs, while maintaining sensitivity to tenofovir and lamivudine. The residue corresponding to Gln151 is strictly conserved as a methionine in HBV Pol. Therefore, the structure of the dNTP-binding pocket of the HIV-1 RT Q151M mutant may reflect that of HBV Pol. Here, the crystal structure of HIV-1 RT Q151M, determined at 2.6 Å resolution, in a new crystal form with space group P321 is presented. Although the structure of HIV-1 RT Q151M superimposes well onto that of HIV-1 RT in a closed conformation, a slight movement of the β-strands (β2–β3) that partially create the dNTP-binding pocket was observed. This movement might be caused by the introduction of the bulky thioether group of Met151. The structure also highlighted the possibility that the hydrogen-bonding network among amino acids and NRTIs is rearranged by the Q151M mutation, leading to a difference in the affinity of NRTIs for HIV-1 RT and HBV Pol.

  4. Structure of the HIV-1 reverse transcriptase Q151M mutant: insights into the inhibitor resistance of HIV-1 reverse transcriptase and the structure of the nucleotide-binding pocket of Hepatitis B virus polymerase.

    Science.gov (United States)

    Nakamura, Akiyoshi; Tamura, Noriko; Yasutake, Yoshiaki

    2015-11-01

    Hepatitis B virus polymerase (HBV Pol) is an important target for anti-HBV drug development; however, its low solubility and stability in vitro has hindered detailed structural studies. Certain nucleotide reverse transcriptase (RT) inhibitors (NRTIs) such as tenofovir and lamivudine can inhibit both HBV Pol and Human immunodeficiency virus 1 (HIV-1) RT, leading to speculation on structural and mechanistic analogies between the deoxynucleotide triphosphate (dNTP)-binding sites of these enzymes. The Q151M mutation in HIV-1 RT, located at the dNTP-binding site, confers resistance to various NRTIs, while maintaining sensitivity to tenofovir and lamivudine. The residue corresponding to Gln151 is strictly conserved as a methionine in HBV Pol. Therefore, the structure of the dNTP-binding pocket of the HIV-1 RT Q151M mutant may reflect that of HBV Pol. Here, the crystal structure of HIV-1 RT Q151M, determined at 2.6 Å resolution, in a new crystal form with space group P321 is presented. Although the structure of HIV-1 RT Q151M superimposes well onto that of HIV-1 RT in a closed conformation, a slight movement of the β-strands (β2-β3) that partially create the dNTP-binding pocket was observed. This movement might be caused by the introduction of the bulky thioether group of Met151. The structure also highlighted the possibility that the hydrogen-bonding network among amino acids and NRTIs is rearranged by the Q151M mutation, leading to a difference in the affinity of NRTIs for HIV-1 RT and HBV Pol.

  5. Free enthalpies of replacing water molecules in protein binding pockets.

    Science.gov (United States)

    Riniker, Sereina; Barandun, Luzi J; Diederich, François; Krämer, Oliver; Steffen, Andreas; van Gunsteren, Wilfred F

    2012-12-01

    Water molecules in the binding pocket of a protein and their role in ligand binding have increasingly raised interest in recent years. Displacement of such water molecules by ligand atoms can be either favourable or unfavourable for ligand binding depending on the change in free enthalpy. In this study, we investigate the displacement of water molecules by an apolar probe in the binding pocket of two proteins, cyclin-dependent kinase 2 and tRNA-guanine transglycosylase, using the method of enveloping distribution sampling (EDS) to obtain free enthalpy differences. In both cases, a ligand core is placed inside the respective pocket and the remaining water molecules are converted to apolar probes, both individually and in pairs. The free enthalpy difference between a water molecule and a CH(3) group at the same location in the pocket in comparison to their presence in bulk solution calculated from EDS molecular dynamics simulations corresponds to the binding free enthalpy of CH(3) at this location. From the free enthalpy difference and the enthalpy difference, the entropic contribution of the displacement can be obtained too. The overlay of the resulting occupancy volumes of the water molecules with crystal structures of analogous ligands shows qualitative correlation between experimentally measured inhibition constants and the calculated free enthalpy differences. Thus, such an EDS analysis of the water molecules in the binding pocket may give valuable insight for potency optimization in drug design.

  6. Identification of potential small molecule binding pockets on Rho family GTPases.

    Directory of Open Access Journals (Sweden)

    Juan Manuel Ortiz-Sanchez

    Full Text Available Rho GTPases are conformational switches that control a wide variety of signaling pathways critical for eukaryotic cell development and proliferation. They represent attractive targets for drug design as their aberrant function and deregulated activity is associated with many human diseases including cancer. Extensive high-resolution structures (>100 and recent mutagenesis studies have laid the foundation for the design of new structure-based chemotherapeutic strategies. Although the inhibition of Rho signaling with drug-like compounds is an active area of current research, very little attention has been devoted to directly inhibiting Rho by targeting potential allosteric non-nucleotide binding sites. By avoiding the nucleotide binding site, compounds may minimize the potential for undesirable off-target interactions with other ubiquitous GTP and ATP binding proteins. Here we describe the application of molecular dynamics simulations, principal component analysis, sequence conservation analysis, and ensemble small-molecule fragment mapping to provide an extensive mapping of potential small-molecule binding pockets on Rho family members. Characterized sites include novel pockets in the vicinity of the conformationaly responsive switch regions as well as distal sites that appear to be related to the conformations of the nucleotide binding region. Furthermore the use of accelerated molecular dynamics simulation, an advanced sampling method that extends the accessible time-scale of conventional simulations, is found to enhance the characterization of novel binding sites when conformational changes are important for the protein mechanism.

  7. On the benzodiazepine binding pocket in GABAA receptors.

    Science.gov (United States)

    Berezhnoy, Dmytro; Nyfeler, Yves; Gonthier, Anne; Schwob, Hervé; Goeldner, Maurice; Sigel, Erwin

    2004-01-30

    Benzodiazepines are used for their sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsive effects. They exert their actions through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the gamma-aminobutyric acid, type A (GABA(A)) receptor channel, where they act as positive allosteric modulators. To start to elucidate the relative positioning of benzodiazepine binding site ligands in their binding pocket, GABA(A) receptor residues thought to reside in the site were individually mutated to cysteine and combined with benzodiazepine analogs carrying substituents reactive to cysteine. Direct apposition of such reactive partners is expected to lead to an irreversible site-directed reaction. We describe here the covalent interaction of alpha(1)H101C with a reactive group attached to the C-7 position of diazepam. This interaction was studied at the level of radioactive ligand binding and at the functional level using electrophysiological methods. Covalent reaction occurs concomitantly with occupancy of the binding pocket. It stabilizes the receptor in its allosterically stimulated conformation. Covalent modification is not observed in wild type receptors or when using mutated alpha(1)H101C-containing receptors in combination with the reactive ligand pre-reacted with a sulfhydryl group, and the modification rate is reduced by the binding site ligand Ro15-1788. We present in addition evidence that gamma(2)Ala-79 is probably located in the access pathway of the ligand to its binding pocket.

  8. Investigating cyclic nucleotide and cyclic dinucleotide binding to HCN channels by surface plasmon resonance.

    Directory of Open Access Journals (Sweden)

    Sebastien Hayoz

    Full Text Available Hyperpolarization-activated cyclic nucleotide-modulated (HCN channels control cardiac and neuronal rhythmicity. HCN channels contain cyclic nucleotide-binding domain (CNBD in their C-terminal region linked to the pore-forming transmembrane segment with a C-linker. The C-linker couples the conformational changes caused by the direct binding of cyclic nucleotides to the HCN pore opening. Recently, cyclic dinucleotides were shown to antagonize the effect of cyclic nucleotides in HCN4 but not in HCN2 channels. Based on the structural analysis and mutational studies it has been proposed that cyclic dinucleotides affect HCN4 channels by binding to the C-linker pocket (CLP. Here, we first show that surface plasmon resonance (SPR can be used to accurately measure cyclic nucleotide binding affinity to the C-linker/CNBD of HCN2 and HCN4 channels. We then used SPR to investigate cyclic dinucleotide binding in HCN channels. To our surprise, we detected no binding of cyclic dinucleotides to the isolated monomeric C-linker/CNBDs of HCN4 channels with SPR. The binding of cyclic dinucleotides was further examined with isothermal calorimetry (ITC, which indicated no binding of cyclic dinucleotides to both monomeric and tetrameric C-linker/CNBDs of HCN4 channels. Taken together, our results suggest that interaction of the C-linker/CNBD with other parts of the channel is necessary for cyclic-dinucleotide binding in HCN4 channels.

  9. The Tomato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor I-2 Couples DNA-Binding to Nucleotide-Binding Domain Nucleotide Exchange

    NARCIS (Netherlands)

    Fenyk, S.; Dixon, C.H.; Gittens, W.H.; Townsend, P.D.; Sharples, G.J.; Pålsson, L.O.; Takken, F.L.W.; Cann, M.J.

    2016-01-01

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognise and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception.

  10. The minor binding pocket: a major player in 7TM receptor activation

    DEFF Research Database (Denmark)

    Rosenkilde, Mette Marie; Benned-Jensen, Tau; Frimurer, Thomas M.

    2010-01-01

    From the deep part of the main ligand-binding crevice, a minor, often shallower pocket extends between the extracellular ends of transmembrane domains (TM)-I, II, III and VII of 7TM receptors. This minor binding pocket is defined by a highly conserved kink in TM-II that is induced by a proline...... residue located in one of two adjacent positions. Here we argue that this minor binding pocket is important for receptor activation. Functional coupling of the receptors seems to be mediated through the hydrogen bond network located between the intracellular segments of these TMs, with the allosteric...

  11. Design and synthesis of ATP-based nucleotide analogues and profiling of nucleotide-binding proteins

    NARCIS (Netherlands)

    Wolters, Justina. C.; Roelfes, Gerard; Poolman, Bert

    Two nucleotide-based probes were designed and synthesized in order to enrich samples for specific classes of proteins by affinity-based protein profiling. We focused on the profiling of adenine nucleotide-binding proteins. Two properties were considered in the design of the probes: the bait needs to

  12. The PickPocket method for predicting binding specificities for receptors based on receptor pocket similarities: application to MHC-peptide binding

    DEFF Research Database (Denmark)

    Zhang, H.; Lund, Ole; Nielsen, M.

    2009-01-01

    of the specificities of MHC molecules in this library weighted by the similarity of their pocket-residues to the query. This PickPocket method is demonstrated to accurately predict MHC-peptide binding for a broad range of MHC alleles, including human and non-human species. In contrast to neural network-based pan-specific......Motivation: Receptor-ligand interactions play an important role in controlling many biological systems. One prominent example is the binding of peptides to the major histocompatibility complex (MHC) molecules controlling the onset of cellular immune responses. Thousands of MHC allelic versions...... exist, making determination of the binding specificity for each variant experimentally infeasible. Here, we present a method that can extrapolate from variants with known binding specificity to those where no experimental data are available. Results: For each position in the peptide ligand, we extracted...

  13. Doubling the Size of the Glucocorticoid Receptor Ligand Binding Pocket by Deacylcortivazol

    Energy Technology Data Exchange (ETDEWEB)

    Suino-Powell, Kelly; Xu, Yong; Zhang, Chenghai; Tao, Yong-guang; Tolbert, W. David; Simons, Jr., S. Stoney; Xu, H. Eric (NIH)

    2010-03-08

    A common feature of nuclear receptor ligand binding domains (LBD) is a helical sandwich fold that nests a ligand binding pocket within the bottom half of the domain. Here we report that the ligand pocket of glucocorticoid receptor (GR) can be continuously extended into the top half of the LBD by binding to deacylcortivazol (DAC), an extremely potent glucocorticoid. It has been puzzling for decades why DAC, which contains a phenylpyrazole replacement at the conserved 3-ketone of steroid hormones that are normally required for activation of their cognate receptors, is a potent GR activator. The crystal structure of the GR LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 reveals that the GR ligand binding pocket is expanded to a size of 1,070 {angstrom}{sup 3}, effectively doubling the size of the GR dexamethasone-binding pocket of 540 {angstrom}{sup 3} and yet leaving the structure of the coactivator binding site intact. DAC occupies only {approx}50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. The dramatic expansion of the DAC-binding pocket thus highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.

  14. A Nucleotide Phosphatase Activity in the Nucleotide Binding Domain of an Orphan Resistance Protein from Rice*

    Science.gov (United States)

    Fenyk, Stepan; de San Eustaquio Campillo, Alba; Pohl, Ehmke; Hussey, Patrick J.; Cann, Martin J.

    2012-01-01

    Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack. PMID:22157756

  15. A nucleotide phosphatase activity in the nucleotide binding domain of an orphan resistance protein from rice.

    Science.gov (United States)

    Fenyk, Stepan; Campillo, Alba de San Eustaquio; Pohl, Ehmke; Hussey, Patrick J; Cann, Martin J

    2012-02-03

    Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack.

  16. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange*

    Science.gov (United States)

    Fenyk, Stepan; Dixon, Christopher H.; Gittens, William H.; Townsend, Philip D.; Sharples, Gary J.; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

    2016-01-01

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. PMID:26601946

  17. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

    Science.gov (United States)

    Fenyk, Stepan; Dixon, Christopher H; Gittens, William H; Townsend, Philip D; Sharples, Gary J; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

    2016-01-15

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. A comprehensive survey of small-molecule binding pockets in proteins.

    Directory of Open Access Journals (Sweden)

    Mu Gao

    2013-10-01

    Full Text Available Many biological activities originate from interactions between small-molecule ligands and their protein targets. A detailed structural and physico-chemical characterization of these interactions could significantly deepen our understanding of protein function and facilitate drug design. Here, we present a large-scale study on a non-redundant set of about 20,000 known ligand-binding sites, or pockets, of proteins. We find that the structural space of protein pockets is crowded, likely complete, and may be represented by about 1,000 pocket shapes. Correspondingly, the growth rate of novel pockets deposited in the Protein Data Bank has been decreasing steadily over the recent years. Moreover, many protein pockets are promiscuous and interact with ligands of diverse scaffolds. Conversely, many ligands are promiscuous and interact with structurally different pockets. Through a physico-chemical and structural analysis, we provide insights into understanding both pocket promiscuity and ligand promiscuity. Finally, we discuss the implications of our study for the prediction of protein-ligand interactions based on pocket comparison.

  19. Nucleotide binding to Na+/K+-ATPase

    Czech Academy of Sciences Publication Activity Database

    Kubala, Martin; Lánský, Zdeněk; Ettrich, R.; Plášek, J.; Teisinger, Jan; Amler, Evžen

    2005-01-01

    Roč. 272, č. S1 (2005), s. 191-191 E-ISSN 1742-4658. [FEBS Congress /30./ and IUBMB Conference /9./. 02.07.2005-07.07.2005, Budapest] Keywords : Na+/K+- ATPase * ATP binding * TNP-ATP Subject RIV: BO - Biophysics

  20. Displacement of disordered water molecules from hydrophobic pocket creates enthalpic signature: binding of phosphonamidate to the S₁'-pocket of thermolysin.

    Science.gov (United States)

    Englert, L; Biela, A; Zayed, M; Heine, A; Hangauer, D; Klebe, G

    2010-11-01

    Prerequisite for the design of tight binding protein inhibitors and prediction of their properties is an in-depth understanding of the structural and thermodynamic details of the binding process. A series of closely related phosphonamidates was studied to elucidate the forces underlying their binding affinity to thermolysin. The investigated inhibitors are identical except for the parts penetrating into the hydrophobic S₁'-pocket. A correlation of structural, kinetic and thermodynamic data was carried out by X-ray crystallography, kinetic inhibition assay and isothermal titration calorimetry. Binding affinity increases with larger ligand hydrophobic P₁'-moieties accommodating the S₁'-pocket. Surprisingly, larger P₁'-side chain modifications are accompanied by an increase in the enthalpic contribution to binding. In agreement with other studies, it is suggested that the release of largely disordered waters from an imperfectly hydrated pocket results in an enthalpically favourable integration of these water molecules into bulk water upon inhibitor binding. This enthalpically favourable process contributes more strongly to the binding energetics than the entropy increase resulting from the release of water molecules from the S₁'-pocket or the formation of apolar interactions between protein and inhibitor. Displacement of highly disordered water molecules from a rather imperfectly hydrated and hydrophobic specificity pocket can reveal an enthalpic signature of inhibitor binding. Copyright © 2010 Elsevier B.V. All rights reserved.

  1. Diversity and evolutionary relationship of nucleotide binding site ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    Most plant disease-resistance genes (R-genes) isolated so far encode proteins with a nucleotide binding site (NBS) domain and belong to a superfamily. NBS domains related to R-genes show a highly conserved backbone of an amino acid motif, which makes it possible to isolate resistance gene analogues (RGAs) by ...

  2. Structural determinants of a conserved enantiomer-selective carvone binding pocket in the human odorant receptor OR1A1.

    Science.gov (United States)

    Geithe, Christiane; Protze, Jonas; Kreuchwig, Franziska; Krause, Gerd; Krautwurst, Dietmar

    2017-11-01

    Chirality is a common phenomenon within odorants. Most pairs of enantiomers show only moderate differences in odor quality. One example for enantiomers that are easily discriminated by their odor quality is the carvones: humans significantly distinguish between the spearmint-like (R)-(-)-carvone and caraway-like (S)-(+)-carvone enantiomers. Moreover, for the (R)-(-)-carvone, an anosmia is observed in about 8% of the population, suggesting enantioselective odorant receptors (ORs). With only about 15% de-orphaned human ORs, the lack of OR crystal structures, and few comprehensive studies combining in silico and experimental approaches to elucidate structure-function relations of ORs, knowledge on cognate odorant/OR interactions is still sparse. An adjusted homology modeling approach considering OR-specific proline-caused conformations, odorant docking studies, single-nucleotide polymorphism (SNP) analysis, site-directed mutagenesis, and subsequent functional studies with recombinant ORs in a cell-based, real-time luminescence assay revealed 11 amino acid positions to constitute an enantioselective binding pocket necessary for a carvone function in human OR1A1 and murine Olfr43, respectively. Here, we identified enantioselective molecular determinants in both ORs that discriminate between minty and caraway odor. Comparison with orthologs from 36 mammalian species demonstrated a hominid-specific carvone binding pocket with about 100% conservation. Moreover, we identified loss-of-function SNPs associated with the carvone binding pocket of OR1A1. Given carvone enantiomer-specific receptor activation patterns including OR1A1, our data suggest OR1A1 as a candidate receptor for constituting a carvone enantioselective phenotype, which may help to explain mechanisms underlying a (R)-(-)-carvone-specific anosmia in humans.

  3. Structural studies on MtRecA-nucleotide complexes: insights into DNA and nucleotide binding and the structural signature of NTP recognition.

    Science.gov (United States)

    Datta, S; Ganesh, N; Chandra, Nagasuma R; Muniyappa, K; Vijayan, M

    2003-02-15

    RecA protein plays a crucial role in homologous recombination and repair of DNA. Central to all activities of RecA is its binding to Mg(+2)-ATP. The active form of the protein is a helical nucleoprotein filament containing the nucleotide cofactor and single-stranded DNA. The stability and structure of the helical nucleoprotein filament formed by RecA are modulated by nucleotide cofactors. Here we report crystal structures of a MtRecA-ADP complex, complexes with ATPgammaS in the presence and absence of magnesium as well as a complex with dATP and Mg+2. Comparison with the recently solved crystal structures of the apo form as well as a complex with ADP-AlF4 confirms an expansion of the P-loop region in MtRecA, compared to its homologue in Escherichia coli, correlating with the reduced affinity of MtRecA for ATP. The ligand bound structures reveal subtle variations in nucleotide conformations among different nucleotides that serve in maintaining the network of interactions crucial for nucleotide binding. The nucleotide binding site itself, however, remains relatively unchanged. The analysis also reveals that ATPgammaS rather than ADP-AlF4 is structurally a better mimic of ATP. From among the complexed structures, a definition for the two DNA-binding loops L1 and L2 has clearly emerged for the first time and provides a basis to understand DNA binding by RecA. The structural information obtained from these complexes correlates well with the extensive biochemical data on mutants available in the literature, contributing to an understanding of the role of individual residues in the nucleotide binding pocket, at the molecular level. Modeling studies on the mutants again point to the relative rigidity of the nucleotide binding site. Comparison with other NTP binding proteins reveals many commonalties in modes of binding by diverse members in the structural family, contributing to our understanding of the structural signature of NTP recognition. Copyright 2003 Wiley

  4. Prediction of Nucleotide Binding Peptides Using Star Graph Topological Indices.

    Science.gov (United States)

    Liu, Yong; Munteanu, Cristian R; Fernández Blanco, Enrique; Tan, Zhiliang; Santos Del Riego, Antonino; Pazos, Alejandro

    2015-11-01

    The nucleotide binding proteins are involved in many important cellular processes, such as transmission of genetic information or energy transfer and storage. Therefore, the screening of new peptides for this biological function is an important research topic. The current study proposes a mixed methodology to obtain the first classification model that is able to predict new nucleotide binding peptides, using only the amino acid sequence. Thus, the methodology uses a Star graph molecular descriptor of the peptide sequences and the Machine Learning technique for the best classifier. The best model represents a Random Forest classifier based on two features of the embedded and non-embedded graphs. The performance of the model is excellent, considering similar models in the field, with an Area Under the Receiver Operating Characteristic Curve (AUROC) value of 0.938 and true positive rate (TPR) of 0.886 (test subset). The prediction of new nucleotide binding peptides with this model could be useful for drug target studies in drug development. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers*

    Science.gov (United States)

    Zoghbi, Maria E.; Altenberg, Guillermo A.

    2013-01-01

    The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides “sandwiched” at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we used luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation. PMID:24129575

  6. Spatial Decomposition of Translational Water–Water Correlation Entropy in Binding Pockets

    Science.gov (United States)

    2015-01-01

    A number of computational tools available today compute the thermodynamic properties of water at surfaces and in binding pockets by using inhomogeneous solvation theory (IST) to analyze explicit-solvent simulations. Such methods enable qualitative spatial mappings of both energy and entropy around a solute of interest and can also be applied quantitatively. However, the entropy estimates of existing methods have, to date, been almost entirely limited to the first-order terms in the IST’s entropy expansion. These first-order terms account for localization and orientation of water molecules in the field of the solute but not for the modification of water–water correlations by the solute. Here, we present an extension of the Grid Inhomogeneous Solvation Theory (GIST) approach which accounts for water–water translational correlations. The method involves rewriting the two-point density of water in terms of a conditional density and utilizes the efficient nearest-neighbor entropy estimation approach. Spatial maps of this second order term, for water in and around the synthetic host cucurbit[7]uril and in the binding pocket of the enzyme Factor Xa, reveal mainly negative contributions, indicating solute-induced water–water correlations relative to bulk water; particularly strong signals are obtained for sites at the entrances of cavities or pockets. This second-order term thus enters with the same, negative, sign as the first order translational and orientational terms. Numerical and convergence properties of the methodology are examined. PMID:26636620

  7. Computational identification and experimental characterization of substrate binding determinants of nucleotide pyrophosphatase/phosphodiesterase 7

    Directory of Open Access Journals (Sweden)

    Parrill Abby L

    2011-12-01

    Full Text Available Abstract Background Nucleotide pyrophosphatase/phosphodiesterase 7 (NPP7 is the only member of the mammalian NPP enzyme family that has been confirmed to act as a sphingomyelinase, hydrolyzing sphingomyelin (SM to form phosphocholine and ceramide. NPP7 additionally hydrolyzes lysophosphatidylcholine (LPC, a substrate preference shared with the NPP2/autotaxin(ATX and NPP6 mammalian family members. This study utilizes a synergistic combination of molecular modeling validated by experimental site-directed mutagenesis to explore the molecular basis for the unique ability of NPP7 to hydrolyze SM. Results The catalytic function of NPP7 against SM, LPC, platelet activating factor (PAF and para-nitrophenylphosphorylcholine (pNPPC is impaired in the F275A mutant relative to wild type NPP7, but different impacts are noted for mutations at other sites. These results are consistent with a previously described role of F275 to interact with the choline headgroup, where all substrates share a common functionality. The L107F mutation showed enhanced hydrolysis of LPC, PAF and pNPPC but reduced hydrolysis of SM. Modeling suggests this difference can be explained by the gain of cation-pi interactions with the choline headgroups of all four substrates, opposed by increased steric crowding against the sphingoid tail of SM. Modeling also revealed that the long and flexible hydrophobic tails of substrates exhibit considerable dynamic flexibility in the binding pocket, reducing the entropic penalty that might otherwise be incurred upon substrate binding. Conclusions Substrate recognition by NPP7 includes several important contributions, ranging from cation-pi interactions between F275 and the choline headgroup of all substrates, to tail-group binding pockets that accommodate the inherent flexibility of the lipid hydrophobic tails. Two contributions to the unique ability of NPP7 to hydrolyze SM were identified. First, the second hydrophobic tail of SM occupies a second

  8. Importance of Residues Outside the Cation Binding Pocket for Na+ and K+ Binding to the Na+/K+-ATPase

    DEFF Research Database (Denmark)

    Christiansen, Line; Toustrup-Jensen, Mads Schak; Einholm, Anja P.

    Mutagenesis studies have identified several oxygen-containing residues in the transmembrane region which are important for the coordination of Na+ and/or K+. These were later confirmed by the high-resolution crystal structures of the Na+/K+-ATPase with bound Na+ or K+. However, more information...... is needed to reveal the translocation pathway through the Na+/K+-ATPase. Mutagenesis studies have shown that several other transmembrane residues located outside the cation binding pocket, including Phe785 and Phe788, affect the ion binding properties of the Na+/K+-ATPase (1,2). It is well...... not significantly affect the Na+ binding properties of the Na+/K+-ATPase. 1. Vilsen, B. (1999) Biochemistry 38, 11389 2. Rodacker, V. et al (2006) JBC 281, 18539 3. Laursen M et al. (2013) PNAS 110, 10958...

  9. The same pocket in menin binds both MLL and JUND but has opposite effects on transcription

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Jing; Gurung, Buddha; Wan, Bingbing; Matkar, Smita; Veniaminova, Natalia A.; Wan, Ke; Merchant, Juanita L.; Hua, Xianxin; Lei, Ming (Michigan); (Michigan-Med); (UPENN-MED)

    2013-04-08

    Menin is a tumour suppressor protein whose loss or inactivation causes multiple endocrine neoplasia 1 (MEN1), a hereditary autosomal dominant tumour syndrome that is characterized by tumorigenesis in multiple endocrine organs. Menin interacts with many proteins and is involved in a variety of cellular processes. Menin binds the JUN family transcription factor JUND and inhibits its transcriptional activity. Several MEN1 missense mutations disrupt the menin-JUND interaction, suggesting a correlation between the tumour-suppressor function of menin and its suppression of JUND-activated transcription. Menin also interacts with mixed lineage leukaemia protein 1 (MLL1), a histone H3 lysine 4 methyltransferase, and functions as an oncogenic cofactor to upregulate gene transcription and promote MLL1-fusion-protein-induced leukaemogenesis. A recent report on the tethering of MLL1 to chromatin binding factor lens epithelium-derived growth factor (LEDGF) by menin indicates that menin is a molecular adaptor coordinating the functions of multiple proteins. Despite its importance, how menin interacts with many distinct partners and regulates their functions remains poorly understood. Here we present the crystal structures of human menin in its free form and in complexes with MLL1 or with JUND, or with an MLL1-LEDGF heterodimer. These structures show that menin contains a deep pocket that binds short peptides of MLL1 or JUND in the same manner, but that it can have opposite effects on transcription. The menin-JUND interaction blocks JUN N-terminal kinase (JNK)-mediated JUND phosphorylation and suppresses JUND-induced transcription. In contrast, menin promotes gene transcription by binding the transcription activator MLL1 through the peptide pocket while still interacting with the chromatin-anchoring protein LEDGF at a distinct surface formed by both menin and MLL1.

  10. Specificity of anion-binding in the substrate-pocket ofbacteriorhodopsin

    Energy Technology Data Exchange (ETDEWEB)

    Facciotti, Marc T.; Cheung, Vincent S.; Lunde, Christopher S.; Rouhani, Shahab; Baliga, Nitin S.; Glaeser, Robert M.

    2003-08-30

    The structure of the D85S mutant of bacteriorhodopsin with a nitrate anion bound in the Schiff-base binding site, and the structure of the anion-free protein have been obtained in the same crystal form. Together with the previously solved structures of this anion pump, in both the anion-free state and bromide-bound state, these new structures provide insight into how this mutant of bacteriorhodopsin is able to bind a variety of different anions in the same binding pocket. The structural analysis reveals that the main structural change that accommodates different anions is the repositioning of the polar side-chain of S85. On the basis of these x-ray crystal structures, the prediction is then made that the D85S/D212N double mutant might bind similar anions and do so over a broader pH range than does the single mutant. Experimental comparison of the dissociation constants, K{sub d}, for a variety of anions confirms this prediction and demonstrates, in addition, that the binding affinity is dramatically improved by the D212N substitution.

  11. Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin

    Energy Technology Data Exchange (ETDEWEB)

    Whittle, James R.R.; Zhang, Ruijun; Khurana, Surender; King, Lisa R.; Manischewitz, Jody; Golding, Hana; Dormitzer, Philip R.; Haynes, Barton F.; Walter, Emmanuel B.; Moody, M. Anthony; Kepler, Thomas B.; Liao, Hua-Xin; Harrison, Stephen C. (Harvard-Med); (Novartis); (US-FDA); (Duke)

    2011-09-20

    Seasonal antigenic drift of circulating influenza virus leads to a requirement for frequent changes in vaccine composition, because exposure or vaccination elicits human antibodies with limited cross-neutralization of drifted strains. We describe a human monoclonal antibody, CH65, obtained by isolating rearranged heavy- and light-chain genes from sorted single plasma cells, coming from a subject immunized with the 2007 trivalent influenza vaccine. The crystal structure of a complex of the hemagglutinin (HA) from H1N1 strain A/Solomon Islands/3/2006 with the Fab of CH65 shows that the tip of the CH65 heavy-chain complementarity determining region 3 (CDR3) inserts into the receptor binding pocket on HA1, mimicking in many respects the interaction of the physiological receptor, sialic acid. CH65 neutralizes infectivity of 30 out of 36 H1N1 strains tested. The resistant strains have a single-residue insertion near the rim of the sialic-acid pocket. We conclude that broad neutralization of influenza virus can be achieved by antibodies with contacts that mimic those of the receptor.

  12. Nucleos: a web server for the identification of nucleotide-binding sites in protein structures.

    Science.gov (United States)

    Parca, Luca; Ferré, Fabrizio; Ausiello, Gabriele; Helmer-Citterich, Manuela

    2013-07-01

    Nucleos is a web server for the identification of nucleotide-binding sites in protein structures. Nucleos compares the structure of a query protein against a set of known template 3D binding sites representing nucleotide modules, namely the nucleobase, carbohydrate and phosphate. Structural features, clustering and conservation are used to filter and score the predictions. The predicted nucleotide modules are then joined to build whole nucleotide-binding sites, which are ranked by their score. The server takes as input either the PDB code of the query protein structure or a user-submitted structure in PDB format. The output of Nucleos is composed of ranked lists of predicted nucleotide-binding sites divided by nucleotide type (e.g. ATP-like). For each ranked prediction, Nucleos provides detailed information about the score, the template structure and the structural match for each nucleotide module composing the nucleotide-binding site. The predictions on the query structure and the template-binding sites can be viewed directly on the web through a graphical applet. In 98% of the cases, the modules composing correct predictions belong to proteins with no homology relationship between each other, meaning that the identification of brand-new nucleotide-binding sites is possible using information from non-homologous proteins. Nucleos is available at http://nucleos.bio.uniroma2.it/nucleos/.

  13. Targeting the Small- and Intermediate-Conductance Ca2+-Activated Potassium Channels: The Drug-Binding Pocket at the Channel/Calmodulin Interface

    Directory of Open Access Journals (Sweden)

    Meng Cui

    2014-10-01

    Full Text Available The small- and intermediate-conductance Ca2+-activated potassium (SK/IK channels play important roles in the regulation of excitable cells in both the central nervous and cardiovascular systems. Evidence from animal models has implicated SK/IK channels in neurological conditions such as ataxia and alcohol use disorders. Further, genome-wide association studies have suggested that cardiovascular abnormalities such as arrhythmias and hypertension are associated with single nucleotide polymorphisms that occur within the genes encoding the SK/IK channels. The Ca2+ sensitivity of the SK/IK channels stems from a constitutively bound Ca2+-binding protein: calmodulin. Small-molecule positive modulators of SK/IK channels have been developed over the past decade, and recent structural studies have revealed that the binding pocket of these positive modulators is located at the interface between the channel and calmodulin. SK/IK channel positive modulators can potentiate channel activity by enhancing the coupling between Ca2+ sensing via calmodulin and mechanical opening of the channel. Here, we review binding pocket studies that have provided structural insight into the mechanism of action for SK/IK channel positive modulators. These studies lay the foundation for structure-based drug discovery efforts that can identify novel SK/IK channel positive modulators. © 2014 S. Karger AG, Basel

  14. Chlamydial entry involves TARP binding of guanine nucleotide exchange factors.

    Directory of Open Access Journals (Sweden)

    B Josh Lane

    2008-03-01

    Full Text Available Chlamydia trachomatis attachment to cells induces the secretion of the elementary body-associated protein TARP (Translocated Actin Recruiting Protein. TARP crosses the plasma membrane where it is immediately phosphorylated at tyrosine residues by unknown host kinases. The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment. We show that TARP participates directly in chlamydial invasion activating the Rac-dependent signaling cascade to recruit actin. TARP functions by binding two distinct Rac guanine nucleotide exchange factors (GEFs, Sos1 and Vav2, in a phosphotyrosine-dependent manner. The tyrosine phosphorylation profile of the sequence YEPISTENIYESI within TARP, as well as the transient activation of the phosphatidylinositol 3-kinase (PI3-K, appears to determine which GEF is utilized to activate Rac. The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate. Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways. Collectively, these data implicate TARP in signaling to the actin cytoskeleton remodeling machinery, demonstrating a mechanism by which C.trachomatis invades non-phagocytic cells.

  15. Identification of the Tolfenamic Acid Binding Pocket in PrbP from Liberibacter asiaticus

    Directory of Open Access Journals (Sweden)

    Lei Pan

    2017-08-01

    Full Text Available In Liberibacter asiaticus, PrbP is an important transcriptional accessory protein that was found to regulate gene expression through interactions with the RNA polymerase β-subunit and a specific sequence on the promoter region. It was found that inactivation of PrbP, using the inhibitor tolfenamic acid, resulted in a significant decrease in the overall transcriptional activity of L. asiaticus, and the suppression of L. asiaticus infection in HLB symptomatic citrus seedlings. The molecular interactions between PrbP and tolfenamic acid, however, were yet to be elucidated. In this study, we modeled the structure of PrbP and identified a ligand binding pocket, TaP, located at the interface of the predicted RNA polymerase interaction domain (N-terminus and the DNA binding domain (C-terminus. The molecular interactions of PrbP with tolfenamic acid were predicted using in silico docking. Site-directed mutagenesis of specific amino acids was followed by electrophoresis mobility shift assays and in vitro transcription assays, where residues N107, G109, and E148 were identified as the primary amino acids involved in interactions with tolfenamic acid. These results provide insight into the binding mechanism of PrbP to a small inhibitory molecule, and a starting scaffold for the identification and development of therapeutics targeting PrbP and other homologs in the CarD_CdnL_TRCF family.

  16. PocketMatch: a new algorithm to compare binding sites in protein structures.

    Science.gov (United States)

    Yeturu, Kalidas; Chandra, Nagasuma

    2008-12-17

    Recognizing similarities and deriving relationships among protein molecules is a fundamental requirement in present-day biology. Similarities can be present at various levels which can be detected through comparison of protein sequences or their structural folds. In some cases similarities obscure at these levels could be present merely in the substructures at their binding sites. Inferring functional similarities between protein molecules by comparing their binding sites is still largely exploratory and not as yet a routine protocol. One of the main reasons for this is the limitation in the choice of appropriate analytical tools that can compare binding sites with high sensitivity. To benefit from the enormous amount of structural data that is being rapidly accumulated, it is essential to have high throughput tools that enable large scale binding site comparison. Here we present a new algorithm PocketMatch for comparison of binding sites in a frame invariant manner. Each binding site is represented by 90 lists of sorted distances capturing shape and chemical nature of the site. The sorted arrays are then aligned using an incremental alignment method and scored to obtain PMScores for pairs of sites. A comprehensive sensitivity analysis and an extensive validation of the algorithm have been carried out. A comparison with other site matching algorithms is also presented. Perturbation studies where the geometry of a given site was retained but the residue types were changed randomly, indicated that chance similarities were virtually non-existent. Our analysis also demonstrates that shape information alone is insufficient to discriminate between diverse binding sites, unless combined with chemical nature of amino acids. A new algorithm has been developed to compare binding sites in accurate, efficient and high-throughput manner. Though the representation used is conceptually simplistic, we demonstrate that along with the new alignment strategy used, it is sufficient to

  17. Exploring the water-binding pocket of the type II dehydroquinase enzyme in the structure-based design of inhibitors.

    Science.gov (United States)

    Blanco, Beatriz; Sedes, Antía; Peón, Antonio; Otero, José M; van Raaij, Mark J; Thompson, Paul; Hawkins, Alastair R; González-Bello, Concepción

    2014-04-24

    Structural and computational studies to explore the WAT1 binding pocket in the structure-based design of inhibitors against the type II dehydroquinase (DHQ2) enzyme are reported. The crystal structures of DHQ2 from M. tuberculosis in complex with four of the reported compounds are described. The electrostatic interaction observed between the guanidinium group of the essential arginine and the carboxylate group of one of the inhibitors in the reported crystal structures supports the recently suggested role of this arginine as the residue that triggers the release of the product from the active site. The results of the structural and molecular dynamics simulation studies revealed that the inhibitory potency is favored by promoting interactions with WAT1 and the residues located within this pocket and, more importantly, by avoiding situations where the ligands occupy the WAT1 binding pocket. The new insights can be used to advantage in the structure-based design of inhibitors.

  18. A phosphoserine/threonine-binding pocket in AGC kinases and PDK1 mediates activation by hydrophobic motif phosphorylation

    DEFF Research Database (Denmark)

    Frödin, Morten; Antal, Torben L; Dümmler, Bettina A

    2002-01-01

    docking site that recruits and activates PDK1, which then phosphorylates the activation loop. Here, we discover a pocket in the kinase domain of PDK1 that recognizes the phosphoserine/phosphothreonine in the hydrophobic motif by identifying two oppositely positioned arginine and lysine residues that bind...

  19. Definition of the G protein-coupled receptor transmembrane bundle binding pocket and calculation of receptor similarities for drug design

    DEFF Research Database (Denmark)

    Gloriam, David Erik Immanuel; Foord, Steven M; Blaney, Frank E

    2009-01-01

    Recent advances in structural biology for G-protein-coupled receptors (GPCRs) have provided new opportunities to improve the definition of the transmembrane binding pocket. Here a reference set of 44 residue positions accessible for ligand binding was defined through detailed analysis of all...... to endogenous ligand types, although it revealed subdivision of certain classes, notably peptide and lipid receptors. The transmembrane binding site reference set, particularly when coupled with a means of identifying the subset of ligand binding residues, provides a general paradigm for understanding...

  20. Structures of BmrR-Drug Complexes Reveal a Rigid Multidrug Binding Pocket And Transcription Activation Through Tyrosine Expulsion

    Energy Technology Data Exchange (ETDEWEB)

    Newberry, K.J.; Huffman, J.L.; Miller, M.C.; Vazquez-Laslop, N.; Neyfakh, A.A.; Brennan, R.G.

    2009-05-22

    BmrR is a member of the MerR family and a multidrug binding transcription factor that up-regulates the expression of the bmr multidrug efflux transporter gene in response to myriad lipophilic cationic compounds. The structural mechanism by which BmrR binds these chemically and structurally different drugs and subsequently activates transcription is poorly understood. Here, we describe the crystal structures of BmrR bound to rhodamine 6G (R6G) or berberine (Ber) and cognate DNA. These structures reveal each drug stacks against multiple aromatic residues with their positive charges most proximal to the carboxylate group of Glu-253 and that, unlike other multidrug binding pockets, that of BmrR is rigid. Substitution of Glu-253 with either alanine (E253A) or glutamine (E253Q) results in unpredictable binding affinities for R6G, Ber, and tetraphenylphosphonium. Moreover, these drug binding studies reveal that the negative charge of Glu-253 is not important for high affinity binding to Ber and tetraphenylphosphonium but plays a more significant, but unpredictable, role in R6G binding. In vitro transcription data show that E253A and E253Q are constitutively active, and structures of the drug-free E253A-DNA and E253Q-DNA complexes support a transcription activation mechanism requiring the expulsion of Tyr-152 from the multidrug binding pocket. In sum, these data delineate the mechanism by which BmrR binds lipophilic, monovalent cationic compounds and suggest the importance of the redundant negative electrostatic nature of this rigid drug binding pocket that can be used to discriminate against molecules that are not substrates of the Bmr multidrug efflux pump.

  1. An induced pocket for the binding of potent fusion inhibitor CL-385319 with H5N1 influenza virus hemagglutinin.

    Directory of Open Access Journals (Sweden)

    Runming Li

    Full Text Available The influenza glycoprotein hemagglutinin (HA plays crucial roles in the early stage of virus infection, including receptor binding and membrane fusion. Therefore, HA is a potential target for developing anti-influenza drugs. Recently, we characterized a novel inhibitor of highly pathogenic H5N1 influenza virus, CL-385319, which specifically inhibits HA-mediated viral entry. Studies presented here identified the critical binding residues for CL-385319, which clustered in the stem region of the HA trimer by site-directed mutagenesis. Extensive computational simulations, including molecular docking, molecular dynamics simulations, molecular mechanics generalized Born surface area (MM_GBSA calculations, charge density and Laplacian calculations, have been carried out to uncover the detailed molecular mechanism that underlies the binding of CL-385319 to H5N1 influenza virus HA. It was found that the recognition and binding of CL-385319 to HA proceeds by a process of "induced fit" whereby the binding pocket is formed during their interaction. Occupation of this pocket by CL-385319 stabilizes the neutral pH structure of hemagglutinin, thus inhibiting the conformational rearrangements required for membrane fusion. This "induced fit" pocket may be a target for structure-based design of more potent influenza fusion inhibitors.

  2. A characteristic back support structure in the bisphenol A-binding pocket in the human nuclear receptor ERRγ.

    Directory of Open Access Journals (Sweden)

    Xiaohui Liu

    Full Text Available The endocrine disruptor bisphenol A (BPA affects various genes and hormones even at merely physiological levels. We recently demonstrated that BPA binds strongly to human nuclear receptor estrogen-related receptor (ERR γ and that the phenol-A group of BPA is in a receptacle pocket with essential amino acid residues to provide structural support at the backside. This led BPA to bind to ERRγ in an induced-fit-type binding mode, for example, with a rotated motion of Val313 to support the Tyr326-binding site. A similar binding mechanism appears to occur at the binding site of the BPA phenol-B ring. X-ray crystal analysis of the ERRγ-ligand-binding domain/BPA complex suggested that the ERRγ receptor residues Leu342, Leu345, Asn346, and Ile349 function as intrinsic binding sites of the BPA phenol-B, whereas Leu265, Leu268, Ile310, Val313, Leu324, Tyr330, Lys430, Ala431, and His434 work as structural elements to assist these binding sites. In the present study, by evaluating the mutant receptors replaced by a series of amino acids, we demonstrated that a finely assembled structural network indeed exists around the two adjacent Leu342-Asn346 and Leu345-Ile349 ridges on the same α-helix 7 (H7, constructing a part of the binding pocket structure with back support residues for the BPA phenol-B ring. The results reveal that the double-layer binding sites, namely, the ordinary ligand binding sites and their back support residues, substantiate the strong binding of BPA to ERRγ. When ERRγ-Asn346 was replaced by the corresponding Gly and Tyr in ERRα and ERRβ, respectively, the binding affinity of BPA and even 4-hydroxytamxifen (4-OHT is much reduced. Asn346 was found to be one of the residues that make ERRγ to be exclusive to BPA.

  3. AcrB drug-binding pocket substitution confers clinically relevant resistance and altered substrate specificity.

    Science.gov (United States)

    Blair, Jessica M A; Bavro, Vassiliy N; Ricci, Vito; Modi, Niraj; Cacciotto, Pierpaolo; Kleinekathӧfer, Ulrich; Ruggerone, Paolo; Vargiu, Attilio V; Baylay, Alison J; Smith, Helen E; Brandon, Yvonne; Galloway, David; Piddock, Laura J V

    2015-03-17

    The incidence of multidrug-resistant bacterial infections is increasing globally and the need to understand the underlying mechanisms is paramount to discover new therapeutics. The efflux pumps of Gram-negative bacteria have a broad substrate range and transport antibiotics out of the bacterium, conferring intrinsic multidrug resistance (MDR). The genomes of pre- and posttherapy MDR clinical isolates of Salmonella Typhimurium from a patient that failed antibacterial therapy and died were sequenced. In the posttherapy isolate we identified a novel G288D substitution in AcrB, the resistance-nodulation division transporter in the AcrAB-TolC tripartite MDR efflux pump system. Computational structural analysis suggested that G288D in AcrB heavily affects the structure, dynamics, and hydration properties of the distal binding pocket altering specificity for antibacterial drugs. Consistent with this hypothesis, recreation of the mutation in standard Escherichia coli and Salmonella strains showed that G288D AcrB altered substrate specificity, conferring decreased susceptibility to the fluoroquinolone antibiotic ciprofloxacin by increased efflux. At the same time, the substitution increased susceptibility to other drugs by decreased efflux. Information about drug transport is vital for the discovery of new antibacterials; the finding that one amino acid change can cause resistance to some drugs, while conferring increased susceptibility to others, could provide a basis for new drug development and treatment strategies.

  4. Molecular sampling of the allosteric binding pocket of the TSH receptor provides discriminative pharmacophores for antagonist and agonists.

    Science.gov (United States)

    Hoyer, Inna; Haas, Ann-Karin; Kreuchwig, Annika; Schülein, Ralf; Krause, Gerd

    2013-02-01

    The TSHR (thyrotropin receptor) is activated endogenously by the large hormone thyrotropin and activated pathologically by auto-antibodies. Both activate and bind at the extracellular domain. Recently, SMLs (small-molecule ligands) have been identified, which bind in an allosteric binding pocket within the transmembrane domain. Modelling driven site-directed mutagenesis of amino acids lining this pocket led to the delineation of activation and inactivation sensitive residues. Modified residues showing CAMs (constitutively activating mutations) indicate signalling-sensitive positions and mark potential trigger points for agonists. Silencing mutations lead to an impairment of basal activity and mark contact points for antagonists. Mapping these residues on to a structural model of TSHR indicates locations where an SML may switch the receptor to an inactive or active conformation. In the present article, we report the effects of SMLs on these signalling-sensitive amino acids at the TSHR. Surprisingly, the antagonistic effect of SML compound 52 was reversed to an agonistic effect, when tested at the CAM Y667A. Switching agonism to antagonism and the reverse by changing either SMLs or residues covering the binding pocket provides detailed knowledge about discriminative pharmacophores. It prepares the basis for rational optimization of new high-affinity antagonists to interfere with the pathogenic activation of the TSHR.

  5. Access Path to the Ligand Binding Pocket May Play a Role in Xenobiotics Selection by AhR

    Science.gov (United States)

    Szöllősi, Dániel; Erdei, Áron; Gyimesi, Gergely; Magyar, Csaba; Hegedűs, Tamás

    2016-01-01

    Understanding of multidrug binding at the atomic level would facilitate drug design and strategies to modulate drug metabolism, including drug transport, oxidation, and conjugation. Therefore we explored the mechanism of promiscuous binding of small molecules by studying the ligand binding domain, the PAS-B domain of the aryl hydrocarbon receptor (AhR). Because of the low sequence identities of PAS domains to be used for homology modeling, structural features of the widely employed HIF-2α and a more recent suitable template, CLOCK were compared. These structures were used to build AhR PAS-B homology models. We performed molecular dynamics simulations to characterize dynamic properties of the PAS-B domain and the generated conformational ensembles were employed in in silico docking. In order to understand structural and ligand binding features we compared the stability and dynamics of the promiscuous AhR PAS-B to other PAS domains exhibiting specific interactions or no ligand binding function. Our exhaustive in silico binding studies, in which we dock a wide spectrum of ligand molecules to the conformational ensembles, suggest that ligand specificity and selection may be determined not only by the PAS-B domain itself, but also by other parts of AhR and its protein interacting partners. We propose that ligand binding pocket and access channels leading to the pocket play equally important roles in discrimination of endogenous molecules and xenobiotics. PMID:26727491

  6. Access Path to the Ligand Binding Pocket May Play a Role in Xenobiotics Selection by AhR.

    Directory of Open Access Journals (Sweden)

    Dániel Szöllősi

    Full Text Available Understanding of multidrug binding at the atomic level would facilitate drug design and strategies to modulate drug metabolism, including drug transport, oxidation, and conjugation. Therefore we explored the mechanism of promiscuous binding of small molecules by studying the ligand binding domain, the PAS-B domain of the aryl hydrocarbon receptor (AhR. Because of the low sequence identities of PAS domains to be used for homology modeling, structural features of the widely employed HIF-2α and a more recent suitable template, CLOCK were compared. These structures were used to build AhR PAS-B homology models. We performed molecular dynamics simulations to characterize dynamic properties of the PAS-B domain and the generated conformational ensembles were employed in in silico docking. In order to understand structural and ligand binding features we compared the stability and dynamics of the promiscuous AhR PAS-B to other PAS domains exhibiting specific interactions or no ligand binding function. Our exhaustive in silico binding studies, in which we dock a wide spectrum of ligand molecules to the conformational ensembles, suggest that ligand specificity and selection may be determined not only by the PAS-B domain itself, but also by other parts of AhR and its protein interacting partners. We propose that ligand binding pocket and access channels leading to the pocket play equally important roles in discrimination of endogenous molecules and xenobiotics.

  7. Access Path to the Ligand Binding Pocket May Play a Role in Xenobiotics Selection by AhR.

    Science.gov (United States)

    Szöllősi, Dániel; Erdei, Áron; Gyimesi, Gergely; Magyar, Csaba; Hegedűs, Tamás

    2016-01-01

    Understanding of multidrug binding at the atomic level would facilitate drug design and strategies to modulate drug metabolism, including drug transport, oxidation, and conjugation. Therefore we explored the mechanism of promiscuous binding of small molecules by studying the ligand binding domain, the PAS-B domain of the aryl hydrocarbon receptor (AhR). Because of the low sequence identities of PAS domains to be used for homology modeling, structural features of the widely employed HIF-2α and a more recent suitable template, CLOCK were compared. These structures were used to build AhR PAS-B homology models. We performed molecular dynamics simulations to characterize dynamic properties of the PAS-B domain and the generated conformational ensembles were employed in in silico docking. In order to understand structural and ligand binding features we compared the stability and dynamics of the promiscuous AhR PAS-B to other PAS domains exhibiting specific interactions or no ligand binding function. Our exhaustive in silico binding studies, in which we dock a wide spectrum of ligand molecules to the conformational ensembles, suggest that ligand specificity and selection may be determined not only by the PAS-B domain itself, but also by other parts of AhR and its protein interacting partners. We propose that ligand binding pocket and access channels leading to the pocket play equally important roles in discrimination of endogenous molecules and xenobiotics.

  8. Recurrent De Novo Mutations Disturbing the GTP/GDP Binding Pocket of RAB11B Cause Intellectual Disability and a Distinctive Brain Phenotype.

    Science.gov (United States)

    Lamers, Ideke J C; Reijnders, Margot R F; Venselaar, Hanka; Kraus, Alison; Jansen, Sandra; de Vries, Bert B A; Houge, Gunnar; Gradek, Gyri Aasland; Seo, Jieun; Choi, Murim; Chae, Jong-Hee; van der Burgt, Ineke; Pfundt, Rolph; Letteboer, Stef J F; van Beersum, Sylvia E C; Dusseljee, Simone; Brunner, Han G; Doherty, Dan; Kleefstra, Tjitske; Roepman, Ronald

    2017-11-02

    The Rab GTPase family comprises ∼70 GTP-binding proteins, functioning in vesicle formation, transport and fusion. They are activated by a conformational change induced by GTP-binding, allowing interactions with downstream effectors. Here, we report five individuals with two recurrent de novo missense mutations in RAB11B; c.64G>A; p.Val22Met in three individuals and c.202G>A; p.Ala68Thr in two individuals. An overlapping neurodevelopmental phenotype, including severe intellectual disability with absent speech, epilepsy, and hypotonia was observed in all affected individuals. Additionally, visual problems, musculoskeletal abnormalities, and microcephaly were present in the majority of cases. Re-evaluation of brain MRI images of four individuals showed a shared distinct brain phenotype, consisting of abnormal white matter (severely decreased volume and abnormal signal), thin corpus callosum, cerebellar vermis hypoplasia, optic nerve hypoplasia and mild ventriculomegaly. To compare the effects of both variants with known inactive GDP- and active GTP-bound RAB11B mutants, we modeled the variants on the three-dimensional protein structure and performed subcellular localization studies. We predicted that both variants alter the GTP/GDP binding pocket and show that they both have localization patterns similar to inactive RAB11B. Evaluation of their influence on the affinity of RAB11B to a series of binary interactors, both effectors and guanine nucleotide exchange factors (GEFs), showed induction of RAB11B binding to the GEF SH3BP5, again similar to inactive RAB11B. In conclusion, we report two recurrent dominant mutations in RAB11B leading to a neurodevelopmental syndrome, likely caused by altered GDP/GTP binding that inactivate the protein and induce GEF binding and protein mislocalization. Copyright © 2017 American Society of Human Genetics. All rights reserved.

  9. Phosphate-binding pocket in Dicer-2 PAZ domain for high-fidelity siRNA production.

    Science.gov (United States)

    Kandasamy, Suresh K; Fukunaga, Ryuya

    2016-12-06

    The enzyme Dicer produces small silencing RNAs such as micro-RNAs (miRNAs) and small interfering RNAs (siRNAs). In Drosophila, Dicer-1 produces ∼22-24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. Here we report that recognition of the 5'-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. We propose that the 5'-monophosphate of a long dsRNA substrate is anchored by the phosphate-binding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing.

  10. MexY-promoted aminoglycoside resistance in Pseudomonas aeruginosa: involvement of a putative proximal binding pocket in aminoglycoside recognition.

    Science.gov (United States)

    Lau, Calvin Ho-Fung; Hughes, Daniel; Poole, Keith

    2014-04-22

    The resistance-nodulation-division (RND) family multidrug efflux system MexXY-OprM is a major determinant of aminoglycoside resistance in Pseudomonas aeruginosa, although the details of aminoglycoside recognition and export by MexY, the substrate-binding RND component of this efflux system, have not been elucidated. To identify regions/residues of MexY important for aminoglycoside resistance, plasmid-borne mexY was mutagenized and mutations that impaired MexY-promoted aminoglycoside (streptomycin) resistance were identified in a ΔmexY strain of P. aeruginosa. Sixty-one streptomycin-sensitive mexY mutants were recovered; among these, 7 unique mutations that yielded wild-type levels of MexY expression were identified. These mutations compromised resistance to additional aminoglycosides and to other antimicrobials and occurred in both the transmembrane and periplasmic regions of the protein. Mapping of the mutated residues onto a 3-dimensional structure of MexY modeled on Escherichia coli AcrB revealed that these tended to occur in regions implicated in general pump operation (transmembrane domain) and MexY trimer assembly (docking domain) and, thus, did not provide insights into aminoglycoside recognition. A region corresponding to a proximal binding pocket connected to a periplasm-linked cleft, part of a drug export pathway of AcrB, was identified in MexY and proposed to play a role in aminoglycoside recognition. To test this, selected residues (K79, D133, and Y613) within this pocket were mutagenized and the impact on aminoglycoside resistance was assessed. Mutations of D133 and Y613 compromised aminoglycoside resistance, while, surprisingly, the K79 mutation enhanced aminoglycoside resistance, confirming a role for this putative proximal binding pocket in aminoglycoside recognition and export. IMPORTANCE Bacterial RND pumps do not typically accommodate highly hydrophilic agents such as aminoglycosides, and it is unclear how those, such as MexY, which accommodate

  11. Nucleotide Binding Site Communication in Arabidopsis thaliana Adenosine 5′-Phosphosulfate Kinase*

    Science.gov (United States)

    Ravilious, Geoffrey E.; Jez, Joseph M.

    2012-01-01

    Adenosine 5′-phosphosulfate kinase (APSK) catalyzes the ATP-dependent synthesis of adenosine 3′-phosphate 5′-phosphosulfate (PAPS), which is an essential metabolite for sulfur assimilation in prokaryotes and eukaryotes. Using APSK from Arabidopsis thaliana, we examine the energetics of nucleotide binary and ternary complex formation and probe active site features that coordinate the order of ligand addition. Calorimetric analysis shows that binding can occur first at either nucleotide site, but that initial interaction at the ATP/ADP site was favored and enhanced affinity for APS in the second site by 50-fold. The thermodynamics of the two possible binding models (i.e. ATP first versus APS first) differs and implies that active site structural changes guide the order of nucleotide addition. The ligand binding analysis also supports an earlier suggestion of intermolecular interactions in the dimeric APSK structure. Crystallographic, site-directed mutagenesis, and energetic analyses of oxyanion recognition by the P-loop in the ATP/ADP binding site and the role of Asp136, which bridges the ATP/ADP and APS/PAPS binding sites, suggest how the ordered nucleotide binding sequence and structural changes are dynamically coordinated for catalysis. PMID:22810229

  12. Disruption of key NADH-binding pocket residues of the Mycobacterium tuberculosis InhA affects DD-CoA binding ability.

    Science.gov (United States)

    Shaw, Daniel J; Robb, Kirsty; Vetter, Beatrice V; Tong, Madeline; Molle, Virginie; Hunt, Neil T; Hoskisson, Paul A

    2017-07-05

    Tuberculosis (TB) is a global health problem that affects over 10 million people. There is an urgent need to develop novel antimicrobial therapies to combat TB. To achieve this, a thorough understanding of key validated drug targets is required. The enoyl reductase InhA, responsible for synthesis of essential mycolic acids in the mycobacterial cell wall, is the target for the frontline anti-TB drug isoniazid. To better understand the activity of this protein a series of mutants, targeted to the NADH co-factor binding pocket were created. Residues P193 and W222 comprise a series of hydrophobic residues surrounding the cofactor binding site and mutation of both residues negatively affect InhA function. Construction of an M155A mutant of InhA results in increased affinity for NADH and DD-CoA turnover but with a reduction in V max for DD-CoA, impairing overall activity. This suggests that NADH-binding geometry of InhA likely permits long-range interactions between residues in the NADH-binding pocket to facilitate substrate turnover in the DD-CoA binding region of the protein. Understanding the precise details of substrate binding and turnover in InhA and how this may affect protein-protein interactions may facilitate the development of improved inhibitors enabling the development of novel anti-TB drugs.

  13. Investigating structural changes induced by nucleotide binding to RecA using difference FTIR.

    OpenAIRE

    Butler, Blaine C; Hanchett, Ross H; Rafailov, Helena; MacDonald, Gina

    2002-01-01

    Nucleotide binding to RecA results in either the high-DNA affinity form (Adenosine 5'-triphosphate (ATP)-bound) or the more inactive protein conformation associated with a lower affinity for DNA (Adenosine 5'-diphosphate (ADP)-bound). Many of the key structural differences between the RecA-ATP and RecA-ADP bound forms have yet to be elucidated. We have used caged-nucleotides and difference FTIR in efforts to obtain a comprehensive understanding of the molecular changes induced by nucleotide b...

  14. Thermodynamics of nucleotide binding to actomyosin V and VI: a positive heat capacity change accompanies strong ADP binding.

    Science.gov (United States)

    Robblee, James P; Cao, Wenxiang; Henn, Arnon; Hannemann, Diane E; De La Cruz, Enrique M

    2005-08-02

    We have measured the energetics of ATP and ADP binding to single-headed actomyosin V and VI from the temperature dependence of the rate and equilibrium binding constants. Nucleotide binding to actomyosin V and VI can be modeled as two-step binding mechanisms involving the formation of collision complexes followed by isomerization to states with high nucleotide affinity. Formation of the actomyosin VI-ATP collision complex is much weaker and slower than for actomyosin V. A three-step binding mechanism where actomyosin VI isomerizes between two conformations, one competent to bind ATP and one not, followed by rapid ATP binding best accounts for the data. ADP binds to actomyosin V more tightly than actomyosin VI. At 25 degrees C, the strong ADP-binding equilibria are comparable for actomyosin V and VI, and the different overall ADP affinities arise from differences in the ADP collision complex affinity. The actomyosin-ADP isomerization leading to strong ADP binding is entropy driven at >15 degrees C and occurs with a large, positive change in heat capacity (DeltaC(P) degrees ) for both actomyosin V and VI. Sucrose slows ADP binding and dissociation from actomyosin V and VI but not the overall equilibrium constants for strong ADP binding, indicating that solvent viscosity dampens ADP-dependent kinetic transitions, presumably a tail swing that occurs with ADP binding and release. We favor a mechanism where strong ADP binding increases the dynamics and flexibility of the actomyosin complex. The heat capacity (DeltaC(P) degrees ) and entropy (DeltaS degrees ) changes are greater for actomyosin VI than actomyosin V, suggesting different extents of ADP-induced structural rearrangement.

  15. Determinants of nucleotide-binding selectivity of malic enzyme.

    Directory of Open Access Journals (Sweden)

    Ju-Yi Hsieh

    Full Text Available Malic enzymes have high cofactor selectivity. An isoform-specific distribution of residues 314, 346, 347 and 362 implies that they may play key roles in determining the cofactor specificity. Currently, Glu314, Ser346, Lys347 and Lys362 in human c-NADP-ME were changed to the corresponding residues of human m-NAD(P-ME (Glu, Lys, Tyr and Gln, respectively or Ascaris suum m-NAD-ME (Ala, Ile, Asp and His, respectively. Kinetic data demonstrated that the S346K/K347Y/K362Q c-NADP-ME was transformed into a debilitated NAD⁺-utilizing enzyme, as shown by a severe decrease in catalytic efficiency using NADP⁺ as the cofactor without a significant increase in catalysis using NAD⁺ as the cofactor. However, the S346K/K347Y/K362H enzyme displayed an enhanced value for k(cat,NAD, suggesting that His at residue 362 may be more beneficial than Gln for NAD⁺ binding. Furthermore, the S346I/K347D/K362H mutant had a very large K(m,NADP value compared to other mutants, suggesting that this mutant exclusively utilizes NAD⁺ as its cofactor. Since the S346K/K347Y/K362Q, S346K/K347Y/K362H and S346I/K347D/K362H c-NADP-ME mutants did not show significant reductions in their K(m,NAD values, the E314A mutation was then introduced into these triple mutants. Comparison of the kinetic parameters of each triple-quadruple mutant pair (for example, S346K/K347Y/K362Q versus E314A/S346K/K347Y/K362Q revealed that all of the K(m values for NAD⁺ and NADP(+ of the quadruple mutants were significantly decreased, while either k(cat,NAD or k(cat,NADP was substantially increased. By adding the E314A mutation to these triple mutant enzymes, the E314A/S346K/K347Y/K362Q, E314A/S346K/K347Y/K362H and E314A/S346I/K347D/K362H c-NADP-ME variants are no longer debilitated but become mainly NAD⁺-utilizing enzymes by a considerable increase in catalysis using NAD⁺ as the cofactor. These results suggest that abolishing the repulsive effect of Glu314 in these quadruple mutants increases

  16. Determinants of nucleotide-binding selectivity of malic enzyme.

    Science.gov (United States)

    Hsieh, Ju-Yi; Chen, Meng-Chun; Hung, Hui-Chih

    2011-01-01

    Malic enzymes have high cofactor selectivity. An isoform-specific distribution of residues 314, 346, 347 and 362 implies that they may play key roles in determining the cofactor specificity. Currently, Glu314, Ser346, Lys347 and Lys362 in human c-NADP-ME were changed to the corresponding residues of human m-NAD(P)-ME (Glu, Lys, Tyr and Gln, respectively) or Ascaris suum m-NAD-ME (Ala, Ile, Asp and His, respectively). Kinetic data demonstrated that the S346K/K347Y/K362Q c-NADP-ME was transformed into a debilitated NAD⁺-utilizing enzyme, as shown by a severe decrease in catalytic efficiency using NADP⁺ as the cofactor without a significant increase in catalysis using NAD⁺ as the cofactor. However, the S346K/K347Y/K362H enzyme displayed an enhanced value for k(cat,NAD), suggesting that His at residue 362 may be more beneficial than Gln for NAD⁺ binding. Furthermore, the S346I/K347D/K362H mutant had a very large K(m,NADP) value compared to other mutants, suggesting that this mutant exclusively utilizes NAD⁺ as its cofactor. Since the S346K/K347Y/K362Q, S346K/K347Y/K362H and S346I/K347D/K362H c-NADP-ME mutants did not show significant reductions in their K(m,NAD) values, the E314A mutation was then introduced into these triple mutants. Comparison of the kinetic parameters of each triple-quadruple mutant pair (for example, S346K/K347Y/K362Q versus E314A/S346K/K347Y/K362Q) revealed that all of the K(m) values for NAD⁺ and NADP(+) of the quadruple mutants were significantly decreased, while either k(cat,NAD) or k(cat,NADP) was substantially increased. By adding the E314A mutation to these triple mutant enzymes, the E314A/S346K/K347Y/K362Q, E314A/S346K/K347Y/K362H and E314A/S346I/K347D/K362H c-NADP-ME variants are no longer debilitated but become mainly NAD⁺-utilizing enzymes by a considerable increase in catalysis using NAD⁺ as the cofactor. These results suggest that abolishing the repulsive effect of Glu314 in these quadruple mutants increases the binding

  17. Different characteristics and nucleotide binding properties of inosine monophosphate dehydrogenase (IMPDH isoforms.

    Directory of Open Access Journals (Sweden)

    Elaine C Thomas

    Full Text Available We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH, a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii communication occurs between the Bateman and catalytic domains and (iii the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.

  18. Location of the hydrophobic pocket in the binding site of fentanyl analogs in the m-opioid receptor

    Directory of Open Access Journals (Sweden)

    LJILJANA DOSEN–MICOVIC

    2007-07-01

    Full Text Available Fentanyl is a highly potent and clinically widely used narcotic analgesic. The synthesis of its analogs remains a challenge in an attempt to develop highly se­lective m-opioid receptor agonists with specific pharmacological properties. In this paper, the use of flexible molecular docking of several specific fentanyl analogs to the m-opioid receptor model, in order to test the hypothesis that the hydrophobic po­cket accommodates alkyl groups at position 3 of the fentanyl skeleton, is descri­bed. The stereoisomers of the following compounds were studied: cis- and trans-3-me­thylfentanyl, 3,3-dimethylfentanyl, cis- and trans-3-ethylfentanyl, cis- and trans-3-propylfentanyl, cis-3-isopropylfentanyl and cis-3-benzylfentanyl. The opti­mal position and orientation of these fentanyl analogs in the binding pocket of the m-receptor, explaining their enantiospecific potency, were determined. It was found that the 3-alkyl group of cis-3R,4S and trans-3S,4S stereoisomers of all the active compounds occupies the hydrophobic pocket between TM5, TM6 and TM7, made up of the amino acids Trp318 (TM7, Ile322 (TM7, Ile301 (TM6 and Phe237 (TM5. However, the fact that this hydrophobic pocket can also accommodate the bulky 3-alkyl substituents of the two inactive compounds: cis-3-isopro­pylfentanyl, and cis-3-benzylfentanyl, indicates that this hydrophobic pocket in the employed receptor model is probably too large.

  19. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    Science.gov (United States)

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-11-01

    Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches.

  20. Split tasks of asymmetric nucleotide-binding sites in the heterodimeric ABC exporter EfrCD.

    Science.gov (United States)

    Hürlimann, Lea M; Hohl, Michael; Seeger, Markus A

    2017-06-01

    Many heterodimeric ATP-binding cassette (ABC) exporters evolved asymmetric ATP-binding sites containing a degenerate site incapable of ATP hydrolysis due to noncanonical substitutions in conserved sequence motifs. Recent studies revealed that nucleotide binding to the degenerate site stabilizes contacts between the nucleotide-binding domains (NBDs) of the inward-facing transporter and regulates ATP hydrolysis at the consensus site via allosteric coupling mediated by the D-loops. However, it is unclear whether nucleotide binding to the degenerate site is strictly required for substrate transport. In this study, we examined the functional consequences of a systematic set of mutations introduced at the degenerate and consensus site of the multidrug efflux pump EfrCD of Enterococcus faecalis. Mutating motifs which differ among the two ATP-binding sites (Walker B, switch loop, and ABC signature) or which are involved in interdomain communication (D-loop and Q-loop) led to asymmetric results in the functional assays and were better tolerated at the degenerate site. This highlights the importance of the degenerate site to allosterically regulate the events at the consensus site. Mutating invariant motifs involved in ATP binding and NBD closure (A-loop and Walker A) resulted in equally reduced transport activities, regardless at which ATP-binding site they were introduced. In contrast to previously investigated heterodimeric ABC exporters, mutation of the degenerate site Walker A lysine completely inactivated ATPase activity and substrate transport, indicating that ATP binding to the degenerate site is essential for EfrCD. This study provides novel insights into the split tasks of asymmetric ATP-binding sites of heterodimeric ABC exporters. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  1. BIND – An algorithm for loss-less compression of nucleotide ...

    Indian Academy of Sciences (India)

    2012-08-13

    Aug 13, 2012 ... an algorithm specialized for compressing nucleotide sequence data. By adopting a unique 'block-length' encoding for representing binary data (as a key step), BIND achieves significant compression gains as compared to the widely used general purpose compression algorithms (gzip, bzip2 and lzma).

  2. eMatchSite: sequence order-independent structure alignments of ligand binding pockets in protein models.

    Directory of Open Access Journals (Sweden)

    Michal Brylinski

    2014-09-01

    Full Text Available Detecting similarities between ligand binding sites in the absence of global homology between target proteins has been recognized as one of the critical components of modern drug discovery. Local binding site alignments can be constructed using sequence order-independent techniques, however, to achieve a high accuracy, many current algorithms for binding site comparison require high-quality experimental protein structures, preferably in the bound conformational state. This, in turn, complicates proteome scale applications, where only various quality structure models are available for the majority of gene products. To improve the state-of-the-art, we developed eMatchSite, a new method for constructing sequence order-independent alignments of ligand binding sites in protein models. Large-scale benchmarking calculations using adenine-binding pockets in crystal structures demonstrate that eMatchSite generates accurate alignments for almost three times more protein pairs than SOIPPA. More importantly, eMatchSite offers a high tolerance to structural distortions in ligand binding regions in protein models. For example, the percentage of correctly aligned pairs of adenine-binding sites in weakly homologous protein models is only 4-9% lower than those aligned using crystal structures. This represents a significant improvement over other algorithms, e.g. the performance of eMatchSite in recognizing similar binding sites is 6% and 13% higher than that of SiteEngine using high- and moderate-quality protein models, respectively. Constructing biologically correct alignments using predicted ligand binding sites in protein models opens up the possibility to investigate drug-protein interaction networks for complete proteomes with prospective systems-level applications in polypharmacology and rational drug repositioning. eMatchSite is freely available to the academic community as a web-server and a stand-alone software distribution at http://www.brylinski.org/ematchsite.

  3. Unveiling a novel transient druggable pocket in BACE-1 through molecular simulations: Conformational analysis and binding mode of multisite inhibitors

    Science.gov (United States)

    Di Pietro, Ornella; Laughton, Charles A.

    2017-01-01

    The critical role of BACE-1 in the formation of neurotoxic ß-amyloid peptides in the brain makes it an attractive target for an efficacious treatment of Alzheimer’s disease. However, the development of clinically useful BACE-1 inhibitors has proven to be extremely challenging. In this study we examine the binding mode of a novel potent inhibitor (compound 1, with IC50 80 nM) designed by synergistic combination of two fragments—huprine and rhein—that individually are endowed with very low activity against BACE-1. Examination of crystal structures reveals no appropriate binding site large enough to accommodate 1. Therefore we have examined the conformational flexibility of BACE-1 through extended molecular dynamics simulations, paying attention to the highly flexible region shaped by loops 8–14, 154–169 and 307–318. The analysis of the protein dynamics, together with studies of pocket druggability, has allowed us to detect the transient formation of a secondary binding site, which contains Arg307 as a key residue for the interaction with small molecules, at the edge of the catalytic cleft. The formation of this druggable “floppy” pocket would enable the binding of multisite inhibitors targeting both catalytic and secondary sites. Molecular dynamics simulations of BACE-1 bound to huprine-rhein hybrid compounds support the feasibility of this hypothesis. The results provide a basis to explain the high inhibitory potency of the two enantiomeric forms of 1, together with the large dependence on the length of the oligomethylenic linker. Furthermore, the multisite hypothesis has allowed us to rationalize the inhibitory potency of a series of tacrine-chromene hybrid compounds, specifically regarding the apparent lack of sensitivity of the inhibition constant to the chemical modifications introduced in the chromene unit. Overall, these findings pave the way for the exploration of novel functionalities in the design of optimized BACE-1 multisite inhibitors

  4. The nucleotide-binding site of the Escherichia coli DnaC protein: molecular topography of DnaC protein-nucleotide cofactor complexes.

    Science.gov (United States)

    Galletto, Roberto; Jezewska, Maria J; Maillard, Rodrigo; Bujalowski, Wlodzimierz

    2005-01-01

    The structure of the nucleotide-binding site of the Escherichia coli replication factor DnaC protein and the effect of the nucleotide cofactor on the protein structure have been examined using ultraviolet, steady-state, and time-dependent fluorescence spectroscopy. Emission spectra and quenching studies of the fluorescent nucleotide analogs, 3'-O-(N-methylantraniloyl)-5'-triphosphate (MANT-ATP) and 3'-O-(N-methylantraniloyl)-5'-diphosphate (MANT-ADP), bound to the DnaC protein indicate that the nucleotide-binding site forms a hydrophobic cleft on the surface of the protein. Fluorescence decays of free and bound MANT-ATP and MANT-ADP indicate that cofactors exist in two different conformations both, free and bound to the protein. However, the two conformations of the bound nucleotides differ in their solvent accessibilities. Moreover, there are significant differences in the solvent accessibility between ATP and ADP complexes. Specific binding of magnesium to the protein controls the structure of the binding site, particularly, in the case of the ATP complex, leading to additional opening of the binding site cleft. Both tyrosine and tryptophan residues are located on the surface of the protein. The tryptophans are clustered at a large distance from the nucleotide-binding site. However, in spite of a large spatial separation, binding of both cofactors induces significant and different changes in the structure of the environment of tryptophans, indicating long-range structural effects through the DnaC molecule. Moreover, only ATP induces changes in the distribution of the tyrosine residues on the surface of the protein. The data reveal that the nucleotide-DnaC protein complex is a sophisticated allosteric system, responding differently to the ATP and ADP binding.

  5. Nucleotide-dependent structural fluctuations and regulation of microtubule-binding affinity of KIF1A.

    Science.gov (United States)

    Kanada, Ryo; Takagi, Fumiko; Kikuchi, Macoto

    2015-05-01

    Molecular motors such as kinesin regulate affinity to a rail protein during the ATP hydrolysis cycle. The regulation mechanism, however, is yet to be determined. To understand this mechanism, we investigated the structural fluctuations of the motor head of the single-headed kinesin called KIF1A in different nucleotide states using molecular dynamics simulations of a Gō-like model. We found that the helix α4 at the microtubule (MT) binding site intermittently exhibits a large structural fluctuation when MT is absent. Frequency of this fluctuation changes systematically according to the nucleotide states and correlates strongly with the experimentally observed binding affinity to MT. We also showed that thermal fluctuation enhances the correlation and the interaction with the nucleotide suppresses the fluctuation of the helix α4. These results suggest that KIF1A regulates affinity to MT by changing the flexibility of the helix α4 during the ATP hydrolysis process: the binding site becomes more flexible in the strong binding state than in the weak binding state. © 2015 Wiley Periodicals, Inc.

  6. Studies on ATP-diphosphohydrolase nucleotide-binding sites by intrinsic fluorescence

    Directory of Open Access Journals (Sweden)

    A.M. Kettlun

    2000-07-01

    Full Text Available Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10 and Desirée (ATPase/ADPase = 1 isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.

  7. Spectroscopic investigations of the B12-binding subunit of glutamate mutase: refined solution structure of the complex with the B12-nucleotide, dynamics and binding studies with two corrinoid cofactors

    International Nuclear Information System (INIS)

    Eichmueller, C.

    2002-06-01

    Glutamate mutase is an enzyme isolated from Clostridium tetanomorphum and Clostridium cochlearum. It catalyses the reversible rearrangement of (2S)-glutamate to (2S,3S)-3-methylaspartate. Coenzyme B12 is required as cofactor for an active enzyme, as the first step of the catalytic cycle is the homolytic cleavage of the cobalt-carbon bond. The rearrangement itself follows a radical mechanism. The holoenzyme is an alpha2beta2 heterotetramer containing two identical catalytic and two B12 binding domains, as well as two coenzyme B12 molecules. The smaller B12 binding domain from Clostridium tetanomorphum, MutS, is known to bind coenzyme B12 in its unusual 'base-off' form. A conserved histidine residue coordinates to the cobalt atom instead of the normally coordinated dimethlybenzimidole in free coenzyme B12. In the present work a refined solution structure of the B12 binding subunit from Clostridium tetanomorphum (MutS) in complex with the detached nucleotide loop of coenzyme B12 has been determined using nuclear magnetic resonance. The found topology is almost identical to the crystal structure of glutamate mutase from C.cochlearum [Reitzer et al., 1999], in contrast to the solution structures obtained for apo-MutS [Hoffmann et al., 2001; Tollinger et al., 1998] and apo-GlmS [Hoffmann et al., 1999]. In these two structures a helix at one side of the B12 nucleotide loop binding pocket is mostly unstructured and shows motions on a microsecond to millisecond timescale. The previously found stabilization of this helix upon B12-nucleotide binding [Tollinger et al., 2001] was confirmed using 13C and 15N labeled MutS. Some differences are found in the structure of the binding pocket and the bound nucleotide loop compared to the crystal structure. This indicates that additional conformational changes occur upon binding of the corrin ring of coenzyme B12. NMR-relaxation measurements performed on apo-MutS showed interesting slow molecular motions not only in the mainly

  8. BIND – An algorithm for loss-less compression of nucleotide ...

    Indian Academy of Sciences (India)

    2012-08-13

    Aug 13, 2012 ... is required for academic or non-profit use. [Bose T, Mohammed MH, Dutta A and Mande SS 2012 .... (c) BIND uses individual binary codes (00, 01, 10 and 11) to represent the four nucleotides (A, T, G and C), ... (v.9.20). A Linux desktop having a dual core 2.33 GHz processor with 2 GB RAM was used for all ...

  9. Exploring the binding nature of pyrrolidine pocket-dependent interactions in the polo-box domain of polo-like kinase 1.

    Directory of Open Access Journals (Sweden)

    Ravichandran N Murugan

    Full Text Available BACKGROUND: Over the years, a great deal of effort has been focused on the design and synthesis of potent, linear peptide inhibitors targeting the polo-like kinase 1 (Plk1, which is critically involved in multiple mitotic processes and has been established as an adverse prognostic marker for tumor patients. Plk1 localizes to its intracellular anchoring sites via its polo-box domain, and inhibiting the Plk1 polo-box domain has been considered as an approach to circumvent the specificity problems associated with inhibiting the conserved adenosine triphosphate-binding pocket. The polo-box domain consists of two different binding regions, such as the unique, broader pyrrolidine-binding pocket and the conserved, narrow, Tyr-rich hydrophobic channel, among the three Plk polo-box domains (Plks 1-3, respectively. Therefore, the studies that provide insights into the binding nature of the unique, broader pyrrolidine-binding pocket might lead to the development of selective Plk1-inhibitory compounds. METHODOLOGY/PRINCIPAL FINDINGS: In an attempt to retain the monospecificity by targeting the unique, broader pyrrolidine-binding pocket, here, for the first time, a systematic approach was undertaken to examine the structure-activity relationship of N-terminal-truncated PLHSpTM derivatives, to apply a site-directed ligand approach using bulky aromatic and non-aromatic systems, and to characterize the binding nature of these analogues using X-ray crystallographic studies. We have identified a new mode of binding interactions, having improved binding affinity and retaining the Plk1 polo-box domain specificity, at the pyrrolidine-binding pocket. Furthermore, our data revealed that the pyrrolidine-binding pocket was very specific to recognize a short and bulky hydrophobic ligand like adamantane, whereas the Tyr-rich hydrophobic channel was specific with lengthy and small hydrophobic groups. CONCLUSION/SIGNIFICANCE: The progress made using our site

  10. Acetylcholine-Binding Protein Engineered to Mimic the α4-α4 Binding Pocket in α4β2 Nicotinic Acetylcholine Receptors Reveals Interface Specific Interactions Important for Binding and Activity

    DEFF Research Database (Denmark)

    Shahsavar, Azadeh; Ahring, Philip K; Olsen, Jeppe A

    2015-01-01

    Neuronal α4β2 nicotinic acetylcholine receptors are attractive drug targets for psychiatric and neurodegenerative disorders and smoking cessation aids. Recently, a third agonist binding site between two α4 subunits in the (α4)(3)(β2)(2) receptor subpopulation was discovered. In particular, three...... specific nicotinic acetylcholine receptor interfaces....... by introduction of three point mutations, R104H, L112Q, and M114T, into the binding pocket of Lymnaea stagnalis acetylcholine-binding protein (Ls-AChBP). Cocrystallization with two agonists possessing distinct pharmacologic profiles, NS3920 [1-(6-bromopyridin-3-yl)-1,4-diazepane] and NS3573 [1-(5-ethoxypyridin-3...

  11. Characterization of the allosteric binding pocket of human liver fructose-1,6-bisphosphatase by protein crystallography and inhibitor activity studies.

    Science.gov (United States)

    Iversen, L F; Brzozowski, M; Hastrup, S; Hubbard, R; Kastrup, J S; Larsen, I K; Naerum, L; Nørskov-Lauridsen, L; Rasmussen, P B; Thim, L; Wiberg, F C; Lundgren, K

    1997-05-01

    The structures of three complexes of human fructose-1,6-bisphosphatase (FB) with the allosteric inhibitor AMP and two AMP analogues have been determined and all fully refined. The data used for structure determination were collected at cryogenic temperature (110 K), and with the use of synchrotron radiation. The structures reveal a common mode of binding for AMP and formycine monophosphate (FMP). 5-Amino-4-carboxamido-1 beta-D-5-phosphate-ribofuranosyl-1H-imidazole (AICAR-P) shows an unexpected mode of binding to FB, different from that of the other two ligands. The imidazole ring of AICAR-P is rotated 180 degrees compared to the AMP and FMP bases. This rotation results in a slightly different hydrogen bonding pattern and minor changes in the water structure in the binding pocket. Common features of binding are seen for the ribose and phosphate moieties of all three compounds. Although binding in a different mode, AICAR-P is still capable of making all the important interactions with the residues building the allosteric binding pocket. The IC50 values of AMP, FMP, and AICAR-P were determined to be 1.7, 1.4, and 20.9 microM, respectively. Thus, the approximately 10 times lower potency of AICAR-P is difficult to explain solely from the variations observed in the binding pocket. Only one water molecule in the allosteric binding pocket was found to be conserved in all four subunits in all three structures. This water molecule coordinates to a phosphate oxygen atom and the N7 atom of the AMP molecule, and to similarly situated atoms in the FMP and AICAR-P complexes. This implies an important role of the conserved water molecule in binding of the ligand.

  12. Inhibition of the acetyl lysine-binding pocket of bromodomain and extraterminal domain proteins interferes with adipogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Goupille, Olivier [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Penglong, Tipparat [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Thalassemia Research Center, Mahidol University (Thailand); Kadri, Zahra; Granger-Locatelli, Marine [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Fucharoen, Suthat [Thalassemia Research Center, Mahidol University (Thailand); Maouche-Chrétien, Leila [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); INSERM, Paris (France); Prost, Stéphane [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Leboulch, Philippe [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); Thalassemia Research Center, Mahidol University (Thailand); Chrétien, Stany, E-mail: stany.chretien@cea.fr [CEA, Institute of Emerging Diseases and Innovative Therapies (IMETI), Fontenay-aux-Roses and Université Paris-Saclay, UMR-E 007 (France); INSERM, Paris (France)

    2016-04-15

    The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors. - Highlights: • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into white adipocytes. • JQ1 affected clonal cell expansion and abolished lipid accumulation. • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into brown adipocytes. • JQ1 treatment did not lead to the conversion of white adipose tissue into brown adipose tissue. • JQ1 decreased STAT5 expression, but STAT5B{sup ca} expression did not restore adipogenesis.

  13. Inhibition of the acetyl lysine-binding pocket of bromodomain and extraterminal domain proteins interferes with adipogenesis

    International Nuclear Information System (INIS)

    Goupille, Olivier; Penglong, Tipparat; Kadri, Zahra; Granger-Locatelli, Marine; Fucharoen, Suthat; Maouche-Chrétien, Leila; Prost, Stéphane; Leboulch, Philippe; Chrétien, Stany

    2016-01-01

    The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors. - Highlights: • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into white adipocytes. • JQ1 affected clonal cell expansion and abolished lipid accumulation. • JQ1 prevented the differentiation of 3T3-L1 preadipocytes into brown adipocytes. • JQ1 treatment did not lead to the conversion of white adipose tissue into brown adipose tissue. • JQ1 decreased STAT5 expression, but STAT5B ca expression did not restore adipogenesis.

  14. A conserved NAD+binding pocket that regulates protein-protein interactions during aging.

    Science.gov (United States)

    Li, Jun; Bonkowski, Michael S; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P; Ling, Alvin J Y; Rajman, Luis A; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L; Steegborn, Clemens; Sinclair, David A

    2017-03-24

    DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD + (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD + to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD + concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD + Thus, NAD + directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging. Copyright © 2017, American Association for the Advancement of Science.

  15. Role of the Ectodomain Serine 275 in Shaping the Binding Pocket of the ATP-Gated P2X3 Receptor

    Czech Academy of Sciences Publication Activity Database

    Petrenko, N.; Khafizov, K.; Tvrdoňová, Vendula; Skorinkin, A.; Giniatullin, R.

    2011-01-01

    Roč. 50, č. 39 (2011), s. 8427-8436 ISSN 0006-2960 Grant - others:Univerzita Karlova(CZ) 3446/2011 Institutional research plan: CEZ:AV0Z50110509 Keywords : P2X3 * purinergic * ATP * ATP-binding pocket * receptor Subject RIV: ED - Physiology Impact factor: 3.422, year: 2011

  16. Residues remote from the binding pocket control the antagonist selectivity towards the corticotropin-releasing factor receptor-1

    Science.gov (United States)

    Sun, Xianqiang; Cheng, Jianxin; Wang, Xu; Tang, Yun; Ågren, Hans; Tu, Yaoquan

    2015-01-01

    The corticotropin releasing factors receptor-1 and receptor-2 (CRF1R and CRF2R) are therapeutic targets for treating neurological diseases. Antagonists targeting CRF1R have been developed for the potential treatment of anxiety disorders and alcohol addiction. It has been found that antagonists targeting CRF1R always show high selectivity, although CRF1R and CRF2R share a very high rate of sequence identity. This has inspired us to study the origin of the selectivity of the antagonists. We have therefore built a homology model for CRF2R and carried out unbiased molecular dynamics and well-tempered metadynamics simulations for systems with the antagonist CP-376395 in CRF1R or CRF2R to address this issue. We found that the side chain of Tyr6.63 forms a hydrogen bond with the residue remote from the binding pocket, which allows Tyr6.63 to adopt different conformations in the two receptors and results in the presence or absence of a bottleneck controlling the antagonist binding to or dissociation from the receptors. The rotameric switch of the side chain of Tyr3566.63 allows the breaking down of the bottleneck and is a perquisite for the dissociation of CP-376395 from CRF1R.

  17. Interaction between the flagellar pocket collar and the hook complex via a novel microtubule-binding protein in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Anna Albisetti

    2017-11-01

    Full Text Available Trypanosoma brucei belongs to a group of unicellular, flagellated parasites that are responsible for human African trypanosomiasis. An essential aspect of parasite pathogenicity is cytoskeleton remodelling, which occurs during the life cycle of the parasite and is accompanied by major changes in morphology and organelle positioning. The flagellum originates from the basal bodies and exits the cell body through the flagellar pocket (FP but remains attached to the cell body via the flagellum attachment zone (FAZ. The FP is an invagination of the pellicular membrane and is the sole site for endo- and exocytosis. The FAZ is a large complex of cytoskeletal proteins, plus an intracellular set of four specialised microtubules (MtQ that elongate from the basal bodies to the anterior end of the cell. At the distal end of the FP, an essential, intracellular, cytoskeletal structure called the flagellar pocket collar (FPC circumvents the flagellum. Overlapping the FPC is the hook complex (HC (a sub-structure of the previously named bilobe that is also essential and is thought to be involved in protein FP entry. BILBO1 is the only functionally characterised FPC protein and is necessary for FPC and FP biogenesis. Here, we used a combination of in vitro and in vivo approaches to identify and characterize a new BILBO1 partner protein-FPC4. We demonstrate that FPC4 localises to the FPC, the HC, and possibly to a proximal portion of the MtQ. We found that the C-terminal domain of FPC4 interacts with the BILBO1 N-terminal domain, and we identified the key amino acids required for this interaction. Interestingly, the FPC4 N-terminal domain was found to bind microtubules. Over-expression studies highlight the role of FPC4 in its association with the FPC, HC and FPC segregation. Our data suggest a tripartite association between the FPC, the HC and the MtQ.

  18. A mutation in the glycine binding pocket of the N-methyl-D-aspartate receptor NR1 subunit alters agonist efficacy.

    Science.gov (United States)

    Wood, M W; VanDongen, H M; VanDongen, A M

    1999-11-10

    Alanine 714 of the NMDA receptor NR1 subunit resides in the glycine binding pocket. The Ala714Leu mutation substantially shifts glycine affinity, but here no effect on antagonism by DCK is detected. Ala714Leu is also found to limit the efficacy of a partial agonist without altering its apparent affinity. The differential sensitivity of Ala714Leu to glycine agonists suggests that alanine 714 may be an intermediary in transducing the ligand binding signal.

  19. Electrostatic modifications of the human leukocyte antigen-DR P9 peptide-binding pocket and susceptibility to primary sclerosing cholangitis.

    Science.gov (United States)

    Hov, Johannes R; Kosmoliaptsis, Vasilis; Traherne, James A; Olsson, Marita; Boberg, Kirsten M; Bergquist, Annika; Schrumpf, Erik; Bradley, J Andrew; Taylor, Craig J; Lie, Benedicte A; Trowsdale, John; Karlsen, Tom H

    2011-06-01

    The strongest genetic risk factors for primary sclerosing cholangitis (PSC) are found in the human leukocyte antigen (HLA) complex at chromosome 6p21. Genes in the HLA class II region encode molecules that present antigen to T lymphocytes. Polymorphisms in these genes are associated with most autoimmune diseases, most likely because they contribute to the specificity of immune responses. The aim of this study was to analyze the structure and electrostatic properties of the peptide-binding groove of HLA-DR in relation to PSC. Thus, four-digit resolution HLA-DRB1 genotyping was performed in 356 PSC patients and 366 healthy controls. Sequence information was used to assign which amino acids were encoded at all polymorphic positions. In stepwise logistic regressions, variations at residues 37 and 86 were independently associated with PSC (P = 1.2 × 10(-32) and P = 1.8 × 10(-22) in single-residue models, respectively). Three-dimensional modeling was performed to explore the effect of these key residues on the HLA-DR molecule. This analysis indicated that residue 37 was a major determinant of the electrostatic properties of pocket P9 of the peptide-binding groove. Asparagine at residue 37, which was associated with PSC, induced a positive charge in pocket P9. Tyrosine, which protected against PSC, induced a negative charge in this pocket. Consistent with the statistical observations, variation at residue 86 also indirectly influenced the electrostatic properties of this pocket. DRB1*13:01, which was PSC-associated, had a positive P9 pocket and DRB1*13:02, protective against PSC, had a negative P9 pocket. The results suggest that in patients with PSC, residues 37 and 86 of the HLA-DRβ chain critically influence the electrostatic properties of pocket P9 and thereby the range of peptides presented. Copyright © 2011 American Association for the Study of Liver Diseases.

  20. Neutron Crystallography Detects Differences in Protein Dynamics: Structure of the PKG II Cyclic Nucleotide Binding Domain in Complex with an Activator.

    Science.gov (United States)

    Gerlits, Oksana; Campbell, James C; Blakeley, Matthew P; Kim, Choel; Kovalevsky, Andrey

    2018-03-27

    As one of the main receptors of a second messenger, cGMP, cGMP-dependent protein kinase (PKG) isoforms I and II regulate distinct physiological processes. The design of isoform-specific activators is thus of great biomedical importance and requires detailed structural information about PKG isoforms bound with activators, including accurate positions of hydrogen atoms and a description of the hydrogen bonding and water architecture. Here, we determined a 2.2 Å room-temperature joint X-ray/neutron (XN) structure of the human PKG II carboxyl cyclic nucleotide binding (CNB-B) domain bound with a potent PKG II activator, 8-pCPT-cGMP. The XN structure directly visualizes intermolecular interactions and reveals changes in hydrogen bonding patterns upon comparison to the X-ray structure determined at cryo-temperatures. Comparative analysis of the backbone hydrogen/deuterium exchange patterns in PKG II:8-pCPT-cGMP and previously reported PKG Iβ:cGMP XN structures suggests that the ability of these agonists to activate PKG is related to how effectively they quench dynamics of the cyclic nucleotide binding pocket and the surrounding regions.

  1. Mutation of lysine residues in the nucleotide binding segments of the poliovirus RNA-dependent RNA polymerase.

    OpenAIRE

    Richards, O C; Baker, S; Ehrenfeld, E

    1996-01-01

    The poliovirus 3D RNA-dependent RNA polymerase contains two peptide segments previously shown to cross-link to nucleotide substrates via lysine residues. To determine which lysine residue(s) might be implicated in catalytic function, we engineered mutations to generate proteins with leucine residues substituted individually for each of the lysine residues in the NTP binding regions. These proteins were expressed in Escherichia coli and were examined for their abilities to bind nucleotides and...

  2. The nucleotide-binding domain of NLRC5 is critical for nuclear import and transactivation activity

    Energy Technology Data Exchange (ETDEWEB)

    Meissner, Torsten B. [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02215 (United States); Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02215 (United States); Li, Amy; Liu, Yuen-Joyce [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02215 (United States); Gagnon, Etienne [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02215 (United States); Institut de Recherche en Immunologie et Cancerologie, Departement de Microbiologie et Immunologie, Universite de Montreal, Montreal, Canada H3T1J4 (Canada); Kobayashi, Koichi S., E-mail: Koichi_Kobayashi@dfci.harvard.edu [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02215 (United States); Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02215 (United States)

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer NLRC5 requires an intact NLS for its function as MHC class I transactivator. Black-Right-Pointing-Pointer Nuclear presence of NLRC5 is required for MHC class I induction. Black-Right-Pointing-Pointer Nucleotide-binding controls nuclear import and transactivation activity of NLRC5. -- Abstract: Major histocompatibility complex (MHC) class I and class II are crucial for the function of the human adaptive immune system. A member of the NLR (nucleotide-binding domain, leucine-rich repeat) protein family, NLRC5, has recently been identified as a transcriptional regulator of MHC class I and related genes. While a 'master regulator' of MHC class II genes, CIITA, has long been known, NLRC5 specifically associates with and transactivates the proximal promoters of MHC class I genes. In this study, we analyzed the molecular requirements of NLRC5 nuclear import and transactivation activity. We show that NLRC5-mediated MHC class I gene induction requires an intact nuclear localization signal and nuclear distribution of NLRC5. In addition, we find that the nucleotide-binding domain (NBD) of NLRC5 is critical not only for nuclear translocation but also for the transactivation of MHC class I genes. Changing the cellular localization of NLRC5 is likely to immediately impact MHC class I expression as well as MHC class I-mediated antigen presentation. NLRC5 may thus provide a promising target for the modulation of MHC class I antigen presentation, especially in the setting of transplant medicine.

  3. DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair.

    Science.gov (United States)

    de Laat, W L; Appeldoorn, E; Sugasawa, K; Weterings, E; Jaspers, N G; Hoeijmakers, J H

    1998-08-15

    The human single-stranded DNA-binding replication A protein (RPA) is involved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3'- or 5'-protruding single-stranded arms, we found that hRPA binds ssDNA with a defined polarity; a strong ssDNA interaction domain of hRPA is positioned at the 5' side of its binding region, a weak ssDNA-binding domain resides at the 3' side. Polarity appears crucial for positioning of the excision repair nucleases XPG and ERCC1-XPF on the DNA. With the 3'-oriented side of hRPA facing a duplex ssDNA junction, hRPA interacts with and stimulates ERCC1-XPF, whereas the 5'-oriented side of hRPA at a DNA junction allows stable binding of XPG to hRPA. Our data pinpoint hRPA to the undamaged strand during nucleotide excision repair. Polarity of hRPA on ssDNA is likely to contribute to the directionality of other hRPA-dependent processes as well.

  4. Immunization with influenza virus hemagglutinin globular region containing the receptor-binding pocket.

    Science.gov (United States)

    Jeon, Sung Ho; Arnon, Ruth

    2002-01-01

    The globular region of hemagglutinin (residues 91-261) membrane glycoprotein of influenza virus that encompasses the binding zone to the oligosaccharide receptor of target cells has been cloned by reverse transcriptase-polymerase chain reaction (RT-PCR). This protein segment (denoted HA91-261 peptide) induced significant immune response in mice. The serum antibodies and lung homogenates from the immunized mice cross-reacted with native virus particles. The cellular immunity was manifested by proliferative splenocyte responses and cytokine release indicating T helper type 1 activity. The plasmid DNA containing this segment (denoted pHA91-261) provoked, in addition, a significant cytotoxic T lymphocyte (CTL) response, whereas the HA91-261 protein fragment led to no such response. Both the DNA and the protein fragment of HA91-261 induced significant protection against viral challenge, although the immune response they induce might be along different pathways. Interestingly, the combined DNA priming-protein boosting immunization regimen did not induce protection against viral challenges even though it led to significant humoral immune responses similar to that induced by the peptide vaccine.

  5. A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase.

    Science.gov (United States)

    Morgan, Hugh P; Walsh, Martin J; Blackburn, Elizabeth A; Wear, Martin A; Boxer, Matthew B; Shen, Min; Veith, Henrike; McNae, Iain W; Nowicki, Matthew W; Michels, Paul A M; Auld, Douglas S; Fothergill-Gilmore, Linda A; Walkinshaw, Malcolm D

    2012-11-15

    PYK (pyruvate kinase) plays a central role in the metabolism of many organisms and cell types, but the elucidation of the details of its function in a systems biology context has been hampered by the lack of specific high-affinity small-molecule inhibitors. High-throughput screening has been used to identify a family of saccharin derivatives which inhibit LmPYK (Leishmania mexicana PYK) activity in a time- (and dose-) dependent manner, a characteristic of irreversible inhibition. The crystal structure of DBS {4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid} complexed with LmPYK shows that the saccharin moiety reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP. Mutation of the lysine residue to an arginine residue eliminated the effect of the inhibitor molecule, providing confirmation of the proposed inhibitor mechanism. This lysine residue is conserved in the active sites of the four human PYK isoenzymes, which were also found to be irreversibly inhibited by DBS. X-ray structures of PYK isoforms show structural differences at the DBS-binding pocket, and this covalent inhibitor of PYK provides a chemical scaffold for the design of new families of potentially isoform-specific irreversible inhibitors.

  6. A Novel Protein Interaction between Nucleotide Binding Domain of Hsp70 and p53 Motif

    Directory of Open Access Journals (Sweden)

    Asita Elengoe

    2015-01-01

    Full Text Available Currently, protein interaction of Homo sapiens nucleotide binding domain (NBD of heat shock 70 kDa protein (PDB: 1HJO with p53 motif remains to be elucidated. The NBD-p53 motif complex enhances the p53 stabilization, thereby increasing the tumor suppression activity in cancer treatment. Therefore, we identified the interaction between NBD and p53 using STRING version 9.1 program. Then, we modeled the three-dimensional structure of p53 motif through homology modeling and determined the binding affinity and stability of NBD-p53 motif complex structure via molecular docking and dynamics (MD simulation. Human DNA binding domain of p53 motif (SCMGGMNR retrieved from UniProt (UniProtKB: P04637 was docked with the NBD protein, using the Autodock version 4.2 program. The binding energy and intermolecular energy for the NBD-p53 motif complex were −0.44 Kcal/mol and −9.90 Kcal/mol, respectively. Moreover, RMSD, RMSF, hydrogen bonds, salt bridge, and secondary structure analyses revealed that the NBD protein had a strong bond with p53 motif and the protein-ligand complex was stable. Thus, the current data would be highly encouraging for designing Hsp70 structure based drug in cancer therapy.

  7. pocketZebra: a web-server for automated selection and classification of subfamily-specific binding sites by bioinformatic analysis of diverse protein families.

    Science.gov (United States)

    Suplatov, Dmitry; Kirilin, Eugeny; Arbatsky, Mikhail; Takhaveev, Vakil; Svedas, Vytas

    2014-07-01

    The new web-server pocketZebra implements the power of bioinformatics and geometry-based structural approaches to identify and rank subfamily-specific binding sites in proteins by functional significance, and select particular positions in the structure that determine selective accommodation of ligands. A new scoring function has been developed to annotate binding sites by the presence of the subfamily-specific positions in diverse protein families. pocketZebra web-server has multiple input modes to meet the needs of users with different experience in bioinformatics. The server provides on-site visualization of the results as well as off-line version of the output in annotated text format and as PyMol sessions ready for structural analysis. pocketZebra can be used to study structure-function relationship and regulation in large protein superfamilies, classify functionally important binding sites and annotate proteins with unknown function. The server can be used to engineer ligand-binding sites and allosteric regulation of enzymes, or implemented in a drug discovery process to search for potential molecular targets and novel selective inhibitors/effectors. The server, documentation and examples are freely available at http://biokinet.belozersky.msu.ru/pocketzebra and there are no login requirements. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. The thermodynamic signature of ligand binding to histone deacetylase-like amidohydrolases is most sensitive to the flexibility in the L2-loop lining the active site pocket.

    Science.gov (United States)

    Meyners, Christian; Krämer, Andreas; Yildiz, Özkan; Meyer-Almes, Franz-Josef

    2017-07-01

    The analysis of the thermodynamic driving forces of ligand-protein binding has been suggested to be a key component for the selection and optimization of active compounds into drug candidates. The binding enthalpy as deduced from isothermal titration calorimetry (ITC) is usually interpreted assuming single-step binding of a ligand to one conformation of the target protein. Although successful in many cases, these assumptions are oversimplified approximations of the reality with flexible proteins and complicated binding mechanism in many if not most cases. The relationship between protein flexibility and thermodynamic signature of ligand binding is largely understudied. Directed mutagenesis, X-ray crystallography, enzyme kinetics and ITC methods were combined to dissect the influence of loop flexibility on the thermodynamics and mechanism of ligand binding to histone deacetylase (HDAC)-like amidohydrolases. The general ligand-protein binding mechanism comprises an energetically demanding gate opening step followed by physical binding. Increased flexibility of the L2-loop in HDAC-like amidohydrolases facilitates access of ligands to the binding pocket resulting in predominantly enthalpy-driven complex formation. The study provides evidence for the great importance of flexibility adjacent to the active site channel for the mechanism and observed thermodynamic driving forces of molecular recognition in HDAC like enzymes. The flexibility or malleability in regions adjacent to binding pockets should be given more attention when designing better drug candidates. The presented case study also suggests that the observed binding enthalpy of protein-ligand systems should be interpreted with caution, since more complicated binding mechanisms may obscure the significance regarding potential drug likeness. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Structural Basis for Nucleotide Binding and Reaction Catalysis in Mevalonate Diphosphate Decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; McWhorter, William J.; Miziorko, Henry M.; Geisbrecht, Brian V. (UMKC)

    2012-09-17

    Mevalonate diphosphate decarboxylase (MDD) catalyzes the final step of the mevalonate pathway, the Mg{sup 2+}-ATP dependent decarboxylation of mevalonate 5-diphosphate (MVAPP), producing isopentenyl diphosphate (IPP). Synthesis of IPP, an isoprenoid precursor molecule that is a critical intermediate in peptidoglycan and polyisoprenoid biosynthesis, is essential in Gram-positive bacteria (e.g., Staphylococcus, Streptococcus, and Enterococcus spp.), and thus the enzymes of the mevalonate pathway are ideal antimicrobial targets. MDD belongs to the GHMP superfamily of metabolite kinases that have been extensively studied for the past 50 years, yet the crystallization of GHMP kinase ternary complexes has proven to be difficult. To further our understanding of the catalytic mechanism of GHMP kinases with the purpose of developing broad spectrum antimicrobial agents that target the substrate and nucleotide binding sites, we report the crystal structures of wild-type and mutant (S192A and D283A) ternary complexes of Staphylococcus epidermidis MDD. Comparison of apo, MVAPP-bound, and ternary complex wild-type MDD provides structural information about the mode of substrate binding and the catalytic mechanism. Structural characterization of ternary complexes of catalytically deficient MDD S192A and D283A (k{sub cat} decreased 10{sup 3}- and 10{sup 5}-fold, respectively) provides insight into MDD function. The carboxylate side chain of invariant Asp{sup 283} functions as a catalytic base and is essential for the proper orientation of the MVAPP C3-hydroxyl group within the active site funnel. Several MDD amino acids within the conserved phosphate binding loop ('P-loop') provide key interactions, stabilizing the nucleotide triphosphoryl moiety. The crystal structures presented here provide a useful foundation for structure-based drug design.

  10. 14-3-3 binding and phosphorylation of neuroglobin during hypoxia modulate six-to-five heme pocket coordination and rate of nitrite reduction to nitric oxide.

    Science.gov (United States)

    Jayaraman, Thottala; Tejero, Jesús; Chen, Bill B; Blood, Arlin B; Frizzell, Sheila; Shapiro, Calli; Tiso, Mauro; Hood, Brian L; Wang, Xunde; Zhao, Xuejun; Conrads, Thomas P; Mallampalli, Rama K; Gladwin, Mark T

    2011-12-09

    Neuroglobin protects neurons from hypoxia in vitro and in vivo; however, the underlying mechanisms for this effect remain poorly understood. Most of the neuroglobin is present in a hexacoordinate state with proximal and distal histidines in the heme pocket directly bound to the heme iron. At equilibrium, the concentration of the five-coordinate neuroglobin remains very low (0.1-5%). Recent studies have shown that post-translational redox regulation of neuroglobin surface thiol disulfide formation increases the open probability of the heme pocket and allows nitrite binding and reaction to form NO. We hypothesized that the equilibrium between the six- and five-coordinate states and secondary reactions with nitrite to form NO could be regulated by other hypoxia-dependent post-translational modification(s). Protein sequence models identified candidate sites for both 14-3-3 binding and phosphorylation. In both in vitro experiments and human SH-SY5Y neuronal cells exposed to hypoxia and glucose deprivation, we observed that 1) neuroglobin phosphorylation and protein-protein interactions with 14-3-3 increase during hypoxic and metabolic stress; 2) neuroglobin binding to 14-3-3 stabilizes and increases the half-life of phosphorylation; and 3) phosphorylation increases the open probability of the heme pocket, which increases ligand binding (CO and nitrite) and accelerates the rate of anaerobic nitrite reduction to form NO. These data reveal a series of hypoxia-dependent post-translational modifications to neuroglobin that regulate the six-to-five heme pocket equilibrium and heme access to ligands. Hypoxia-regulated reactions of nitrite and neuroglobin may contribute to the cellular adaptation to hypoxia.

  11. Paying the Price of Desolvation in Solvent-Exposed Protein Pockets: Impact of Distal Solubilizing Groups on Affinity and Binding Thermodynamics in a Series of Thermolysin Inhibitors.

    Science.gov (United States)

    Cramer, Jonathan; Krimmer, Stefan G; Heine, Andreas; Klebe, Gerhard

    2017-07-13

    In lead optimization, open, solvent-exposed protein pockets are often disregarded as prospective binding sites. Because of bulk-solvent proximity, researchers are instead enticed to attach charged polar groups at inhibitor scaffolds to improve solubility and pharmacokinetic properties. It is rarely considered that solvent effects from water reorganization in the first hydration shell of protein-ligand complexes can have a significant impact on binding. We investigate the thermodynamic fingerprint of thermolysin inhibitors featuring terminal charged ammonium groups that are gradually pulled from a distal, solvent-exposed position into the flat, bowl-shaped S 2 ' pocket. Even for the most remote attachment, costs for partial desolvation of the polar group next to the protein-solvent interface are difficult to compensate by interactions with the protein or surrounding water molecules. Through direct comparison with hydrophobic analogues, a significant 180-fold affinity loss was recorded, which questions popular strategies to attach polar ligand-solubilizing groups at the exposed terminus of substituents accommodated in flat open pockets.

  12. Five Fatty Aldehyde Dehydrogenase Enzymes from Marinobacter and Acinetobacter spp. and Structural Insights into the Aldehyde Binding Pocket

    Energy Technology Data Exchange (ETDEWEB)

    Bertram, Jonathan H.; Mulliner, Kalene M.; Shi, Ke; Plunkett, Mary H.; Nixon, Peter; Serratore, Nicholas A.; Douglas, Christopher J.; Aihara, Hideki; Barney, Brett M.; Parales, Rebecca E.

    2017-04-07

    ABSTRACT

    Enzymes involved in lipid biosynthesis and metabolism play an important role in energy conversion and storage and in the function of structural components such as cell membranes. The fatty aldehyde dehydrogenase (FAldDH) plays a central function in the metabolism of lipid intermediates, oxidizing fatty aldehydes to the corresponding fatty acid and competing with pathways that would further reduce the fatty aldehydes to fatty alcohols or require the fatty aldehydes to produce alkanes. In this report, the genes for four putative FAldDH enzymes fromMarinobacter aquaeoleiVT8 and an additional enzyme fromAcinetobacter baylyiwere heterologously expressed inEscherichia coliand shown to display FAldDH activity. Five enzymes (Maqu_0438, Maqu_3316, Maqu_3410, Maqu_3572, and the enzyme reported under RefSeq accession no.WP_004927398) were found to act on aldehydes ranging from acetaldehyde to hexadecanal and also acted on the unsaturated long-chain palmitoleyl and oleyl aldehydes. A comparison of the specificities of these enzymes with various aldehydes is presented. Crystallization trials yielded diffraction-quality crystals of one particular FAldDH (Maqu_3316) fromM. aquaeoleiVT8. Crystals were independently treated with both the NAD+cofactor and the aldehyde substrate decanal, revealing specific details of the likely substrate binding pocket for this class of enzymes. A likely model for how catalysis by the enzyme is accomplished is also provided.

    IMPORTANCEThis study provides a comparison of multiple enzymes with the ability

  13. Structural and functional studies of conserved nucleotide-binding protein LptB in lipopolysaccharide transport

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zhongshan [Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, NR4 7TJ (United Kingdom); College of Life Sciences, Sichuan University, Chengdu 610065 (China); Biomedical Sciences Research Complex, School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST (United Kingdom); Xiang, Quanju [College of Life Sciences, Sichuan University, Chengdu 610065 (China); Biomedical Sciences Research Complex, School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST (United Kingdom); Department of Microbiology, College of Resource and Environment Science, Sichuan Agriculture University, Yaan 625000 (China); Zhu, Xiaofeng [College of Life Sciences, Sichuan University, Chengdu 610065 (China); Dong, Haohao [Biomedical Sciences Research Complex, School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST (United Kingdom); He, Chuan [School of Electronics and Information, Wuhan Technical College of Communications, No. 6 Huangjiahu West Road, Hongshan District, Wuhan, Hubei 430065 (China); Wang, Haiyan; Zhang, Yizheng [College of Life Sciences, Sichuan University, Chengdu 610065 (China); Wang, Wenjian, E-mail: Wenjian166@gmail.com [Laboratory of Department of Surgery, The First Affiliated Hospital, Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong 510080 (China); Dong, Changjiang, E-mail: C.Dong@uea.ac.uk [Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, NR4 7TJ (United Kingdom)

    2014-09-26

    Highlights: • Determination of the structure of the wild-type LptB in complex with ATP and Mg{sup 2+}. • Demonstrated that ATP binding residues are essential for LptB’s ATPase activity and LPS transport. • Dimerization is required for the LptB’s function and LPS transport. • Revealed relationship between activity of the LptB and the vitality of E. coli cells. - Abstract: Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane by seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg{sup 2+}, which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB’s ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics.

  14. Structural and functional studies of conserved nucleotide-binding protein LptB in lipopolysaccharide transport

    International Nuclear Information System (INIS)

    Wang, Zhongshan; Xiang, Quanju; Zhu, Xiaofeng; Dong, Haohao; He, Chuan; Wang, Haiyan; Zhang, Yizheng; Wang, Wenjian; Dong, Changjiang

    2014-01-01

    Highlights: • Determination of the structure of the wild-type LptB in complex with ATP and Mg 2+ . • Demonstrated that ATP binding residues are essential for LptB’s ATPase activity and LPS transport. • Dimerization is required for the LptB’s function and LPS transport. • Revealed relationship between activity of the LptB and the vitality of E. coli cells. - Abstract: Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane by seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg 2+ , which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB’s ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics

  15. Single-nucleotide mutation matrix: a new model for predicting the NF-κB DNA binding sites.

    Science.gov (United States)

    Du, Wenxin; Gao, Jing; Wang, Tingting; Wang, Jinke

    2014-01-01

    In this study, we established a single nucleotide mutation matrix (SNMM) model based on the relative binding affinities of NF-κB p50 homodimer to a wild-type binding site (GGGACTTTCC) and its all single-nucleotide mutants detected with the double-stranded DNA microarray. We evaluated this model by scoring different groups of 10-bp DNA sequences with this model and analyzing the correlations between the scores and the relative binding affinities detected with three wet experiments, including the electrophoresis mobility shift assay (EMSA), the protein-binding microarray (PBM) and the systematic evolution of ligands by exponential enrichment-sequencing (SELEX-Seq). The results revealed that the SNMM scores were strongly correlated with the detected binding affinities. We also scored the DNA sequences with other three models, including the principal coordinate (PC) model, the position weight matrix scoring algorithm (PWMSA) model and the Match model, and analyzed the correlations between the scores and the detected binding affinities. In comparison with these models, the SNMM model achieved reliable results. We finally determined 0.747 as the optimal threshold for predicting the NF-κB DNA-binding sites with the SNMM model. The SNMM model thus provides a new alternative model for scoring the relative binding affinities of NF-κB to the 10-bp DNA sequences and predicting the NF-κB DNA-binding sites.

  16. Interaction between nucleotide binding sites on chloroplast coupling factor 1 during ATP hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Leckband, D.; Hammes, G.G.

    1987-04-21

    The initial hydrolysis of radioactively-labelled CaATP by chloroplast coupling factor 1 was studied with the quenched-flow method. The time course of hydrolysis can be described as a first-order conversion of the enzyme to an active form followed by steady-state formation of product. The rate constant for the first-order process is independent of substrate concentration but increased hyperbolically to a limiting value of 0.43 s/sup -1/ with increasing concentrations of free Ca/sup 2 +/. A mechanism involving a Ca/sup 2 +/-triggered conversion to an active form of the enzyme is consistent with the data. The steady-state rate varied sigmoidally with the CaATP concentration. Initial exchange of tightly bound ADP is complex: approx. 50% of the bound nucleotide is lost within 30 s, with complete exchange requiring several minutes. The first-order rate constant characterizing the rapid phase of the reaction increases hyperbolically to a limiting value of 0.26 s/sup -1/ as the concentration of CaATP is increased, indicating that the binding of CaATP to the enzyme promotes the exchange process. Modification of the quenched-flow apparatus permitted measurement of the rate of nucleotide exchange during steady-state catalysis. The value of the first-order rate constant characterizing this process is similar to the catalytic rate constant determined under identical conditions. When MgATP is tightly bound to the enzyme, none of the kinetic properties of the enzyme described above were significantly changes. The results obtained suggest a mechanism in which two sites on the enzyme participate in catalysis. Several possible mechanisms consistent with the data are discussed.

  17. Supramolecular Properties of Triazole-containing Two Armed Peptidomimetics: From Organogelators to Nucleotide-binding Tweezers

    Science.gov (United States)

    Chui, Tin Ki

    obtain a clearer picture on the mode of association of these two series of branched peptidomimetics, the length of the tripeptidomimetic arms was truncated to a dipeptide, and the amino acid, valine, was used for further studies. Both the two new candidates, 88-K-V2 and 89-B-V2, were shown to dimerize in chloroform as shown from vapor pressure osmometry (VPO) studies. 1H NMR titration experiments indicated a better dimerization strength for the latter candidate due to the intermolecular pi-pi interactions offered by its benzene ring in addition to the intermolecular hydrogen bonding by the amides and triazole units. H/D exchange and 2D NMR experiments, and molecular modeling revealed that 88-K-V2 dimerized through the formation of antiparallel beta-strands whereas formation of parallel beta-strands took place in 89-B-V2. Compound 88-K-V2 was found to form 1:1 complexes with chloride (Ka 640 M-1) and monobasic diethyl phosphate (DEP) ion (Ka 810 M-1) in chloroform. Interestingly, 89-B-V 2 was shown to form the usual 1:1 complex with the former ion (Ka 970 M-1) while forming an unexpected 2:1 complex with the latter with positive cooperativity. It was observed that both the amides and triazole protons were involved in anion-binding. In the 88-K-V2-DEP complex, the host formed a helix-like structure that wrapped around the anion located at the center of the complex as determined by 2D NMR and molecular modeling studies. Finally, further structural modification of 88-K-V2 gave a water-soluble nucleotide-binding tweezer 93-K-R2·4TFA . This tweezer consisted of four arginines (R), two triazole units, two pyrene probes and a small hydrophilic ethanolamine tail. Fluorescence study showed that this tweezer was able to form 1:1 complexes with different nucleotides in water with similar binding strength regardless of the number of phosphate groups present in the nucleotides. Moleular modeling suggested that such a charge-independent binding behavior was due to the similar number

  18. Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C

    International Nuclear Information System (INIS)

    Anholt, R.R.H.; Mumby, S.M.; Stoffers, D.A.; Girard, P.R.; Kuo, J.F.; Snyder, S.H.

    1987-01-01

    The authors have analyzed guanine nucleotide binding proteins (G-proteins) in the olfactory epithelium of Rana catesbeiana using subunit-specific antisera. The olfactory epithelium contained the α subunits of three G-proteins, migrating on polyacrylamide gels in SDS with apparent molecular weights of 45,000, 42,000, and 40,000, corresponding to G/sub s/, G/sub i/, and G/sub o/, respectively. A single β subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the α subunit of retinal transducin failed to detect immunoreactive proteins in olfactory cilia detached from the epithelium. The olfactory cilia appeared to be enriched in immunoreactive G/sub sα/ relative to G/sub ichemical bond/ and G/sub ochemical bond/ when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. Bound antibody was detected by autoradiography after incubation with [ 125 I]protein. Immunohistochemical studies using an antiserum against the β subunit of G-proteins revealed intense staining of the ciliary surface of the olfactory epithelium and of the axon bundles in the lamina propria. In contrast, an antiserum against a common sequence of the α subunits preferentially stained the cell membranes of the olfactory receptor cells and the acinar cells of Bowman's glands and the deep submucosal glands. In addition to G-proteins, they have identified protein kinase C in olfactory cilia via a protein kinase C specific antiserum and via phorbol ester binding. However, in contrast to the G-proteins, protein kinase C occurred also in cilia isolated from respiratory epithelium

  19. Genome Wide Analysis of Nucleotide-Binding Site Disease Resistance Genes in Brachypodium distachyon

    Directory of Open Access Journals (Sweden)

    Shenglong Tan

    2012-01-01

    Full Text Available Nucleotide-binding site (NBS disease resistance genes play an important role in defending plants from a variety of pathogens and insect pests. Many R-genes have been identified in various plant species. However, little is known about the NBS-encoding genes in Brachypodium distachyon. In this study, using computational analysis of the B. distachyon genome, we identified 126 regular NBS-encoding genes and characterized them on the bases of structural diversity, conserved protein motifs, chromosomal locations, gene duplications, promoter region, and phylogenetic relationships. EST hits and full-length cDNA sequences (from Brachypodium database of 126 R-like candidates supported their existence. Based on the occurrence of conserved protein motifs such as coiled-coil (CC, NBS, leucine-rich repeat (LRR, these regular NBS-LRR genes were classified into four subgroups: CC-NBS-LRR, NBS-LRR, CC-NBS, and X-NBS. Further expression analysis of the regular NBS-encoding genes in Brachypodium database revealed that these genes are expressed in a wide range of libraries, including those constructed from various developmental stages, tissue types, and drought challenged or nonchallenged tissue.

  20. A genomic analysis of disease-resistance genes encoding nucleotide binding sites in Sorghum bicolor

    Directory of Open Access Journals (Sweden)

    Xiao Cheng

    2010-01-01

    Full Text Available A large set of candidate nucleotide-binding site (NBS-encoding genes related to disease resistance was identified in the sorghum (Sorghum bicolor genome. These resistance (R genes were characterized based on their structural diversity, physical chromosomal location and phylogenetic relationships. Based on their N-terminal motifs and leucine-rich repeats (LRR, 50 non-regular NBS genes and 224 regular NBS genes were identified in 274 candidate NBS genes. The regular NBS genes were classified into ten types: CNL, CN, CNLX, CNX, CNXL, CXN, NX, N, NL and NLX. The vast majority (97% of NBS genes occurred in gene clusters, indicating extensive gene duplication in the evolution of S. bicolor NBS genes. Analysis of the S. bicolor NBS phylogenetic tree revealed two major clades. Most NBS genes were located at the distal tip of the long arms of the ten sorghum chromosomes, a pattern significantly different from rice and Arabidopsis, the NBS genes of which have a random chromosomal distribution.

  1. Molecular cloning and functional characterization of duck nucleotide-binding oligomerization domain 1 (NOD1).

    Science.gov (United States)

    Li, Huilin; Jin, Hui; Li, Yaqian; Liu, Dejian; Foda, Mohamed Frahat; Jiang, Yunbo; Luo, Rui

    2017-09-01

    Nucleotide-binding oligomerization domain 1 (NOD1) is an imperative cytoplasmic pattern recognition receptor (PRR) and considered as a key member of the NOD-like receptor (NLR) family which plays a critical role in innate immunity through sensing microbial components derived from bacterial peptidoglycan. In the current study, the full-length of duck NOD1 (duNOD1) cDNA from duck embryo fibroblasts (DEFs) was cloned. Multiple sequence alignment and phylogenetic analysis demonstrated that duNOD1 exhibited a strong evolutionary relationship with chicken and rock pigeon NOD1. Tissue-specific expression analysis showed that duNOD1 was widely distributed in various organs, with the highest expression observed in the liver. Furthermore, duNOD1 overexpression induced NF-κB activation in DEFs and the CARD domain is crucial for duNOD1-mediated NF-κB activation. In addition, silencing the duNOD1 decreased the activity of NF-κB in DEFs stimulated by iE-DAP. Overexpression of duNOD1 significantly increased the expression of TNF-α, IL-6, and RANTES in DEFs. These findings highlight the crucial role of duNOD1 as an intracellular sensor in duck innate immune system. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Cross-Neutralising Nanobodies Bind to a Conserved Pocket in the Hemagglutinin Stem Region Identified Using Yeast Display and Deep Mutational Scanning.

    Directory of Open Access Journals (Sweden)

    Tiziano Gaiotto

    Full Text Available Cross-neutralising monoclonal antibodies against influenza hemagglutinin (HA are of considerable interest as both therapeutics and diagnostic tools. We have recently described five different single domain antibodies (nanobodies which share this cross-neutralising activity and suggest their small size, high stability, and cleft binding properties may present distinct advantages over equivalent conventional antibodies. We have used yeast display in combination with deep mutational scanning to give residue level resolution of positions in the antibody-HA interface which are crucial for binding. In addition, we have mapped positions within HA predicted to have minimal effect on antibody binding when mutated. Our cross-neutralising nanobodies were shown to bind to a highly conserved pocket in the HA2 domain of A(H1N1pdm09 influenza virus overlapping with the fusion peptide suggesting their mechanism of action is through the inhibition of viral membrane fusion. We also note that the epitope overlaps with that of CR6261 and F10 which are human monoclonal antibodies in clinical development as immunotherapeutics. Although all five nanobodies mapped to the same highly conserved binding pocket we observed differences in the size of the epitope footprint which has implications in comparing the relative genetic barrier each nanobody presents to a rapidly evolving influenza virus. To further refine our epitope map, we have re-created naturally occurring mutations within this HA stem epitope and tested their effect on binding using yeast display. We have shown that a D46N mutation in the HA2 stem domain uniquely interferes with binding of R2b-E8. Further testing of this substitution in the context of full length purified HA from 1918 H1N1 pandemic (Spanish flu, 2009 H1N1 pandemic (swine flu and highly pathogenic avian influenza H5N1 demonstrated binding which correlated with D46 whereas binding to seasonal H1N1 strains carrying N46 was absent. In addition, our

  3. Structural and mechanistic investigations on Salmonella typhimurium acetate kinase (AckA: identification of a putative ligand binding pocket at the dimeric interface

    Directory of Open Access Journals (Sweden)

    Chittori Sagar

    2012-10-01

    Full Text Available Abstract Background Bacteria such as Escherichia coli and Salmonella typhimurium can utilize acetate as the sole source of carbon and energy. Acetate kinase (AckA and phosphotransacetylase (Pta, key enzymes of acetate utilization pathway, regulate flux of metabolites in glycolysis, gluconeogenesis, TCA cycle, glyoxylate bypass and fatty acid metabolism. Results Here we report kinetic characterization of S. typhimurium AckA (StAckA and structures of its unliganded (Form-I, 2.70 Å resolution and citrate-bound (Form-II, 1.90 Å resolution forms. The enzyme showed broad substrate specificity with kcat/Km in the order of acetate > propionate > formate. Further, the Km for acetyl-phosphate was significantly lower than for acetate and the enzyme could catalyze the reverse reaction (i.e. ATP synthesis more efficiently. ATP and Mg2+ could be substituted by other nucleoside 5′-triphosphates (GTP, UTP and CTP and divalent cations (Mn2+ and Co2+, respectively. Form-I StAckA represents the first structural report of an unliganded AckA. StAckA protomer consists of two domains with characteristic βββαβαβα topology of ASKHA superfamily of proteins. These domains adopt an intermediate conformation compared to that of open and closed forms of ligand-bound Methanosarcina thermophila AckA (MtAckA. Spectroscopic and structural analyses of StAckA further suggested occurrence of inter-domain motion upon ligand-binding. Unexpectedly, Form-II StAckA structure showed a drastic change in the conformation of residues 230–300 compared to that of Form-I. Further investigation revealed electron density corresponding to a citrate molecule in a pocket located at the dimeric interface of Form-II StAckA. Interestingly, a similar dimeric interface pocket lined with largely conserved residues could be identified in Form-I StAckA as well as in other enzymes homologous to AckA suggesting that ligand binding at this pocket may influence the function of these

  4. The importance of highly conserved nucleotides in the binding region of chloramphenicol at the peptidyl transfer centre of Escherichia coli 23S ribosomal RNA

    DEFF Research Database (Denmark)

    Vester, Birte; Garrett, Roger Antony

    1988-01-01

    conserved nucleotides. With a view to investigate the function of this RNA region further, four of these conserved nucleotides, including one indirectly implicated in chloramphenicol binding, were selected for mutation in Escherichia coli 23S rRNA using oligonucleotide primers. Mutant RNAs were expressed...... of highly conserved nucleotides in the chloramphenicol binding region. A mechanistic model is also presented to explain the disruptive effect of chloramphenicol (and other antibiotics) on peptide bond formation at the ribosomal subunit interface....

  5. Redox-linked Gating of Nucleotide Binding by the N-terminal Domain of Adenosine 5′-Phosphosulfate Kinase*

    Science.gov (United States)

    Ravilious, Geoffrey E.; Westfall, Corey S.; Jez, Joseph M.

    2013-01-01

    Adenosine 5′-phosphosulfate kinase (APSK) catalyzes the phosphorylation of adenosine 5′-phosphosulfate (APS) to 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Crystallographic studies of APSK from Arabidopsis thaliana revealed the presence of a regulatory intersubunit disulfide bond (Cys86–Cys119). The reduced enzyme displayed improved catalytic efficiency and decreased effectiveness of substrate inhibition by APS compared with the oxidized form. Here we examine the effect of disulfide formation and the role of the N-terminal domain on nucleotide binding using isothermal titration calorimetry (ITC) and steady-state kinetics. Formation of the disulfide bond in A. thaliana APSK (AtAPSK) inverts the binding affinities at the ATP/ADP and APS/PAPS sites from those observed in the reduced enzyme, consistent with initial binding of APS as inhibitory, and suggests a role for the N-terminal domain in guiding nucleotide binding order. To test this, an N-terminal truncation variant (AtAPSKΔ96) was generated. The resulting protein was completely insensitive to substrate inhibition by APS. ITC analysis of AtAPSKΔ96 showed decreased affinity for APS binding, although the N-terminal domain does not directly interact with this ligand. Moreover, AtAPSKΔ96 displayed reduced affinity for ADP, which corresponds to a loss of substrate inhibition by formation of an E·ADP·APS dead end complex. Examination of the AtAPSK crystal structure suggested Arg93 as important for positioning of the N-terminal domain. ITC and kinetic analysis of the R93A mutant also showed a complete loss of substrate inhibition and altered nucleotide binding affinities, which mimics the effect of the N-terminal deletion. These results show how thiol-linked changes in AtAPSK alter the energetics of binding equilibria to control its activity. PMID:23322773

  6. Fluoroketone inhibition of Ca(2+)-independent phospholipase A2 through binding pocket association defined by hydrogen/deuterium exchange and molecular dynamics.

    Science.gov (United States)

    Hsu, Yuan-Hao; Bucher, Denis; Cao, Jian; Li, Sheng; Yang, Sheng-Wei; Kokotos, George; Woods, Virgil L; McCammon, J Andrew; Dennis, Edward A

    2013-01-30

    The mechanism of inhibition of group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)) by fluoroketone (FK) ligands is examined by a combination of deuterium exchange mass spectrometry (DXMS) and molecular dynamics (MD). Models for iPLA(2) were built by homology with the known structure of patatin and equilibrated by extensive MD simulations. Empty pockets were identified during the simulations and studied for their ability to accommodate FK inhibitors. Ligand docking techniques showed that the potent inhibitor 1,1,1,3-tetrafluoro-7-phenylheptan-2-one (PHFK) forms favorable interactions inside an active-site pocket, where it blocks the entrance of phospholipid substrates. The polar fluoroketone headgroup is stabilized by hydrogen bonds with residues Gly486, Gly487, and Ser519. The nonpolar aliphatic chain and aromatic group are stabilized by hydrophobic contacts with Met544, Val548, Phe549, Leu560, and Ala640. The binding mode is supported by DXMS experiments showing an important decrease of deuteration in the contact regions in the presence of the inhibitor. The discovery of the precise binding mode of FK ligands to the iPLA(2) should greatly improve our ability to design new inhibitors with higher potency and selectivity.

  7. An alanine residue in the M3-M4 linker lines the glycine binding pocket of the N-methyl-D-aspartate receptor.

    Science.gov (United States)

    Wood, M W; VanDongen, H M; VanDongen, A M

    1997-02-07

    While attempting to map a central region in the M3-M4 linker of the N-methyl-D-aspartate receptor NR1 subunit, we found that mutation of a single position, Ala-714, greatly reduced the apparent affinity for glycine. Proximal N-glycosylation localized this region to the extracellular space. Glycine affinities of additional Ala-714 mutations correlated with side chain volume. Substitution of alanine 714 with cysteine did not alter glycine sensitivity, although this mutant was rapidly inhibited by dithionitrobenzoate. Glycine protected the A714C mutant from modification by dithionitrobenzoate, whereas the co-agonist L-glutamate was ineffective. These experiments place Ala-714 in the glycine binding pocket of the N-methyl-D-aspartate receptor, a determination not predicted by previous structural models based on bacterial periplasmic binding protein homology.

  8. Nucleoporin NUP153 phenylalanine-glycine motifs engage a common binding pocket within the HIV-1 capsid protein to mediate lentiviral infectivity.

    Directory of Open Access Journals (Sweden)

    Kenneth A Matreyek

    Full Text Available Lentiviruses can infect non-dividing cells, and various cellular transport proteins provide crucial functions for lentiviral nuclear entry and integration. We previously showed that the viral capsid (CA protein mediated the dependency on cellular nucleoporin (NUP 153 during HIV-1 infection, and now demonstrate a direct interaction between the CA N-terminal domain and the phenylalanine-glycine (FG-repeat enriched NUP153 C-terminal domain (NUP153(C. NUP153(C fused to the effector domains of the rhesus Trim5α restriction factor (Trim-NUP153(C potently restricted HIV-1, providing an intracellular readout for the NUP153(C-CA interaction during retroviral infection. Primate lentiviruses and equine infectious anemia virus (EIAV bound NUP153(C under these conditions, results that correlated with direct binding between purified proteins in vitro. These binding phenotypes moreover correlated with the requirement for endogenous NUP153 protein during virus infection. Mutagenesis experiments concordantly identified NUP153(C and CA residues important for binding and lentiviral infectivity. Different FG motifs within NUP153(C mediated binding to HIV-1 versus EIAV capsids. HIV-1 CA binding mapped to residues that line the common alpha helix 3/4 hydrophobic pocket that also mediates binding to the small molecule PF-3450074 (PF74 inhibitor and cleavage and polyadenylation specific factor 6 (CPSF6 protein, with Asn57 (Asp58 in EIAV playing a particularly important role. PF74 and CPSF6 accordingly each competed with NUP153(C for binding to the HIV-1 CA pocket, and significantly higher concentrations of PF74 were needed to inhibit HIV-1 infection in the face of Trim-NUP153(C expression or NUP153 knockdown. Correlation between CA mutant viral cell cycle and NUP153 dependencies moreover indicates that the NUP153(C-CA interaction underlies the ability of HIV-1 to infect non-dividing cells. Our results highlight similar mechanisms of binding for disparate host factors

  9. Decipher the mechanisms of protein conformational changes induced by nucleotide binding through free-energy landscape analysis: ATP binding to Hsp70.

    Directory of Open Access Journals (Sweden)

    Adrien Nicolaï

    Full Text Available ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD of Hsp70 propagates a signal to its substrate-binding domain (SBD. Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in

  10. Immunolocalization of Upstream binding factor and pocket protein p130 during final stages of bovine oocyte growth

    Czech Academy of Sciences Publication Activity Database

    Baran, Vladimír; Pavlok, Antonín; Bjerregaard, B.; Wrenzycki, C.; Hermann, D.; Philimonenko, V. V.; Lapathitis, Georgios; Hozák, Pavel; Niemann, H.; Motlík, Jan

    2004-01-01

    Roč. 70, č. 4 (2004), s. 877-886 ISSN 0006-3363 R&D Projects: GA MŠk ME 573; GA MŠk LN00A065; GA AV ČR IPP1050128 Grant - others:Slovenská Akademie věd(SK) Vega 2/3065/23; Evropská unie(XE) QLK3-CT1999-00104 Institutional research plan: CEZ:AV0Z5045916 Keywords : pocket protein p130 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.550, year: 2004

  11. Decoding ChIP-seq with a double-binding signal refines binding peaks to single-nucleotides and predicts cooperative interaction

    Science.gov (United States)

    Gomes, Antonio L.C.; Abeel, Thomas; Peterson, Matthew; Azizi, Elham; Lyubetskaya, Anna; Carvalho, Luís

    2014-01-01

    The comprehension of protein and DNA binding in vivo is essential to understand gene regulation. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) provides a global map of the regulatory binding network. Most ChIP-seq analysis tools focus on identifying binding regions from coverage enrichment. However, less work has been performed to infer the physical and regulatory details inside the enriched regions. This research extends a previous blind-deconvolution approach to develop a post-peak–calling algorithm that improves binding site resolution and predicts cooperative interactions. At the core of our new method is a physically motivated model that characterizes the binding signal as an extreme value distribution. This model suggests a mathematical framework to study physical properties of DNA shearing from the ChIP-seq coverage. The model explains the ChIP-seq coverage with two signals: The first considers DNA fragments with only a single binding event, whereas the second considers fragments with two binding events (a double-binding signal). The model incorporates motif discovery and is able to detect multiple sites in an enriched region with single-nucleotide resolution, high sensitivity, and high specificity. Our method improves peak caller sensitivity, from less than 45% up to 94%, at a false positive rate ChIP-seq analysis: the identification of cooperative interaction. Predictions of known cooperative binding sites show a 0.85 area under an ROC curve. PMID:25024162

  12. Microarray study of single nucleotide polymorphisms and expression of ATP-binding cassette genes in breast tumors

    Science.gov (United States)

    Tsyganov, M. M.; Ibragimova, M. K.; Karabut, I. V.; Freydin, M. B.; Choinzonov, E. L.; Litvyakov, N. V.

    2015-11-01

    Our previous research establishes that changes of expression of the ATP-binding cassette genes family is connected with the neoadjuvant chemotherapy effect. However, the mechanism of regulation of resistance gene expression remains unclear. As many researchers believe, single nucleotide polymorphisms can be involved in this process. Thereupon, microarray analysis is used to study polymorphisms in ATP-binding cassette genes. It is thus found that MDR gene expression is connected with 5 polymorphisms, i.e. rs241432, rs241429, rs241430, rs3784867, rs59409230, which participate in the regulation of expression of own genes.

  13. Graph-Theoretic Models of Mutations in the Nucleotide Binding Domain 1 of the Cystic Fibrosis Transmembrane Conductance Regulator

    Directory of Open Access Journals (Sweden)

    Debra J. Knisley

    2013-01-01

    Full Text Available Cystic fibrosis is one of the most common inherited diseases and is caused by a mutation in a membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR. This protein serves as a chloride channel and regulates the viscosity of mucus lining the ducts of a number of organs. Although much has been learned about the consequences of mutations on the energy landscape and the resulting disrupted folding pathway of CFTR, a level of understanding needed to correct the misfolding has not been achieved. The most common mutations of CFTR are located in one of two nucleotide binding domains, namely, the nucleotide binding domain 1 (NBD1. We model NBD1 using a nested graph model. The vertices in the lowest layer each represent an atom in the structure of an amino acid residue, while the vertices in the mid layer each represent the residue. The vertices in the top layer each represent a subdomain of the nucleotide binding domain. We use this model to quantify the effects of a single point mutation on the protein domain. We compare the wildtype structure with eight of the most common mutations. The graph-theoretic model provides insight into how a single point mutation can have such profound structural consequences.

  14. Binding water clusters to an aromatic-rich hydrophobic pocket: [2.2.2]paracyclophane-(H2O)n, n = 1-5.

    Science.gov (United States)

    Buchanan, Evan G; Zwier, Timothy S

    2014-09-18

    [2.2.2]Paracylcophane (tricyclophane, TCP) is a macrocycle with three phenyl substituents linked by ethyl bridges (-CH2CH2-) in the para-position, forming an aromatic-rich pocket capable of binding various substituents, including nature's solvent, water. Building on previous work [Buchanan, E. G.; et al. J. Chem. Phys. 2013, 138, 064308] that reported on the ground state conformational preferences of TCP, the focus of the present study is on the infrared and ultraviolet spectroscopy of TCP-(H2O)n clusters with n = 1-5. Resonant two-photon ionization (R2PI) was used to interrogate the mass selected electronic spectrum of the clusters, reporting on the perturbations imposed on the electronic states of TCP as the size of the water clusters bound to it vary in size from n = 1-5. The TCP-(H2O)n S0-S1 origins are shifted to lower frequency from the monomer, indicating an increased binding energy of the water or water network in the excited state. Ground state resonant ion-dip infrared (RIDIR) spectra of TCP-(H2O)n (n = 1-5) clusters were recorded in the OH stretch region, which probes the H-bonded water networks present and the perturbations imposed on them by TCP. The experimental frequencies are compared with harmonic vibrational frequencies calculated using density functional theory (DFT) with the dispersion-corrected functional ωB97X-D and a 6-311+g(d,p) basis set, providing firm assignments for their H-bonding structures. The H2O molecule in TCP-(H2O)1 sits on top of the binding pocket, donating both of its hydrogen atoms to the aromatic-rich interior of the monomer. The antisymmetric stretch fundamental of H2O in the complex is composed of a closely spaced set of transitions that likely reflect contributions from both para- and ortho-forms of H2O due to internal rotation of the H2O in the binding pocket. TCP-(H2O)2 also exists in a single conformational isomer that retains the same double-donor binding motif for the first water molecule, with the second H2O acting

  15. Novel drug design for Chagas disease via targeting Trypanosoma cruzi tubulin: Homology modeling and binding pocket prediction on Trypanosoma cruzi tubulin polymerization inhibition by naphthoquinone derivatives.

    Science.gov (United States)

    Ogindo, Charles O; Khraiwesh, Mozna H; George, Matthew; Brandy, Yakini; Brandy, Nailah; Gugssa, Ayele; Ashraf, Mohammad; Abbas, Muneer; Southerland, William M; Lee, Clarence M; Bakare, Oladapo; Fang, Yayin

    2016-08-15

    Chagas disease, also called American trypanosomiasis, is a parasitic disease caused by Trypanosoma cruzi (T. cruzi). Recent findings have underscored the abundance of the causative organism, (T. cruzi), especially in the southern tier states of the US and the risk burden for the rural farming communities there. Due to a lack of safe and effective drugs, there is an urgent need for novel therapeutic options for treating Chagas disease. We report here our first scientific effort to pursue a novel drug design for treating Chagas disease via the targeting of T. cruzi tubulin. First, the anti T. cruzi tubulin activities of five naphthoquinone derivatives were determined and correlated to their anti-trypanosomal activities. The correlation between the ligand activities against the T. cruzi organism and their tubulin inhibitory activities was very strong with a Pearson's r value of 0.88 (P value cruzi tubulin polymerization inhibition. Subsequent molecular modeling studies were carried out to understand the mechanisms of the anti-tubulin activities, wherein, the homology model of T. cruzi tubulin dimer was generated and the putative binding site of naphthoquinone derivatives was predicted. The correlation coefficient for ligand anti-tubulin activities and their binding energies at the putative pocket was found to be r=0.79, a high correlation efficiency that was not replicated in contiguous candidate pockets. The homology model of T. cruzi tubulin and the identification of its putative binding site lay a solid ground for further structure based drug design, including molecular docking and pharmacophore analysis. This study presents a new opportunity for designing potent and selective drugs for Chagas disease. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. Functional Mutations Form at CTCF-Cohesin Binding Sites in Melanoma Due to Uneven Nucleotide Excision Repair across the Motif.

    Science.gov (United States)

    Poulos, Rebecca C; Thoms, Julie A I; Guan, Yi Fang; Unnikrishnan, Ashwin; Pimanda, John E; Wong, Jason W H

    2016-12-13

    CTCF binding sites are frequently mutated in cancer, but how these mutations accumulate and whether they broadly perturb CTCF binding are not well understood. Here, we report that skin cancers exhibit a highly specific asymmetric mutation pattern within CTCF motifs attributable to ultraviolet irradiation and differential nucleotide excision repair (NER). CTCF binding site mutations form independently of replication timing and are enriched at sites of CTCF/cohesin complex binding, suggesting a role for cohesin in stabilizing CTCF-DNA binding and impairing NER. Performing CTCF ChIP-seq in a melanoma cell line, we show CTCF binding site mutations to be functional by demonstrating allele-specific reduction of CTCF binding to mutant alleles. While topologically associating domains with mutated CTCF anchors in melanoma contain differentially expressed cancer-associated genes, CTCF motif mutations appear generally under neutral selection. However, the frequency and potential functional impact of such mutations in melanoma highlights the need to consider their impact on cellular phenotype in individual genomes. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  17. Molecular dynamics and binding selectivity of nucleotides and polynucleotide substrates with EIF2C2/Ago2 PAZ domain.

    Science.gov (United States)

    Kandeel, Mahmoud; Kitade, Yukio

    2018-02-01

    RNA interference (RNAi) constitutes a major target in drug discovery. Recently, we reported that the Argonaute protein 2 (Ago2) PAZ domain selectively binds with all ribonucleotides except adenine and poorly recognizes deoxyribonucleotides. The binding properties of the PAZ domain with polynucleotides and the molecular mechanisms of substrates' selectivity remains unclear. In this study, the binding potencies of polynucleotides and the associated conformational and dynamic changes in PAZ domain are investigated. Coinciding with nucleotides' binding profile with the PAZ domain, polyuridylate (PolyU) and polycytidylate (PolyC) were potent binders. However, K dPolyU and K dPolyC were 15.8 and 9.3μM, respectively. In contrast, polyadenylate (PolyA) binding was not detectable. Molecular dynamics (MD) simulation revealed the highest change in root mean square deviation (RMSD) with ApoPAZ or PAZ domain bound with experimentally approved, low affinity substrates, whereas stronger binding substrates such as UMP or PolyU showed minimal RMSD changes. The loop between α3 and β5 in the β-hairpin subdomain showed the most responsive change in RMSD, being highly movable in the ApoPAZ and PAZ-AMP complex. Favorable substrate recognition was associate with moderate change in secondary structure content. In conclusion, the PAZ domain retains differential substrate selectivity associated with corresponding dynamic and structural changes upon binding. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Auxin-binding pocket of ABP1 is crucial for its gain-of-function cellular and developmental roles

    Czech Academy of Sciences Publication Activity Database

    Grones, P.; Chen, X.; Simon, S.; Kaufmann, W.A.; De Rycke, R.; Nodzyński, T.; Zažímalová, Eva; Friml, J.

    2015-01-01

    Roč. 66, č. 16 (2015), s. 5055-5065 ISSN 0022-0957 Institutional support: RVO:61389030 Keywords : Auxin * ABP1 * Auxin binding Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.677, year: 2015

  19. The importance of highly conserved nucleotides in the binding region of chloramphenicol at the peptidyl transfer centre of Escherichia coli 23S ribosomal RNA

    DEFF Research Database (Denmark)

    Vester, Birte; Garrett, Roger Antony

    1988-01-01

    conserved nucleotides. With a view to investigate the function of this RNA region further, four of these conserved nucleotides, including one indirectly implicated in chloramphenicol binding, were selected for mutation in Escherichia coli 23S rRNA using oligonucleotide primers. Mutant RNAs were expressed...

  20. Importance of the alphaC-helix in the cyclic nucleotide binding domain for the stable channel regulation and function of cyclic nucleotide gated ion channels in Arabidopsis.

    Science.gov (United States)

    Chin, Kimberley; Moeder, Wolfgang; Abdel-Hamid, Huda; Shahinas, Dea; Gupta, Deepali; Yoshioka, Keiko

    2010-05-01

    The involvement of cyclic nucleotide gated ion channels (CNGCs) in the signal transduction of animal light and odorant perception is well documented. Although plant CNGCs have recently been revealed to mediate multiple stress responses and developmental pathways, studies that aim to elucidate their structural and regulatory properties are still very much in their infancy. The structure-function relationship of plant CNGCs was investigated here by using the chimeric Arabidopsis AtCNGC11/12 gene that induces multiple defence responses in the Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22) for the identification of functionally essential residues. A genetic screen for mutants that suppress cpr22-conferred phenotypes identified over 20 novel mutant alleles in AtCNGC11/12. One of these mutants, suppressor S58 possesses a single amino acid substitution, arginine 557 to cysteine, in the alphaC-helix of the cyclic nucleotide-binding domain (CNBD). The suppressor S58 lost all cpr22 related phenotypes, such as spontaneous cell death formation under ambient temperature conditions. However, these phenotypes were recovered at 16 degrees C suggesting that the stability of channel function is affected by temperature. In silico modelling and site-directed mutagenesis analyses suggest that arginine 557 in the alphaC-helix of the CNBD is important for channel regulation, but not for basic function. Furthermore, another suppressor mutant, S136 that lacks the entire alphaC-helix due to a premature stop codon, lost channel function completely. Our data presented here indicate that the alphaC-helix is functionally important in plant CNGCs.

  1. Two types of antibodies are induced by vaccination with A/California/2009 pdm virus: binding near the sialic acid-binding pocket and neutralizing both H1N1 and H5N1 viruses.

    Directory of Open Access Journals (Sweden)

    Nobuko Ohshima

    Full Text Available Many people have a history of catching the flu several times during childhood but no additional flu in adulthood, even without vaccination. We analyzed the total repertoire of antibodies (Abs against influenza A group 1 viruses induced in such a flu-resistant person after vaccination with 2009 H1N1 pandemic influenza virus. They were classified into two types, with no exceptions. The first type, the products of B cells newly induced through vaccination, binds near the sialic acid-binding pocket. The second type, the products of long-lived memory B cells established before vaccination, utilizes the 1-69 VH gene, binds to the stem of HA, and neutralizes both H1N1 and H5N1 viruses with few exceptions. These observations indicate that the sialic acid-binding pocket and its surrounding region are immunogenically very potent and majority of the B cells whose growth is newly induced by vaccination produce Abs that recognize these regions. However, they play a role in protection against influenza virus infection for a short period since variant viruses that have acquired resistance to these Abs become dominant. On the other hand, although the stem of HA is immunogenically not potent, the second type of B cells eventually becomes dominant. Thus, a selection system should function in forming the repertoire of long-lived memory B cells and the stability of the epitope would greatly affect the fate of the memory cells. Acquisition of the ability to produce Abs that bind to the stable epitope could be a major factor of flu resistance.

  2. Two types of antibodies are induced by vaccination with A/California/2009 pdm virus: binding near the sialic acid-binding pocket and neutralizing both H1N1 and H5N1 viruses.

    Science.gov (United States)

    Ohshima, Nobuko; Kubota-Koketsu, Ritsuko; Iba, Yoshitaka; Okuno, Yoshinobu; Kurosawa, Yoshikazu

    2014-01-01

    Many people have a history of catching the flu several times during childhood but no additional flu in adulthood, even without vaccination. We analyzed the total repertoire of antibodies (Abs) against influenza A group 1 viruses induced in such a flu-resistant person after vaccination with 2009 H1N1 pandemic influenza virus. They were classified into two types, with no exceptions. The first type, the products of B cells newly induced through vaccination, binds near the sialic acid-binding pocket. The second type, the products of long-lived memory B cells established before vaccination, utilizes the 1-69 VH gene, binds to the stem of HA, and neutralizes both H1N1 and H5N1 viruses with few exceptions. These observations indicate that the sialic acid-binding pocket and its surrounding region are immunogenically very potent and majority of the B cells whose growth is newly induced by vaccination produce Abs that recognize these regions. However, they play a role in protection against influenza virus infection for a short period since variant viruses that have acquired resistance to these Abs become dominant. On the other hand, although the stem of HA is immunogenically not potent, the second type of B cells eventually becomes dominant. Thus, a selection system should function in forming the repertoire of long-lived memory B cells and the stability of the epitope would greatly affect the fate of the memory cells. Acquisition of the ability to produce Abs that bind to the stable epitope could be a major factor of flu resistance.

  3. Mutation of lysine residues in the nucleotide binding segments of the poliovirus RNA-dependent RNA polymerase.

    Science.gov (United States)

    Richards, O C; Baker, S; Ehrenfeld, E

    1996-12-01

    The poliovirus 3D RNA-dependent RNA polymerase contains two peptide segments previously shown to cross-link to nucleotide substrates via lysine residues. To determine which lysine residue(s) might be implicated in catalytic function, we engineered mutations to generate proteins with leucine residues substituted individually for each of the lysine residues in the NTP binding regions. These proteins were expressed in Escherichia coli and were examined for their abilities to bind nucleotides and to catalyze RNA chain elongation in vitro. Replacement of each lysine residue in the NTP binding segment located in the central portion of the 3D molecule (Lys-276, -278, or -283) with leucine produced no impairment of GTP binding or polymerase activity. Substitution of leucine for Lys-61 in the N-terminal portion of the protein, however, abolished the binding of protein to GTP-agarose and all detectable polymerase activity. A nearby lysine replacement with leucine at position 66 had no effect on enzyme activity. The three mutations in the central region of 3D were introduced into full-length viral cDNAs, and the infectivities of RNA transcripts were examined in transfected HeLa cells. Growth of virus containing 3D with a mutation at residue 278 (3Dmu278) or 3Dmu283 was indistinguishable from that of the wild type; however, 3Dmu276 generated extremely slow-growing, small-plaque virus. Polyprotein processing by 3CDmu276 was unaffected. Large-plaque variants, in which the Leu-276 codon had mutated again to an arginine codon, emerged at high frequency. The results suggest that a lysine residue at position 61 of 3Dpol is essential for polymerase catalytic function and that a basic (lysine or arginine) residue at position 276 is required for some other function of 3D important for virus growth but not for RNA chain elongation or polyprotein processing.

  4. Modulation of nucleotide binding to the catalytic sites of thermophilic F(1)-ATPase by the epsilon subunit: implication for the role of the epsilon subunit in ATP synthesis.

    Science.gov (United States)

    Yasuno, Taichi; Muneyuki, Eiro; Yoshida, Masasuke; Kato-Yamada, Yasuyuki

    2009-12-11

    Effect of epsilon subunit on the nucleotide binding to the catalytic sites of F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) has been tested by using alpha(3)beta(3)gamma and alpha(3)beta(3)gammaepsilon complexes of TF(1) containing betaTyr341 to Trp substitution. The nucleotide binding was assessed with fluorescence quenching of the introduced Trp. The presence of the epsilon subunit weakened ADP binding to each catalytic site, especially to the highest affinity site. This effect was also observed when GDP or IDP was used. The ratio of the affinity of the lowest to the highest nucleotide binding sites had changed two orders of magnitude by the epsilon subunit. The differences may relate to the energy required for the binding change in the ATP synthesis reaction and contribute to the efficient ATP synthesis.

  5. Asn792 participates in the hydrogen bond network around the K+-binding pocket of gastric H,K-ATPase.

    NARCIS (Netherlands)

    Swarts, H.G.P.; Koenderink, J.B.; Willems, P.H.G.M.; Krieger, E.; Pont, J.J.H.H.M. de

    2005-01-01

    Asn792 present in M5 of gastric H,K-ATPase is highly conserved within the P-type ATPase family. A direct role in K+ binding was postulated for Na,K-ATPase but was not found in a recent model for gastric H,K-ATPase (Koenderink, J. B., Swarts, H. G. P., Willems, P. H. G. M., Krieger, E., and De Pont,

  6. Guanine nucleotide binding proteins in zucchini seedlings: Characterization and interactions with the NPA receptor

    International Nuclear Information System (INIS)

    Lindeberg, M.; Jacobs, M.

    1989-01-01

    A microsomal membrane preparation from hypocotyls of dark-grown Cucurbita pepo L. seedlings contains specific high-affinity binding sites for the non-hydrolyzable GTP analog guanosine 5'-[γ-thio] triphosphate (GTP-γ-S). Both the binding affinity and the pattern of binding specificity for GTP and GTP analogs are similar to animal G-proteins, and two zucchini membrane proteins are recognized in western blots by antiserum specific for the σ subunit of platelet G s protein. GTP-γ-S can increase specific naphthylphthalamic acid (NPA) binding in zucchini microsomal membrane preparations, with its stimulation increasing with large tissue age. Al +3 and F - agents known to activate G-proteins - decreased NPA specific binding by ca. 15%. In tests of in vitro auxin transport employing zucchini plasma membrane vesicles, AlF - 4 strongly inhibited 3 H-indoleacetic acid nor accumulation; GTP-γ-S effects on this system will be discussed

  7. Ligand binding study of human PEBP1/RKIP: interaction with nucleotides and Raf-1 peptides evidenced by NMR and mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Laurette Tavel

    Full Text Available BACKGROUND: Human Phosphatidylethanolamine binding protein 1 (hPEBP1 also known as Raf kinase inhibitory protein (RKIP, affects various cellular processes, and is implicated in metastasis formation and Alzheimer's disease. Human PEBP1 has also been shown to inhibit the Raf/MEK/ERK pathway. Numerous reports concern various mammalian PEBP1 binding ligands. However, since PEBP1 proteins from many different species were investigated, drawing general conclusions regarding human PEBP1 binding properties is rather difficult. Moreover, the binding site of Raf-1 on hPEBP1 is still unknown. METHODS/FINDINGS: In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands: GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants K(D for different ligands. Native mass spectrometry was used as an alternative method for measuring K(D values. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.

  8. Three-dimensional structures of the mammalian multidrug resistance P-glycoprotein demonstrate major conformational changes in the transmembrane domains upon nucleotide binding.

    Science.gov (United States)

    Rosenberg, Mark F; Kamis, Alhaji Bukar; Callaghan, Richard; Higgins, Christopher F; Ford, Robert C

    2003-03-07

    P-glycoprotein is an ATP-binding cassette transporter that is associated with multidrug resistance and the failure of chemotherapy in human patients. We have previously shown, based on two-dimensional projection maps, that P-glycoprotein undergoes conformational changes upon binding of nucleotide to the intracellular nucleotide binding domains. Here we present the three-dimensional structures of P-glycoprotein in the presence and absence of nucleotide, at a resolution limit of approximately 2 nm, determined by electron crystallography of negatively stained crystals. The data reveal a major reorganization of the transmembrane domains throughout the entire depth of the membrane upon binding of nucleotide. In the absence of nucleotide, the two transmembrane domains form a single barrel 5-6 nm in diameter and about 5 nm deep with a central pore that is open to the extracellular surface and spans much of the membrane depth. Upon binding nucleotide, the transmembrane domains reorganize into three compact domains that are each 2-3 nm in diameter and 5-6 nm deep. This reorganization opens the central pore along its length in a manner that could allow access of hydrophobic drugs (transport substrates) directly from the lipid bilayer to the central pore of the transporter.

  9. Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket*

    Science.gov (United States)

    Tam, Ming F.; Rice, Natalie W.; Maillett, David H.; Simplaceanu, Virgil; Ho, Nancy T.; Tam, Tsuey Chyi S.; Shen, Tong-Jian; Ho, Chien

    2013-01-01

    The E11 valine in the distal heme pocket of either the α- or β-subunit of human adult hemoglobin (Hb A) was replaced by leucine, isoleucine, or phenylalanine. Recombinant proteins were expressed in Escherichia coli and purified for structural and functional studies. 1H NMR spectra were obtained for the CO and deoxy forms of Hb A and the mutants. The mutations did not disturb the α1β2 interface in either form, whereas the H-bond between αHis-103 and βGln-131 in the α1β1 interfaces of the deoxy α-subunit mutants was weakened. Localized structural changes in the mutated heme pocket were detected for the CO form of recombinant Hb (rHb) (αV62F), rHb (βV67I), and rHb (βV67F) compared with Hb A. In the deoxy form the proximal histidyl residue in the β-subunit of rHb (βV67F) has been altered. Furthermore, the interactions between the porphyrin ring and heme pocket residues have been perturbed in rHb (αV62I), rHb (αV62F), and rHb (βV67F). Functionally, the oxygen binding affinity (P50), cooperativity (n50), and the alkaline Bohr Effect of the three α-subunit mutants and rHb (βV67L) are similar to those of Hb A. rHb (βV67I) and rHb (βV67F) exhibit low and high oxygen affinity, respectively. rHb (βV67F) has P50 values lower that those reported for rHb (αL29F), a B10 mutant studied previously in our laboratory (Wiltrout, M. E., Giovannelli, J. L., Simplaceanu, V., Lukin, J. A., Ho, N. T., and Ho, C. (2005) Biochemistry 44, 7207–7217). These E11 mutations do not slow down the autoxidation and azide-induced oxidation rates of the recombinant proteins. Results from this study provide new insights into the roles of E11 mutants in the structure-function relationship in hemoglobin. PMID:23867463

  10. Thermodynamics of the ATPase cycle of GlcV, the nucleotide-binding domain of the glucose ABC transporter of Sulfolobus solfataricus

    NARCIS (Netherlands)

    Pretz, Monika G.; Albers, Sonja-Verena; Schuurman-Wolters, Gea; Tampe, Robert; Driessen, Arnold J. M.; van der Does, Chris

    2006-01-01

    ATP-binding cassette transporters drive the transport of substrates across the membrane by the hydrolysis of ATP. They typically have a conserved domain structure with two membrane-spanning domains that form the transport channel and two cytosolic nucleotide-binding domains ( NBDs) that energize the

  11. Investigating the Importance of the Pocket-estimation Method in Pocket-based Approaches: An Illustration Using Pocket-ligand Classification.

    Science.gov (United States)

    Caumes, Géraldine; Borrel, Alexandre; Abi Hussein, Hiba; Camproux, Anne-Claude; Regad, Leslie

    2017-09-01

    Small molecules interact with their protein target on surface cavities known as binding pockets. Pocket-based approaches are very useful in all of the phases of drug design. Their first step is estimating the binding pocket based on protein structure. The available pocket-estimation methods produce different pockets for the same target. The aim of this work is to investigate the effects of different pocket-estimation methods on the results of pocket-based approaches. We focused on the effect of three pocket-estimation methods on a pocket-ligand (PL) classification. This pocket-based approach is useful for understanding the correspondence between the pocket and ligand spaces and to develop pharmacological profiling models. We found pocket-estimation methods yield different binding pockets in terms of boundaries and properties. These differences are responsible for the variation in the PL classification results that can have an impact on the detected correspondence between pocket and ligand profiles. Thus, we highlighted the importance of the pocket-estimation method choice in pocket-based approaches. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Biochemical and structural characterization of Penicillium purpurogenum α-D galactosidase: Binding of galactose to an alternative pocket may explain enzyme inhibition.

    Science.gov (United States)

    Morales-Quintana, Luis; Faúndez, Carolina; Herrera, Raúl; Zavaleta, Vasni; Ravanal, María Cristina; Eyzaguirre, Jaime; Moya-León, María Alejandra

    2017-08-07

    The fungus Penicillium purpurogenum degrades plant cell walls by the action of cellulolytic, xylanolytic and pectinolytic enzymes. The α-D-galactosidase is one of the enzymes which may act on pectin degradation. This enzyme has several biotechnological and medical applications. The aim of this work was to better understand the molecular mechanism of α-D-galactosidase from P. purpurogenum (GALP1). For this purpose, a gene coding for the enzyme was identified from the fungal genome and heterologously expressed in Pichia pastoris. The enzyme belongs to glycosyl hydrolase family 27. The protein of 435 amino acids has an optimum pH and temperature for activity of 5.0 and 50 °C, respectively. The K M for p-nitrophenyl-α-D-galactopyranoside (GalαpNP) is 0.138 mM. The enzyme is inhibited by GalαpNP at concentrations higher than 1 mM, and by the product galactose. A kinetic analysis of product inhibition shows that it is of mixed type, suggesting the presence of an additional binding site in the enzyme. To confirm this hypothesis, a structural model for GALP1 was built by comparative modelling methodology, which was validated and refined by molecular dynamics simulation. The data suggest that galactose may bind to an enzyme alternative pocket promoting structural changes of the active site, thus explaining its inhibitory effect. In silico site-directed mutagenesis experiments highlighted key residues involved in the maintenance of the alternative binding site, and their mutations for Ala predict the formation of proteins which should not be inhibited by galactose. The availability of an α-galactosidase with different kinetic properties to the existent proteins may be of interest for biotechnological applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Probing the binding pocket of the active site of aromatase with 2-phenylaliphatic androsta-1,4-diene-3,17-dione steroids.

    Science.gov (United States)

    Takahashi, Madoka; Yamashita, Kouwa; Numazawa, Mitsuteru

    2010-04-01

    A series of 2-phenylaliphatic-substituted androsta-1,4-diene-3,17-diones (6) as well as their androstenedione derivatives (5) were synthesized as aromatase inhibitors to gain insights of structure-activity relationships of varying the alkyl moiety (C(1) to C(4)) of the 2-phenylaliphatic substituents as well as introducing a methyl- or trifluoromethyl function to p-position of a phenethyl moiety to the inhibitory activity. The inhibitors examined showed a competitive type inhibition. The 2-phenpropylandrosta-1,4-diene 6c was the most powerful inhibitor (K(i): 16.1nM) among them. Compounds 6c along with the phenethyl derivative 6b caused a time-dependent inactivation of aromatase (k(inact): 0.0293 and 0.0454min(-1) for 6b and 6c, respectively). The inactivation was prevented by the substrate androstenedione, and no significant effect of l-cysteine on the inactivation was observed in each case. Molecular docking of the phenpropyl compound 6c to aromatase was conducted to demonstrate that the phenpropyl group orients to a hydrophobic binding pocket in the active site to result in the formation of thermodynamically stable enzyme-inhibitor complex.

  14. Design, synthesis and anti-HIV evaluation of novel diarylnicotinamide derivatives (DANAs) targeting the entrance channel of the NNRTI binding pocket through structure-guided molecular hybridization.

    Science.gov (United States)

    Liu, Zhaoqiang; Chen, Wenmin; Zhan, Peng; De Clercq, Erik; Pannecouque, Christophe; Liu, Xinyong

    2014-11-24

    Through a structure-based molecular hybridization approach, a novel series of diarylnicotinamide derivatives (DANAs) targeting the entrance channel of HIV-1 NNRTIs binding pocket (NNIBP) were rationally designed, synthesized and evaluated for their anti-HIV activities in MT-4 cells together with the inhibition against the reverse transcriptase (RT) in an enzymatic assay. Encouragingly, most of the new DANAs were found to be active against wild-type HIV-1 with an EC50 in the range of 0.027-4.54 μM. Among them, compound 6b11 (EC50 = 0.027 μM, SI > 12518) and 6b5 (EC50 = 0.029 μM, SI = 2471) were identified as the most potent inhibitors, which were more potent than the reference drugs nevirapine (EC50 = 0.31 μM) and delavirdine (EC50 = 0.66 μM). Some DANAs were also active at micromolar concentrations against the K103N + Y181C resistant mutant. Compound 6b11 exhibited the highest enzymatic inhibition activity (IC50 = 20 nM), which is equal to that of efavirenz (EC50 = 20 nM) and 31 times higher than that of nevirapine (EC50 = 0.62 μM). Preliminary structure-activity relationships (SARs) and molecular modeling of these new DANAs have been discussed. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  15. Active-site modification of mammalian DNA polymerase β with pyridoxal 5'-phosphate: Mechanism of inhibition and identification of lysine 71 in the deoxynucleoside triphosphate binding pocket

    International Nuclear Information System (INIS)

    Basu, A.; Kedar, P.; Wilson, S.H.; Modak, M.J.

    1989-01-01

    Pyridoxal 5'-phosphate is a potent inhibitor of the DNA polymerase activity of recombinant rat DNA polymerase β. Kinetic studies indicate that the mechanism of PLP inhibition is complex. In a lower range of PLP concentration, inhibition is competitive with respect to substrate dNTP, whereas at higher levels of PLP several forms of enzyme combine with PLP and are involved in the overall inhibition, and a possible model for these interactions during the catalytic process is suggested. Reduction of the PLP-treated enzyme with sodium [ 3 H]borohydride results in covalent incorporation of about 4 mol of PLP/mol of enzyme, and the modified enzyme is not capable of DNA polymerase activity. The presence of dNTP during the modification reaction blocks incorporation of 1 mol of PLP/mol of enzyme, and the enzyme so modified is almost fully active. This protective effect is not observed in the absence of template-primer. Tryptic peptide mapping of the PLP-modified enzyme reveals four major sites of modification. Of these four sites, only one is protected by dNTP from pyridoxylation. Sequence analysis of the tryptic peptide corresponding to the protected site reveals that it spans residues 68-80 in the amino acid sequence of the enzyme, with Lys 71 as the site of pyridoxylation. These results indicate that Lys 71 is at or near the binding pocket for the dNTP substrate

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

    Science.gov (United States)

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

    2009-08-01

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

  17. Identification of MDP (muramyl dipeptide)-binding key domains in NOD2 (nucleotide-binding and oligomerization domain-2) receptor of Labeo rohita.

    Science.gov (United States)

    Maharana, Jitendra; Swain, Banikalyan; Sahoo, Bikash R; Dikhit, Manas R; Basu, Madhubanti; Mahapatra, Abhijit S; Jayasankar, Pallipuram; Samanta, Mrinal

    2013-08-01

    In lower eukaryotes-like fish, innate immunity contributed by various pattern recognition receptor (PRR) plays an essential role in protection against diseases. Nucleotide-binding and oligomerization domain (NOD)-2 is a cytoplasmic PRR that recognizes MDP (muramyl dipeptide) of the Gram positive and Gram negative bacteria as ligand and activates signalling to induce innate immunity. Hypothesizing a similar NOD2 signalling pathway of higher eukaryotes, the peripheral blood leucocytes (PBLs) of rohu (Labeo rohita) was stimulated with MDP. The data of quantitative real-time PCR (qRT-PCR) revealed MDP-mediated inductive expression of NOD2 and its down-stream molecule RICK/RIP2 (receptor-interacting serine-threonine protein kinase-2). This observation suggested the existence of MDP-binding sites in rohu NOD2 (rNOD2). To investigate it, 3D model of ligand-binding leucine-rich repeat (LRR) region of rNOD2 (rNOD2-LRR) was constructed following ab initio and threading approaches in I-TASSER web server. Structural refinement of the model was performed by energy minimization, and MD (molecular dynamics) simulation was performed in GROMACS (Groningen Machine for Chemical Simulations). The refined model of rNOD2-LRR was validated through SAVES, ProSA, ProQ, WHAT IF and MolProbity servers, and molecular docking with MDP was carried out in GOLD 4.1. The result of docking identified LRR3-7 comprising Lys820, Phe821, Asn822, Arg847, Gly849, Trp877, Trp901 and Trp931 as MDP-binding critical amino acids in rNOD2. This is the first study in fish to provide an insight into the 3D structure of NOD2-LRR region and its important motifs that are expected to be engaged in MDP binding and innate immunity.

  18. Polymorphisms in miRNA binding sites of nucleotide excision repair genes and colorectal cancer risk

    Czech Academy of Sciences Publication Activity Database

    Naccarati, Alessio; Pardini, Barbara; Landi, S.; Landi, D.; Slyšková, Jana; Novotný, J.; Levý, M.; Poláková, Veronika; Lipská, L.; Vodička, Pavel

    2012-01-01

    Roč. 33, č. 7 (2012), s. 1346-1351 ISSN 0143-3334 R&D Projects: GA ČR GAP304/10/1286; GA ČR GP305/09/P194 Institutional research plan: CEZ:AV0Z50390703 Keywords : DNA repair * polymorphisms * miRNA binding sites Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.635, year: 2012

  19. Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins

    International Nuclear Information System (INIS)

    Negishi, M.; Ito, S.; Yokohama, H.; Hayashi, H.; Katada, T.; Ui, M.; Hayaishi, O.

    1988-01-01

    Prostaglandin E 2 (PEG 2 ) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet. In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, the authors purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its α subunit from two known pertussis toxin substrate G-proteins (G/sub i/ and G 0 ) purified from bovine brain. The molecular weight of the α subunit was 40,000, which is between those of G/sub i/ and G 0 . The purified protein was also distinguished immunologically from G/sub i/ and G 0 and was referred to as G/sub am/. Reconstitution of the PGE receptor with pure C/sub am/, G/sub i/, or G 0 in phospholipid vesicles resulted in a remarkable restoration of [ 3 H]PGE 2 binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. These results indicate that the PGE receptor can couple functionally with G/sub am/, G/sub i/, or G 0 in phospholipid vesicles and suggest that G/sub am/ may be involved in signal transduction of the PGE receptor in bovine adrenal medulla

  20. Novel Nucleotide Variations, Haplotypes Structure and Associations with Growth Related Traits of Goat AT Motif-Binding Factor ( Gene

    Directory of Open Access Journals (Sweden)

    Xiaoyan Zhang

    2015-10-01

    Full Text Available The AT motif-binding factor (ATBF1 not only interacts with protein inhibitor of activated signal transducer and activator of transcription 3 (STAT3 (PIAS3 to suppress STAT3 signaling regulating embryo early development and cell differentiation, but is required for early activation of the pituitary specific transcription factor 1 (Pit1 gene (also known as POU1F1 critically affecting mammalian growth and development. The goal of this study was to detect novel nucleotide variations and haplotypes structure of the ATBF1 gene, as well as to test their associations with growth-related traits in goats. Herein, a total of seven novel single nucleotide polymorphisms (SNPs (SNP 1-7 within this gene were found in two well-known Chinese native goat breeds. Haplotypes structure analysis demonstrated that there were four haplotypes in Hainan black goat while seventeen haplotypes in Xinong Saanen dairy goat, and both breeds only shared one haplotype (hap1. Association testing revealed that the SNP2, SNP5, SNP6, and SNP7 loci were also found to significantly associate with growth-related traits in goats, respectively. Moreover, one diplotype in Xinong Saanen dairy goats significantly linked to growth related traits. These preliminary findings not only would extend the spectrum of genetic variations of the goat ATBF1 gene, but also would contribute to implementing marker-assisted selection in genetics and breeding in goats.

  1. Homology modeling, molecular docking and DNA binding studies of nucleotide excision repair UvrC protein from M. tuberculosis.

    Science.gov (United States)

    Parulekar, Rishikesh S; Barage, Sagar H; Jalkute, Chidambar B; Dhanavade, Maruti J; Fandilolu, Prayagraj M; Sonawane, Kailas D

    2013-08-01

    Mycobacterium tuberculosis is a Gram positive, acid-fast bacteria belonging to genus Mycobacterium, is the leading causative agent of most cases of tuberculosis. The pathogenicity of the bacteria is enhanced by its developed DNA repair mechanism which consists of machineries such as nucleotide excision repair. Nucleotide excision repair consists of excinuclease protein UvrABC endonuclease, multi-enzymatic complex which carries out repair of damaged DNA in sequential manner. UvrC protein is a part of this complex and thus helps to repair the damaged DNA of M. tuberculosis. Hence, structural bioinformatics study of UvrC protein from M. tuberculosis was carried out using homology modeling and molecular docking techniques. Assessment of the reliability of the homology model was carried out by predicting its secondary structure along with its model validation. The predicted structure was docked with the ATP and the interacting amino acid residues of UvrC protein with the ATP were found to be TRP539, PHE89, GLU536, ILE402 and ARG575. The binding of UvrC protein with the DNA showed two different domains. The residues from domain I of the protein VAL526, THR524 and LEU521 interact with the DNA whereas, amino acids interacting from the domain II of the UvrC protein included ARG597, GLU595, GLY594 and GLY592 residues. This predicted model could be useful to design new inhibitors of UvrC enzyme to prevent pathogenesis of Mycobacterium and so the tuberculosis.

  2. The Potato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor Rx1 Is a Pathogen-dependent DNA-deforming Protein.

    Science.gov (United States)

    Fenyk, Stepan; Townsend, Philip D; Dixon, Christopher H; Spies, Gerhard B; de San Eustaquio Campillo, Alba; Slootweg, Erik J; Westerhof, Lotte B; Gawehns, Fleur K K; Knight, Marc R; Sharples, Gary J; Goverse, Aska; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

    2015-10-09

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus; however, conserved nuclear targets that support their role in immunity are unknown. Previously, we noted a structural homology between the nucleotide-binding domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC (nucleotide-binding, Apaf-1, R-proteins, and CED-4) domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro, dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger an Rx1-DNA interaction. DNA binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signaling and define DNA as a molecular target of an activated NLR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. The Potato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor Rx1 Is a Pathogen-dependent DNA-deforming Protein*

    Science.gov (United States)

    Fenyk, Stepan; Townsend, Philip D.; Dixon, Christopher H.; Spies, Gerhard B.; de San Eustaquio Campillo, Alba; Slootweg, Erik J.; Westerhof, Lotte B.; Gawehns, Fleur K. K.; Knight, Marc R.; Sharples, Gary J.; Goverse, Aska; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

    2015-01-01

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus; however, conserved nuclear targets that support their role in immunity are unknown. Previously, we noted a structural homology between the nucleotide-binding domain of NLRs and DNA replication origin-binding Cdc6/Orc1 proteins. Here we show that the NB-ARC (nucleotide-binding, Apaf-1, R-proteins, and CED-4) domain of the Rx1 NLR of potato binds nucleic acids. Rx1 induces ATP-dependent bending and melting of DNA in vitro, dependent upon a functional P-loop. In situ full-length Rx1 binds nuclear DNA following activation by its cognate pathogen-derived effector protein, the coat protein of potato virus X. In line with its obligatory nucleocytoplasmic distribution, DNA binding was only observed when Rx1 was allowed to freely translocate between both compartments and was activated in the cytoplasm. Immune activation induced by an unrelated NLR-effector pair did not trigger an Rx1-DNA interaction. DNA binding is therefore not merely a consequence of immune activation. These data establish a role for DNA distortion in Rx1 immune signaling and define DNA as a molecular target of an activated NLR. PMID:26306038

  4. Cyclic nucleotide binding and structural changes in the isolated GAF domain of Anabaena adenylyl cyclase, CyaB2

    Directory of Open Access Journals (Sweden)

    Kabir Hassan Biswas

    2015-04-01

    Full Text Available GAF domains are a large family of regulatory domains, and a subset are found associated with enzymes involved in cyclic nucleotide (cNMP metabolism such as adenylyl cyclases and phosphodiesterases. CyaB2, an adenylyl cyclase from Anabaena, contains two GAF domains in tandem at the N-terminus and an adenylyl cyclase domain at the C-terminus. Cyclic AMP, but not cGMP, binding to the GAF domains of CyaB2 increases the activity of the cyclase domain leading to enhanced synthesis of cAMP. Here we show that the isolated GAFb domain of CyaB2 can bind both cAMP and cGMP, and enhanced specificity for cAMP is observed only when both the GAFa and the GAFb domains are present in tandem (GAFab domain. In silico docking and mutational analysis identified distinct residues important for interaction with either cAMP or cGMP in the GAFb domain. Structural changes associated with ligand binding to the GAF domains could not be detected by bioluminescence resonance energy transfer (BRET experiments. However, amide hydrogen-deuterium exchange mass spectrometry (HDXMS experiments provided insights into the structural basis for cAMP-induced allosteric regulation of the GAF domains, and differences in the changes induced by cAMP and cGMP binding to the GAF domain. Thus, our findings could allow the development of molecules that modulate the allosteric regulation by GAF domains present in pharmacologically relevant proteins.

  5. Nucleotide binding induces conformational changes apparent on the Na+/K+ -ATPase large cytoplasmic loop

    Czech Academy of Sciences Publication Activity Database

    Šimunová, Lenka; Kubala, M.; Lánský, Zdeněk; Teisinger, Jan; Amler, Evžen

    Roč.13, č.1 (2006), s. 37-37 ISSN 1211-5894. [Discussions on Structural Molecular Biology /5./. 16.03.2006-18.03.2006, Nové Hrady] R&D Projects: GA MŠk(CZ) LC554; GA AV ČR 1ET400110403; GA ČR GD305/03/H148 Grant - others:Grantová agentura UK(CZ) 200053 Keywords : Na+/K+ - ATPase * ATP binding * TNP-ATP Subject RIV: BO - Biophysics

  6. Affinity chromatography and binding studies on immobilized 5'-monophosphate and adenosine 2',5'-bisphosphate of nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa.

    Science.gov (United States)

    Höjeberg, B; Brodelius, P; Rydström, J; Mosbach, K

    1976-07-15

    1. Nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa was purified to apparent homogeneity with an improved method employing affinity chromatography on N6-(6aminohexyl)-adenosine 2', 5'-bisphosphate-Sepharose 4B. 2. Polyacrylamide gel electrophoresis of the purified transhydrogenase carried out in the presence of sodium dodecyl sulphate, indicated a minimal molecular weight of 55000 +/- 2000. 3. The kinetic and regulatory properties of the purified transhydrogenase resembled those of the crude enzyme, i.e., NADPH, adenosine 2'-monophosphate and Ca2+ were activators whereas NADP+ was inhibitory. 4. Nicotinamide nucleotide-specific release of binding of the transhydrogenase to N6-(6-aminohexyl)-adenosine-2',5'-bisphosphate-Sepharose and N6-(-aminohexyl)-adenosine-5'-monophosphate-Sepharose suggests the presence of at least two separate binding sites for nicotinamide nucleotides, one that is specific for NADP(H) and one that binds both NAD(H) and NADP(H). 5. Binding of transhydrogenase to N6-)6-aminohexyl)-adenosine-2',5'-bisphosphate-Sepharose and activation of the enzyme by adenosine-2',5'-bisphophate showed a marked pH dependence. In contrast, inhibition of the Ca2+-activated enzyme by adenosine 2',5'-bisphosphate was virtually constant at various pH values. This descrepancy was interpreted to indicate the existence of separate nucleotide-binding effector and active sites.

  7. Specific binding of (/sup 3/H)substance P to the rat submaxillary gland. The effects of ions and guanine nucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Bahouth, S.W.; Musacchio, J.M.

    1985-08-01

    (/sup 3/H)Substance P ((/sup 3/H)SP), in a high ionic strength incubation medium, binds to a single class of saturable, noninteracting binding sites on rat submaxillary gland membranes with a KD = 2.8 +/- 0.34 nM and maximum binding (Bmax) = 220 +/- 31 fmol/mg of protein. The rank order of potency of various tachykinins, SP fragments and analogs to compete against (/sup 3/H)SP is correlated with their potency to induce salivation. These findings indicate that, under the conditions described, (/sup 3/H)SP binds to a physiologically relevant tachykinin receptor of the SP-P subtype. (/sup 3/H)SP binding increases by 35% in the presence of optimal concentrations of Mn++ and Mg++ whereas guanine nucleotides reduce (/sup 3/H)SP binding. The effect produced by either divalent cations or guanine nucleotides is due to increasing or decreasing the Bmax, respectively, without changing the affinity of (/sup 3/H)SP. Guanine nucleotides reduce the Bmax of (/sup 3/H)SP to the same level in the presence or absence of divalent cations, indicating that divalent cations increase the population of SP receptors that are sensitive to guanine nucleotides. In low ionic strength media, and when the nonspecific binding is defined by 1 microM SP, (/sup 3/H)SP binds to two sites: a high affinity site with a KD of 0.14 nM and a Bmax of 370 fmol/mg of protein and a low affinity high capacity site. When the nonspecific binding is defined by 1 microM physalaemin, the high affinity is the only detectable site. However, in low ionic strength media, physalaemin has about one-fiftieth the potency of SP in competing with (/sup 3/H)SP.

  8. The E6AP binding pocket of the HPV16 E6 oncoprotein provides a docking site for a small inhibitory peptide unrelated to E6AP, indicating druggability of E6.

    Directory of Open Access Journals (Sweden)

    Katia Zanier

    Full Text Available The HPV E6 oncoprotein maintains the malignant phenotype of HPV-positive cancer cells and represents an attractive therapeutic target. E6 forms a complex with the cellular E6AP ubiquitin ligase, ultimately leading to p53 degradation. The recently elucidated x-ray structure of a HPV16 E6/E6AP complex showed that HPV16 E6 forms a distinct binding pocket for E6AP. This discovery raises the question whether the E6AP binding pocket is druggable, i. e. whether it provides a docking site for functional E6 inhibitors. To address these issues, we performed a detailed analysis of the HPV16 E6 interactions with two small peptides: (i E6APpep, corresponding to the E6 binding domain of E6AP, and (ii pep11**, a peptide that binds to HPV16 E6 and, in contrast to E6APpep, induces apoptosis, specifically in HPV16-positive cancer cells. Surface plasmon resonance, NMR chemical shift perturbation, and mammalian two-hybrid analyses coupled to mutagenesis indicate that E6APpep contacts HPV16 E6 amino acid residues within the E6AP pocket, both in vitro and intracellularly. Many of these amino acids were also important for binding to pep11**, suggesting that the binding sites for the two peptides on HPV16 E6 overlap. Yet, few E6 amino acids were differentially involved which may contribute to the higher binding affinity of pep11**. Data from the HPV16 E6/pep11** interaction allowed the rational design of single amino acid exchanges in HPV18 and HPV31 E6 that enabled their binding to pep11**. Taken together, these results suggest that E6 molecular surfaces mediating E6APpep binding can also accommodate pro-apoptotic peptides that belong to different sequence families. As proof of concept, this study provides the first experimental evidence that the E6AP binding pocket is druggable, opening new possibilities for rational, structure-based drug design.

  9. Mutational analysis of the nucleotide binding site of Escherichia coli dCTP deaminase

    DEFF Research Database (Denmark)

    Thymark, Majbritt; Johansson, Eva; Larsen, Sine

    2007-01-01

    In Escherichia coli and Salmonella typhimurium about 80% of the dUMP used for dTMP synthesis is derived from deamination of dCTP. The dCTP deaminase produces dUTP that subsequently is hydrolyzed by dUTPase to dUMP and diphosphate. The dCTP deaminase is regulated by dTTP that inhibits the enzyme...... by binding to the active site and induces an inactive conformation of the trimeric enzyme. We have analyzed the role of residues previously suggested to play a role in catalysis. The mutant enzymes R115Q, S111C, S111T and E138D were all purified and analyzed for activity. Only S111T and E138D displayed...... detectable activity with a 30- and 140-fold reduction in k(cat), respectively. Furthermore, S111T and E138D both showed altered dTTP inhibition compared to wild-type enzyme. S111T was almost insensitive to the presence of dTTP. With the E138D enzyme the dTTP dependent increase in cooperativity of d...

  10. Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Meisheng; Tran, V.T.; Fong, H.K.W. (Univ. of Southern California, Los Angeles (United States)); Pandey, S. (Doheny Eye Inst., Los Angeles, CA (United States))

    1991-05-01

    The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein {alpha} subunits (G{alpha}) including G{sub s}{alpha}, G{sub i-1}{alpha}, G{sub i-2}{alpha}, G{sub i-3}{alpha}, and G{sub z}{alpha} (or G{sub x}{alpha}), where G{sub s} and G{sub i} are proteins that stimulate or inhibit adenylyl cyclase, respectively, and G{sub z} is a protein that may mediate pertussis toxin-insensitive events. Other G{alpha}-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to G{sub i-2}{alpha} and G{sub s}{alpha} protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G{alpha} gene family, at least two other G{alpha} subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G{alpha} subunits were isolated and characterized. The results indicate that this G{alpha} subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.

  11. Translocation of microfilament-associated inhibitory guanine-nucleotide-binding proteins to the plasma membrane in myeloid differentiated human leukemia (HL-60) cells

    NARCIS (Netherlands)

    Meyer zu Heringdorf, D.; Liedel, K.; Kaldenberg-Stasch, S.; Michel, M. C.; Jakobs, K. H.; Wieland, T.

    1996-01-01

    The cytoskeletal localization of inhibitory guanine-nucleotide-binding (Gi) proteins and the coupling of these proteins to formyl peptide receptors were studied in myeloid differentiated human leukemia (HL-60) cells. Treatment of HL-60 cells with cytochalasin B or botulinum C2 toxin, which leads to

  12. Native mass spectrometry provides direct evidence for DNA mismatch-induced regulation of asymmetric nucleotide binding in mismatch repair protein MutS

    NARCIS (Netherlands)

    M.C. Monti; S.X. Cohen (Serge); A. Fish (Alexander); H.H.K. Winterwerp (Herrie); A. Barendregt (Arjan); P. Friedhoff (Peter); A. Perrakis (Anastassis); A.J.R. Heck (Albert); T.K. Sixma (Titia); R.H.H. van den Heuvel (Robert); J.H.G. Lebbink (Joyce)

    2011-01-01

    textabstractThe DNA mismatch repair protein MutS recognizes mispaired bases in DNA and initiates repair in an ATP-dependent manner. Understanding of the allosteric coupling between DNA mismatch recognition and two asymmetric nucleotide binding sites at opposing sides of the MutS dimer requires

  13. The UV-damaged DNA binding protein mediates efficient targeting of the nucleotide excision repair complex to UV-induced photo lesions

    NARCIS (Netherlands)

    Moser, J; Volker, M; Kool, H; Alekseev, S; Vrieling, H; Yasui, A; van Zeeland, AA; Mullenders, LHF

    2005-01-01

    Previous studies point to the XPC-hHR23B complex as the principal initiator of global genome nucleotide excision repair (NER) pathway, responsible for the repair of UV-induced cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) in human cells. However, the UV-damaged DNA binding

  14. Prediction of mono- and di-nucleotide-specific DNA-binding sites in proteins using neural networks

    Directory of Open Access Journals (Sweden)

    Mizuguchi Kenji

    2009-05-01

    Full Text Available Abstract Background DNA recognition by proteins is one of the most important processes in living systems. Therefore, understanding the recognition process in general, and identifying mutual recognition sites in proteins and DNA in particular, carries great significance. The sequence and structural dependence of DNA-binding sites in proteins has led to the development of successful machine learning methods for their prediction. However, all existing machine learning methods predict DNA-binding sites, irrespective of their target sequence and hence, none of them is helpful in identifying specific protein-DNA contacts. In this work, we formulate the problem of predicting specific DNA-binding sites in terms of contacts between the residue environments of proteins and the identity of a mononucleotide or a dinucleotide step in DNA. The aim of this work is to take a protein sequence or structural features as inputs and predict for each amino acid residue if it binds to DNA at locations identified by one of the four possible mononucleotides or one of the 10 unique dinucleotide steps. Contact predictions are made at various levels of resolution viz. in terms of side chain, backbone and major or minor groove atoms of DNA. Results Significant differences in residue preferences for specific contacts are observed, which combined with other features, lead to promising levels of prediction. In general, PSSM-based predictions, supported by secondary structure and solvent accessibility, achieve a good predictability of ~70–80%, measured by the area under the curve (AUC of ROC graphs. The major and minor groove contact predictions stood out in terms of their poor predictability from sequences or PSSM, which was very strongly (>20 percentage points compensated by the addition of secondary structure and solvent accessibility information, revealing a predominant role of local protein structure in the major/minor groove DNA-recognition. Following a detailed

  15. Global alteration of the drug-binding pocket of human P-glycoprotein (ABCB1) by substitution of fifteen conserved residues reveals a negative correlation between substrate size and transport efficiency.

    Science.gov (United States)

    Vahedi, Shahrooz; Chufan, Eduardo E; Ambudkar, Suresh V

    2017-11-01

    P-glycoprotein (P-gp), an ATP-dependent efflux pump, is linked to the development of multidrug resistance in cancer cells. However, the drug-binding sites and translocation pathways of this transporter are not yet well-characterized. We recently demonstrated the important role of tyrosine residues in regulating P-gp ATP hydrolysis via hydrogen bond formations with high affinity modulators. Since tyrosine is both a hydrogen bond donor and acceptor, and non-covalent interactions are key in drug transport, in this study we investigated the global effect of enrichment of tyrosine residues in the drug-binding pocket on the drug binding and transport function of P-gp. By employing computational analysis, 15 conserved residues in the drug-binding pocket of human P-gp that interact with substrates were identified and then substituted with tyrosine, including 11 phenylalanine (F72, F303, F314, F336, F732, F759, F770, F938, F942, F983, F994), two leucine (L339, L975), one isoleucine (I306), and one methionine (M949). Characterization of the tyrosine-rich P-gp mutant in HeLa cells demonstrated that this major alteration in the drug-binding pocket by introducing fifteen additional tyrosine residues is well tolerated and has no measurable effect on total or cell surface expression of this mutant. Although the tyrosine-enriched mutant P-gp could transport small to moderate size (transport large (>1000 Daltons) substrates such as NBD-cyclosporine A, Bodipy-paclitaxel and Bodipy-vinblastine was significantly decreased. This was further supported by the physico-chemical characterization of seventeen tested substrates, which revealed a negative correlation between drug transport and molecular size for the tyrosine-enriched P-gp mutant. Published by Elsevier Inc.

  16. Role of Nucleotide-Binding Oligomerization Domain-Containing (NOD 2 in Host Defense during Pneumococcal Pneumonia.

    Directory of Open Access Journals (Sweden)

    Tijmen J Hommes

    Full Text Available Streptococcus (S. pneumoniae is the most common causative pathogen in community-acquired pneumonia. Nucleotide-binding oligomerization domain-containing (NOD 2 is a pattern recognition receptor located in the cytosol of myeloid cells that is able to detect peptidoglycan fragments of S. pneumoniae. We here aimed to investigate the role of NOD2 in the host response during pneumococcal pneumonia. Phagocytosis of S. pneumoniae was studied in NOD2 deficient (Nod2-/- and wild-type (Wt alveolar macrophages and neutrophils in vitro. In subsequent in vivo experiments Nod2-/- and Wt mice were inoculated with serotype 2 S. pneumoniae (D39, an isogenic capsule locus deletion mutant (D39Δcps or serotype 3 S. pneumoniae (6303 via the airways, and bacterial growth and dissemination and the lung inflammatory response were evaluated. Nod2-/- alveolar macrophages and blood neutrophils displayed a reduced capacity to internalize pneumococci in vitro. During pneumonia caused by S. pneumoniae D39 Nod2-/- mice were indistinguishable from Wt mice with regard to bacterial loads in lungs and distant organs, lung pathology and neutrophil recruitment. While Nod2-/- and Wt mice also had similar bacterial loads after infection with the more virulent S. pneumoniae 6303 strain, Nod2-/- mice displayed a reduced bacterial clearance of the normally avirulent unencapsulated D39Δcps strain. These results suggest that NOD2 does not contribute to host defense during pneumococcal pneumonia and that the pneumococcal capsule impairs recognition of S. pneumoniae by NOD2.

  17. Silencing of a unique integrated domain nucleotide-binding leucine-rich repeat gene in wheat abolishes Diuraphis noxia resistance.

    Science.gov (United States)

    Nicolis, Vittorio Filippo; Venter, Eduard

    2018-03-13

    Plants respond in a similar manner to aphid feeding as to pathogen attack. Diuraphis noxia is a specialist aphid, feeding only on selected grasses that include wheat, barley, and oats. The wheat-Diuraphis noxia interaction is characterized by very similar responses as seen in wheat-pathogen interactions with none of the underlying resistance pathways and genes characterized yet. From wheat harboring the Dn1 resistance gene, we have identified a nucleotide-binding leucine-rich repeat (NLR) gene containing two integrated domains (IDs). These are three C-terminus ankyrin repeat-domains and an N-terminus WRKY domain. The NLR core of the gene can be traced through speciation events within the grass family, with a recent WRKY domain integration that is Triticum specific. Virus induced gene silencing of the gene in a resistant wheat line resulted in the abolishment of the resistance response and induced a highly susceptible phenotype. Silenced plants supported a higher number of aphids similar to the susceptible NIL and the intrinsic rate of increase of the aphids matched that of aphids feeding on the susceptible NIL. The presence of the gene is necessary for Dn1 resistance and we have named the gene Associated with Dn resistance 1 (Adr1) to reflect this function.

  18. Nucleotide-Binding Oligomerization Domain-1 and -2 Play No Role in Controlling Brucella abortus Infection in Mice

    Directory of Open Access Journals (Sweden)

    Fernanda S. Oliveira

    2012-01-01

    Full Text Available Nucleotide-binding oligomerization domain proteins (NODs are modular cytoplasmic proteins implicated in the recognition of peptidoglycan-derived molecules. Further, several in vivo studies have demonstrated a role for Nod1 and Nod2 in host defense against bacterial pathogens. Here, we demonstrated that macrophages from NOD1-, NOD2-, and Rip2-deficient mice produced lower levels of TNF-α following infection with live Brucella abortus compared to wild-type mice. Similar reduction on cytokine synthesis was not observed for IL-12 and IL-6. However, NOD1, NOD2, and Rip2 knockout mice were no more susceptible to infection with virulent B. abortus than wild-type mice. Additionally, spleen cells from NOD1-, NOD2-, and Rip2-deficient mice showed unaltered production of IFN-γ compared to C57BL/6 mice. Taken together, this study demonstrates that NOD1, NOD2 and Rip2 are dispensable for the control of B. abortus during in vivo infection.

  19. Cyclosporine A Impairs Nucleotide Binding Oligomerization Domain (Nod1)-Mediated Innate Antibacterial Renal Defenses in Mice and Human Transplant Recipients

    Science.gov (United States)

    Tourneur, Emilie; Ben Mkaddem, Sanae; Chassin, Cécilia; Bens, Marcelle; Goujon, Jean-Michel; Charles, Nicolas; Pellefigues, Christophe; Aloulou, Meryem; Hertig, Alexandre; Monteiro, Renato C.; Girardin, Stephen E.; Philpott, Dana J.; Rondeau, Eric

    2013-01-01

    Acute pyelonephritis (APN), which is mainly caused by uropathogenic Escherichia coli (UPEC), is the most common bacterial complication in renal transplant recipients receiving immunosuppressive treatment. However, it remains unclear how immunosuppressive drugs, such as the calcineurin inhibitor cyclosporine A (CsA), decrease renal resistance to UPEC. Here, we investigated the effects of CsA in host defense against UPEC in an experimental model of APN. We show that CsA-treated mice exhibit impaired production of the chemoattractant chemokines CXCL2 and CXCL1, decreased intrarenal recruitment of neutrophils, and greater susceptibility to UPEC than vehicle-treated mice. Strikingly, renal expression of Toll-like receptor 4 (Tlr4) and nucleotide-binding oligomerization domain 1 (Nod1), neutrophil migration capacity, and phagocytic killing of E. coli were significantly reduced in CsA-treated mice. CsA inhibited lipopolysaccharide (LPS)-induced, Tlr4-mediated production of CXCL2 by epithelial collecting duct cells. In addition, CsA markedly inhibited Nod1 expression in neutrophils, macrophages, and renal dendritic cells. CsA, acting through inhibition of the nuclear factor of activated T-cells (NFATs), also markedly downregulated Nod1 in neutrophils and macrophages. Silencing the NFATc1 isoform mRNA, similar to CsA, downregulated Nod1 expression in macrophages, and administration of the 11R-VIVIT peptide inhibitor of NFATs to mice also reduced neutrophil bacterial phagocytosis and renal resistance to UPEC. Conversely, synthetic Nod1 stimulating agonists given to CsA-treated mice significantly increased renal resistance to UPEC. Renal transplant recipients receiving CsA exhibited similar decrease in NOD1 expression and neutrophil phagocytosis of E. coli. The findings suggest that such mechanism of NFATc1-dependent inhibition of Nod1-mediated innate immune response together with the decrease in Tlr4-mediated production of chemoattractant chemokines caused by CsA may

  20. Cyclosporine A impairs nucleotide binding oligomerization domain (Nod1-mediated innate antibacterial renal defenses in mice and human transplant recipients.

    Directory of Open Access Journals (Sweden)

    Emilie Tourneur

    2013-01-01

    Full Text Available Acute pyelonephritis (APN, which is mainly caused by uropathogenic Escherichia coli (UPEC, is the most common bacterial complication in renal transplant recipients receiving immunosuppressive treatment. However, it remains unclear how immunosuppressive drugs, such as the calcineurin inhibitor cyclosporine A (CsA, decrease renal resistance to UPEC. Here, we investigated the effects of CsA in host defense against UPEC in an experimental model of APN. We show that CsA-treated mice exhibit impaired production of the chemoattractant chemokines CXCL2 and CXCL1, decreased intrarenal recruitment of neutrophils, and greater susceptibility to UPEC than vehicle-treated mice. Strikingly, renal expression of Toll-like receptor 4 (Tlr4 and nucleotide-binding oligomerization domain 1 (Nod1, neutrophil migration capacity, and phagocytic killing of E. coli were significantly reduced in CsA-treated mice. CsA inhibited lipopolysaccharide (LPS-induced, Tlr4-mediated production of CXCL2 by epithelial collecting duct cells. In addition, CsA markedly inhibited Nod1 expression in neutrophils, macrophages, and renal dendritic cells. CsA, acting through inhibition of the nuclear factor of activated T-cells (NFATs, also markedly downregulated Nod1 in neutrophils and macrophages. Silencing the NFATc1 isoform mRNA, similar to CsA, downregulated Nod1 expression in macrophages, and administration of the 11R-VIVIT peptide inhibitor of NFATs to mice also reduced neutrophil bacterial phagocytosis and renal resistance to UPEC. Conversely, synthetic Nod1 stimulating agonists given to CsA-treated mice significantly increased renal resistance to UPEC. Renal transplant recipients receiving CsA exhibited similar decrease in NOD1 expression and neutrophil phagocytosis of E. coli. The findings suggest that such mechanism of NFATc1-dependent inhibition of Nod1-mediated innate immune response together with the decrease in Tlr4-mediated production of chemoattractant chemokines caused by Cs

  1. Systematic Analysis and Comparison of Nucleotide-Binding Site Disease Resistance Genes in a Diploid Cotton Gossypium raimondii

    Science.gov (United States)

    Wei, Hengling; Li, Wei; Sun, Xiwei; Zhu, Shuijin; Zhu, Jun

    2013-01-01

    Plant disease resistance genes are a key component of defending plants from a range of pathogens. The majority of these resistance genes belong to the super-family that harbors a Nucleotide-binding site (NBS). A number of studies have focused on NBS-encoding genes in disease resistant breeding programs for diverse plants. However, little information has been reported with an emphasis on systematic analysis and comparison of NBS-encoding genes in cotton. To fill this gap of knowledge, in this study, we identified and investigated the NBS-encoding resistance genes in cotton using the whole genome sequence information of Gossypium raimondii. Totally, 355 NBS-encoding resistance genes were identified. Analyses of the conserved motifs and structural diversity showed that the most two distinct features for these genes are the high proportion of non-regular NBS genes and the high diversity of N-termini domains. Analyses of the physical locations and duplications of NBS-encoding genes showed that gene duplication of disease resistance genes could play an important role in cotton by leading to an increase in the functional diversity of the cotton NBS-encoding genes. Analyses of phylogenetic comparisons indicated that, in cotton, the NBS-encoding genes with TIR domain not only have their own evolution pattern different from those of genes without TIR domain, but also have their own species-specific pattern that differs from those of TIR genes in other plants. Analyses of the correlation between disease resistance QTL and NBS-encoding resistance genes showed that there could be more than half of the disease resistance QTL associated to the NBS-encoding genes in cotton, which agrees with previous studies establishing that more than half of plant resistance genes are NBS-encoding genes. PMID:23936305

  2. Guanine nucleotide-binding protein subunit beta-2-like 1, a new Annexin A7 interacting protein

    Energy Technology Data Exchange (ETDEWEB)

    Du, Yue; Meng, Jinyi; Huang, Yuhong; Wu, Jun; Wang, Bo; Ibrahim, Mohammed M.; Tang, Jianwu, E-mail: jianwutdlmedu@163.com

    2014-02-28

    Highlights: • RACK1 formed a complex with Annexin A7. • Depletion of RACK1 inhibited the proliferation, migration and invasion. • RACK1 RNAi abolished RACK1-Annexin A7 interaction. • RACK1-Annexin A7 may play a role in regulating the metastatic potentials. - Abstract: We report for the first time that Guanine nucleotide-binding protein subunit beta-2-like 1 (RACK1) formed a complex with Annexin A7. Hca-F and Hca-P are a pair of syngeneic mouse hepatocarcinoma cell lines established and maintained in our laboratory. Our previous study showed that both Annexin A7 and RACK1 were expressed higher in Hca-F (lymph node metastasis >70%) than Hca-P (lymph node metastasis <30%). Suppression of Annexin A7 expression in Hca-F cells induced decreased migration and invasion ability. In this study, knockdown of RACK1 by RNA interference (RNAi) had the same impact on metastasis potential of Hca-F cells as Annexin A7 down-regulation. Furthermore, by co-immunoprecipitation and double immunofluorescence confocal imaging, we found that RACK1 was in complex with Annexin A7 in control cells, but not in the RACK1-down-regulated cells, indicating the abolishment of RACK1-Annexin A7 interaction in Hca-F cells by RACK1 RNAi. Taken together, these results suggest that RACK1-Annexin A7 interaction may be one of the means by which RACK1 and Annexin A7 influence the metastasis potential of mouse hepatocarcinoma cells in vitro.

  3. Efficacy of the nucleotide-binding oligomerzation domain 1 inhibitor Nodinhibit-1 on corneal alkali burns in rats

    Directory of Open Access Journals (Sweden)

    Xu Huang

    2015-10-01

    Full Text Available AIM:To evaluate the therapeutic effect of Nodinhibit-1 on alkali-burn-induced corneal neovascularization (CNV and inflammation.The nucleotide-binding oligomerzation domain 1 (NOD1 is a potent angiogenic gene.METHODS:The alkali-burned rat corneas (32 right eyes were treated with eye drops containing Nodinhibit-1 or phosphate buffered solution (PBS, PH 7.4 only, four times per day. CNV and inflammation were monitored using slit lamp microscopy, and the area of CNV was measured by formula. Vascular endothelial growth factor (VEGF and pigment epithelium-derived factor (PEDF was determined by Western blot analysis. The TUNEL assay was used to assess the corneal apoptosis cells.RESULTS:Alkali-burn-induced progressive CNV and inflammation in the cornea. After treatment for 7d and 14d, there were statistically significant differences in the CNV areas and inflammatory index on that between two group(P<0.05, respectively. Epithelial defect quantification showed a significant difference between the two groups at days 4 and 7 after the alkali burns (P<0.05. The apoptotic cells on days 1, 4, and 7 between the two groups showed significant differences at all time points (P<0.05, respectively. Compared to that in control group, the protein level of VEGF expression was significantly reduced whereas the PEDF expression was increase in the Nodinhibit-1 groups on day 14 (P<0.05, respectively=.CONCLUSION:Topical application of 10.0 μg/mL Nodinhibit-1 may have potential effect for the alkali burn-induced CNV and inflammation. The effect of Nodinhibit-1 on CNV may be by regulation the equilibrium of VEGF and PEDF in the wounded cornea.

  4. ΔF508-CFTR Modulator Screen Based on Cell Surface Targeting of a Chimeric Nucleotide Binding Domain 1 Reporter.

    Science.gov (United States)

    Phuan, Puay-Wah; Veit, Guido; Tan, Joseph-Anthony; Roldan, Ariel; Finkbeiner, Walter E; Haggie, Peter M; Lukacs, Gergely L; Verkman, Alan S

    2018-03-01

    The most common cystic fibrosis-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine at residue 508 (∆F508). The ∆F508 mutation impairs folding of nucleotide binding domain 1 (NBD1) and interfacial interactions of NBD1 and the membrane spanning domains. Here, we report a domain-targeted screen to identify ∆F508-CFTR modulators that act on NBD1. A biochemical screen for ΔF508-NBD1 cell surface expression was done in Madin-Darby canine kidney cells expressing a chimeric reporter consisting of ΔF508-NBD1, the CD4 transmembrane domain, and an extracellular horseradish peroxidase (HRP) reporter. Using a luminescence readout of HRP activity, the screen was robust with a Z' factor of 0.7. The screening of ~20,000 synthetic small molecules allowed the identification of compounds from four chemical classes that increased ∆F508-NBD1 cell surface expression by up to 4-fold; for comparison, a 12-fold increased cell surface expression was found for a wild-type NBD1 chimera. While the compounds were inactive as correctors of full-length ΔF508-CFTR, several carboxamide-benzothiophenes had potentiator activity with low micromolar EC 50 . Interestingly, the potentiators did not activate G551D or wild-type CFTR. Our results provide a proof of concept for a cell-based NBD1 domain screen to identify ∆F508-CFTR modulators that target the NBD1 domain.

  5. A primary survey on bryophyte species reveals two novel classes of nucleotide-binding site (NBS genes.

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    Jia-Yu Xue

    Full Text Available Due to their potential roles in pathogen defense, genes encoding nucleotide-binding site (NBS domain have been particularly surveyed in many angiosperm genomes. Two typical classes were found: one is the TIR-NBS-LRR (TNL class and the other is the CC-NBS-LRR (CNL class. It is seldom known, however, what kind of NBS-encoding genes are mainly present in other plant groups, especially the most ancient groups of land plants, that is, bryophytes. To fill this gap of knowledge, in this study, we mainly focused on two bryophyte species: the moss Physcomitrella patens and the liverwort Marchantia polymorpha, to survey their NBS-encoding genes. Surprisingly, two novel classes of NBS-encoding genes were discovered. The first novel class is identified from the P. patens genome and a typical member of this class has a protein kinase (PK domain at the N-terminus and a LRR domain at the C-terminus, forming a complete structure of PK-NBS-LRR (PNL, reminiscent of TNL and CNL classes in angiosperms. The second class is found from the liverwort genome and a typical member of this class possesses an α/β-hydrolase domain at the N-terminus and also a LRR domain at the C-terminus (Hydrolase-NBS-LRR, HNL. Analysis on intron positions and phases also confirmed the novelty of HNL and PNL classes, as reflected by their specific intron locations or phase characteristics. Phylogenetic analysis covering all four classes of NBS-encoding genes revealed a closer relationship among the HNL, PNL and TNL classes, suggesting the CNL class having a more divergent status from the others. The presence of specific introns highlights the chimerical structures of HNL, PNL and TNL genes, and implies their possible origin via exon-shuffling during the quick lineage separation processes of early land plants.

  6. Study of the nucleotide binding site of the yeast Schizosaccharomyces pombe plasma membrane H+-ATPase using formycin triphosphate-terbium complex

    International Nuclear Information System (INIS)

    Ronjat, M.; Lacapere, J.J.; Dufour, J.P.; Dupont, Y.

    1987-01-01

    The plasma membrane of yeasts contains an H+-ATPase similar to the other cation transport ATPases of eukaryotic organisms. This enzyme has been purified and shows H+ transport in reconstituted vesicles. In the presence of Mg2+, formycin triphosphate (FTP) is hydrolyzed by the H+-ATPase and supports H+ transport. When combined with terbium ion, FTP (Tb-FTP) and ATP (Tb-ATP) are no longer hydrolyzed. Competition between Mg-ATP and Tb-FTP for ATP hydrolysis indicates that terbium-associated nucleotides bind to the catalytic site of the H+-ATPase. The fluorescent properties of the Tb-FTP complex were used to study the active site of the H+-ATPase. Fluorescence of Tb-FTP is greatly enhanced upon binding into the nucleotide site of H+-ATPase with a dissociation constant of 1 microM. Tb-ATP, Tb-ADP, and Tb-ITP are competitive inhibitors of Tb-FTP binding with Ki = 4.5, 5.0, and 6.0 microM, respectively. Binding of Tb-FTP is observed only in the presence of an excess of Tb3+ with an activation constant Ka = 25 microM for Tb3+. Analysis of the data reveals that the sites for Tb-FTP and Tb3+ binding are independent entities. In standard conditions these sites would be occupied by Mg-ATP and Mg2+, respectively. These findings suggest an important regulatory role of divalent cations on the activity of H+-ATPase. Replacement of H 2 O by D 2 O in the medium suggests the existence of two types of nucleotide binding sites differing by the hydration state of the Tb3+ ion in the bound Tb-FTP complex

  7. Design of pocket foundations

    OpenAIRE

    Kastelec, Dejan

    2010-01-01

    Graduation thesis consists of three parts. The first part is theoretical. General facts about shallow and deep foundations are presented with an emphasis on shallow foundations. Pocket foundations are explained in detail. The calculation of stresses below footing slab under different types of loads is given. In the second part the procedure for design of pocket foundation is explained. Two examples of design of pocket foundations are presented: pocket foundation with flat inner surface of foo...

  8. Genome-Wide Comparative Analyses Reveal the Dynamic Evolution of Nucleotide-Binding Leucine-Rich Repeat Gene Family among Solanaceae Plants

    OpenAIRE

    Seo, Eunyoung; Kim, Seungill; Yeom, Seon-In; Choi, Doil

    2016-01-01

    Plants have evolved an elaborate innate immune system against invading pathogens. Within this system, intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors are known play critical roles in effector-triggered immunity (ETI) plant defense. We performed genome-wide identification and classification of NLR-coding sequences from the genomes of pepper, tomato, and potato using fixed criteria. We then compared genomic duplication and evolution features. We identified intact 267...

  9. Backbone NMR resonance assignments of the nucleotide binding domain of the ABC multidrug transporter LmrA from Lactococcus lactis in its ADP-bound state.

    Science.gov (United States)

    Hellmich, Ute A; Duchardt-Ferner, Elke; Glaubitz, Clemens; Wöhnert, Jens

    2012-04-01

    LmrA from Lactococcus lactis is a multidrug transporter and a member of the ATP binding cassette (ABC) transporter family. ABC transporters consist of a transmembrane domain (TMD) and a nucleotide binding domain (NBD). The NBD contains the highly conserved signature motifs of this transporter superfamily. In the case of LmrA, the TMD and the NBD are expressed as a single polypeptide. LmrA catalyzes the extrusion of hydrophobic compounds including antibiotics from the cell membrane at the expense of ATP hydrolysis. ATP binds to the NBD, where binding and hydrolysis induce conformational changes that lead to the extrusion of the substrate via the TMD. Here, we report the (1)H, (13)C and (15)N backbone chemical shift assignments of the isolated 263 amino acid containing NBD of LmrA in its ADP bound state.

  10. Global expression analysis of nucleotide binding site-leucine rich repeat-encoding and related genes in Arabidopsis

    Science.gov (United States)

    Tan, Xiaoping; Meyers, Blake C; Kozik, Alexander; West, Marilyn AL; Morgante, Michele; St Clair, Dina A; Bent, Andrew F; Michelmore, Richard W

    2007-01-01

    Background Nucleotide binding site-leucine rich repeat (NBS-LRR)-encoding genes comprise the largest class of plant disease resistance genes. The 149 NBS-LRR-encoding genes and the 58 related genes that do not encode LRRs represent approximately 0.8% of all ORFs so far annotated in Arabidopsis ecotype Col-0. Despite their prevalence in the genome and functional importance, there was little information regarding expression of these genes. Results We analyzed the expression patterns of ~170 NBS-LRR-encoding and related genes in Arabidopsis Col-0 using multiple analytical approaches: expressed sequenced tag (EST) representation, massively parallel signature sequencing (MPSS), microarray analysis, rapid amplification of cDNA ends (RACE) PCR, and gene trap lines. Most of these genes were expressed at low levels with a variety of tissue specificities. Expression was detected by at least one approach for all but 10 of these genes. The expression of some but not the majority of NBS-LRR-encoding and related genes was affected by salicylic acid (SA) treatment; the response to SA varied among different accessions. An analysis of previously published microarray data indicated that ten NBS-LRR-encoding and related genes exhibited increased expression in wild-type Landsberg erecta (Ler) after flagellin treatment. Several of these ten genes also showed altered expression after SA treatment, consistent with the regulation of R gene expression during defense responses and overlap between the basal defense response and salicylic acid signaling pathways. Enhancer trap analysis indicated that neither jasmonic acid nor benzothiadiazole (BTH), a salicylic acid analog, induced detectable expression of the five NBS-LRR-encoding genes and one TIR-NBS-encoding gene tested; however, BTH did induce detectable expression of the other TIR-NBS-encoding gene analyzed. Evidence for alternative mRNA polyadenylation sites was observed for many of the tested genes. Evidence for alternative splicing

  11. Global expression analysis of nucleotide binding site-leucine rich repeat-encoding and related genes in Arabidopsis

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    St Clair Dina A

    2007-10-01

    Full Text Available Abstract Background Nucleotide binding site-leucine rich repeat (NBS-LRR-encoding genes comprise the largest class of plant disease resistance genes. The 149 NBS-LRR-encoding genes and the 58 related genes that do not encode LRRs represent approximately 0.8% of all ORFs so far annotated in Arabidopsis ecotype Col-0. Despite their prevalence in the genome and functional importance, there was little information regarding expression of these genes. Results We analyzed the expression patterns of ~170 NBS-LRR-encoding and related genes in Arabidopsis Col-0 using multiple analytical approaches: expressed sequenced tag (EST representation, massively parallel signature sequencing (MPSS, microarray analysis, rapid amplification of cDNA ends (RACE PCR, and gene trap lines. Most of these genes were expressed at low levels with a variety of tissue specificities. Expression was detected by at least one approach for all but 10 of these genes. The expression of some but not the majority of NBS-LRR-encoding and related genes was affected by salicylic acid (SA treatment; the response to SA varied among different accessions. An analysis of previously published microarray data indicated that ten NBS-LRR-encoding and related genes exhibited increased expression in wild-type Landsberg erecta (Ler after flagellin treatment. Several of these ten genes also showed altered expression after SA treatment, consistent with the regulation of R gene expression during defense responses and overlap between the basal defense response and salicylic acid signaling pathways. Enhancer trap analysis indicated that neither jasmonic acid nor benzothiadiazole (BTH, a salicylic acid analog, induced detectable expression of the five NBS-LRR-encoding genes and one TIR-NBS-encoding gene tested; however, BTH did induce detectable expression of the other TIR-NBS-encoding gene analyzed. Evidence for alternative mRNA polyadenylation sites was observed for many of the tested genes. Evidence for

  12. Global expression analysis of nucleotide binding site-leucine rich repeat-encoding and related genes in Arabidopsis.

    Science.gov (United States)

    Tan, Xiaoping; Meyers, Blake C; Kozik, Alexander; West, Marilyn A L; Morgante, Michele; St Clair, Dina A; Bent, Andrew F; Michelmore, Richard W

    2007-10-23

    Nucleotide binding site-leucine rich repeat (NBS-LRR)-encoding genes comprise the largest class of plant disease resistance genes. The 149 NBS-LRR-encoding genes and the 58 related genes that do not encode LRRs represent approximately 0.8% of all ORFs so far annotated in Arabidopsis ecotype Col-0. Despite their prevalence in the genome and functional importance, there was little information regarding expression of these genes. We analyzed the expression patterns of approximately 170 NBS-LRR-encoding and related genes in Arabidopsis Col-0 using multiple analytical approaches: expressed sequenced tag (EST) representation, massively parallel signature sequencing (MPSS), microarray analysis, rapid amplification of cDNA ends (RACE) PCR, and gene trap lines. Most of these genes were expressed at low levels with a variety of tissue specificities. Expression was detected by at least one approach for all but 10 of these genes. The expression of some but not the majority of NBS-LRR-encoding and related genes was affected by salicylic acid (SA) treatment; the response to SA varied among different accessions. An analysis of previously published microarray data indicated that ten NBS-LRR-encoding and related genes exhibited increased expression in wild-type Landsberg erecta (Ler) after flagellin treatment. Several of these ten genes also showed altered expression after SA treatment, consistent with the regulation of R gene expression during defense responses and overlap between the basal defense response and salicylic acid signaling pathways. Enhancer trap analysis indicated that neither jasmonic acid nor benzothiadiazole (BTH), a salicylic acid analog, induced detectable expression of the five NBS-LRR-encoding genes and one TIR-NBS-encoding gene tested; however, BTH did induce detectable expression of the other TIR-NBS-encoding gene analyzed. Evidence for alternative mRNA polyadenylation sites was observed for many of the tested genes. Evidence for alternative splicing was

  13. KRAS G12C Drug Development: Discrimination between Switch II Pocket Configurations Using Hydrogen/Deuterium-Exchange Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Jia; Harrison, Rane A.; Li, Lianbo; Zeng, Mei; Gondi, Sudershan; Scott, David; Gray, Nathanael S.; Engen, John R.; Westover, Kenneth D. (NEU); (DFCI); (UTSMC); (Harvard-Med)

    2017-09-01

    KRAS G12C, the most common RAS mutation found in non-small-cell lung cancer, has been the subject of multiple recent covalent small-molecule inhibitor campaigns including efforts directed at the guanine nucleotide pocket and separate work focused on an inducible pocket adjacent to the switch motifs. Multiple conformations of switch II have been observed, suggesting that switch II pocket (SIIP) binders may be capable of engaging a range of KRAS conformations. Here we report the use of hydrogen/deuterium-exchange mass spectrometry (HDX MS) to discriminate between conformations of switch II induced by two chemical classes of SIIP binders. We investigated the structural basis for differences in HDX MS using X-ray crystallography and discovered a new SIIP configuration in response to binding of a quinazoline chemotype. These results have implications for structure-guided drug design targeting the RAS SIIP.

  14. Role of a guanine nucleotide-binding protein in α1-adrenergic receptor-mediated Ca2+ mobilization in DDT1 MF-2 cells

    International Nuclear Information System (INIS)

    Cornett, L.E.; Norris, J.S.

    1987-01-01

    In this study the mechanisms involved in α 1 -adrenergic receptor-mediated Ca 2+ mobilization at the level of the plasma membrane were investigated. Stimulation of 45 Ca 2+ efflux from saponin-permeabilized DDT 1 MF-2 cells was observed with the addition of either the α 1 -adrenergic agonist phenylephrine and guanosine-5'-triphosphate or the nonhydrolyzable guanine nucleotide guanylyl-imidodiphosphate. In the presence of [ 32 P] NAD, pertussis toxin was found to catalyze ADP-ribosylation of a M/sub r/ = 40,500 (n = 8) peptide in membranes prepared from DDT 1 , MF-2 cells, possibly the α-subunit of N/sub i/. However, stimulation of unidirectional 45 Ca 2+ efflux by phenylephrine was not affected by previous treatment of cells with 100 ng/ml pertussis toxin. These data suggest that the putative guanine nucleotide-binding protein which couples the α 1 -adrenergic receptor to Ca 2+ mobilization in DDT 1 MF-2 cells is not a pertussis toxin substrate and may possibly be an additional member of guanine nucleotide binding protein family

  15. Structures of a minimal human CFTR first nucleotide-binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant

    Energy Technology Data Exchange (ETDEWEB)

    Atwell, Shane; Brouillette, Christie G.; Conners, Kris; Emtage, Spencer; Gheyi, Tarun; Guggino, William B.; Hendle, Jorg; Hunt, John F.; Lewis, Hal A.; Lu, Frances; Protasevich, Irina I.; Rodgers, Logan A.; Romero, Rich; Wasserman, Stephen R.; Weber, Patricia C.; Wetmore, Diana; Zhang, Feiyu F.; Zhao, Xun (Cystic); (UAB); (JHU); (Columbia); (Lilly)

    2010-04-26

    Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646({Delta}405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-{angstrom} resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The {Delta}F508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542-547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.

  16. Recurrent De Novo Mutations Disturbing the GTP/GDP Binding Pocket of RAB11B Cause Intellectual Disability and a Distinctive Brain Phenotype

    NARCIS (Netherlands)

    Lamers, I.J.C.; Reijnders, M.R.F.; Venselaar, H.; Kraus, A.; Jansen, S; Vries, B.B.A. de; Houge, G.; Gradek, G.A.; Seo, J.; Choi, M.; Chae, J.H.; Burgt, I. van der; Pfundt, R.P.; Letteboer, S.J.F.; Beersum, S.E.C. van; Dusseljee, S.; Brunner, H.G.; Doherty, D.; Kleefstra, T.; Roepman, R.

    2017-01-01

    The Rab GTPase family comprises approximately 70 GTP-binding proteins, functioning in vesicle formation, transport and fusion. They are activated by a conformational change induced by GTP-binding, allowing interactions with downstream effectors. Here, we report five individuals with two recurrent de

  17. Reconstruction of the complete ouabain-binding pocket of Na,K-ATPase in gastric H,K-ATPase by substitution of only seven amino acids

    NARCIS (Netherlands)

    Qiu, L.Y.; Krieger, E.; Schaftenaar, G.; Swarts, H.G.P.; Willems, P.H.G.M.; Pont, J.J.H.H.M. de; Koenderink, J.B.

    2005-01-01

    Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na, K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we

  18. Reconstruction of the complete ouabain-binding pocket of Na,K-ATPase in gastric H,K-ATPase by substitution of only seven amino acids.

    NARCIS (Netherlands)

    Qiu, L.; Krieger, E.; Schaftenaar, G.; Swarts, H.G.P.; Willems, P.H.G.M.; Pont, J.J.H.H.M. de; Koenderink, J.B.

    2005-01-01

    Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we

  19. (1)H, (13)C and (15)N chemical shift assignments for the cyclic-nucleotide binding homology domain of a KCNH channel.

    Science.gov (United States)

    Li, Qingxin; Ng, Hui Qi; Kang, CongBao

    2015-04-01

    The KCNH family of ion channels plays important roles in heart and nerve cells. The C-terminal region of the KCNH channel contains a cyclic-nucleotide binding homology domain (CNBHD) which is important for channel gating through interaction with the eag domain. To study the solution structure of CNBHD of the KCNH channel of zebrafish, we over-expressed and purified this domain from E. coli. We report the resonance assignments of the CNBHD. The assignments will allow us to perform structural and dynamic studies for this domain, which will shed light on its role in channel gating.

  20. Nucleotide-binding oligomerization domain containing 1 (NOD1 haplotypes and single nucleotide polymorphisms modify susceptibility to inflammatory bowel diseases in a New Zealand caucasian population: a case-control study

    Directory of Open Access Journals (Sweden)

    Barclay Murray L

    2009-03-01

    Full Text Available Abstract Background The nucleotide-binding oligomerization domain containing 1 (NOD1 gene encodes a pattern recognition receptor that senses pathogens, leading to downstream responses characteristic of innate immunity. We investigated the role of NOD1 single nucleotide polymorphisms (SNPs on IBD risk in a New Zealand Caucasian population, and studied Nod1 expression in response to bacterial invasion in the Caco2 cell line. Findings DNA samples from 388 Crohn's disease (CD, 405 ulcerative colitis (UC, 27 indeterminate colitis patients and 201 randomly selected controls, from Canterbury, New Zealand were screened for 3 common SNPs in NOD1, using the MassARRAY® iPLEX Gold assay. Transcriptional activation of the protein produced by NOD1 (Nod1 was studied after infection of Caco2 cells with Escherichia coli LF82. Carrying the rs2075818 G allele decreased the risk of CD (OR = 0.66, 95% CI = 0.50–0.88, p Conclusion The NOD1 gene is important in signalling invasion of colonic cells by pathogenic bacteria, indicative of its' key role in innate immunity. Carrying specific SNPs in this gene significantly modifies the risk of CD and/or UC in a New Zealand Caucasian population.

  1. The CK2 alpha/CK2 beta interface of human protein kinase CK2 harbors a binding pocket for small molecules

    DEFF Research Database (Denmark)

    Raaf, Jennifer; Brunstein, Elena; Issinger, Olaf-Georg

    2008-01-01

    . Inhibition kinetic studies corroborate the dual binding mode of the inhibitor. Structural comparisons reveal a surprising conformational plasticity of human CK2 alpha around both DRB binding sites. After local rearrangement, the allosteric site serves as a CK2 beta interface. This opens the potential......, selective CK2 inhibitors are required. An often-used CK2 inhibitor is 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). In a complex structure with human CK2 alpha, DRB binds to the canonical ATP cleft, but additionally it occupies an allosteric site that can be alternatively filled by glycerol...

  2. The periodontal pocket.

    Science.gov (United States)

    Donos, Nikolaos

    2018-02-01

    Periodontal disease is synonymous with the presence of periodontal pockets, and very often the clinical success of periodontal therapy is based on periodontal pocket depth reduction. Therefore, in the fields of periodontology and implant dentistry, significant research effort has been placed on the etiopathogenesis, diagnosis and treatment of periodontal/peri-implant disease and as a consequence on pocket pathology. In this volume of Periodontology 2000, the in-depth reviews include topics ranging from preclinical models, anatomy and structure of tissues, and molecular and bacterial components, to treatments of pockets around teeth and implants. These reviews aim to provide the readers with current and future perspectives on the different areas of research into the periodontal pocket. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Absence of zero-temperature transmission rate of a double-chain tight-binding model for DNA with random sequence of nucleotides in thermodynamic limit

    International Nuclear Information System (INIS)

    Xiong Gang; Wang, X.R.

    2005-01-01

    The zero-temperature transmission rate spectrum of a double-chain tight-binding model for real DNA is calculated. It is shown that a band of extended-like states exists only for finite chain length with strong inter-chain coupling. While the whole spectrum tends to zero in thermodynamic limit, regardless of the strength of inter-chain coupling. It is also shown that a more faithful model for real DNA with periodic sugar-phosphate chains in backbone structures can be mapped into the above simple double-chain tight-binding model. Combined with above results, the transmission rate of real DNA with long random sequence of nucleotides is expected to be poor

  4. Insulin: its binding to specific receptors and its stimulation of DNA synthesis and 2',3'-cyclic nucleotide phosphohydrolase in embryonic mouse brain cell cultures

    International Nuclear Information System (INIS)

    Shanker, G.; Pieringer, R.A.

    1986-01-01

    Previously, the authors demonstrated that ornithine decarboxylase was stimulated by insulin in cultures of embryonic mouse brain cells. In the present work, they have investigated the presence and specificity of insulin receptors in these cultures. A time study showed that maximum binding of 125 [I] labelled insulin was around 75 min. Other studies measured the influence of concentration and age on insulin binding. A displacement study using increasing concentrations of cold insulin, glucagon or growth hormone demonstrated that the specificity of the receptors for insulin was rather high. It was also found that insulin displayed a clear dose-dependent stimulation of thymidine incorporation into the brain cells. Insulin also stimulated the glial enzyme 2':3'-cyclic nucleotide phosphohydrolase (CNP-ase). The results suggest a dual role for insulin; it regulates both cell proliferation as well as differentiation

  5. A two-nucleotide deletion renders the mannose-binding lectin 2 (MBL2) gene nonfunctional in Danish Landrace and Duroc pigs

    DEFF Research Database (Denmark)

    Bergman, Ingrid-Maria; Edman, K; van As, P

    2014-01-01

    The mannose-binding lectins (MBLs) are central components of innate immunity, facilitating phagocytosis and inducing the lectin activation pathway of the complement system. Previously, it has been found that certain single-nucleotide polymorphisms (SNPs) in porcine MBL1 and MBL2 (pMBL1, pMBL2...... = 30) and Duroc (N = 10) pigs. Furthermore, the combined pMBL haplotypes of 89 Piètrain × (Large White × Landrace) crossbred pigs were studied, and the genotypes of 67 crossbreds challenged with Escherichia coli were compared to their individual disease records. In the purebred animals, three non......-synonymous SNPs and a two-nucleotide deletion were detected in the coding sequence of pMBL2. The two-nucleotide deletion was present at a frequency of 0.88 in the Landrace pigs and 0.90 in the Duroc pigs, respectively. In the crossbreds, the T allele of the SNP G949T in pMBL1—previously shown to have profound...

  6. Water's role in reshaping a macrocycle's binding pocket: infrared and ultraviolet spectroscopy of benzo-15-crown-5-(H(2)O)(n) and 4'-aminobenzo-15-crown-5-(H(2)O)(n), n = 1, 2.

    Science.gov (United States)

    Shubert, V Alvin; Müller, Christian W; Zwier, Timothy S

    2009-07-16

    Laser-induced fluorescence (LIF), resonant two-photon ionization (R2PI), ultraviolet hole-burning (UVHB), resonant ion-dip infrared (RIDIR), and infrared-infrared ultraviolet hole-burning (IR-IR-UV) spectroscopies were carried out on benzo-15-crown-5 ether-(H(2)O)(n) (B15C-(H(2)O)(n)) and 4'-amino-benzo-15-crown-5 ether-(H(2)O)(n) (ABC-(H(2)O)(n)) clusters with n = 1,2 formed in a supersonic expansion. Two isomers of B15C-(H(2)O)(1) with S(0)-S(1) origins at 35,628 and 35,685 cm(-1) (B15C-(H(2)O)(1)(A) and B15C-(H(2)O)(1)(B), respectively) were identified and, on the basis of the combined evidence from the single-isomer UV and IR spectra, assigned to structures in which the H(2)O molecule donates both its OH groups to H-bonds to the crown oxygens. Both isomers share the same open, chairlike C(s) symmetry structure for the crown ether that exposes the crown oxygen lone pairs to binding to H(2)O on the interior of the crown. This crown conformation is not among those represented in the observed conformers in the absence of the H(2)O molecule, indicating that even a single water molecule is capable of reshaping the crown binding pocket in binding to it. In B15C-(H(2)O)(1)(A), the water molecule takes up a position parallel to the crown plane of symmetry, using one OH group to bind to the two benzo oxygens, while the other OH binds to a single crown oxygen on the opposite side of the crown. The H(2)O molecule in B15C-(H(2)O)(1)(B) binds to the other two crown oxygens, in an orientation perpendicular to the crown's symmetry plane. B15C-(H(2)O)(2) also has two isomers. The first, B15C-(H(2)O)(2)(A) with S(0)-S(1) origin at 35,813 cm(-1), is assigned to a structure in which the two water molecules take up the two positions occupied by individual water molecules in B15C-(H(2)O)(1) A and B. The second isomer, with S(0)-S(1) origin at 35,665 cm(-1), has an OH stretch RIDIR spectrum that reflects a water-water H-bond, with the second water molecule binding to the crown

  7. Influences of energization and nucleotide binding on the reaction of Lucifer Yellow vinyl sulfone with the alpha subunits of the chloroplast ATP synthase.

    Science.gov (United States)

    Cunningham, K M; McCarty, R E

    2000-04-18

    The catalytic portion of the chloroplast ATP synthase (CF(1)) consists of five different polypeptides in the stoichiometry alpha(3)beta(3)gammadeltaepsilon and is structurally asymmetric. Asymmetry is readily apparent in the properties of the six nucleotide binding sites and the single-copy, smaller subunits. Asymmetry is also detected in the alpha subunits by the rapid and covalent binding of Lucifer Yellow vinyl sulfone (LY) to one of the three chemically identical alpha subunits. The binding of LY to a single alpha subunit has allowed the investigation of whether asymmetry in the alpha subunits is a permanent feature of CF(1). The development of an electrochemical proton gradient across illuminated thylakoid membranes and the preincubation of CF(1) in solution with Mg(2+)-ATP were found to alter the LY distribution such that multiple alpha subunits were labeled with LY. Illumination of thylakoid membranes doubled the extent of LY labeling, and fluorescence resonance energy transfer measurements indicated that LY was bound to more than one alpha subunit. Since the change in LY distribution was inhibited by proton ionophores (uncouplers), alteration of alpha conformation by illumination is a result of the generation of a proton gradient. Preincubation of CF(1) in solution with Mg(2+)-ATP had no effect on the extent of LY labeling but resulted in multiple alpha subunits binding LY as determined by fluorescence resonance energy transfer measurements. Adenine nucleotides at substrate level concentrations inhibit the reaction of LY with the alpha subunits. No increase in LY labeling was observed when thylakoids were illuminated under conditions in which CF(1) was catalytically active.

  8. Structural Insights into the Nucleotide-Binding Domains of the P1B-type ATPases HMA6 and HMA8 from Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Hubert Mayerhofer

    Full Text Available Copper is a crucial ion in cells, but needs to be closely controlled due to its toxic potential and ability to catalyse the formation of radicals. In chloroplasts, an important step for the proper functioning of the photosynthetic electron transfer chain is the delivery of copper to plastocyanin in the thylakoid lumen. The main route for copper transport to the thylakoid lumen is driven by two PIB-type ATPases, Heavy Metal ATPase 6 (HMA6 and HMA8, located in the inner membrane of the chloroplast envelope and in the thylakoid membrane, respectively. Here, the crystal structures of the nucleotide binding domain of HMA6 and HMA8 from Arabidopsis thaliana are reported at 1.5Å and 1.75Å resolution, respectively, providing the first structural information on plants Cu+-ATPases. The structures reveal a compact domain, with two short helices on both sides of a twisted beta-sheet. A double mutant, aiding in the crystallization, provides a new crystal contact, but also avoids an internal clash highlighting the benefits of construct modifications. Finally, the histidine in the HP motif of the isolated domains, unable to bind ATP, shows a side chain conformation distinct from nucleotide bound structures.

  9. Nucleotide-Binding Oligomerization Domain 2 Contributes to Limiting Growth of Mycobacterium abscessus in the Lung of Mice by Regulating Cytokines and Nitric Oxide Production

    Directory of Open Access Journals (Sweden)

    Jun-Young Lee

    2017-11-01

    Full Text Available Mycobacterium abscessus is a prominent cause of pulmonary infection in immunosuppressed patients and those with cystic fibrosis. Nucleotide-binding oligomerization domain (NOD 2 is a cytosolic receptor which senses a bacterial peptidoglycan component, muramyl dipeptide (MDP. Although nucleotide-binding oligomerization domain 2 (NOD2 contributes to protect host against various microbial infections, it is still unclear whether NOD2 is essential to regulate host immune responses against M. abscessus infection. In this study, we sought to clarify the role of NOD2 and the underlying mechanism in host defense against M. abscessus infection. Mice were infected intranasally with M. abscessus and sacrificed at indicated time points. Bacterial survival, cytokines production, and pathology in the lungs were determined. Bone marrow-derived macrophages were used to clarify cellular mechanism of NOD2-mediated immune response. Bacterial clearance was impaired, and pathology was more severe in the lungs of NOD2-deficient mice compared with the wild-type mice. In macrophages, NOD2-mediated activation of p38 and JNK were required for production of proinflammatory cytokines and nitric oxide (NO and expression of iNOS in response to M. abscessus. NO was critical for limiting intracellular growth of the pathogen. Intranasal administration of MDP reduced in vivo bacterial replication and thus improved lung pathology in M. abscessus-infected mice. This study offers important new insights into the potential roles of the NOD2 in initiating and potentiating innate immune response against M. abscessus pulmonary infection.

  10. Inositol phospholipids regulate the guanine-nucleotide-exchange factor Tiam1 by facilitating its binding to the plasma membrane and regulating GDP/GTP exchange on Rac1.

    Science.gov (United States)

    Fleming, Ian N; Batty, Ian H; Prescott, Alan R; Gray, Alex; Kular, Gursant S; Stewart, Hazel; Downes, C Peter

    2004-09-15

    Binding of the Rac1-specific guanine-nucleotide-exchange factor, Tiam1, to the plasma membrane requires the N-terminal pleckstrin homology domain. In the present study, we show that membrane-association is mediated by binding of PtdIns(4,5)P(2) to the pleckstrin homology domain. Moreover, in 1321N1 astrocytoma cells, translocation of Tiam1 to the cytosol, following receptor-mediated stimulation of PtdIns(4,5)P(2) breakdown, correlates with decreased Rac1-GTP levels, indicating that membrane-association is required for GDP/GTP exchange on Rac1. In addition, we show that platelet-derived growth factor activates Rac1 in vivo by increasing PtdIns(3,4,5)P(3) concentrations, rather than the closely related lipid, PtdIns(3,4)P(2). Finally, the data demonstrate that PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) bind to the same pleckstrin homology domain in Tiam1 and that soluble inositol phosphates appear to compete with lipids for this binding. Together, these novel observations provide strong evidence that distinct phosphoinositides regulate different functions of this enzyme, indicating that local concentrations of signalling lipids and the levels of cytosolic inositol phosphates will play crucial roles in determining its activity in vivo.

  11. The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf

    Directory of Open Access Journals (Sweden)

    Geldner Niko

    2005-02-01

    Full Text Available Abstract Background Small G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown. Results Based on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (TBC-Sec7. Conclusion Our analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains.

  12. Reconstruction of the complete ouabain-binding pocket of Na,K-ATPase in gastric H,K-ATPase by substitution of only seven amino acids.

    Science.gov (United States)

    Qiu, Li Yan; Krieger, Elmar; Schaftenaar, Gijs; Swarts, Herman G P; Willems, Peter H G M; De Pont, Jan Joep H H M; Koenderink, Jan B

    2005-09-16

    Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we started from the fact that a closely related enzyme, the gastric H,K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na,K-ATPase, bound ouabain with high affinity (Koenderink, J. B., Hermsen, H. P. H., Swarts, H. G. P., Willems, P. H. G. M., and De Pont, J. J. H. H. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11209-11214). We also demonstrated that only three amino acids (Phe(783), Thr(797), and Asp(804)) present in the M5-M6 hairpin of Na,K-ATPase were sufficient to confer high affinity ouabain binding to a chimera which contained in addition the M3-M4 hairpin of Na,K-ATPase (Qiu, L. Y., Koenderink, J. B., Swarts, H. G., Willems, P. H., and De Pont, J. J. H. H. M. (2003) J. Biol. Chem. 278, 47240-47244). To further pinpoint the ouabain-binding site here we used a chimera-based loss-of-function strategy and identified four amino acids (Glu(312), Val(314), Ile(315), Gly(319)), all present in M4, as being important for ouabain binding. In a final gain-of-function study we showed that a gastric H,K-ATPase that contained Glu(312), Val(314), Ile(315), Gly(319), Phe(783), Thr(797), and Asp(804) of Na,K-ATPase bound ouabain with the same affinity as the native enzyme. Based on the E(2)P crystal structure of Ca(2+)-ATPase we constructed a homology model for the ouabain-binding site of Na,K-ATPase involving all seven amino acids as well as several earlier postulated amino acids.

  13. Identification of an hexapeptide that binds to a surface pocket in cyclin A and inhibits the catalytic activity of the complex cyclin-dependent kinase 2-cyclin A.

    Science.gov (United States)

    Canela, Núria; Orzáez, Mar; Fucho, Raquel; Mateo, Francesca; Gutierrez, Ricardo; Pineda-Lucena, Antonio; Bachs, Oriol; Pérez-Payá, Enrique

    2006-11-24

    The protein-protein complexes formed between different cyclins and cyclin-dependent kinases (CDKs) are central to cell cycle regulation. These complexes represent interesting points of chemical intervention for the development of antineoplastic molecules. Here we describe the identification of an all d-amino acid hexapeptide, termed NBI1, that inhibits the kinase activity of the cyclin-dependent kinase 2 (cdk2)-cyclin A complex through selective binding to cyclin A. The mechanism of inhibition is non-competitive for ATP and non-competitive for protein substrates. In contrast to the existing CDKs peptide inhibitors, the hexapeptide NBI1 interferes with the formation of the cdk2-cyclin A complex. Furthermore, a cell-permeable derivative of NBI1 induces apoptosis and inhibits proliferation of tumor cell lines. Thus, the NBI1-binding site on cyclin A may represent a new target site for the selective inhibition of activity cdk2-cyclin A complex.

  14. A KAS2 cDNA complements the phenotypes of the Arabidopsis fab1 mutant that differs in a single residue bordering the substrate binding pocket

    DEFF Research Database (Denmark)

    Carlsson, A.S.; LaBrie, S.T.; Kinney, A.J.

    2002-01-01

    in Arabidopsis KAS2 that results in a Leu337Phe substitution. The Leu337 residue is conserved among plant and bacterial KAS proteins, and in the crystal structures of E. coli KAS I and KAS II, this leucine abuts a phenylalanine whose imidazole ring extends into the substrate binding cavity causing the fatty acid...... chain to bend. For functional analysis the equivalent Leu207Phe mutation was introduced into the fabB gene encoding the E. coli KAS I enzyme. Compared to wild-type, the Leu207Phe protein showed a 10-fold decrease in binding affinity for the fatty acid substrate, exhibited a modified behavior during size...

  15. Simulation of the coupling between nucleotide binding and transmembrane domains in the ABC transporter BtuCD

    DEFF Research Database (Denmark)

    Sonne, Jacob; Kandt, C.; Peters, Günther H.j.

    2007-01-01

    binding domains and the shared role of ATP in ABC transporters. The rearrangements in the cytoplasmic gate region do not provide enough space for B12 to diffuse from the transporter pore into the cytoplasm, which could suggest that peristaltic forces are needed to exclude B12 from the transporter pore....

  16. Amino acid substitution at peptide-binding pockets of HLA class I molecules increases risk of severe acute GVHD and mortality

    Science.gov (United States)

    Wang, Tao; Haagenson, Michael; Spellman, Stephen R.; Askar, Medhat; Battiwalla, Minoo; Baxter-Lowe, Lee Ann; Bitan, Menachem; Fernandez-Viña, Marcelo; Gandhi, Manish; Jakubowski, Ann A.; Maiers, Martin; Marino, Susana R.; Marsh, Steven G. E.; Oudshoorn, Machteld; Palmer, Jeanne; Prasad, Vinod K.; Reddy, Vijay; Ringden, Olle; Saber, Wael; Santarone, Stella; Schultz, Kirk R.; Setterholm, Michelle; Trachtenberg, Elizabeth; Turner, E. Victoria; Woolfrey, Ann E.; Lee, Stephanie J.; Anasetti, Claudio

    2013-01-01

    HLA disparity has a negative impact on the outcomes of hematopoietic cell transplantation (HCT). We studied the independent impact of amino acid substitution (AAS) at peptide-binding positions 9, 99, 116, and 156, and killer immunoglobulin-like receptor binding position 77 of HLA-A, B, or C, on the risks for grade 3-4 acute graft-versus-host disease (GVHD), chronic GVHD, treatment-related mortality (TRM), relapse, and overall survival. In multivariate analysis, a mismatch at HLA-C position 116 was associated with increased risk for severe acute GVHD (hazard ratio [HR] = 1.45, 95% confidence interval [CI] = 1.15-1.82, P = .0016). Mismatch at HLA-C position 99 was associated with increased transplant-related mortality (HR = 1.37, 95% CI = 1.1-1.69, P = .0038). Mismatch at HLA-B position 9 was associated with increased chronic GVHD (HR = 2.28, 95% CI = 1.36-3.82, P = .0018). No AAS were significantly associated with outcome at HLA-A. Specific AAS pair combinations with a frequency >30 were tested for association with HCT outcomes. Cysteine to tyrosine substitution at position 99 of HLA-C was associated with increased TRM (HR = 1.78, 95% = CI 1.27-2.51, P = .0009). These results demonstrate that donor-recipient mismatch for certain peptide-binding residues of the HLA class I molecule is associated with increased risk for acute and chronic GVHD and death. PMID:23982174

  17. Computational studies on receptor-ligand interactions between novel buffalo (Bubalus bubalis) nucleotide-binding oligomerization domain-containing protein 2 (NOD2) variants and muramyl dipeptide (MDP).

    Science.gov (United States)

    Brahma, Biswajit; Patra, Mahesh Chandra; Mishra, Purusottam; De, Bidhan Chandra; Kumar, Sushil; Maharana, Jitendra; Vats, Ashutosh; Ahlawat, Sonika; Datta, Tirtha Kumar; De, Sachinandan

    2016-04-01

    Nucleotide binding and oligomerization domain 2 (NOD2), a member of intracellular NOD-like receptors (NLRs) family, recognizes the bacterial peptidoglycan, muramyl dipeptide (MDP) and initiates host immune response. The precise ligand recognition mechanism of NOD2 has remained elusive, although studies have suggested leucine rich repeat (LRR) region of NOD2 as the possible binding site of MDP. In this study, we identified multiple transcripts of NOD2 gene in buffalo (buNOD2) and at least five LRR variants (buNOD2-LRRW (wild type), buNOD2-LRRV1-V4) were found to be expressed in buffalo peripheral blood mononuclear cells. The newly identified buNOD2 transcripts were shorter in lengths as a result of exon-skipping and frame-shift mutations. Among the variants, buNOD2-LRRW, V1, and V3 were expressed more frequently in the animals studied. A comparative receptor-ligand interaction study through modeling of variants, docking, and molecular dynamics simulation revealed that the binding affinity of buNOD2-LRRW towards MDP was greater than that of the shorter variants. The absence of a LRR segment in the buNOD2 variants had probably affected their affinity toward MDP. Notwithstanding a high homology among the variants, the amino acid residues that interact with MDP were located on different LRR motifs. The binding free energy calculation revealed that the amino acids Arg850(LRR4) and Glu932(LRR7) of buNOD2-LRRW, Lys810(LRR3) of buNOD2-LRRV1, and Lys830(LRR3) of buNOD2-LRRV3 largely contributed towards MDP recognition. The knowledge of MDP recognition and binding modes on buNOD2 variants could be useful to understand the regulation of NOD-mediated immune response as well as to develop next generation anti-inflammatory compounds. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Secondary and tertiary structure of nucleotide-binding domain of alpha subunit of Na+/K+-ATPase

    Czech Academy of Sciences Publication Activity Database

    Hofbauerová, Kateřina; Kopecký ml., Vladimír; Ettrich, Rüdiger; Ettrichová, Olga; Amler, Evžen

    2002-01-01

    Roč. 67, 4-5 (2002), s. 242-246 ISSN 0006-3525 R&D Projects: GA ČR GA204/01/0254; GA ČR GA204/01/1001 Grant - others:Volkswagen Foundation(DE) I/74 679 Institutional research plan: CEZ:AV0Z5011922 Keywords : Na+/K+- ATPase * ATP binding * molecular modeling Subject RIV: BO - Biophysics Impact factor: 2.372, year: 2002

  19. Structure of an Odorant-Vinding Protein form the Mosquito Aedes aegypti Suggests a Binding Pocket Covered by a pH-Sensitive

    Energy Technology Data Exchange (ETDEWEB)

    N Leite; R Krogh; W Xu; Y Ishida; J Iulek; W Leal; G Oliva

    2011-12-31

    The yellow fever mosquito, Aedes aegypti, is the primary vector for the viruses that cause yellow fever, mostly in tropical regions of Africa and in parts of South America, and human dengue, which infects 100 million people yearly in the tropics and subtropics. A better understanding of the structural biology of olfactory proteins may pave the way for the development of environmentally-friendly mosquito attractants and repellents, which may ultimately contribute to reduction of mosquito biting and disease transmission. Previously, we isolated and cloned a major, female-enriched odorant-binding protein (OBP) from the yellow fever mosquito, AaegOBP1, which was later inadvertently renamed AaegOBP39. We prepared recombinant samples of AaegOBP1 by using an expression system that allows proper formation of disulfide bridges and generates functional OBPs, which are indistinguishable from native OBPs. We crystallized AaegOBP1 and determined its three-dimensional structure at 1.85 {angstrom} resolution by molecular replacement based on the structure of the malaria mosquito OBP, AgamOBP1, the only mosquito OBP structure known to date. The structure of AaegOBP1 (= AaegOBP39) shares the common fold of insect OBPs with six {alpha}-helices knitted by three disulfide bonds. A long molecule of polyethylene glycol (PEG) was built into the electron-density maps identified in a long tunnel formed by a crystallographic dimer of AaegOBP1. Circular dichroism analysis indicated that delipidated AaegOBP1 undergoes a pH-dependent conformational change, which may lead to release of odorant at low pH (as in the environment in the vicinity of odorant receptors). A C-terminal loop covers the binding cavity and this 'lid' may be opened by disruption of an array of acid-labile hydrogen bonds thus explaining reduced or no binding affinity at low pH.

  20. Cap-proximal nucleotides via differential eIF4E binding and alternative promoter usage mediate translational response to energy stress.

    Science.gov (United States)

    Tamarkin-Ben-Harush, Ana; Vasseur, Jean-Jacques; Debart, Françoise; Ulitsky, Igor; Dikstein, Rivka

    2017-02-08

    Transcription start-site (TSS) selection and alternative promoter (AP) usage contribute to gene expression complexity but little is known about their impact on translation. Here we performed TSS mapping of the translatome following energy stress. Assessing the contribution of cap-proximal TSS nucleotides, we found dramatic effect on translation only upon stress. As eIF4E levels were reduced, we determined its binding to capped-RNAs with different initiating nucleotides and found the lowest affinity to 5'cytidine in correlation with the translational stress-response. In addition, the number of differentially translated APs was elevated following stress. These include novel glucose starvation-induced downstream transcripts for the translation regulators eIF4A and Pabp, which are also translationally-induced despite general translational inhibition. The resultant eIF4A protein is N-terminally truncated and acts as eIF4A inhibitor. The induced Pabp isoform has shorter 5'UTR removing an auto-inhibitory element. Our findings uncovered several levels of coordination of transcription and translation responses to energy stress.

  1. Interaction Pattern of Arg 62 in the A-Pocket of Differentially Disease-Associated HLA-B27 Subtypes Suggests Distinct TCR Binding Modes

    Science.gov (United States)

    Cauli, Alberto; Mathieu, Alessandro; Tedeschi, Valentina; Caristi, Silvana; Sorrentino, Rosa; Böckmann, Rainer A.; Fiorillo, Maria Teresa

    2012-01-01

    The single amino acid replacement Asp116His distinguishes the two subtypes HLA-B*2705 and HLA-B*2709 which are, respectively, associated and non-associated with Ankylosing Spondylitis, an autoimmune chronic inflammatory disease. The reason for this differential association is so far poorly understood and might be related to subtype-specific HLA:peptide conformations as well as to subtype/peptide-dependent dynamical properties on the nanoscale. Here, we combine functional experiments with extensive molecular dynamics simulations to investigate the molecular dynamics and function of the conserved Arg62 of the α1-helix for both B27 subtypes in complex with the self-peptides pVIPR (RRKWRRWHL) and TIS (RRLPIFSRL), and the viral peptides pLMP2 (RRRWRRLTV) and NPflu (SRYWAIRTR). Simulations of HLA:peptide systems suggest that peptide-stabilizing interactions of the Arg62 residue observed in crystal structures are metastable for both B27 subtypes under physiological conditions, rendering this arginine solvent-exposed and, probably, a key residue for TCR interaction more than peptide-binding. This view is supported by functional experiments with conservative (R62K) and non-conservative (R62A) B*2705 and B*2709 mutants that showed an overall reduction in their capability to present peptides to CD8+ T cells. Moreover, major subtype-dependent differences in the peptide recognition suggest distinct TCR binding modes for the B*2705 versus the B*2709 subtype. PMID:22403718

  2. Google Pocket Guide

    CERN Document Server

    Calishain, Tara; Adams, DJ

    2003-01-01

    Beneath its deceptively simple search form, Google is a remarkably powerful and flexible search engine that indexes billions of web pages, handling more than 150 million searches a day. You know that what you're looking for must be in there somewhere, but how do you make Google work for you? Crafted from our best-selling Google Hacks title, the Google Pocket Guide provides exactly the information you need to make your searches faster and more effective, right from the start. The Google Pocket Guide unleashes the power behind that blinking cursor by delivering: A thorough but concise tour o

  3. Newnes electrical pocket book

    CERN Document Server

    Reeves, E A

    2013-01-01

    Newnes Electrical Pocket Book, Twenty-first Edition, provides engineers with convenient access to various facts, tables, and formulae relating to the particular branch of engineering being dealt with. In the case of electrical engineering, it is essential that the engineer have a clear understanding of the methods by which the various formulae are derived to ensure that any particular formulae is applicable to the conditions being considered. The first section of the Pocket Book is devoted to the theoretical groundwork upon which all the practical applications are based. This covers symbols,

  4. HTML & XHTML Pocket Reference

    CERN Document Server

    Robbins, Jennifer

    2010-01-01

    After years of using spacer GIFs, layers of nested tables, and other improvised solutions for building your web sites, getting used to the more stringent standards-compliant design can be intimidating. HTML and XHTML Pocket Reference is the perfect little book when you need answers immediately. Jennifer Niederst-Robbins, author Web Design in a Nutshell, has revised and updated the fourth edition of this pocket guide by taking the top 20% of vital reference information from her Nutshell book, augmenting it judiciously, cross-referencing everything, and organizing it according to the most com

  5. Multiple single nucleotide polymorphisms in the first intron of the IL2RA gene affect transcription factor binding and enhancer activity.

    Science.gov (United States)

    Schwartz, Anton M; Demin, Denis E; Vorontsov, Ilya E; Kasyanov, Artem S; Putlyaeva, Lidia V; Tatosyan, Karina A; Kulakovskiy, Ivan V; Kuprash, Dmitry V

    2017-02-20

    IL2RA gene encodes the alpha subunit of a high-affinity receptor for interleukin-2 which is expressed by several distinct populations of lymphocytes involved in autoimmune processes. A large number of polymorphic alleles of the IL2RA locus are associated with the development of various autoimmune diseases. With bioinformatics analysis we the dissected the first intron of the IL2RA gene and selected several single nucleotide polymorphisms (SNPs) that may influence the regulation of the IL2RA gene in cell types relevant to autoimmune pathology. We described five enhancers containing the selected SNPs that stimulated activity of the IL2RA promoter in a cell-type specific manner, and tested the effect of specific SNP alleles on activity of the respective enhancers (E1 to E5, labeled according to the distance to the promoter). The E4 enhancer with minor T variant of rs61839660 SNP demonstrated reduced activity due to disrupted binding of MEF2A/C transcription factors (TFs). Neither rs706778 nor rs706779 SNPs, both associated with a number of autoimmune diseases, had any effect on the activity of the enhancer E2. However, rare variants of several SNPs (rs139767239, rs115133228, rs12722502, rs12722635) genetically linked to either rs706778 and/or rs706779 significantly influenced the activity of E1, E3 and E5 enhancers, presumably by disrupting EBF1, GABPA and ELF1 binding sites. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. The stem rust resistance gene Rpg5 encodes a protein with nucleotide-binding-site, leucine-rich, and protein kinase domains.

    Science.gov (United States)

    Brueggeman, R; Druka, A; Nirmala, J; Cavileer, T; Drader, T; Rostoks, N; Mirlohi, A; Bennypaul, H; Gill, U; Kudrna, D; Whitelaw, C; Kilian, A; Han, F; Sun, Y; Gill, K; Steffenson, B; Kleinhofs, A

    2008-09-30

    We isolated the barley stem rust resistance genes Rpg5 and rpg4 by map-based cloning. These genes are colocalized on a 70-kb genomic region that was delimited by recombination. The Rpg5 gene consists of an unusual structure encoding three typical plant disease resistance protein domains: nucleotide-binding site, leucine-rich repeat, and serine threonine protein kinase. The predicted RPG5 protein has two putative transmembrane sites possibly involved in membrane binding. The gene is expressed at low but detectable levels. Posttranscriptional gene silencing using VIGS resulted in a compatible reaction with a normally incompatible stem rust pathogen. Allele sequencing also validated the candidate Rpg5 gene. Allele and recombinant sequencing suggested that the probable rpg4 gene encoded an actin depolymerizing factor-like protein. Involvement of actin depolymerizing factor genes in nonhost resistance has been documented, but discovery of their role in gene-for-gene interaction would be novel and needs to be further substantiated.

  7. Total Syntheses and Initial Evaluation of [Ψ[C(=S)NH]Tpg4]vancomycin, [Ψ[C(=NH)NH]Tpg4]vancomycin, [Ψ[CH2NH]Tpg4]vancomycin and their (4-Chlorobiphenyl)methyl Derivatives: Synergistic Binding Pocket and Peripheral Modifications for the Glycopeptide Antibiotics

    Science.gov (United States)

    Okano, Akinori; Nakayama, Atsushi; Wu, Kejia; Lindsey, Erick A.; Schammel, Alex W.; Feng, Yiqing; Collins, Karen C.

    2015-01-01

    Full details of studies are disclosed on the total synthesis of binding pocket analogues of vancomycin, bearing the peripheral L-vancosaminyl-1,2-D-glucosyl disaccharide, that contain changes to a key single atom in the residue 4 amide (residue 4 carbonyl O → S, NH, H2) designed to directly address the underlying molecular basis of resistance to vancomycin. Also disclosed are studies piloting the late stage transformations conducted on the synthetically more accessible C-terminus hydroxymethyl aglycon derivatives and full details of the peripheral chlorobiphenyl functionalization of all the binding pocket modified vancomycin analogues designed for dual D-Ala-D-Ala/D-Ala-D-Lac binding are reported. Their collective assessment indicate that combined binding pocket and chlorobiphenyl peripherally modified analogues exhibit a remarkable spectrum of antimicrobial activity (VSSA, MRSA, VanA and VanB VRE) and impressive potencies against both vancomycin-sensitive and vancomycin-resistant bacteria (MICs = 0.06–0.005 μg/mL and 0.5–0.06 μg/mL for the amidine and methylene analogues, respectively) and likely benefit from two independent and synergistic mechanisms of action, only one of which is dependent on D-Ala-D-Ala/D-Ala-D-Lac binding. Such analogues are likely to display especially durable antibiotic activity not prone to rapidly acquired clinical resistance. PMID:25750995

  8. Guanine nucleotide-binding protein (Gα) endocytosis by a cascade of ubiquitin binding domain proteins is required for sustained morphogenesis and proper mating in yeast.

    Science.gov (United States)

    Dixit, Gauri; Baker, Rachael; Sacks, Carly M; Torres, Matthew P; Dohlman, Henrik G

    2014-05-23

    Heterotrimeric G proteins are well known to transmit signals from cell surface receptors to intracellular effector proteins. There is growing appreciation that G proteins are also present at endomembrane compartments, where they can potentially interact with a distinct set of signaling proteins. Here, we examine the cellular trafficking function of the G protein α subunit in yeast, Gpa1. Gpa1 contains a unique 109-amino acid insert within the α-helical domain that undergoes a variety of posttranslational modifications. Among these is monoubiquitination, catalyzed by the NEDD4 family ubiquitin ligase Rsp5. Using a newly optimized method for G protein purification together with biophysical measures of structure and function, we show that the ubiquitination domain does not influence enzyme activity. By screening a panel of 39 gene deletion mutants, each lacking a different ubiquitin binding domain protein, we identify seven that are necessary to deliver Gpa1 to the vacuole compartment including four proteins (Ede1, Bul1, Ddi1, and Rup1) previously not known to be involved in this process. Finally, we show that proper endocytosis of the G protein is needed for sustained cellular morphogenesis and mating in response to pheromone stimulation. We conclude that a cascade of ubiquitin-binding proteins serves to deliver the G protein to its final destination within the cell. In this instance and in contrast to the previously characterized visual system, endocytosis from the plasma membrane is needed for proper signal transduction rather than for signal desensitization. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Data communications pocket book

    CERN Document Server

    Tooley, Michael

    2014-01-01

    Data Communications Pocket Book, Second Edition presents information relevant to data communication. The book provides tabulated reference materials with a brief description and diagrams. The coverage of the text includes abbreviations, terminal control codes, and conversion tables. The text will be of great use to individuals involved in the interconnection of computer systems.

  10. Pocket Guide to Transportation 2017

    Science.gov (United States)

    2017-05-01

    The BTS Pocket Guide to Transportation is a quick reference guide to significant transportation statistics. All the previous seven sections plus a new Major Trends section are included. This year marks the 20th anniversary of the Pocket Guide, which ...

  11. SQL Pocket Guide

    CERN Document Server

    Gennick, Jonathan

    2010-01-01

    If you're a programmer or database administrator who uses SQL in your day-to-day work, this popular pocket guide is the ideal on-the-job reference. You'll find many examples that address the language's complexity, along with key aspects of SQL used in IBM DB2 Release 9.7, MySQL 5.1, Oracle Database 11g Release 2, PostgreSQL 9.0, and Microsoft SQL Server 2008 Release 2. SQL Pocket Guide describes how these database systems implement SQL syntax for querying, managing transactions, and making changes to data. It also shows how the systems use SQL functions, regular expression syntax, and type c

  12. Software engineer's pocket book

    CERN Document Server

    Tooley, Michael

    2013-01-01

    Software Engineer's Pocket Book provides a concise discussion on various aspects of software engineering. The book is comprised of six chapters that tackle various areas of concerns in software engineering. Chapter 1 discusses software development, and Chapter 2 covers programming languages. Chapter 3 deals with operating systems. The book also tackles discrete mathematics and numerical computation. Data structures and algorithms are also explained. The text will be of great use to individuals involved in the specification, design, development, implementation, testing, maintenance, and qualit

  13. Python pocket reference

    CERN Document Server

    Lutz, Mark

    2014-01-01

    Updated for both Python 3.4 and 2.7, this convenient pocket guide is the perfect on-the-job quick reference. You’ll find concise, need-to-know information on Python types and statements, special method names, built-in functions and exceptions, commonly used standard library modules, and other prominent Python tools. The handy index lets you pinpoint exactly what you need.

  14. GDB Pocket Reference

    CERN Document Server

    Robbins, Arnold

    2009-01-01

    The GNU debugger is valuable for testing, fixing, and retesting software because it allows you to see exactly what's going on inside of a program as it's executing. This new pocket reference shows you how to specify a target for debugging, perform a careful examination to find the cause of program failure, and make quick changes for further testing. The guide covers several popular programming languages.

  15. Isolation and characterization of nucleotide-binding site and C-terminal leucine-rich repeat-resistance gene candidates in bananas.

    Science.gov (United States)

    Lu, Y; Xu, W H; Xie, Y X; Zhang, X; Pu, J J; Qi, Y X; Li, H P

    2011-12-15

    Commercial banana varieties are highly susceptible to fungal pathogens, as well as bacterial pathogens, nematodes, viruses, and insect pests. The largest known family of plant resistance genes encodes proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for the isolation of candidate genes in banana that may be involved in plant defense. Six degenerate PCR primers were designed to target NBS and additional domains were tested on commercial banana species Musa acuminata subsp malaccensis and the Musa AAB Group propagated in vitro and plants maintained in a greenhouse. Total DNA was isolated by a modified CTAB extraction technique. Four resistance gene analogs were amplified and deposited in GenBank and assigned numbers HQ199833-HQ199836. The predicted amino acid sequences compared to the amino acid sequences of known resistance genes (MRGL1, MRGL2, MRGL3, and MRGL4) revealed significant sequence similarity. The presence of consensus domains, namely kinase-1a, kinase-2 and hydrophobic domain, provided evidence that the cloned sequences belong to the typical non-Toll/interleukin-1 receptor-like domain NBS-LRR gene family.

  16. Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations.

    Science.gov (United States)

    Vernon, Robert M; Chong, P Andrew; Lin, Hong; Yang, Zhengrong; Zhou, Qingxian; Aleksandrov, Andrei A; Dawson, Jennifer E; Riordan, John R; Brouillette, Christie G; Thibodeau, Patrick H; Forman-Kay, Julie D

    2017-08-25

    Characterization of the second nucleotide-binding domain (NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR) has lagged behind research into the NBD1 domain, in part because NBD1 contains the F508del mutation, which is the dominant cause of cystic fibrosis. Research on NBD2 has also been hampered by the overall instability of the domain and the difficulty of producing reagents. Nonetheless, multiple disease-causing mutations reside in NBD2, and the domain is critical for CFTR function, because channel gating involves NBD1/NBD2 dimerization, and NBD2 contains the catalytically active ATPase site in CFTR. Recognizing the paucity of structural and biophysical data on NBD2, here we have defined a bioinformatics-based method for manually identifying stabilizing substitutions in NBD2, and we used an iterative process of screening single substitutions against thermal melting points to both produce minimally mutated stable constructs and individually characterize mutations. We present a range of stable constructs with minimal mutations to help inform further research on NBD2. We have used this stabilized background to study the effects of NBD2 mutations identified in cystic fibrosis (CF) patients, demonstrating that mutants such as N1303K and G1349D are characterized by lower stability, as shown previously for some NBD1 mutations, suggesting a potential role for NBD2 instability in the pathology of CF. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Association between single nucleotide polymorphisms of sterol regulatory element binding protein-2 gene and risk of knee osteoarthritis in a Chinese Han population.

    Science.gov (United States)

    Qiu, Xiao-Ming; Jin, Cheng-Tao; Wang, Wei

    2014-04-01

    To investigate associations between single nucleotide polymorphisms (SNPs) rs2228314 and rs2267443 in the sterol regulatory element binding protein-2 gene (SREBP-2) and knee osteoarthritis (OA) susceptibility in a Chinese Han population. SREBP-2 rs2228314 and rs2267443 polymorphisms were genotyped in patients with knee OA and age- and sex-matched OA-free controls from a Chinese Han population. A total of 402 patients with knee OA and 410 controls were enrolled in the study. GC and CC genotypes of rs2228314, and variant C, were associated with a significantly increased risk of knee OA. On stratification analysis, the association between the risk of OA and rs2228314 GC heterozygotes compared with GG homozygotes was stronger in females and those aged >65 years. In contrast, the GA and AA genotypes of rs2267443 were not significantly associated with the risk of knee OA, even after further stratification analysis according to age or sex. SREBP-2 rs2228314 G to C change and variant C genotype may contribute to knee OA risk in a Chinese Han population.

  18. Listeria monocytogenes activated p38 MAPK and induced IL-8 secretion in a nucleotide-binding oligomerization domain 1-dependent manner in endothelial cells.

    Science.gov (United States)

    Opitz, Bastian; Püschel, Anja; Beermann, Wiebke; Hocke, Andreas C; Förster, Stefanie; Schmeck, Bernd; van Laak, Vincent; Chakraborty, Trinad; Suttorp, Norbert; Hippenstiel, Stefan

    2006-01-01

    Nucleotide-binding oligomerization domain (Nod) proteins serve as intracellular pattern recognition molecules recognizing peptidoglycans. To further examine intracellular immune recognition, we used Listeria monocytogenes as an organism particularly amenable for studying innate immunity to intracellular pathogens. In contrast to wild-type L. monocytogenes, the nonpathogenic Listeria innocua, or L. monocytogenes mutants lacking internalin B or listeriolysin O, poorly invaded host cells and escaped into host cell cytoplasm, respectively, and were therefore used as controls. In this study, we show that only the invasive wild-type L. monocytogenes, but not the listeriolysin O- or internalin B-negative L. monocytogenes mutants or L. innocua, substantially induced IL-8 production in HUVEC. RNA interference and Nod1-overexpression experiments demonstrated that Nod1 is critically involved in chemokine secretion and NF-kappaB activation initiated by L. monocytogenes in human endothelial cells. Moreover, we show for the first time that Nod1 mediated activation of p38 MAPK signaling induced by L. monocytogenes. Finally, L. monocytogenes- and Nod1-induced IL-8 production was blocked by a specific p38 inhibitor. In conclusion, L. monocytogenes induced a Nod1-dependent activation of p38 MAPK signaling and NF-kappaB which resulted in IL-8 production in endothelial cells. Thus, Nod1 is an important component of a cytoplasmic surveillance pathway.

  19. Genome-Wide Comparative Analyses Reveal the Dynamic Evolution of Nucleotide-Binding Leucine-Rich Repeat Gene Family among Solanaceae Plants

    Science.gov (United States)

    Seo, Eunyoung; Kim, Seungill; Yeom, Seon-In; Choi, Doil

    2016-01-01

    Plants have evolved an elaborate innate immune system against invading pathogens. Within this system, intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors are known play critical roles in effector-triggered immunity (ETI) plant defense. We performed genome-wide identification and classification of NLR-coding sequences from the genomes of pepper, tomato, and potato using fixed criteria. We then compared genomic duplication and evolution features. We identified intact 267, 443, and 755 NLR-encoding genes in tomato, potato, and pepper genomes, respectively. Phylogenetic analysis and classification of Solanaceae NLRs revealed that the majority of NLR super family members fell into 14 subgroups, including a TIR-NLR (TNL) subgroup and 13 non-TNL subgroups. Specific subgroups have expanded in each genome, with the expansion in pepper showing subgroup-specific physical clusters. Comparative analysis of duplications showed distinct duplication patterns within pepper and among Solanaceae plants suggesting subgroup- or species-specific gene duplication events after speciation, resulting in divergent evolution. Taken together, genome-wide analysis of NLR family members provide insights into their evolutionary history in Solanaceae. These findings also provide important foundational knowledge for understanding NLR evolution and will empower broader characterization of disease resistance genes to be used for crop breeding. PMID:27559340

  20. Genome-wide identification and tissue-specific expression analysis of nucleotide binding site-leucine rich repeat gene family in Cicer arietinum (kabuli chickpea).

    Science.gov (United States)

    Sharma, Ranu; Rawat, Vimal; Suresh, C G

    2017-12-01

    The nucleotide binding site-leucine rich repeat (NBS-LRR) proteins play an important role in the defense mechanisms against pathogens. Using bioinformatics approach, we identified and annotated 104 NBS-LRR genes in chickpea. Phylogenetic analysis points to their diversification into two families namely TIR-NBS-LRR and non-TIR-NBS-LRR. Gene architecture revealed intron gain/loss events in this resistance gene family during their independent evolution into two families. Comparative genomics analysis elucidated its evolutionary relationship with other fabaceae species. Around 50% NBS-LRRs reside in macro-syntenic blocks underlining positional conservation along with sequence conservation of NBS-LRR genes in chickpea. Transcriptome sequencing data provided evidence for their transcription and tissue-specific expression. Four cis -regulatory elements namely WBOX, DRE, CBF, and GCC boxes, that commonly occur in resistance genes, were present in the promoter regions of these genes. Further, the findings will provide a strong background to use candidate disease resistance NBS-encoding genes and identify their specific roles in chickpea.

  1. Genome-wide Comparative Analyses Reveal the Dynamic Evolution of Nucleotide-Binding Leucine-Rich Repeat Gene Family among Solanaceae Plants

    Directory of Open Access Journals (Sweden)

    Eunyoung Seo

    2016-08-01

    Full Text Available Plants have evolved an elaborate innate immune system against invading pathogens. Within this system, intracellular nucleotide-binding leucine-rich repeat (NLR immune receptors are known play critical roles in effector-triggered immunity (ETI plant defense. We performed genome-wide identification and classification of NLR-coding sequences from the genomes of pepper, tomato, and potato using fixed criteria. We then compared genomic duplication and evolution features. We identified intact 267, 443, and 755 NLR-encoding genes in tomato, potato, and pepper genomes, respectively. Phylogenetic analyses and classification of Solanaceae NLRs revealed that the majority of NLR super family members fell into 14 subgroups, including a TIR-NLR (TNL subgroup and 13 non-TNL subgroups. Specific subgroups have expanded in each genome, with the expansion in pepper showing subgroup-specific physical clusters. Comparative analysis of duplications showed distinct duplication patterns within pepper and among Solanaceae plants suggesting subgroup- or species-specific gene duplication events after speciation, resulting in divergent evolution. Taken together, genome-wide analyses of NLR family members provide insights into their evolutionary history in Solanaceae. These findings also provide important foundational knowledge for understanding NLR evolution and will empower broader characterization of disease resistance genes to be used for crop breeding.

  2. A Cluster of Nucleotide-Binding Site–Leucine-Rich Repeat Genes Resides in a Barley Powdery Mildew Resistance Quantitative Trait Loci on 7HL

    Directory of Open Access Journals (Sweden)

    Carlos P. Cantalapiedra

    2016-07-01

    Full Text Available Powdery mildew causes severe yield losses in barley production worldwide. Although many resistance genes have been described, only a few have already been cloned. A strong QTL (quantitative trait locus conferring resistance to a wide array of powdery mildew isolates was identified in a Spanish barley landrace on the long arm of chromosome 7H. Previous studies narrowed down the QTL position, but were unable to identify candidate genes or physically locate the resistance. In this study, the exome of three recombinant lines from a high-resolution mapping population was sequenced and analyzed, narrowing the position of the resistance down to a single physical contig. Closer inspection of the region revealed a cluster of closely related NBS-LRR (nucleotide-binding site–leucine-rich repeat containing protein genes. Large differences were found between the resistant lines and the reference genome of cultivar Morex, in the form of PAV (presence-absence variation in the composition of the NBS-LRR cluster. Finally, a template-guided assembly was performed and subsequent expression analysis revealed that one of the new assembled candidate genes is transcribed. In summary, the results suggest that NBS-LRR genes, absent from the reference and the susceptible genotypes, could be functional and responsible for the powdery mildew resistance. The procedure followed is an example of the use of NGS (next-generation sequencing tools to tackle the challenges of gene cloning when the target gene is absent from the reference genome.

  3. Genome-Wide Comparative Analyses Reveal the Dynamic Evolution of Nucleotide-Binding Leucine-Rich Repeat Gene Family among Solanaceae Plants.

    Science.gov (United States)

    Seo, Eunyoung; Kim, Seungill; Yeom, Seon-In; Choi, Doil

    2016-01-01

    Plants have evolved an elaborate innate immune system against invading pathogens. Within this system, intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors are known play critical roles in effector-triggered immunity (ETI) plant defense. We performed genome-wide identification and classification of NLR-coding sequences from the genomes of pepper, tomato, and potato using fixed criteria. We then compared genomic duplication and evolution features. We identified intact 267, 443, and 755 NLR-encoding genes in tomato, potato, and pepper genomes, respectively. Phylogenetic analysis and classification of Solanaceae NLRs revealed that the majority of NLR super family members fell into 14 subgroups, including a TIR-NLR (TNL) subgroup and 13 non-TNL subgroups. Specific subgroups have expanded in each genome, with the expansion in pepper showing subgroup-specific physical clusters. Comparative analysis of duplications showed distinct duplication patterns within pepper and among Solanaceae plants suggesting subgroup- or species-specific gene duplication events after speciation, resulting in divergent evolution. Taken together, genome-wide analysis of NLR family members provide insights into their evolutionary history in Solanaceae. These findings also provide important foundational knowledge for understanding NLR evolution and will empower broader characterization of disease resistance genes to be used for crop breeding.

  4. Nucleotide Metabolism

    DEFF Research Database (Denmark)

    Martinussen, Jan; Willemoës, M.; Kilstrup, Mogens

    2011-01-01

    Metabolic pathways are connected through their utilization of nucleotides as supplier of energy, allosteric effectors, and their role in activation of intermediates. Therefore, any attempt to exploit a given living organism in a biotechnological process will have an impact on nucleotide metabolism...

  5. Rails Pocket Reference

    CERN Document Server

    Berry, Eric

    2008-01-01

    Rails 2.1 brings a new level of stability and power to this acclaimed web development framework, but keeping track of its numerous moving parts is still a chore. Rails Pocket Reference offers you a painless alternative to hunting for resources online, with brief yet thorough explanations of the most frequently used methods and structures supported by Rails 2.1, along with key concepts you need to work through the framework's most tangled corners. Organized to help you quickly find what you need, this book will not only get you up to speed on how Rails works, it also provides a handy referenc

  6. LINQ Pocket Reference

    CERN Document Server

    Albahari, Joseph

    2008-01-01

    Ready to take advantage of LINQ with C# 3.0? This guide has the detail you need to grasp Microsoft's new querying technology, and concise explanations to help you learn it quickly. And once you begin to apply LINQ, the book serves as an on-the-job reference when you need immediate reminders. All the examples in the LINQ Pocket Reference are preloaded into LINQPad, the highly praised utility that lets you work with LINQ interactively. Created by the authors and free to download, LINQPad will not only help you learn LINQ, it will have you thinking in LINQ. This reference explains: LINQ's ke

  7. Regular Expression Pocket Reference

    CERN Document Server

    Stubblebine, Tony

    2007-01-01

    This handy little book offers programmers a complete overview of the syntax and semantics of regular expressions that are at the heart of every text-processing application. Ideal as a quick reference, Regular Expression Pocket Reference covers the regular expression APIs for Perl 5.8, Ruby (including some upcoming 1.9 features), Java, PHP, .NET and C#, Python, vi, JavaScript, and the PCRE regular expression libraries. This concise and easy-to-use reference puts a very powerful tool for manipulating text and data right at your fingertips. Composed of a mixture of symbols and text, regular exp

  8. CSS Pocket Reference

    CERN Document Server

    Meyer, Eric A

    2007-01-01

    They say that good things come in small packages, and it's certainly true for this edition of CSS Pocket Reference. Completely revised and updated to reflect the latest Cascading Style Sheet specifications in CSS 2.1, this indispensable little book covers the most essential information that web designers and developers need to implement CSS effectively across all browsers. Inside, you'll find: A short introduction to the key concepts of CSS A complete alphabetical reference to all CSS 2.1 selectors and properties A chart displaying detailed information about CSS support for every style ele

  9. CSS Pocket Reference

    CERN Document Server

    Meyer, Eric

    2011-01-01

    When you're working with CSS and need a quick answer, CSS Pocket Reference delivers. This handy, concise book provides all of the essential information you need to implement CSS on the fly. Ideal for intermediate to advanced web designers and developers, the 4th edition is revised and updated for CSS3, the latest version of the Cascading Style Sheet specification. Along with a complete alphabetical reference to CSS3 selectors and properties, you'll also find a short introduction to the key concepts of CSS. Based on Cascading Style Sheets: The Definitive Guide, this reference is an easy-to-us

  10. NUnit Pocket Reference

    CERN Document Server

    Hamilton, Bill

    2009-01-01

    The open source NUnit framework is an excellent way to test .NET code as it is written, saving hundreds of QA hours and headaches. Unfortunately, some of those hours saved can be wasted trying to master this popular but under-documented framework. Proof that good things come in small packages, the NUnit Pocket Reference is everything you need to get NUnit up and working for you. It's the only book you'll need on this popular and practical new open source framework.

  11. Linux Desktop Pocket Guide

    CERN Document Server

    Brickner, David

    2005-01-01

    While Mac OS X garners all the praise from pundits, and Windows XP attracts all the viruses, Linux is quietly being installed on millions of desktops every year. For programmers and system administrators, business users, and educators, desktop Linux is a breath of fresh air and a needed alternative to other operating systems. The Linux Desktop Pocket Guide is your introduction to using Linux on five of the most popular distributions: Fedora, Gentoo, Mandriva, SUSE, and Ubuntu. Despite what you may have heard, using Linux is not all that hard. Firefox and Konqueror can handle all your web bro

  12. Newnes microprocessor pocket book

    CERN Document Server

    Money, Steve

    2014-01-01

    Newnes Microprocessor Pocket Book explains the basic hardware operation of a microprocessor and describes the actions of the various types of instruction that can be executed. A summary of the characteristics of many of the popular microprocessors is presented. Apart from the popular 8- and 16-bit microprocessors, some details are also given of the popular single chip microcomputers and of the reduced instruction set computer (RISC) type processors such as the Transputer, Novix FORTH processor, and Acorn ARM processor.Comprised of 15 chapters, this book discusses the principles involved in bot

  13. JDBC Pocket Reference

    CERN Document Server

    Bales, Donald

    2003-01-01

    JDBC--the Java Database Connectivity specification--is a complex set of application programming interfaces (APIs) that developers need to understand if they want their Java applications to work with databases. JDBC is so complex that even the most experienced developers need to refresh their memories from time to time on specific methods and details. But, practically speaking, who wants to stop and thumb through a weighty tutorial volume each time a question arises? The answer is the JDBC Pocket Reference, a data-packed quick reference that is both a time-saver and a lifesaver. The JDBC P

  14. Electronics pocket book

    CERN Document Server

    Parr, E A

    1981-01-01

    Electronics Pocket Book, Fourth Edition is a nonmathematical presentation of the many varied topics covered by electronics. The book tackles electron physics, electronic components (i.e. resistors, capacitors, and conductors), integrated circuits, and the principles of a.c. and d.c. amplifiers. The text also discusses oscillators, digital circuits, digital computers, and optoelectronics (i.e., sensors, emitters, and devices that utilize light). Communications (such as line and radio communications, transmitters, receivers, and digital techniques); the principles and examples of servosystems; a

  15. Windows Powershell Pocket Reference

    CERN Document Server

    Holmes, Lee

    2009-01-01

    This portable reference to Windows PowerShell summarizes both the command shell and scripting language, and provides a concise reference to the major tasks that make PowerShell so successful. Written by Microsoft PowerShell team member Lee Holmes, and excerpted from his Windows PowerShell Cookbook, Windows PowerShell Pocket Reference offers up-to-date coverage of PowerShell's 1.0 release. It's an ideal on-the-job tool for Windows administrators who don't have time to plow through huge books or search online.

  16. XSLT 10 Pocket Reference

    CERN Document Server

    Lenz, Evan

    2008-01-01

    XSLT is an essential tool for converting XML into other kinds of documents: HTML, PDF file, and many others. It's a critical technology for XML-based platforms such as Microsoft .NET, Sun Microsystems' Sun One, as well as for most web browsers and authoring tools. As useful as XSLT is, however, most people have a difficult time getting used to its peculiar characteristics. The ability to use advanced techniques depends on a clear and exact understanding of how XSLT templates work and interact. The XSLT 1.0 Pocket Reference from O'Reilly wants to make sure you achieve that level of understan

  17. Python pocket reference

    CERN Document Server

    Lutz, Mark

    2010-01-01

    This is the book to reach for when you're coding on the fly and need an answer now. It's an easy-to-use reference to the core language, with descriptions of commonly used modules and toolkits, and a guide to recent changes, new features, and upgraded built-ins -- all updated to cover Python 3.X as well as version 2.6. You'll also quickly find exactly what you need with the handy index. Written by Mark Lutz -- widely recognized as the world's leading Python trainer -- Python Pocket Reference, Fourth Edition, is the perfect companion to O'Reilly's classic Python tutorials, also written by Mark

  18. RTF Pocket Guide

    CERN Document Server

    Burke, Sean

    2008-01-01

    Rich Text Format, or RTF, is the internal markup language used by Microsoft Word and understood by dozens of other word processors. RTF is a universal file format that pervades practically every desktop. Because RTF is text, it's much easier to generate and process than binary .doc files. Any programmer working with word processing documents needs to learn enough RTF to get around, whether it's to format text for Word (or almost any other word processor), to make global changes to an existing document, or to convert Word files to (or from) another format. RTF Pocket Guide is a concise and e

  19. LRRK2 kinase activity is dependent on LRRK2 GTP binding capacity but independent of LRRK2 GTP binding.

    Directory of Open Access Journals (Sweden)

    Jean-Marc Taymans

    Full Text Available Leucine rich repeat kinase 2 (LRRK2 is a Parkinson's disease (PD gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.

  20. Large-Scale Analyses of Angiosperm Nucleotide-Binding Site-Leucine-Rich Repeat Genes Reveal Three Anciently Diverged Classes with Distinct Evolutionary Patterns1

    Science.gov (United States)

    Shao, Zhu-Qing; Xue, Jia-Yu; Wu, Ping; Zhang, Yan-Mei; Wu, Yue; Hang, Yue-Yu; Wang, Bin; Chen, Jian-Qun

    2016-01-01

    Nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes make up the largest plant disease resistance gene family (R genes), with hundreds of copies occurring in individual angiosperm genomes. However, the expansion history of NBS-LRR genes during angiosperm evolution is largely unknown. By identifying more than 6,000 NBS-LRR genes in 22 representative angiosperms and reconstructing their phylogenies, we present a potential framework of NBS-LRR gene evolution in the angiosperm. Three anciently diverged NBS-LRR classes (TNLs, CNLs, and RNLs) were distinguished with unique exon-intron structures and DNA motif sequences. A total of seven ancient TNL, 14 CNL, and two RNL lineages were discovered in the ancestral angiosperm, from which all current NBS-LRR gene repertoires were evolved. A pattern of gradual expansion during the first 100 million years of evolution of the angiosperm clade was observed for CNLs. TNL numbers remained stable during this period but were eventually deleted in three divergent angiosperm lineages. We inferred that an intense expansion of both TNL and CNL genes started from the Cretaceous-Paleogene boundary. Because dramatic environmental changes and an explosion in fungal diversity occurred during this period, the observed expansions of R genes probably reflect convergent adaptive responses of various angiosperm families. An ancient whole-genome duplication event that occurred in an angiosperm ancestor resulted in two RNL lineages, which were conservatively evolved and acted as scaffold proteins for defense signal transduction. Overall, the reconstructed framework of angiosperm NBS-LRR gene evolution in this study may serve as a fundamental reference for better understanding angiosperm NBS-LRR genes. PMID:26839128

  1. Large-Scale Analyses of Angiosperm Nucleotide-Binding Site-Leucine-Rich Repeat Genes Reveal Three Anciently Diverged Classes with Distinct Evolutionary Patterns.

    Science.gov (United States)

    Shao, Zhu-Qing; Xue, Jia-Yu; Wu, Ping; Zhang, Yan-Mei; Wu, Yue; Hang, Yue-Yu; Wang, Bin; Chen, Jian-Qun

    2016-04-01

    Nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes make up the largest plant disease resistance gene family (R genes), with hundreds of copies occurring in individual angiosperm genomes. However, the expansion history of NBS-LRR genes during angiosperm evolution is largely unknown. By identifying more than 6,000 NBS-LRR genes in 22 representative angiosperms and reconstructing their phylogenies, we present a potential framework of NBS-LRR gene evolution in the angiosperm. Three anciently diverged NBS-LRR classes (TNLs, CNLs, and RNLs) were distinguished with unique exon-intron structures and DNA motif sequences. A total of seven ancient TNL, 14 CNL, and two RNL lineages were discovered in the ancestral angiosperm, from which all current NBS-LRR gene repertoires were evolved. A pattern of gradual expansion during the first 100 million years of evolution of the angiosperm clade was observed for CNLs. TNL numbers remained stable during this period but were eventually deleted in three divergent angiosperm lineages. We inferred that an intense expansion of both TNL and CNL genes started from the Cretaceous-Paleogene boundary. Because dramatic environmental changes and an explosion in fungal diversity occurred during this period, the observed expansions of R genes probably reflect convergent adaptive responses of various angiosperm families. An ancient whole-genome duplication event that occurred in an angiosperm ancestor resulted in two RNL lineages, which were conservatively evolved and acted as scaffold proteins for defense signal transduction. Overall, the reconstructed framework of angiosperm NBS-LRR gene evolution in this study may serve as a fundamental reference for better understanding angiosperm NBS-LRR genes. © 2016 American Society of Plant Biologists. All Rights Reserved.

  2. Invasive Streptococcus mutans induces inflammatory cytokine production in human aortic endothelial cells via regulation of intracellular toll-like receptor 2 and nucleotide-binding oligomerization domain 2.

    Science.gov (United States)

    Nagata, E; Oho, T

    2017-04-01

    Streptococcus mutans, the primary etiologic agent of dental caries, can gain access to the bloodstream and has been associated with cardiovascular disease. However, the roles of S. mutans in inflammation in cardiovascular disease remain unclear. The aim of this study was to examine cytokine production induced by S. mutans in human aortic endothelial cells (HAECs) and to evaluate the participation of toll-like receptors (TLRs) and cytoplasmic nucleotide-binding oligomerization domain (NOD) -like receptors in HAECs. Cytokine production by HAECs was determined using enzyme-linked immunosorbent assays, and the expression of TLRs and NOD-like receptors was evaluated by real-time polymerase chain reaction, flow cytometry and immunocytochemistry. The involvement of TLR2 and NOD2 in cytokine production by invaded HAECs was examined using RNA interference. The invasion efficiencies of S. mutans strains were evaluated by means of antibiotic protection assays. Five of six strains of S. mutans of various serotypes induced interleukin-6, interleukin-8 and monocyte chemoattractant protein-1 production by HAECs. All S. mutans strains upregulated TLR2 and NOD2 mRNA levels in HAECs. Streptococcus mutans Xc upregulated the intracellular TLR2 and NOD2 protein levels in HAECs. Silencing of the TLR2 and NOD2 genes in HAECs invaded by S. mutans Xc led to a reduction in interleukin-6, interleukin-8 and monocyte chemoattractant protein-1 production. Cytokine production induced by invasive S. mutans via intracellular TLR2 and NOD2 in HAECs may be associated with inflammation in cardiovascular disease. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Impact of the [delta]F508 Mutation in First Nucleotide-binding Domain of Human Cystic Fibrosis Transmembrane Conductance Regulator on Domain Folding and Structure

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, Hal A.; Zhao, Xun; Wang, Chi; Sauder, J. Michael; Rooney, Isabelle; Noland, Brian W.; Lorimer, Don; Kearins, Margaret C.; Conners, Kris; Condon, Brad; Maloney, Peter C.; Guggino, William B.; Hunt, John F.; Emtage, Spencer (SG); (Columbia); (JHU)

    2010-07-19

    Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.

  4. Suppression among alleles encoding nucleotide-binding-leucine-rich repeat resistance proteins interferes with resistance in F1 hybrid and allele-pyramided wheat plants.

    Science.gov (United States)

    Stirnweis, Daniel; Milani, Samira D; Brunner, Susanne; Herren, Gerhard; Buchmann, Gabriele; Peditto, David; Jordan, Tina; Keller, Beat

    2014-09-01

    The development of high-yielding varieties with broad-spectrum durable disease resistance is the ultimate goal of crop breeding. In plants, immune receptors of the nucleotide-binding-leucine-rich repeat (NB-LRR) class mediate race-specific resistance against pathogen attack. When employed in agriculture this type of resistance is often rapidly overcome by newly adapted pathogen races. The stacking of different resistance genes or alleles in F1 hybrids or in pyramided lines is a promising strategy for achieving more durable resistance. Here, we identify a molecular mechanism which can negatively interfere with the allele-pyramiding approach. We show that pairwise combinations of different alleles of the powdery mildew resistance gene Pm3 in F1 hybrids and stacked transgenic wheat lines can result in suppression of Pm3-based resistance. This effect is independent of the genetic background and solely dependent on the Pm3 alleles. Suppression occurs at the post-translational level, as levels of RNA and protein in the suppressed alleles are unaffected. Using a transient expression system in Nicotiana benthamiana, the LRR domain was identified as the domain conferring suppression. The results of this study suggest that the expression of closely related NB-LRR resistance genes or alleles in the same genotype can lead to dominant-negative interactions. These findings provide a molecular explanation for the frequently observed ineffectiveness of resistance genes introduced from the secondary gene pool into polyploid crop species and mark an important step in overcoming this limitation. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  5. Characterization of Rabbit Nucleotide-Binding Oligomerization Domain 1 (NOD1 and the Role of NOD1 Signaling Pathway during Bacterial Infection

    Directory of Open Access Journals (Sweden)

    Mengjiao Guo

    2017-10-01

    Full Text Available Nucleotide-binding oligomerization domain 1 (NOD1 is the most prominent of all NOD-like receptors, which in the mammalian innate immune system, serve as intracellular receptors for pathogens and endogenous molecules during tissue injury. From rabbit kidney cells, we cloned rabbit NOD1 (rNOD1 and identified an N-terminal caspase activation and recruitment domain, a central NACHT domain, and C-terminal leucine-rich repeat domains. rNOD1 was expressed in all tested tissues; infection with Escherichia coli induced significantly higher expression in the spleen, liver, and kidney compared to other tissues. The overexpression of rNOD1 induced the expression of proinflammatory cytokines Il1b, Il6, Il8, Ifn-γ, and Tnf and defensins, including Defb124, Defb125, Defb128, Defb135, and Np5 via activation of the nuclear factor (NF-κB pathway. Overexpression of rNOD1 inhibited the growth of E. coli, whereas knockdown of rNOD1 or inhibition of the NF-κB pathway promoted the growth of E. coli. rNOD1 colocalized with LC3, upregulated autophagy pathway protein LC3-II, and increased autolysosome formation in RK-13 cells infected with E. coli. In summary, our results explain the primary signaling pathway and antibacterial ability of rNOD1, as well as the induction of autophagy that it mediates. Such findings suggest that NOD1 could contribute to therapeutic strategies such as targets of new vaccine adjuvants or drugs.

  6. Inhibition by divalent metal ions of human histidine triad nucleotide binding protein1 (hHint1), a regulator of opioid analgesia and neuropathic pain.

    Science.gov (United States)

    Shah, Rachit; Chou, Tsui-Fen; Maize, Kimberly M; Strom, Alexander; Finzel, Barry C; Wagner, Carston R

    2017-09-23

    Human histidine triad nucleotide binding protein 1 (hHint1) is a purine nucleoside phosphoramidase and adenylate hydrolase that has emerged as a potential therapeutic target for the management of pain. However, the molecular mechanism of Hint1 in the signaling pathway has remained less clear. The role of metal ions in regulating postsynaptic transmission is well known, and the active site of hHint1 contains multiple histidines. Here we have investigated the effect of divalent metal ions (Cd 2+ , Cu 2+ , Mg 2+ , Mn 2+ , Ni 2+ , and Zn 2+ ) on the structural integrity and catalytic activity of hHint1. With the exception of Mg 2+ , all the divalent ions inhibited hHint1, the rank of order was found to be Cu 2+ >Zn 2+ >Cd 2+ ≥Ni 2+ >Mn 2+ based on their IC 50 and k in /K I values. A crystal structure of hHint1 with bound Cu 2+ is described to explain the competitive reversible inactivation of hHint1 by divalent cations. All the metal ions exhibited time- and concentration- dependent inhibition, with the rate of inactivation highly dependent on alterations of the C-terminus. With the exception of Cu 2+ ; restoration of inhibition was observed for all the metal ions after treatment with EDTA. Our studies reveal a loss in secondary structure and aggregation of hHint1 upon incubation with 10-fold excess of copper. Thus, hHint1 appears to be structurally sensitive to irreversible inactivation by copper, which may be of neurotoxicological and pharmacological significance. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. The arabidopsis cyclic nucleotide interactome

    KAUST Repository

    Donaldson, Lara Elizabeth

    2016-05-11

    Background Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotide-dependent signaling mechanisms in plants since the downstream target proteins remain unknown. This is largely due to the fact that bioinformatics searches fail to identify plant homologs of protein kinases and phosphodiesterases that are the main targets of cyclic nucleotides in animals. Methods An affinity purification technique was used to identify cyclic nucleotide binding proteins in Arabidopsis thaliana. The identified proteins were subjected to a computational analysis that included a sequence, transcriptional co-expression and functional annotation analysis in order to assess their potential role in plant cyclic nucleotide signaling. Results A total of twelve cyclic nucleotide binding proteins were identified experimentally including key enzymes in the Calvin cycle and photorespiration pathway. Importantly, eight of the twelve proteins were shown to contain putative cyclic nucleotide binding domains. Moreover, the identified proteins are post-translationally modified by nitric oxide, transcriptionally co-expressed and annotated to function in hydrogen peroxide signaling and the defence response. The activity of one of these proteins, GLYGOLATE OXIDASE 1, a photorespiratory enzyme that produces hydrogen peroxide in response to Pseudomonas, was shown to be repressed by a combination of cGMP and nitric oxide treatment. Conclusions We propose that the identified proteins function together as points of cross-talk between cyclic nucleotide, nitric oxide and reactive oxygen species signaling during the defence response.

  8. In silico docking of forchlorfenuron (FCF to septins suggests that FCF interferes with GTP binding.

    Directory of Open Access Journals (Sweden)

    Dimitrios Angelis

    Full Text Available Septins are GTP-binding proteins that form cytoskeleton-like filaments, which are essential for many functions in eukaryotic organisms. Small molecule compounds that disrupt septin filament assembly are valuable tools for dissecting septin functions with high temporal control. To date, forchlorfenuron (FCF is the only compound known to affect septin assembly and functions. FCF dampens the dynamics of septin assembly inducing the formation of enlarged stable polymers, but the underlying mechanism of action is unknown. To investigate how FCF binds and affects septins, we performed in silico simulations of FCF docking to all available crystal structures of septins. Docking of FCF with SEPT2 and SEPT3 indicated that FCF interacts preferentially with the nucleotide-binding pockets of septins. Strikingly, FCF is predicted to form hydrogen bonds with residues involved in GDP-binding, mimicking nucleotide binding. FCF docking with the structure of SEPT2-GppNHp, a nonhydrolyzable GTP analog, and SEPT7 showed that FCF may assume two alternative non-overlapping conformations deeply into and on the outer side of the nucleotide-binding pocket. Surprisingly, FCF was predicted to interact with the P-loop Walker A motif GxxxxGKS/T, which binds the phosphates of GTP, and the GTP specificity motif AKAD, which interacts with the guanine base of GTP, and highly conserved amino acids including a threonine, which is critical for GTP hydrolysis. Thus, in silico FCF exhibits a conserved mechanism of binding, interacting with septin signature motifs and residues involved in GTP binding and hydrolysis. Taken together, our results suggest that FCF stabilizes septins by locking them into a conformation that mimics a nucleotide-bound state, preventing further GTP binding and hydrolysis. Overall, this study provides the first insight into how FCF may bind and stabilize septins, and offers a blueprint for the rational design of FCF derivatives that could target septins with

  9. DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα-encoding (GNAS genomic imprinting domain are associated with performance traits

    Directory of Open Access Journals (Sweden)

    Mullen Michael P

    2011-01-01

    Full Text Available Abstract Background Genes which are epigenetically regulated via genomic imprinting can be potential targets for artificial selection during animal breeding. Indeed, imprinted loci have been shown to underlie some important quantitative traits in domestic mammals, most notably muscle mass and fat deposition. In this candidate gene study, we have identified novel associations between six validated single nucleotide polymorphisms (SNPs spanning a 97.6 kb region within the bovine guanine nucleotide-binding protein Gs subunit alpha gene (GNAS domain on bovine chromosome 13 and genetic merit for a range of performance traits in 848 progeny-tested Holstein-Friesian sires. The mammalian GNAS domain consists of a number of reciprocally-imprinted, alternatively-spliced genes which can play a major role in growth, development and disease in mice and humans. Based on the current annotation of the bovine GNAS domain, four of the SNPs analysed (rs43101491, rs43101493, rs43101485 and rs43101486 were located upstream of the GNAS gene, while one SNP (rs41694646 was located in the second intron of the GNAS gene. The final SNP (rs41694656 was located in the first exon of transcripts encoding the putative bovine neuroendocrine-specific protein NESP55, resulting in an aspartic acid-to-asparagine amino acid substitution at amino acid position 192. Results SNP genotype-phenotype association analyses indicate that the single intronic GNAS SNP (rs41694646 is associated (P ≤ 0.05 with a range of performance traits including milk yield, milk protein yield, the content of fat and protein in milk, culled cow carcass weight and progeny carcass conformation, measures of animal body size, direct calving difficulty (i.e. difficulty in calving due to the size of the calf and gestation length. Association (P ≤ 0.01 with direct calving difficulty (i.e. due to calf size and maternal calving difficulty (i.e. due to the maternal pelvic width size was also observed at the rs

  10. DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα)-encoding (GNAS) genomic imprinting domain are associated with performance traits

    Science.gov (United States)

    2011-01-01

    Background Genes which are epigenetically regulated via genomic imprinting can be potential targets for artificial selection during animal breeding. Indeed, imprinted loci have been shown to underlie some important quantitative traits in domestic mammals, most notably muscle mass and fat deposition. In this candidate gene study, we have identified novel associations between six validated single nucleotide polymorphisms (SNPs) spanning a 97.6 kb region within the bovine guanine nucleotide-binding protein Gs subunit alpha gene (GNAS) domain on bovine chromosome 13 and genetic merit for a range of performance traits in 848 progeny-tested Holstein-Friesian sires. The mammalian GNAS domain consists of a number of reciprocally-imprinted, alternatively-spliced genes which can play a major role in growth, development and disease in mice and humans. Based on the current annotation of the bovine GNAS domain, four of the SNPs analysed (rs43101491, rs43101493, rs43101485 and rs43101486) were located upstream of the GNAS gene, while one SNP (rs41694646) was located in the second intron of the GNAS gene. The final SNP (rs41694656) was located in the first exon of transcripts encoding the putative bovine neuroendocrine-specific protein NESP55, resulting in an aspartic acid-to-asparagine amino acid substitution at amino acid position 192. Results SNP genotype-phenotype association analyses indicate that the single intronic GNAS SNP (rs41694646) is associated (P ≤ 0.05) with a range of performance traits including milk yield, milk protein yield, the content of fat and protein in milk, culled cow carcass weight and progeny carcass conformation, measures of animal body size, direct calving difficulty (i.e. difficulty in calving due to the size of the calf) and gestation length. Association (P ≤ 0.01) with direct calving difficulty (i.e. due to calf size) and maternal calving difficulty (i.e. due to the maternal pelvic width size) was also observed at the rs43101491 SNP. Following

  11. PockDrug-Server: a new web server for predicting pocket druggability on holo and apo proteins.

    Science.gov (United States)

    Hussein, Hiba Abi; Borrel, Alexandre; Geneix, Colette; Petitjean, Michel; Regad, Leslie; Camproux, Anne-Claude

    2015-07-01

    Predicting protein pocket's ability to bind drug-like molecules with high affinity, i.e. druggability, is of major interest in the target identification phase of drug discovery. Therefore, pocket druggability investigations represent a key step of compound clinical progression projects. Currently computational druggability prediction models are attached to one unique pocket estimation method despite pocket estimation uncertainties. In this paper, we propose 'PockDrug-Server' to predict pocket druggability, efficient on both (i) estimated pockets guided by the ligand proximity (extracted by proximity to a ligand from a holo protein structure) and (ii) estimated pockets based solely on protein structure information (based on amino atoms that form the surface of potential binding cavities). PockDrug-Server provides consistent druggability results using different pocket estimation methods. It is robust with respect to pocket boundary and estimation uncertainties, thus efficient using apo pockets that are challenging to estimate. It clearly distinguishes druggable from less druggable pockets using different estimation methods and outperformed recent druggability models for apo pockets. It can be carried out from one or a set of apo/holo proteins using different pocket estimation methods proposed by our web server or from any pocket previously estimated by the user. PockDrug-Server is publicly available at: http://pockdrug.rpbs.univ-paris-diderot.fr. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Substitution of Active Site Tyrosines with Tryptophan Alters the Free Energy for Nucleotide Flipping by Human Alkyladenine DNA Glycosylase†

    Science.gov (United States)

    Hendershot, Jenna M.; Wolfe, Abigail E.; O'Brien, Patrick J.

    2011-01-01

    Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of structurally diverse alkylated and oxidized purine lesions from DNA to initiate the base excision repair pathway. Recognition of a base lesion requires flipping of the damaged nucleotide into a relatively open active site pocket between two conserved tyrosine residues, Y127 and Y159. We have mutated each of these amino acids to tryptophan and measured the kinetic effects on the nucleotide flipping and base excision steps. The Y127W and Y159W mutant proteins have robust glycosylase activity toward DNA containing 1,N6-ethenoadenine (εA), within 4-fold of that of the wildtype enzyme, raising the possibility that tryptophan fluorescence could be used to probe the DNA binding and nucleotide flipping steps. Stopped-flow fluorescence was used to compare the time-dependent changes in tryptophan fluorescence and εA fluorescence. For both mutants, the tryptophan fluorescence exhibited two-step binding with essentially identical rate constants as were observed for the εA fluorescence changes. These results provide evidence that AAG forms an initial recognition complex in which the active site pocket is perturbed and the stacking of the damaged base is disrupted. Upon complete nucleotide flipping, there is further quenching of the tryptophan fluorescence with coincident quenching of the εA fluorescence. Although these mutations do not have large effects on the rate constant for excision of εA, there are dramatic effects on the rate constants for nucleotide flipping that result in 40 to 100-fold decreases in the flipping equilibrium relative to wildtype. Most of this effect is due to an increased rate of unflipping, but surprisingly the Y159W mutation causes a 5-fold increase in the rate constant for flipping. The large effect on the equilibrium for nucleotide flipping explains the greater deleterious effects that these mutations have on the glycosylase activity toward base lesions that are in

  13. Pocket Guide to Transportation 2018

    Science.gov (United States)

    2018-01-01

    The 2018 BTS Pocket Guide to Transportation is a quick reference guide that provides transportation statistics at your fingertips. It provides key information and highlights major trends on the U.S. transportation system. This year features a new and...

  14. Pocket Guide to Transportation 2012

    Science.gov (United States)

    2012-01-01

    The Bureau of Transportation Statistics (BTS) of the Research and Innovative Technology Administration produces the Pocket Guide to Transportation as a compact resource that provides snapshots of the U.S. transportation system and highlights major tr...

  15. Nucleotide sequences within the U5 region of the viral RNA genome are the major determinants for an human immunodeficiency virus type 1 to maintain a primer binding site complementary to tRNA(His).

    Science.gov (United States)

    Zhang, Z; Kang, S M; LeBlanc, A; Hajduk, S L; Morrow, C D

    1996-12-15

    The initiation of reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome requires cellular tRNA(Lys,3) as a primer and occurs at a site in the viral RNA genome, designated as the primer binding site (PBS), which is complementary to the 3'-terminal 18 nucleotides of tRNA(Lys,3). We previously described an HIV-1 virus [designated as HXB2(His-AC)], which contained a sequence within the U5 region complementary to the anticodon region of tRNA(His) in addition to a PBS complementary to the 3'-terminal 18 nucleotides of the tRNA(His). That virus maintained a PBS complementary to tRNA(His) after extended in vitro culture (Wakefield et al., J. Virol. 70, 966-975, 1996). In the present study, we report that subcloning a 200-base-pair DNA fragment encompassing the U5 and PBS regions from an integrated provirus of HXB2(His-AC) back into the wild-type genome (pHXB2) resulted in an infectious virus, designated as HXB2(His-AC-gac), which again stably maintained a PBS complementary to tRNA(His). DNA sequence analysis of the 200-base-pair region revealed only three nucleotide changes from HXB2(His-AC): a T-to-G change at nucleotide 174, a G-to-A change at nucleotide 181, and a T-to-C change at nucleotide 200. The new mutant virus replicated in CD4+ Sup T1 cells similarly to the wild-type virus. Comparison of the nucleotide sequence of nucleocapsid gene of the wild-type and HXB2 (His-AC-gac) virus revealed no differences. Although we found numerous mutations in the reverse transcriptase gene in proviral clones derived from HXB2 (His-AC-gac), no common mutations were found among the 13 clones examined. Comparison of the virion-associated tRNAs of HXB2(His-AC-gac) with those of the wild type revealed that both viruses incorporated a similar subset of cellular tRNAs, with tRNA(Lys,3) being the predominant tRNA found within virions. There was no selective enrichment for tRNA(His) within virions of HXB2(His-AC-gac) virus which selectively use tRNA(His) to

  16. Phosphorylation- and nucleotide-binding-induced changes to the stability and hydrogen exchange patterns of JNK1ß1 provide insight into its mechanisms of activation

    CSIR Research Space (South Africa)

    Owen, GR

    2014-10-01

    Full Text Available binding on both inactive and active JNK1ß1 using the ATP analogue, 5'-adenylyl-imidodiphosphate (AMP-PNP). JNK1ß1 phosphorylation alters HX in regions involved in catalysis and substrate binding, changes that can be ascribed to functional modifications...

  17. Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the. alpha. subunit of the stimulatory guanine nucleotide-binding regulatory protein

    Energy Technology Data Exchange (ETDEWEB)

    Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G. (Burroughs Wellcome Co., Research Triangle Park, NC (USA))

    1988-08-01

    ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5{prime}-({alpha}-{sup 32}P)triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an {alpha} subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera.

  18. Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the α subunit of the stimulatory guanine nucleotide-binding regulatory protein

    International Nuclear Information System (INIS)

    Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G.

    1988-01-01

    ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the α subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5'-[α- 32 P]triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an α subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera

  19. Rad and Rem are non-canonical G-proteins with respect to the regulatory role of guanine nucleotide binding in Ca(V)1.2 channel regulation.

    Science.gov (United States)

    Chang, Donald D; Colecraft, Henry M

    2015-12-01

    Rad and Rem are Ras-like G-proteins linked to diverse cardiovascular functions and pathophysiology. Understanding how Rad and Rem are regulated is important for deepened insights into their pathophysiological roles. As in other Ras-like G-proteins, Rad and Rem contain a conserved guanine-nucleotide binding domain (G-domain). Canonically, G-domains are key control modules, functioning as nucleotide-regulated switches of G-protein activity. Whether Rad and Rem G-domains conform to this canonical paradigm is ambiguous. Here, we used multiple functional measurements in HEK293 cells and cardiomyocytes (Ca(V)1.2 currents, Ca(2+) transients, Ca(V)β binding) as biosensors to probe the role of the G-domain in regulation of Rad and Rem function. We utilized Rad(S105N) and Rem(T94N), which are the cognate mutants to Ras(S17N), a dominant-negative variant of Ras that displays decreased nucleotide binding affinity. In HEK293 cells, over-expression of either Rad(S105N) or Rem(T94N) strongly inhibited reconstituted Ca(V)1.2 currents to the same extent as their wild-type (wt) counterparts, contrasting with reports that Rad(S105N) is functionally inert in HEK293 cells. Adenovirus-mediated expression of either wt Rad or Rad(S105N) in cardiomyocytes dramatically blocked L-type calcium current (I(Ca,L)) and inhibited Ca(2+)-induced Ca(2+) release, contradicting reports that Rad(S105N) acts as a dominant negative in heart. By contrast, Rem(T94N) was significantly less effective than wt Rem at inhibiting I(Ca,L) and Ca(2+) transients in cardiomyocytes. FRET analyses in cardiomyocytes revealed that both Rad(S105N) and Rem(T94N) had moderately reduced binding affinity for Ca(V)βs relative to their wt counterparts. The results indicate Rad and Rem are non-canonical G-proteins with respect to the regulatory role of their G-domain in Ca(V)1.2 regulation. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  20. Characterization of the Escherichia coli prsA1-encoded mutant phosphoribosylpyrophosphate synthetase identifies a divalent cation-nucleotide binding site

    DEFF Research Database (Denmark)

    Bower, Stanley G.; Harlow, Kenneth W.; Switzer, Robert L.

    1989-01-01

    by chemical determination of the amino acid sequence of a tryptic peptide derived from the purified mutant enzyme. The mutation lies at the N-terminal end of a 16 residue sequence that is highly conserved in E. coli, Bacillus subtilis, and rat PRPP synthetases and has the following consensus sequence......The prsA1 allele, specifying a mutant Escherichia coli phosphoribosylpyrophosphate (PRPP) synthetase, has been cloned. The mutation was shown by nucleotide sequence analysis to result from substitution of Asp-128 (GAT) in the wild type by Ala (GCT) in prsA1. This alteration was confirmed...

  1. Reasons not to ''pocket shoot''

    International Nuclear Information System (INIS)

    McCarroll, K.A.; Fisher, D.R.; Cawthon, L.A.; Donovan, K.R.; Roszler, M.H.; Kling, G.A.

    1987-01-01

    The authors' large population of intravenous drug abusers (IVDA) has increasingly resorted to supraclavicular central venous injection for vascular access. Few reports of complications associated with the practice of supraclavicular ''pocket'' injection have appeared in the radiologic literature. This exhibit demonstrates the complications associated with this practice, including pneumothorax, mycotic aneurysm, arteriovenous fistual, jugular vein thrombosis, cellulitis, foreign body and neck abscess. In addition, they show examples of sternoclavicular osteomyelities. The anatomy of the ''pocket'' and the pathophysiology and radiographic manifestations of these complications are reviewed

  2. Nucleotide Excision Repair Lesion-Recognition Protein Rad4 Captures a Pre-Flipped Partner Base in a Benzo[a]pyrene-Derived DNA Lesion: How Structure Impacts the Binding Pathway

    Science.gov (United States)

    2017-01-01

    The xeroderma pigmentosum C protein complex (XPC) recognizes a variety of environmentally induced DNA lesions and is the key in initiating their repair by the nucleotide excision repair (NER) pathway. When bound to a lesion, XPC flips two nucleotide pairs that include the lesion out of the DNA duplex, yielding a productively bound complex that can lead to successful lesion excision. Interestingly, the efficiencies of NER vary greatly among different lesions, influencing their toxicity and mutagenicity in cells. Though differences in XPC binding may influence NER efficiency, it is not understood whether XPC utilizes different mechanisms to achieve productive binding with different lesions. Here, we investigated the well-repaired 10R-(+)-cis-anti-benzo[a]pyrene-N2-dG (cis-B[a]P-dG) DNA adduct in a duplex containing normal partner C opposite the lesion. This adduct is derived from the environmental pro-carcinogen benzo[a]pyrene and is likely to be encountered by NER in the cell. We have extensively investigated its binding to the yeast XPC orthologue, Rad4, using umbrella sampling with restrained molecular dynamics simulations and free energy calculations. The NMR solution structure of this lesion in duplex DNA has shown that the dC complementary to the adducted dG is flipped out of the DNA duplex in the absence of XPC. However, it is not known whether the “pre-flipped” base would play a role in its recognition by XPC. Our results show that Rad4 first captures the displaced dC, which is followed by a tightly coupled lesion-extruding pathway for productive binding. This binding path differs significantly from the one deduced for the small cis-syn cyclobutane pyrimidine dimer lesion opposite mismatched thymines [MuH., (2015) Biochemistry, 54(34), 5263−726270861]. The possibility of multiple paths that lead to productive binding to XPC is consistent with the versatile lesion recognition by XPC that is required for successful NER. PMID:28460163

  3. Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes

    Science.gov (United States)

    Neely, Robert K.; Tamulaitis, Gintautas; Chen, Kai; Kubala, Marta; Siksnys, Virginijus; Jones, Anita C.

    2009-01-01

    Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition and adjust the DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy of 2-aminopurine-labelled DNA in complex with each of these enzymes in solution to explore the nucleotide flipping mechanism and to obtain a detailed picture of the molecular environment of the extrahelical bases. We also report the first study of the 7-bp cutter, PfoI, whose recognition sequence (T/CCNGGA) overlaps with that of the Ecl18kI-type enzymes, and for which the crystal structure is unknown. The time-resolved fluorescence experiments reveal that PfoI also uses base flipping as part of its DNA recognition mechanism and that the extrahelical bases are captured by PfoI in binding pockets whose structures are quite different to those of the structurally characterized enzymes Ecl18kI, PspGI and EcoRII-C. The fluorescence decay parameters of all the enzyme-DNA complexes are interpreted to provide insight into the mechanisms used by these four restriction enzymes to flip and recognize bases and the relationship between nucleotide flipping and DNA cleavage. PMID:19740769

  4. Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes.

    Science.gov (United States)

    Neely, Robert K; Tamulaitis, Gintautas; Chen, Kai; Kubala, Marta; Siksnys, Virginijus; Jones, Anita C

    2009-11-01

    Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition and adjust the DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy of 2-aminopurine-labelled DNA in complex with each of these enzymes in solution to explore the nucleotide flipping mechanism and to obtain a detailed picture of the molecular environment of the extrahelical bases. We also report the first study of the 7-bp cutter, PfoI, whose recognition sequence (T/CCNGGA) overlaps with that of the Ecl18kI-type enzymes, and for which the crystal structure is unknown. The time-resolved fluorescence experiments reveal that PfoI also uses base flipping as part of its DNA recognition mechanism and that the extrahelical bases are captured by PfoI in binding pockets whose structures are quite different to those of the structurally characterized enzymes Ecl18kI, PspGI and EcoRII-C. The fluorescence decay parameters of all the enzyme-DNA complexes are interpreted to provide insight into the mechanisms used by these four restriction enzymes to flip and recognize bases and the relationship between nucleotide flipping and DNA cleavage.

  5. Newnes electronics assembly pocket book

    CERN Document Server

    Brindley, Keith

    2013-01-01

    Produced in association with the Engineering Training Authority with contributions from dozens of people in the electronics industry. The material covers common skills in electrical and electronic engineering and concentrates mainly on wiring and assembly. 'Newnes Electronics Assembly Pocket Book' is for electronics technicians, students and apprentices.

  6. Ecology-A Pocket Guide

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 5; Issue 8. Ecology – A Pocket Guide. Renee M Borges. Book Review Volume 5 Issue 8 August 2000 pp 99-102. Fulltext. Click here to view fulltext PDF. Permanent link: http://www.ias.ac.in/article/fulltext/reso/005/08/0099-0102. Author Affiliations.

  7. The Potato Nucleotide-Binding Leucine-Rich Repeat (NLR) Immune Receptor Rx1 is a Pathogen Dependent DNA-Deforming Protein

    NARCIS (Netherlands)

    Fenyk, S.; Townsend, P.D.; Dixon, C.H.; Spies, G.B.; Campillo, A.S.E.; Slootweg, E.J.; Westerhof, L.B.; Gawehns, F.K.K.; Knight, M.R.; Sharples, G.J.; Goverse, A.; Palsson, L.O.; Takken, F.L.W.; Cann, M.J.

    2015-01-01

    Plant NLR proteins enable cells to respond to pathogen attack. Several NLRs act in the nucleus, however, conserved nuclear targets that support their role in immunity are unknown. Previously we noted a structural homology between the NB domain of NLRs and DNA replication origin-binding Cdc6/Orc1

  8. The roles of the RIIβ linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of the type IIβ protein kinase A: a small angle x-ray and neutron scattering study.

    Science.gov (United States)

    Blumenthal, Donald K; Copps, Jeffrey; Smith-Nguyen, Eric V; Zhang, Ping; Heller, William T; Taylor, Susan S

    2014-10-10

    Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1-280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Novel somatic single nucleotide variants within the RNA binding protein hnRNP A1 in multiple sclerosis patients [v1; ref status: indexed, http://f1000r.es/3nv

    Directory of Open Access Journals (Sweden)

    Sangmin Lee

    2014-06-01

    Full Text Available Some somatic single nucleotide variants (SNVs are thought to be pathogenic, leading to neurological disease. We hypothesized that heterogeneous nuclear ribonuclear protein A1 (hnRNP A1, an autoantigen associated with multiple sclerosis (MS would contain SNVs. MS patients develop antibodies to hnRNP A1293-304, an epitope within the M9 domain (AA268-305 of hnRNP A1. M9 is hnRNP A1’s nucleocytoplasmic transport domain, which binds transportin-1 (TPNO-1 and allows for hnRNP A1’s transport into and out of the nucleus. Genomic DNA sequencing of M9 revealed nine novel SNVs that resulted in an amino acid substitution in MS patients that were not present in controls. SNVs occurred within the TPNO-1 binding domain (hnRNP A1268-289 and the MS IgG epitope (hnRNP A1293-304, within M9.  In contrast to the nuclear localization of wild type (WT hnRNP A1, mutant hnRNP A1 mis-localized to the cytoplasm, co-localized with stress granules and caused cellular apoptosis. Whilst WT hnRNP A1 bound TPNO-1, mutant hnRNP A1 showed reduced TPNO-1 binding. These data suggest SNVs in hnRNP A1 might contribute to pathogenesis of MS.

  10. Novel somatic single nucleotide variants within the RNA binding protein hnRNP A1 in multiple sclerosis patients [v2; ref status: indexed, http://f1000r.es/4dh

    Directory of Open Access Journals (Sweden)

    Sangmin Lee

    2014-09-01

    Full Text Available Some somatic single nucleotide variants (SNVs are thought to be pathogenic, leading to neurological disease. We hypothesized that heterogeneous nuclear ribonuclear protein A1 (hnRNP A1, an autoantigen associated with multiple sclerosis (MS would contain SNVs. MS patients develop antibodies to hnRNP A1293-304, an epitope within the M9 domain (AA268-305 of hnRNP A1. M9 is hnRNP A1’s nucleocytoplasmic transport domain, which binds transportin-1 (TPNO-1 and allows for hnRNP A1’s transport into and out of the nucleus. Genomic DNA sequencing of M9 revealed nine novel SNVs that resulted in an amino acid substitution in MS patients that were not present in controls. SNVs occurred within the TPNO-1 binding domain (hnRNP A1268-289 and the MS IgG epitope (hnRNP A1293-304, within M9.  In contrast to the nuclear localization of wild type (WT hnRNP A1, mutant hnRNP A1 mis-localized to the cytoplasm, co-localized with stress granules and caused cellular apoptosis. Whilst WT hnRNP A1 bound TPNO-1, mutant hnRNP A1 showed reduced TPNO-1 binding. These data suggest SNVs in hnRNP A1 might contribute to pathogenesis of MS.

  11. Structure and site-directed mutagenesis of a flavoprotein from Escherichia coli that reduces nitrocompounds: alteration of pyridine nucleotide binding by a single amino acid substitution.

    Science.gov (United States)

    Kobori, T; Sasaki, H; Lee, W C; Zenno, S; Saigo, K; Murphy, M E; Tanokura, M

    2001-01-26

    The crystal structure of a major oxygen-insensitive nitroreductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-A resolution. This enzyme is a homodimeric flavoprotein with one FMN cofactor per monomer and catalyzes reduction of nitrocompounds using NADPH. The structure exhibits an alpha + beta-fold, and is comprised of a central domain and an excursion domain. The overall structure of NfsA is similar to the NADPH-dependent flavin reductase of Vibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site. On the basis of the crystal structure of NfsA and its alignment with the V. harveyi flavin reductase and the NADPH-dependent nitro/flavin reductase of Bacillus subtilis, residues Arg(203) and Arg(208) of the loop region between helices I and J in the vicinity of the catalytic center FMN is predicted as a determinant for NADPH binding. The R203A mutant results in a 33-fold increase in the K(m) value for NADPH indicating that the side chain of Arg(203) plays a key role in binding NADPH possibly to interact with the 2'-phosphate group.

  12. Exploration of electrostatic interaction in the hydrophobic pocket of lysozyme: Importance of ligand-induced perturbation of the secondary structure on the mode of binding of exogenous ligand and possible consequences.

    Science.gov (United States)

    Panja, Sudipta; Halder, Mintu

    2016-08-01

    Exogenous ligand binding can be adequate to alter the secondary structure of biomolecules besides other external stimuli. In such cases, structural alterations can complicate on the nature of interaction with the exogenous molecules. In order to accommodate the exogenous ligand, the biomolecule has to unfold resulting in a considerable change to its properties. If the bound ligand can be unbound, the biomolecule gets the opportunity to refold back and return to its native state. Keeping this in mind, we have purposely investigated the interaction of tartrazine (TZ), a well abundant azo food colorant, with two homologous lysozymes, namely, human lysozyme (HLZ) and chicken egg white lysozyme (CEWLZ) in physiological pH condition. The binding of TZ with lysozymes has been identified to accompany a ligand-induced secondary structure alteration as indicated by the circular dichroism spectra, and the reduction of α-helical content is more with HLZ than CEWLZ. Interestingly, the binding is identified to occur in the electronic ground state of TZ with lysozyme in its hydrophobic cavity, containing excess of positive charge, predominantly via electrostatic interaction. With increase of salinity of the medium the protein tends to refold back due to wakening of electrostatic forces and consequent reduction of strength of ligand interaction and unbinding. The entropy enthalpy compensation (EEC) has been probed to understand the binding features and it is found that CEWLZ-TZ shows better compensation than HLZ-TZ complex. This is presumably due to the fact that with CEWLZ the binding does not accompany substantial change in the protein secondary structure and hence ineffective to scramble the EEC. The present study initiates the importance of ligand-perturbed structural alteration of biomolecule in controlling the thermodynamics of binding. If there is a considerable alteration of the protein secondary structure due to binding, it is indicative that such changes should bring in

  13. Developmental changes in the role of a pertussis toxin sensitive guanine nucleotide binding protein in the rat cardiac alpha1-adrenergic system

    International Nuclear Information System (INIS)

    Han, H.M.

    1989-01-01

    During development, the cardiac alpha 1 -adrenergic chronotropic response changes from positive in the neonate to negative in the adult. This thesis examined the possibility of a developmental change in coupling of a PT-sensitive G-protein to the alpha 1 -adrenergic receptor. Radioligand binding experiments performed with the iodinated alpha 1 -selective radioligand [ 125 I]-I-2-[β-(4-hydroxphenyl)ethylaminomethyl]tetralone ([ 125 I]-IBE 2254) demonstrated that the alpha 1 -adrenergic receptor is coupled to a G-protein in both neonatal and adult rat hearts. However, in the neonate the alpha 1 -adrenergic receptor is coupled to a PT-insensitive G-protein, whereas in the adult the alpha 1 -adrenergic receptor is coupled to both a PT-insensitive and a PT-sensitive G-protein. Consistent with the results from binding experiments, PT did not have any effect on the alpha 1 -mediated positive chronotropic response in the neonate, whereas in the adult the alpha 1 -mediated negative chronotropic response was completely converted to a positive one after PT-treatment. This thesis also examined the possibility of an alteration in coupling of the alpha 1 -adrenergic receptor to its effector under certain circumstances such as high potassium (K + ) depolarization in nerve-muscle (NM) co-cultures, a system which has been previously shown to be a convenient in vitro model to study the mature inhibitory alpha 1 -response

  14. In vitro studies of immunoglobulin heavy-chain binding protein (BiP, GRP78). Interactions of BiP with newly synthesized proteins and adenine nucleotides

    International Nuclear Information System (INIS)

    Kassenbrock, C.K.

    1988-01-01

    Here we examine the interaction of BiP with newly synthesized polypeptides in an in vitro protein translations-translocation system. We find that BiP forms tight complexes with nonglycosylated yeast invertase and incorrectly disulfide-bonded prolactin but not with glycosylated invertase or correctly disulfide-bonded prolactin. Moreover, BiP associates detectably only with completed chains of prolactin, not with chains undergoing synthesis. We conclude that BiP recognizes and binds with high affinity to aberrantly folded or aberrantly glycosylated polypeptides in vitro, but not to all nascent chains as they are folding. BiP also binds APT and can be purified by APT affinity chromatography. We show that submicromolar levels of ATP or ADP decrease the rate of absorption of 125 I-BiP to nitrocellulose filters coated with protein or nonionic detergents. ATP and ADP also protect portions of BiP from proteolytic degradation. In contrast, micromolar levels of AMP increase the rate of adsorption and the rate of proteolytic degradation of BiP. We also show that an ATPase activity co-purifies with BiP, but its slow turnover number suggests a regulatory, rather than a functional role. The BiP-associated ATPase shares several properties with the related cytoplasmic protein, HSC70/clathrin uncoating ATPase

  15. In Silico Molecular Modeling and Docking Studies on Novel Mutants (E229V, H225P and D230C) of the Nucleotide-Binding Domain of Homo sapiens Hsp70.

    Science.gov (United States)

    Elengoe, Asita; Hamdan, Salehhuddin

    2017-12-01

    In this study, we explored the possibility of determining the synergistic interactions between nucleotide-binding domain (NBD) of Homo sapiens heat-shock 70 kDa protein (Hsp70) and E1A 32 kDa of adenovirus serotype 5 motif (PNLVP) in the efficiency of killing of tumor cells in cancer treatment. At present, the protein interaction between NBD and PNLVP motif is still unknown, but believed to enhance the rate of virus replication in tumor cells. Three mutant models (E229V, H225P and D230C) were built and simulated, and their interactions with PNLVP motif were studied. The PNLVP motif showed the binding energy and intermolecular energy values with the novel E229V mutant at -7.32 and -11.2 kcal/mol. The E229V mutant had the highest number of hydrogen bonds (7). Based on the root mean square deviation, root mean square fluctuation, hydrogen bonds, salt bridge, secondary structure, surface-accessible solvent area, potential energy and distance matrices analyses, it was proved that the E229V had the strongest and most stable interaction with the PNLVP motif among all the four protein-ligand complex structures. The knowledge of this protein-ligand complex model would help in designing Hsp70 structure-based drug for cancer therapy.

  16. Mechanisms of haplotype divergence at the RGA08 nucleotide-binding leucine-rich repeat gene locus in wild banana (Musa balbisiana).

    Science.gov (United States)

    Baurens, Franc-Christophe; Bocs, Stéphanie; Rouard, Mathieu; Matsumoto, Takashi; Miller, Robert N G; Rodier-Goud, Marguerite; MBéguié-A-MBéguié, Didier; Yahiaoui, Nabila

    2010-07-16

    Comparative sequence analysis of complex loci such as resistance gene analog clusters allows estimating the degree of sequence conservation and mechanisms of divergence at the intraspecies level. In banana (Musa sp.), two diploid wild species Musa acuminata (A genome) and Musa balbisiana (B genome) contribute to the polyploid genome of many cultivars. The M. balbisiana species is associated with vigour and tolerance to pests and disease and little is known on the genome structure and haplotype diversity within this species. Here, we compare two genomic sequences of 253 and 223 kb corresponding to two haplotypes of the RGA08 resistance gene analog locus in M. balbisiana "Pisang Klutuk Wulung" (PKW). Sequence comparison revealed two regions of contrasting features. The first is a highly colinear gene-rich region where the two haplotypes diverge only by single nucleotide polymorphisms and two repetitive element insertions. The second corresponds to a large cluster of RGA08 genes, with 13 and 18 predicted RGA genes and pseudogenes spread over 131 and 152 kb respectively on each haplotype. The RGA08 cluster is enriched in repetitive element insertions, in duplicated non-coding intergenic sequences including low complexity regions and shows structural variations between haplotypes. Although some allelic relationships are retained, a large diversity of RGA08 genes occurs in this single M. balbisiana genotype, with several RGA08 paralogs specific to each haplotype. The RGA08 gene family has evolved by mechanisms of unequal recombination, intragenic sequence exchange and diversifying selection. An unequal recombination event taking place between duplicated non-coding intergenic sequences resulted in a different RGA08 gene content between haplotypes pointing out the role of such duplicated regions in the evolution of RGA clusters. Based on the synonymous substitution rate in coding sequences, we estimated a 1 million year divergence time for these M. balbisiana haplotypes. A

  17. Mechanisms of haplotype divergence at the RGA08 nucleotide-binding leucine-rich repeat gene locus in wild banana (Musa balbisiana

    Directory of Open Access Journals (Sweden)

    Miller Robert NG

    2010-07-01

    Full Text Available Abstract Background Comparative sequence analysis of complex loci such as resistance gene analog clusters allows estimating the degree of sequence conservation and mechanisms of divergence at the intraspecies level. In banana (Musa sp., two diploid wild species Musa acuminata (A genome and Musa balbisiana (B genome contribute to the polyploid genome of many cultivars. The M. balbisiana species is associated with vigour and tolerance to pests and disease and little is known on the genome structure and haplotype diversity within this species. Here, we compare two genomic sequences of 253 and 223 kb corresponding to two haplotypes of the RGA08 resistance gene analog locus in M. balbisiana "Pisang Klutuk Wulung" (PKW. Results Sequence comparison revealed two regions of contrasting features. The first is a highly colinear gene-rich region where the two haplotypes diverge only by single nucleotide polymorphisms and two repetitive element insertions. The second corresponds to a large cluster of RGA08 genes, with 13 and 18 predicted RGA genes and pseudogenes spread over 131 and 152 kb respectively on each haplotype. The RGA08 cluster is enriched in repetitive element insertions, in duplicated non-coding intergenic sequences including low complexity regions and shows structural variations between haplotypes. Although some allelic relationships are retained, a large diversity of RGA08 genes occurs in this single M. balbisiana genotype, with several RGA08 paralogs specific to each haplotype. The RGA08 gene family has evolved by mechanisms of unequal recombination, intragenic sequence exchange and diversifying selection. An unequal recombination event taking place between duplicated non-coding intergenic sequences resulted in a different RGA08 gene content between haplotypes pointing out the role of such duplicated regions in the evolution of RGA clusters. Based on the synonymous substitution rate in coding sequences, we estimated a 1 million year

  18. Multisite contacts involved in coupling of the beta-adrenergic receptor with the stimulatory guanine-nucleotide-binding regulatory protein. Structural and functional studies by beta-receptor-site-specific synthetic peptides.

    Science.gov (United States)

    Münch, G; Dees, C; Hekman, M; Palm, D

    1991-06-01

    Synthetic peptides, 12-22 amino acid residues long, comprising the presumed coupling sites of the beta-adrenergic receptor with the stimulatory guanine-nucleotide-binding regulatory protein (Gs), were examined for their ability to modulate Gs activation in turkey erythrocyte membranes. Three peptides corresponding to the second cytoplasmic loop, the N-terminal region of the third cytoplasmic loop, and the N-terminal region of the putative fourth cytoplasmic loop, compete synergistically with the hormone-stimulated receptor for Gs activation with median effector concentrations of 15-35 microM, or 3-4 microM for combinations of two peptides. One peptide, corresponding to the C-terminal region of the third cytoplasmic loop, carries the unique ability to activate the Gs-adenylate-cyclase complex independent of the signalling state of the receptor. These observations are consistent with a dynamic model of receptor-mediated G-protein activation in membranes, where domains composed of the second, third and fourth intracellular loop of the receptor bind to and are interactive with the G-protein heterotrimer, resulting in ligand-induced conformational changes of the receptor. In response to hormone binding, the extent or the number of sites involved in interaction with Gs may be readjusted using a fourth site. Modulation of coupling sites may elicit congruent conformational changes within the Gs heterotrimer, with qualitatively different effects on GTP/GDP exchange in the alpha subunit of Gs and downstream effector regulation. This model corroborates and expands a similar model suggested for activated rhodopsin-transducin interaction [König, B., Arendt, A., McDowell, J. H., Kahlert, M., Hargrave, P. A. & Hofmann, K. P. (1989) Proc. Natl Acad. Sci. USA 86, 6878-6882].

  19. Identification of the SNP (Single Nucleotide Polymorphism for Fatty Acid Composition Associated with Beef Flavor-related FABP4 (Fatty Acid Binding Protein 4 in Korean Cattle

    Directory of Open Access Journals (Sweden)

    Dong-yep Oh

    2012-07-01

    Full Text Available In this study, we investigated the relationship between unsaturated fatty acids influencing beef flavor and four types of SNPs (c.280A>G, c.388G>A, c.408G>C and c.456A>G located at exon 2, 3 and 4 of the FABP4 gene, which is a fatty acid binding protein 4 in Korean cattle (n = 513. When analyzing the relationship between single genotype, fatty acids and carcass trait, individuals of GG, GG, CC and GG genotypes that are homozygotes, had a higher content of unsaturated fatty acids and marbling scores than other genotypes (p<0.05. Then, haplotype block showed strong significant relationships not only with unsaturated fatty acids (54.73%, but also with marbling scores (5.82 in ht1×ht1 group (p<0.05. This ht1×ht1 group showed significant differences with unsaturated fatty acids and marbling scores that affected beef flavor in Korean cattle. Therefore, it can be inferred that the ht1×ht1 types might be valuable new markers for use in the improvement of Korean cattle.

  20. Newnes electronics engineers pocket book

    CERN Document Server

    Brindley, Keith

    2013-01-01

    This book is packed with information and material which everyone involved in electronics will find indispensable. Now when you need to know a transistor's characteristics, or an integrated circuit's pinout details, simply look it up! The book is full of tables, symbols, formulae, conversions and illustrations.Promotion via the new Newnes Pocket Book catalogue to the electronics trade will drive sales into the book trade Covers component data; encapsulations; pin-outs; symbols & codings Extensive material on conversion factors, formulae; units and relationships

  1. JavaScript Pocket Reference

    CERN Document Server

    Flanagan, David

    1998-01-01

    JavaScript is a powerful, object-based scripting language that can be embedded directly in HTML pages. It allows you to create dynamic, interactive Web-based applications that run completely within a Web browser -- JavaScript is the language of choice for developing Dynamic HTML (DHTML) content. JavaScript can be integrated effectively with CGI and Java to produce sophisticated Web applications, although, in many cases, JavaScript eliminates the need for complex CGI scripts and Java applets altogether. The JavaScript Pocket Reference is a companion volume to JavaScript: The Definitive Guide

  2. Electrical engineering a pocket reference

    CERN Document Server

    Schmidt-Walter, Heinz

    2007-01-01

    This essential reference offers you a well-organized resource for accessing the basic electrical engineering knowledge you need for your work. Whether you're an experienced engineer who appreciates an occasional refresher in key areas, or a student preparing to enter the field, Electrical Engineering: A Pocket Reference provides quick and easy access to fundamental principles and their applications. You also find an extensive collection of time-saving equations that help simplify your daily projects.Supported with more than 500 diagrams and figures, 60 tables, and an extensive index, this uniq

  3. Windows Vista Administrator's Pocket Guide

    CERN Document Server

    Stanek, William R

    2007-01-01

    Portable and precise, this pocket-sized guide delivers immediate answers for the day-to-day administration of Windows Vista. Zero in on core support and maintenance tasks using quick-reference tables, instructions, and lists. You'll get the precise information you need to solve problems and get the job done-whether you're at your desk or in the field! Get fast facts to: Install and configure Windows Vista-and optimize the user workspaceMaintain operating system components, hardware devices, and driversCreate user and group accounts-and control rights and permissionsAdminister group policy se

  4. Perl/Tk Pocket Reference

    CERN Document Server

    Lidie, Stephen

    1998-01-01

    The Perl/Tk Pocket Reference is a companion volume to Learning Perl/Tk, an O'Reilly Animal Guide. Learning Perl/Tk is a tutorial for Perl/Tk, the extension to Perl for creating graphical user interfaces. With Tk, Perl programs can be window-based rather than command-line based, with buttons, entry fields, listboxes, menus, scrollbars, balloons, tables, dialogs, and more. And Perl/Tk programs run on UNIX and Windows-based computers. This small book is a handy reference guide geared toward the advanced Perl/Tk programmer. Novice Perl/Tk programmers will find that its compact size gives th

  5. Oracle Data Dictionary Pocket Reference

    CERN Document Server

    Kreines, David

    2003-01-01

    If you work with Oracle, then you don't need to be told that the data dictionary is large and complex, and grows larger with each new Oracle release. It's one of the basic elements of the Oracle database you interact with regularly, but the sheer number of tables and views makes it difficult to remember which view you need, much less the name of the specific column. Want to make it simpler? The Oracle Data Dictionary Pocket Reference puts all the information you need right at your fingertips. Its handy and compact format lets you locate the table and view you need effortlessly without stoppin

  6. Pocket atlas of radiographic anatomy

    International Nuclear Information System (INIS)

    Moeller, T.B.; Reif, E.; Stark, P.

    1993-01-01

    The 'Pocket Atlas of Radiographic Anatomy' presents 170 radiographs of the various body regions of adults, showing only the normal radiographic anatomy. Each radiograph is supplemented on the opposite page by a drawing of the particular body region. There is no commenting text, but the drawings are provided with captions in English. The atlas is a useful guide for interpreting radiographs. The pictures are arranged in chapters entitled as follows: Skeletal Imaging (skull, spine, upper extremity), lower extremity; Miscellaneous Plain Films (chest, mammogram, trachea, lung tomograms); Contrast Examinations (gastrointestinal tract, intravenous contrast examinations, arthrography, angiography); Special Examinations (myelograms, lymphangiograms, bronchograms, sialograms). (UWA). 348 figs [de

  7. Probing the Metabotropic Glutamate Receptor 5 (mGlu5) Positive Allosteric Modulator (PAM) Binding Pocket: Discovery of Point Mutations That Engender a “Molecular Switch” in PAM Pharmacology

    Science.gov (United States)

    Gregory, Karen J.; Nguyen, Elizabeth D.; Reiff, Sean D.; Squire, Emma F.; Stauffer, Shaun R.; Lindsley, Craig W.; Meiler, Jens

    2013-01-01

    Positive allosteric modulation of metabotropic glutamate receptor subtype 5 (mGlu5) is a promising novel approach for the treatment of schizophrenia and cognitive disorders. Allosteric binding sites are topographically distinct from the endogenous ligand (orthosteric) binding site, allowing for co-occupation of a single receptor with the endogenous ligand and an allosteric modulator. Negative allosteric modulators (NAMs) inhibit and positive allosteric modulators (PAMs) enhance the affinity and/or efficacy of the orthosteric agonist. The molecular determinants that govern mGlu5 modulator affinity versus cooperativity are not well understood. Focusing on the modulators based on the acetylene scaffold, we sought to determine the molecular interactions that contribute to PAM versus NAM pharmacology. Generation of a comparative model of the transmembrane-spanning region of mGlu5 served as a tool to predict and interpret the impact of mutations in this region. Application of an operational model of allosterism allowed for determination of PAM and NAM affinity estimates at receptor constructs that possessed no detectable radioligand binding as well as delineation of effects on affinity versus cooperativity. Novel mutations within the transmembrane domain (TM) regions were identified that had differential effects on acetylene PAMs versus 2-methyl-6-(phenylethynyl)-pyridine, a prototypical NAM. Three conserved amino acids (Y658, T780, and S808) and two nonconserved residues (P654 and A809) were identified as key determinants of PAM activity. Interestingly, we identified two point mutations in TMs 6 and 7 that, when mutated, engender a mode switch in the pharmacology of certain PAMs. PMID:23444015

  8. NUCLEOTIDES IN INFANT FEEDING

    Directory of Open Access Journals (Sweden)

    L.G. Mamonova

    2007-01-01

    Full Text Available The article reviews the application of nucleotides-metabolites, playing a key role in many biological processes, for the infant feeding. The researcher provides the date on the nucleotides in the women's milk according to the lactation stages. She also analyzes the foreign experience in feeding newborns with nucleotides-containing milk formulas. The article gives a comparison of nucleotides in the adapted formulas represented in the domestic market of the given products.Key words: children, feeding, nucleotides.

  9. The Type IV Pilus Assembly ATPase PilB of Myxococcus xanthus Interacts with the Inner Membrane Platform Protein PilC and the Nucleotide-binding Protein PilM.

    Science.gov (United States)

    Bischof, Lisa Franziska; Friedrich, Carmen; Harms, Andrea; Søgaard-Andersen, Lotte; van der Does, Chris

    2016-03-25

    Type IV pili (T4P) are ubiquitous bacterial cell surface structures, involved in processes such as twitching motility, biofilm formation, bacteriophage infection, surface attachment, virulence, and natural transformation. T4P are assembled by machinery that can be divided into the outer membrane pore complex, the alignment complex that connects components in the inner and outer membrane, and the motor complex in the inner membrane and cytoplasm. Here, we characterize the inner membrane platform protein PilC, the cytosolic assembly ATPase PilB of the motor complex, and the cytosolic nucleotide-binding protein PilM of the alignment complex of the T4P machinery ofMyxococcus xanthus PilC was purified as a dimer and reconstituted into liposomes. PilB was isolated as a monomer and bound ATP in a non-cooperative manner, but PilB fused to Hcp1 ofPseudomonas aeruginosaformed a hexamer and bound ATP in a cooperative manner. Hexameric but not monomeric PilB bound to PilC reconstituted in liposomes, and this binding stimulated PilB ATPase activity. PilM could only be purified when it was stabilized by a fusion with a peptide corresponding to the first 16 amino acids of PilN, supporting an interaction between PilM and PilN(1-16). PilM-N(1-16) was isolated as a monomer that bound but did not hydrolyze ATP. PilM interacted directly with PilB, but only with PilC in the presence of PilB, suggesting an indirect interaction. We propose that PilB interacts with PilC and with PilM, thus establishing the connection between the alignment and the motor complex. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Two disparate ligand binding sites in the human P2Y1 receptor

    Science.gov (United States)

    Zhang, Dandan; Gao, Zhan-Guo; Zhang, Kaihua; Kiselev, Evgeny; Crane, Steven; Wang, Jiang; Paoletta, Silvia; Yi, Cuiying; Ma, Limin; Zhang, Wenru; Han, Gye Won; Liu, Hong; Cherezov, Vadim; Katritch, Vsevolod; Jiang, Hualiang; Stevens, Raymond C.; Jacobson, Kenneth A.; Zhao, Qiang; Wu, Beili

    2015-01-01

    In response to adenosine 5′-diphosphate, the P2Y1 receptor (P2Y1R) facilitates platelet aggregation, and thus serves as an important antithrombotic drug target. Here we report the crystal structures of the human P2Y1R in complex with a nucleotide antagonist MRS2500 at 2.7Å resolution, and with a non-nucleotide antagonist BPTU at 2.2Å resolution. The structures reveal two distinct ligand binding sites, providing atomic details of P2Y1R’s unique ligand binding modes. MRS2500 recognizes a binding site within the seven transmembrane bundle of P2Y1R, which, however, is different in shape and location from the nucleotide binding site in previously determined P2Y12R structure. BPTU binds to an allosteric pocket on the external receptor interface with the lipid bilayer, making it the first structurally characterized selective G protein-coupled receptor (GPCR) ligand located entirely outside of the helical bundle. These high-resolution insights into P2Y1R should enable discovery of new orthosteric and allosteric antithrombotic drugs with reduced adverse effects. PMID:25822790

  11. Computer simulation of heterogeneous single nucleotide polymorphisms in the catalase gene indicates structural changes in the enzyme active site, NADPH-binding and tetramerization domains: a genetic predisposition for an altered catalase in patients with vitiligo?

    Science.gov (United States)

    Wood, John M; Gibbons, Nicholas C J; Chavan, Bhaven; Schallreuter, Karin U

    2008-04-01

    Patients with vitiligo have low levels/activities of catalase in their lesional and non-lesional epidermis as well as in their epidermal melanocytes under in vitro conditions while the levels of catalase mRNA are unaltered. This defect leads to a build-up of hydrogen peroxide (H(2)O(2)) in the 10(-3) m range in the epidermis of these patients. In this context, it was realized that 10(-3) m H(2)O(2) deactivates catalase. Along this line, it was also suspected that catalase in patients with vitiligo possesses a special sensitivity to this reactive oxygen species (ROS), and indeed several heterozygous single nucleotide polymorphisms (SNPs) have been documented in the cat gene of these patients. Based on the 3D structure of human catalase monomer, we have modelled the influence of three selected SNPs on the enzyme active site, on the NADPH- as well as the tetramerization-binding domains. Our results show that these SNPs severely alter catalase structurally, which in turn should make the enzyme more susceptible to ROS compared with wild-type enzyme. Taken together, the work presented herein together with the earlier results on SNPs in the cat gene suggests a genetic predisposition for an altered catalase in patients with vitiligo.

  12. Clinical characteristics of the eardrum retraction pocket

    Directory of Open Access Journals (Sweden)

    Ješić Snežana

    2003-01-01

    Full Text Available Development of the eardrum retraction pocket, as pathologic finding, depends on Eustachian tube dysfunction, onset of the middle ear infection and site of development of retraction on the eardrum. The study is aimed at: 1. Determining the incidence of eardrum retraction pocket and cholesteatoma within it, as well as at the degree of eardrum retraction; 2. Determining of association between eardrum retraction pocket and changes of the eardrum mucosaand parstensa of the tympanic membrane; 3. Determining of onset and intensity of the bone destruction in eardrum retraction pocket; 4. Examining of Eustachian tube function based on time of mucocilliary transport according to the type of the eardrum retraction pocket. The study is based on the retrospective analysis of the results obtained from the patients treated at the Institute of Oto-Rhino-Laryngology and Maxillofacial Surgery Clinical Centre of Serbia in Belgrade for the diagnosis of the chronic suppurative otitis who underwent otosurgical procedures during the six-year period, from 1996-2001. In our series of 540 patients subjected to otosurgical treatment, the incidence of the retraction pocket of the eardrum was 11.23%. Onset of more severe degree of eardrum retraction was most frequent in the attic. Cholesteatoma was detected in 82.2% of patients of the group with the attic-retraction pocket of the eardrum, as well as in 25% of patients of the group of tensa-sinus retraction pocket of the eardrum. Atrophic changes of the tympanic membrane pars tensa were detected in almost all tensa-sinus retraction pockets of the eardrum. Approximately one half of the attic-retraction pockets of the eardrum were accompanied by eardrum atrophy. Bone destruction of the auditory ossicles was limited to the long process of incus and superior structures of stapes. Time of the mucocilliary transport was significantly longer (p<0.01 in attic-retraction pocket of the eardrum than in tensa-sinus retraction pocket of

  13. ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2-Msh3 are differentially modulated by Mismatch and Double-strand Break Repair DNA substrates

    Science.gov (United States)

    Kumar, Charanya; Eichmiller, Robin; Wang, Bangchen; Williams, Gregory M.; Bianco, Piero R.; Surtees, Jennifer A.

    2014-01-01

    In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3′ non-homologous tail removal (3′NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3′ NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3′ NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3′ NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype. PMID:24746922

  14. GPR17: Molecular modeling and dynamics studies of the 3-D structure and purinergic ligand binding features in comparison with P2Y receptors

    Directory of Open Access Journals (Sweden)

    Ranghino Graziella

    2008-06-01

    Full Text Available Abstract Background GPR17 is a G-protein-coupled receptor located at intermediate phylogenetic position between two distinct receptor families: the P2Y and CysLT receptors for extracellular nucleotides and cysteinyl-LTs, respectively. We previously showed that GPR17 can indeed respond to both classes of endogenous ligands and to synthetic compounds active at the above receptor families, thus representing the first fully characterized non-peptide "hybrid" GPCR. In a rat brain focal ischemia model, the selective in vivo knock down of GPR17 by anti-sense technology or P2Y/CysLT antagonists reduced progression of ischemic damage, thus highlighting GPR17 as a novel therapeutic target for stroke. Elucidation of the structure of GPR17 and of ligand binding mechanisms are the necessary steps to obtain selective and potent drugs for this new potential target. On this basis, a 3-D molecular model of GPR17 embedded in a solvated phospholipid bilayer and refined by molecular dynamics simulations has been the first aim of this study. To explore the binding mode of the "purinergic" component of the receptor, the endogenous agonist UDP and two P2Y receptor antagonists demonstrated to be active on GPR17 (MRS2179 and cangrelor were then modeled on the receptor. Results Molecular dynamics simulations suggest that GPR17 nucleotide binding pocket is similar to that described for the other P2Y receptors, although only one of the three basic residues that have been typically involved in ligand recognition is conserved (Arg255. The binding pocket is enclosed between the helical bundle and covered at the top by EL2. Driving interactions are H-bonds and salt bridges between the 6.55 and 6.52 residues and the phosphate moieties of the ligands. An "accessory" binding site in a region formed by the EL2, EL3 and the Nt was also found. Conclusion Nucleotide binding to GPR17 occurs on the same receptor regions identified for already known P2Y receptors. Agonist

  15. A harpin elicitor induces the expression of a coiled-coil nucleotide binding leucine rich repeat (CC-NB-LRR) defense signaling gene and others functioning during defense to parasitic nematodes.

    Science.gov (United States)

    Aljaafri, Weasam A R; McNeece, Brant T; Lawaju, Bisho R; Sharma, Keshav; Niruala, Prakash M; Pant, Shankar R; Long, David H; Lawrence, Kathy S; Lawrence, Gary W; Klink, Vincent P

    2017-12-01

    The bacterial effector harpin induces the transcription of the Arabidopsis thaliana NON-RACE SPECIFIC DISEASE RESISTANCE 1/HARPIN INDUCED1 (NDR1/HIN1) coiled-coil nucleotide binding leucine rich repeat (CC-NB-LRR) defense signaling gene. In Glycine max, Gm-NDR1-1 transcripts have been detected within root cells undergoing a natural resistant reaction to parasitism by the syncytium-forming nematode Heterodera glycines, functioning in the defense response. Expressing Gm-NDR1-1 in Gossypium hirsutum leads to resistance to Meloidogyne incognita parasitism. In experiments presented here, the heterologous expression of Gm-NDR1-1 in G. hirsutum impairs Rotylenchulus reniformis parasitism. These results are consistent with the hypothesis that Gm-NDR1-1 expression functions broadly in generating a defense response. To examine a possible relationship with harpin, G. max plants topically treated with harpin result in induction of the transcription of Gm-NDR1-1. The result indicates the topical treatment of plants with harpin, itself, may lead to impaired nematode parasitism. Topical harpin treatments are shown to impair G. max parasitism by H. glycines, M. incognita and R. reniformis and G. hirsutum parasitism by M. incognita and R. reniformis. How harpin could function in defense has been examined in experiments showing it also induces transcription of G. max homologs of the proven defense genes ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), TGA2, galactinol synthase, reticuline oxidase, xyloglucan endotransglycosylase/hydrolase, alpha soluble N-ethylmaleimide-sensitive fusion protein (α-SNAP) and serine hydroxymethyltransferase (SHMT). In contrast, other defense genes are not directly transcriptionally activated by harpin. The results indicate harpin induces pathogen associated molecular pattern (PAMP) triggered immunity (PTI) and effector-triggered immunity (ETI) defense processes in the root, activating defense to parasitic nematodes. Copyright © 2017. Published by Elsevier

  16. Follicle-stimulating hormone receptor-mediated uptake of 45Ca2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase

    International Nuclear Information System (INIS)

    Grasso, P.; Reichert, L.E. Jr.

    1990-01-01

    We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel

  17. Follicle-stimulating hormone receptor-mediated uptake of sup 45 Ca sup 2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase

    Energy Technology Data Exchange (ETDEWEB)

    Grasso, P.; Reichert, L.E. Jr. (Albany Medical College, NY (USA))

    1990-08-01

    We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel.

  18. Pocket companion to PMI's PMBOK guide

    CERN Document Server

    Snijders, Paul; Zandhuis, Anton

    2010-01-01

    This pocket guide is based on the PMBOK Guide® Fourth Edition.This pocket guide supplies a summary of the PMBOK Guide® , to provide a quick introduction as well as a structured overview of this method for project management.This pocket guide deals with the key issues and themes within project management and PMBOK:A short overview of the activities of PMI Inc., The organization and its standards: PMBOK Guide®, Standard for Project Portfolio Management, Standard for Program Management, OPM3.The essentials of the Project Lifecycle and Organization.What are the key project management knowledge ar

  19. Macintosh Troubleshooting Pocket Guide for Mac OS

    CERN Document Server

    Lerner, David; Corporation, Tekserve

    2009-01-01

    The Macintosh Troubleshooting Pocket Guide covers the most common user hardware and software trouble. It's not just a book for Mac OS X (although it includes tips for OS X and Jaguar), it's for anyone who owns a Mac of any type-- there are software tips going back as far as OS 6. This slim guide distills the answers to the urgent questions that Tekserve's employee's answer every week into a handy guide that fits in your back pocket or alongside your keyboard.

  20. TOGAF version 9 a pocket guide

    CERN Document Server

    Josey, Andrew

    2008-01-01

    This Pocket Guide is based on TOGAF Version 9 Enterprise Edition. It gives a concise introduction to TOGAF 9. What's more, it's authoritative, with material derived from The Open Group's TOGAF 9 documentation and contributions from members of The Open Group Architecture Forum. The audience for this Pocket Guide is enterprise architects, business architects, IT architects, data architects, systems architects, solutions architects, and senior managers seeking a ?rst introduction to TOGAF.

  1. Disease Mutations in Rab7 Result in Unregulated Nucleotide Exchange and Inappropriate Activation

    Energy Technology Data Exchange (ETDEWEB)

    B McCray; E Skordalakes; J Taylor

    2011-12-31

    Rab GTPases are molecular switches that orchestrate vesicular trafficking, maturation and fusion by cycling between an active, GTP-bound form, and an inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is tightly controlled by regulatory proteins. Missense mutations of the GTPase Rab7 cause a dominantly inherited axonal degeneration known as Charcot-Marie-Tooth type 2B through an unknown mechanism. We present the 2.8 A crystal structure of GTP-bound L129F mutant Rab7 which reveals normal conformations of the effector binding regions and catalytic site, but an alteration to the nucleotide binding pocket that is predicted to alter GTP binding. Through extensive biochemical analysis, we demonstrate that disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect, but permit unregulated nucleotide exchange leading to both excessive activation and hydrolysis-independent inactivation. Consistent with augmented activity, mutant Rab7 shows significantly enhanced interaction with a subset of effector proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is abnormally retained on target membranes. However, we show that the increased activation of mutant Rab7 is counterbalanced by unregulated, GTP hydrolysis-independent membrane cycling. Notably, disease mutations are able to rescue the membrane cycling of a GTPase-deficient mutant. Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and temporal control normally imposed by regulatory proteins and cause disease not by a gain of novel toxic function, but by misregulation of native Rab7 activity.

  2. GPCR crystal structures: Medicinal chemistry in the pocket.

    Science.gov (United States)

    Shonberg, Jeremy; Kling, Ralf C; Gmeiner, Peter; Löber, Stefan

    2015-07-15

    Recent breakthroughs in GPCR structural biology have significantly increased our understanding of drug action at these therapeutically relevant receptors, and this will undoubtedly lead to the design of better therapeutics. In recent years, crystal structures of GPCRs from classes A, B, C and F have been solved, unveiling a precise snapshot of ligand-receptor interactions. Furthermore, some receptors have been crystallized in different functional states in complex with antagonists, partial agonists, full agonists, biased agonists and allosteric modulators, providing further insight into the mechanisms of ligand-induced GPCR activation. It is now obvious that there is enormous diversity in the size, shape and position of the ligand binding pockets in GPCRs. In this review, we summarise the current state of solved GPCR structures, with a particular focus on ligand-receptor interactions in the binding pocket, and how this can contribute to the design of GPCR ligands with better affinity, subtype selectivity or efficacy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Nucleotide-binding oligomerization domain 1 regulates Porphyromonas gingivalis-induced vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 expression in endothelial cells through NF-κB pathway.

    Science.gov (United States)

    Wan, M; Liu, J; Ouyang, X

    2015-04-01

    Porphyromonas gingivalis has been shown to actively invade endothelial cells and induce vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) overexpression. Nucleotide-binding oligomerization domain 1 (NOD1) is an intracellular pattern recognition reporter, and its involvement in this process was unknown. This study focused on endothelial cells infected with P. gingivalis, the detection of NOD1 expression and the role that NOD1 plays in the upregulation of VCAM-1 and ICAM-1. The human umbilical vein endothelial cell line (ECV-304) was intruded by P. gingivalis W83, and cells without any treatment were the control group. Expression levels of NOD1, VCAM-1, ICAM-1, phosphorylated P65 between cells with and without treatment on both mRNA and protein levels were compared. Then we examined whether mesodiaminopimelic acid (NOD1 agonist) could increase VCAM-1 and ICAM-1 expression, meanwhile, NOD1 gene silence by RNA interference could reduce VCAM-1, ICAM-1 and phosphorylated P65 release. At last, we examined whether inhibition of NF-κB by Bay117082 could reduce VCAM-1 and ICAM- 1 expression. The mRNA levels were measured by real-time polymerase chain reaction, and protein levels by western blot or electrophoretic mobility shift assays (for phosphorylated P65). P. gingivalis invasion showed significant upregulation of NOD1, VCAM-1 and ICAM-1. NOD1 activation by meso-diaminopimelic acid increased VCAM-1 and ICAM-1 expression, and NOD1 gene silence reduced VCAM-1 and ICAM-1 release markedly. The NF-κB signaling pathway was activated by P. gingivalis, while NOD1 gene silence decreased the activation of NF-κB. Moreover, inhibition of NF-κB reduced VCAM-1 and ICAM-1 expression induced by P. gingivalis in endothelial cells. The results revealed that P. gingivalis induced NOD1 overexpression in endothelial cells and that NOD1 played an important role in the process of VCAM-1 and ICAM-1 expression in endothelial cells infected with P

  4. Bay11-7082 attenuates neuropathic pain via inhibition of nuclear factor-kappa B and nucleotide-binding domain-like receptor protein 3 inflammasome activation in dorsal root ganglions in a rat model of lumbar disc herniation

    Directory of Open Access Journals (Sweden)

    Zhang AL

    2017-02-01

    Full Text Available Ailiang Zhang, Kun Wang, Lianghua Ding, Xinnan Bao, Xuan Wang, Xubin Qiu, Jinbo Liu Spine Surgery, Third Affiliated Hospital of Soochow University, Changzhou, People’s Republic of China Abstract: Lumbar disc herniation (LDH is an important cause of radiculopathy, but the underlying mechanisms are incompletely understood. Many studies suggested that local inflammation, rather than mechanical compression, results in radiculopathy induced by LDH. On the molecular and cellular level, nuclear factor-kappa B (NF-κB and nucleotide-binding domain-like receptor protein 3 (NLRP3 inflammasome have been implicated in the regulation of neuroinflammation formation and progression. In this study, the autologous nucleus pulposus (NP was implanted in the left L5 dorsal root ganglion (DRG to mimic LDH in rats. We investigated the expression of NF-κB and the components of NLRP3 inflammasome in the DRG neurons in rats. Western blotting and immunofluorescence for the related molecules, including NLRP3, apoptosis-associated speck-like protein containing caspase-1 activator domain (ASC, caspase-1, interleukin (IL-1β, IL-18, IκBα, p-IκBα, p65, p-p65, and calcitonin gene-related peptide (CGRP were examined. In the NP-treated group, the activations of NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65 in DRG neurons in rats were elevated at 1 day after surgery, and the peak occurred at 7 days. Treatment with Bay11-7082, an inhibitor of the actions of IKK-β, was able to inhibit expression and activation of the molecules (NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65 and relieve the pain in rats. Our study shows that NF-κB and NLRP3 inflammasome are involved in the maintenance of NP-induced pain, and that Bay11-7082 could alleviate mechanical allodynia and thermal hyperalgesia by inhibiting NF-κB and NLRP3 inflammasome activation. Keywords: pain, NLRP3, NF-κB, dorsal root ganglion, nucleus pulposus

  5. Flexibility of the myosin heavy chain: direct evidence that the region containing SH/sub 1/ and SH/sub 2/ can move 10 /Angstrom/ under the influence of nucleotide binding

    Energy Technology Data Exchange (ETDEWEB)

    Huston, E.E.; Grammer, J.C.; Yount, R.G.

    1988-12-13

    Previous experiments demonstrated that two thiols of skeletal myosin subfragment 1 (SF/sub 1/) could be oxidized to a disulfide bond by treatment with a 2-fold excess of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) in the presence of MgADP. The resulting characteristic changes in the ATPase activities of SF/sub 1/ and the fact that MgADP was stably trapped at the active site, suggested that the two thiols cross-linked were SH/sub 1/ (Cys-707) and SH/sub 2/ (Cys-697) from the myosin heavy chain. To verify this suggestion, SF/sub 1/, after DTNB treatment as above, was treated with an excess of N-ethylmaleimide to block all accessible thiols. The single protein disulfide produced by DTNB oxidation was reduced with dithioerythritol and the modified SF/sub 1/ internally cross-linked with equimolar (/sup 14/C)p-phenylenedimaleimide (pPDM) in the presence of MgADP. After extensive trypsinization, the major /sup 14/C-labeled peptide was isolated, characterized, and shown to be Cys-Asn-Gly-Val-Leu-Gly-Ile-Arg-Ile-Cys-Arg, in which the two cysteines were cross-linked by pPDM. This peptide is known to contain SH/sub 2/ and SH/sub 1/ in this order and to come from residues 697-708 in the rabbit skeletal myosin heavy chain. Parallel experiments with (/sup 14/C)pPDM and unmodified SF/sub 1/ similar to those above gave an identical SH/sub 1/, SH/sub 2/ tryptic peptide, verifying earlier labeling results. These combined results demonstrate that SH/sub 1/ and SH/sub 2/ cross-linked by pPDM (12-13 /Angstrom/, S to S) or by oxidation with DTNB (2 /Angstrom/, S to S) can move a minimum of 10 /Angstrom/ under the influence of nucleotide binding. Because these residues are separated by only nine amino acids in the primary sequence, this small section of the heavy chain must possess extraordinary flexibility.

  6. Condensing the information in DNA with double-headed nucleotides

    DEFF Research Database (Denmark)

    Hornum, Mick; Sharma, Pawan K; Reslow-Jacobsen, Charlotte

    2017-01-01

    A normal duplex holds as many Watson-Crick base pairs as the number of nucleotides in its constituent strands. Here we establish that single nucleotides can be designed to functionally imitate dinucleotides without compromising binding affinity. This effectively allows sequence information...

  7. Replantation of multi-level fingertip amputation using the pocket principle (palmar pocket method).

    Science.gov (United States)

    Arata, J; Ishikawa, K; Soeda, H; Kitayama, T

    2003-07-01

    Two cases of multi-level fingertip amputation are presented. In each case, replantation was achieved in a two-stage procedure, involving reattachment, de-epithelialisation and insertion into a palmar pocket in stage 1, followed by removal from the palmar pocket 16 days later. The cases are described and the technique is discussed.

  8. Pharmacophore Selection and Redesign of Non-nucleotide Inhibitors of Anthrax Edema Factor

    Directory of Open Access Journals (Sweden)

    Maria Estrella Jimenez

    2012-11-01

    Full Text Available Antibiotic treatment may fail to protect individuals, if not started early enough, after infection with Bacillus anthracis, due to the continuing activity of toxins that the bacterium produces. Stable and easily stored inhibitors of the edema factor toxin (EF, an adenylyl cyclase, could save lives in the event of an outbreak, due to natural causes or a bioweapon attack. The toxin’s basic activity is to convert ATP to cAMP, and it is thus in principle a simple phosphatase, which means that many mammalian enzymes, including intracellular adenylcyclases, may have a similar activity. While nucleotide based inhibitors, similar to its natural substrate, ATP, were identified early, these compounds had low activity and specificity for EF. We used a combined structural and computational approach to choose small organic molecules in large, web-based compound libraries that would, based on docking scores, bind to residues within the substrate binding pocket of EF. A family of fluorenone-based inhibitors was identified that inhibited the release of cAMP from cells treated with EF. The lead inhibitor was also shown to inhibit the diarrhea caused by enterotoxigenic E. coli (ETEC in a murine model, perhaps by serving as a quorum sensor. These inhibitors are now being tested for their ability to inhibit Anthrax infection in animal models and may have use against other pathogens that produce toxins similar to EF, such as Bordetella pertussis or Vibrio cholera.

  9. Main: Nucleotide Analysis [KOME

    Lifescience Database Archive (English)

    Full Text Available Nucleotide Analysis Japonica genome blast search result Result of blastn search against jap...onica genome sequence kome_japonica_genome_blast_search_result.zip kome_japonica_genome_blast_search_result ...

  10. Review of Pocket Guide Megaliths [app

    Directory of Open Access Journals (Sweden)

    Barney Harris

    2017-09-01

    Full Text Available The Pocket Guide Megaliths app was developed by Senet Mobile UK in collaboration with The Megalithic Portal website. The app was released in 2016 and is currently available for iOS devices, such as iPhone and iPads. Pocket Guide Megaliths presents the Megalithic Portal's burgeoning worldwide database of ancient and prehistoric sites in a variety of innovative and engaging ways. It aims to act primarily as a guide to enjoying and exploring the rich prehistoric heritage of the world, though offers additional functionality for would-be monument explorers too.

  11. Supplementary Material for: The arabidopsis cyclic nucleotide interactome

    KAUST Repository

    Donaldson, Lara

    2016-01-01

    Abstract Background Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotide-dependent signaling mechanisms in plants since the downstream target proteins remain unknown. This is largely due to the fact that bioinformatics searches fail to identify plant homologs of protein kinases and phosphodiesterases that are the main targets of cyclic nucleotides in animals. Methods An affinity purification technique was used to identify cyclic nucleotide binding proteins in Arabidopsis thaliana. The identified proteins were subjected to a computational analysis that included a sequence, transcriptional co-expression and functional annotation analysis in order to assess their potential role in plant cyclic nucleotide signaling. Results A total of twelve cyclic nucleotide binding proteins were identified experimentally including key enzymes in the Calvin cycle and photorespiration pathway. Importantly, eight of the twelve proteins were shown to contain putative cyclic nucleotide binding domains. Moreover, the identified proteins are post-translationally modified by nitric oxide, transcriptionally co-expressed and annotated to function in hydrogen peroxide signaling and the defence response. The activity of one of these proteins, GLYGOLATE OXIDASE 1, a photorespiratory enzyme that produces hydrogen peroxide in response to Pseudomonas, was shown to be repressed by a combination of cGMP and nitric oxide treatment. Conclusions We propose that the identified proteins function together as points of cross-talk between cyclic nucleotide, nitric oxide and reactive oxygen species signaling during the defence response.

  12. Implantable cardioverter defibrillator pocket infection caused by ...

    African Journals Online (AJOL)

    Like any other foreign bodies, implanted cardiac devices can become infected. Staphylococcus aureus and coagulase-negative Staphilococci are the most common causes of infections of pacemaker and defibrillator systems. In this case an implantable cardioverter defibrillator pocket infection caused by an extremely rare ...

  13. Dermal pocketing following distal finger replantation.

    Science.gov (United States)

    Puhaindran, Mark E; Paavilainen, Pasi; Tan, David M K; Peng, Yeong Pin; Lim, Aymeric Y T

    2010-08-01

    Replantation is an ideal technique for reconstruction following fingertip amputation as it provides 'like for like' total reconstruction of the nail complex, bone pulp tissue and skin with no donor-site morbidity. However, fingertips are often not replanted because veins cannot be found or are thought to be too small to repair. Attempts at 'cap-plasty' or pocketing of replanted tips with and without microvascular anastomosis have been done in the past with varying degrees of success. We prospectively followed up a group of patients who underwent digital replantation and dermal pocketing in the palm to evaluate the outcome of this procedure. There were 10 patients with 14 amputated digits (two thumbs, five index, four middle, two ring and one little) who underwent dermal pocketing of the amputated digit following replantation. Among the 14 digits that were treated with dermal pocketing, 11 survived completely, one had partial atrophy and two were completely lost. Complications encountered included finger stiffness (two patients) and infection of the replanted fingertip with osteomyelitis of the distal phalanx (one patient). We believe that this technique can help increase the chance of survival for distal replantation with an acceptable salvage rate of 85% in our series. Copyright 2009 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

  14. E-mail security a pocket guide

    CERN Document Server

    Furnell, Steven

    2010-01-01

    This pocket guide will help businesses to address the most important issues. Its comprehensive approach covers both the technical and the managerial aspects of the subject, offering valuable insights for IT professionals, managers and executives, as well as for individual users of e-mail.

  15. Pocket Checklists of Indonesian timber trees

    NARCIS (Netherlands)

    Prawira, Soewanda A.; Tantra, I.G.M.; Whitmore, T.C.

    1984-01-01

    Indonesia as yet does not have a comprehensive account of the forest trees which reach timber size (35 cm dbh = 14 inch or 105 cm gbh = 42 inch). A project has been started in August 1983 by the Botany Section of the Forest Research Institute in Bogor, Indonesia, to prepare pocket checklists of the

  16. Fingertip replantation using the subdermal pocket procedure.

    Science.gov (United States)

    Lin, Tsan-Shiun; Jeng, Seng-Feng; Chiang, Yuan-Cheng

    2004-01-01

    Restoration of finger length and function are the goals of replantation after fingertip amputation. Methods include microsurgical replantation and nonmicrosurgical replantation, such as composite graft techniques. To increase the survival rates for composite grafts, the subcutaneous pocket procedure has been used as a salvage procedure. The subdermal pocket procedure, which is a modification of the subcutaneous pocket procedure, was used for replantation of 17 fingertips in 16 consecutive patients. Eight fingertips experienced guillotine injuries and the other nine fingertips experienced crush injuries. Revascularization of one digital artery without available venous outflow was performed for six fingers, and composite graft techniques were used for the other 11 fingers. The success rate was 16 of 17 cases. The difference in success rates for guillotine versus crush injuries was statistically significant. Comparison of patients with arterial anastomoses and patients without arterial anastomoses also indicated a statistically significant difference. Thirteen fingertips survived completely. One finger, demonstrating complete loss and early termination of the pocketing procedure, was amputated on the eighth postoperative day. Two fingers were partially lost because of severe crushing injuries. One finger demonstrated partial loss of more than one quarter of the fingertip, which required secondary revision, because the patient was a heavy smoker. The pocketing period was 8 +/- 1 days (mean +/- SD, n = 6) for the fingers revascularized with one digital arterial anastomosis and 13.3 +/- 1.9 days (n = 10) for the fingers successfully replanted with composite graft techniques. The mean active range of motion of the interphalangeal joint of the three thumbs was 65 +/- 5 degrees, and that of the distal interphalangeal joint of the other 11 fingers was 51 +/- 11 degrees. The static two-point discrimination result was 6.4 +/- 1.0 mm (n = 14) after an average of 11 +/- 5 months

  17. Cyclic nucleotides and radioresistnace

    International Nuclear Information System (INIS)

    Kulinskij, V.I.; Mikheeva, G.A.; Zel'manovich, B.M.

    1982-01-01

    The addition of glucose to meat-peptone broth does not change the radiosensitizing effect (RSE) of cAMP at the logarithmic phase (LP) and the radioprotective effect (RPE) at the stationary phase (SP), but sensitization, characteristic of cGMP, disappears in SP and turns into RPE in LP. Introduction of glucose into the broth for 20 min eliminates all the effects of both cyclic nucleotides in the cya + strain while cya - mutant exhibits RSE. RSE of both cyclic nucleotides is only manifested on minimal media. These data brought confirmation of the dependence of the influence of cyclic media. These data brought confirmation of the dependence of the influence of cyclic nucleotides on radioresistance upon the metabolic status of the cell [ru

  18. Nucleotide-dependence of G-actin conformation from multiple molecular dynamics simulations and observation of a putatively polymerization-competent superclosed state.

    Science.gov (United States)

    Splettstoesser, Thomas; Noé, Frank; Oda, Toshiro; Smith, Jeremy C

    2009-08-01

    The assembly of monomeric G-actin into filamentous F-actin is nucleotide dependent: ATP-G-actin is favored for filament growth at the "barbed end" of F-actin, whereas ADP-G-actin tends to dissociate from the "pointed end." Structural differences between ATP- and ADP-G-actin are examined here using multiple molecular dynamics simulations. The "open" and "closed" conformational states of G-actin in aqueous solution are characterized, with either ATP or ADP in the nucleotide binding pocket. With both ATP and ADP bound, the open state closes in the absence of actin-bound profilin. The position of the nucleotide in the protein is found to be correlated with the degree of opening of the active site cleft. Further, the simulations reveal the existence of a structurally well-defined, compact, "superclosed" state of ATP-G-actin, as yet unseen crystallographically and absent in the ADP-G-actin simulations. The superclosed state resembles structurally the actin monomer in filament models derived from fiber diffraction and is putatively the polymerization competent conformation of ATP-G-actin. 2008 Wiley-Liss, Inc.

  19. Pengembangan Pocket Mobile Learning Berbasis Android

    Directory of Open Access Journals (Sweden)

    Dasmo Dasmo

    2017-10-01

    Full Text Available Penelitian ini bertujuan untuk mengembangkan media pembelajaran pocket mobile learning berbasis android. Metode penelitian ini menggunakan model pengembangan ADDIE (Analysis, Design, Development, Implementation, dan Evaluation. Penelitian ini telah menghasilkan sebuah luaran berupa media pembelajaran pocket mobile learning berbasis android pada mata kuliah fisika. Media ini layak digunakan berdasarkan validasi dari ahli materi, ahli media dan respon mahasiswa terhadap media pembelajaran yang dibuat. Berdasarkan penilaian validasi ahli materi didapatkan rata-rata skor total sebesar 3,22 pada 14 butir pernyataan, dan termasuk pada kriteria “baik”. Sementara itu, berdasarkan validasi ahli media didapatkan rata-rata skor total sebesar 3,43 pada 15 butir pernyataan, dan termasuk pada kriteria “sangat baik”. Dan berdasarkan hasil analisis respon mahasiswa terhadap media pembelajaran diperoleh rata-rata skor total sebesar 4,0 atau 80%, dengan kategori “kuat”. Dengan demikian dapat disimpulkan bahwa media pembelajaran pocket mobile learning berbasis android layak untuk digunakan dan hampir semua mahasiswa menanggapi respon positif.The current research aims to develop the pocket mobile learning android based. It uses the Research and Development (R&D method with the ADDIE model (analysis, design, development, implementation, and evaluating. This research has resulted from an outcome of learning media pocket mobile android based on physics subject. This instructional media is feasible to be used based on validation from material experts, media experts, and student responses. Based on the assessment of material expert, the validation obtains an average total score of 3,22 at 14 statements and considered as "good" criteria. Meanwhile, based on the validation of media experts, the average total score reaches 3,43 at 15 statements and considered as "very good" criteria. And based on the analysis of students’ response to learning media, the

  20. Single Nucleotide Polymorphism

    DEFF Research Database (Denmark)

    Børsting, Claus; Pereira, Vania; Andersen, Jeppe Dyrberg

    2014-01-01

    Single nucleotide polymorphisms (SNPs) are the most frequent DNA sequence variations in the genome. They have been studied extensively in the last decade with various purposes in mind. In this chapter, we will discuss the advantages and disadvantages of using SNPs for human identification...

  1. Newnes radio and electronics engineer's pocket book

    CERN Document Server

    Moorshead, H W; Perry, J

    1978-01-01

    Newnes Radio and Electronics Engineer's Pocket Book, Fifteenth Edition provides reference of the information relevant in radio and electronics engineering. The book presents tables, illustrations, and diagrams of various data used in radio and electronics engineering. The coverage of the text includes abbreviations and symbols, electrical equations, and code conversions. The text will be useful to engineers, technicians, and other professionals who require a reference about the different aspects of radio and electronics.

  2. ISO27001 / ISO27002 a pocket guide

    CERN Document Server

    Calder, Alan

    2013-01-01

    Information is one of your organisation's most important resources. Keeping it secure is therefore vital to your business. This handy pocket guide is an essential overview of two key information security standards that cover the formal requirements (ISO27001:2013) for creating an Information Security Management System (ISMS), and the best-practice recommendations (ISO27002:2013) for those responsible for initiating, implementing or maintaining it.

  3. Windows® 7 Administrator's Pocket Consultant

    CERN Document Server

    Stanek, William

    2009-01-01

    Portable and precise, this pocket-sized guide delivers immediate answers for the day-to-day administration of Windows 7-from desktop configuration and management to networking and security issues. Zero in on core support and maintenance tasks by using quick-reference tables, instructions, and lists. You'll get the precise information you need to solve problems and get the job done-whether at your desk or in the field!

  4. Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide-protein complexes.

    Science.gov (United States)

    Kondo, Jiro; Westhof, Eric

    2011-10-01

    Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide-protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson-Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson-Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues.

  5. Targeting the Central Pocket in Human Transcription Factor TEAD as a Potential Cancer Therapeutic Strategy.

    Science.gov (United States)

    Pobbati, Ajaybabu V; Han, Xiao; Hung, Alvin W; Weiguang, Seetoh; Huda, Nur; Chen, Guo-Ying; Kang, CongBao; Chia, Cheng San Brian; Luo, Xuelian; Hong, Wanjin; Poulsen, Anders

    2015-11-03

    The human TEAD family of transcription factors (TEAD1-4) is required for YAP-mediated transcription in the Hippo pathway. Hyperactivation of TEAD's co-activator YAP contributes to tissue overgrowth and human cancers, suggesting that pharmacological interference of TEAD-YAP activity may be an effective strategy for anticancer therapy. Here we report the discovery of a central pocket in the YAP-binding domain (YBD) of TEAD that is targetable by small-molecule inhibitors. Our X-ray crystallography studies reveal that flufenamic acid, a non-steroidal anti-inflammatory drug (NSAID), binds to the central pocket of TEAD2 YBD. Our biochemical and functional analyses further demonstrate that binding of NSAIDs to TEAD inhibits TEAD-YAP-dependent transcription, cell migration, and proliferation, indicating that the central pocket is important for TEAD function. Therefore, our studies discover a novel way of targeting TEAD transcription factors and set the stage for therapeutic development of specific TEAD-YAP inhibitors against human cancers. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. ALLO: A tool to discriminate and prioritize allosteric pockets.

    Science.gov (United States)

    Akbar, Rahmad; Helms, Volkhard

    2018-04-01

    Allosteric proteins make up a substantial proportion of human drug targets. Thus, rational design of small molecule binders that target these proteins requires the identification of putative allosteric pockets and an understanding of their potential activity. Here, we characterized allosteric pockets using a set of physicochemical descriptors and compared them to pockets that are found on the surface of a protein. Further, we trained predictive models capable of discriminating allosteric pockets from orthosteric pockets and models capable of prioritizing allosteric pockets in a set of pockets found on a given protein. Such models might be useful for identifying novel allosteric sites and in turn, potentially new allosteric drug targets. Datasets along with a Python program encapsulating the predictive models are available at http://github.com/fibonaccirabbits/allo. © 2017 John Wiley & Sons A/S.

  7. Approaches for Identification of HIV-1 Entry Inhibitors Targeting gp41 Pocket

    Directory of Open Access Journals (Sweden)

    Asim K. Debnath

    2013-01-01

    Full Text Available The hydrophobic pocket in the HIV-1 gp41 N-terminal heptad repeat (NHR domain plays an important role in viral fusion and entry into the host cell, and serves as an attractive target for development of HIV-1 fusion/entry inhibitors. The peptide anti-HIV drug targeting gp41 NHR, T-20 (generic name: enfuvirtide; brand name: Fuzeon, was approved by the U.S. FDA in 2003 as the first HIV fusion/entry inhibitor for treatment of HIV/AIDS patients who fail to respond to the current antiretroviral drugs. However, because T20 lacks the pocket-binding domain (PBD, it exhibits low anti-HIV-1 activity and short half-life. Therefore, several next-generation HIV fusion inhibitory peptides with PBD have been developed. They possess longer half-life and more potent antiviral activity against a broad spectrum of HIV-1 strains, including the T-20-resistant variants. Nonetheless, the clinical application of these peptides is still limited by the lack of oral availability and the high cost of production. Thus, development of small molecule compounds targeting the gp41 pocket with oral availability has been promoted. This review describes the main approaches for identification of HIV fusion/entry inhibitors targeting the gp41 pocket and summarizes the latest progress in developing these inhibitors as a new class of anti-HIV drugs.

  8. Conformational dynamics and role of the acidic pocket in ASIC pH-dependent gating.

    Science.gov (United States)

    Vullo, Sabrina; Bonifacio, Gaetano; Roy, Sophie; Johner, Niklaus; Bernèche, Simon; Kellenberger, Stephan

    2017-04-04

    Acid-sensing ion channels (ASICs) are proton-activated Na + channels expressed in the nervous system, where they are involved in learning, fear behaviors, neurodegeneration, and pain sensation. In this work, we study the role in pH sensing of two regions of the ectodomain enriched in acidic residues: the acidic pocket, which faces the outside of the protein and is the binding site of several animal toxins, and the palm, a central channel domain. Using voltage clamp fluorometry, we find that the acidic pocket undergoes conformational changes during both activation and desensitization. Concurrently, we find that, although proton sensing in the acidic pocket is not required for channel function, it does contribute to both activation and desensitization. Furthermore, protonation-mimicking mutations of acidic residues in the palm induce a dramatic acceleration of desensitization followed by the appearance of a sustained current. In summary, this work describes the roles of potential pH sensors in two extracellular domains, and it proposes a model of acidification-induced conformational changes occurring in the acidic pocket of ASIC1a.

  9. Pocket radiation dosimeter: dosimeter charger assembly

    Science.gov (United States)

    Manning, F.W.

    1982-03-17

    This invention is a novel pocket-type radiation dosimeter comprising an electrometric radiation dosimeter and a charging circuit therefor. The instrument is especially designed to be amenable to mass production, to have a long shelf life, and to be compact, lightweight, and usable by the layman. The dosimeter proper may be of conventional design. The charging circuit includes a shake-type electrostatic generator, a voltage doubler for integrating generator output voltages of one polarity, and a switch operated by an external permanent magnet.

  10. Pocket radiation dosimeter--dosimeter charger assembly

    Science.gov (United States)

    Manning, Frank W.

    1984-01-01

    This invention is a novel pocket-type radiation dosimeter comprising an electrometric radiation dosimeter and a charging circuit therefor. The instrument is especially designed to be amenable to mass production, to have a long shelf life, and to be compact, lightweight, and usable by the layman. The dosimeter proper may be of conventional design. The charging circuit includes a shake-type electrostatic generator, a voltage doubler for integrating generator output voltages of one polarity, and a switch operated by an external permanent magnet.

  11. Python pocket reference, version 2.4

    CERN Document Server

    Lutz, Mark

    2005-01-01

    Python is optimized for quality, productivity, portability, and integration. Hundreds of thousands of Python developers around the world rely on Python for general-purpose tasks, Internet scripting, systems programming, user interfaces, and product customization. Available on all major computing platforms, including commercial versions of Unix, Linux, Windows, and Mac OS X, Python is portable, powerful and remarkable easy to use. With its convenient, quick-reference format, Python Pocket Reference, 3rd Edition is the perfect on-the-job reference. More importantly, it's now been refreshed

  12. Newnes passive and discrete circuits pocket book

    CERN Document Server

    MARSTON, R M

    2000-01-01

    Newnes Passive and Discrete Circuits Pocket Book is aimed at all engineers, technicians, students and experimenters who can build a design directly from a circuit diagram. In a highly concise form Ray Marston presents a huge compendium of circuits that can be built as they appear, adapted or used as building blocks. The devices used have been carefully chosen for their ease of availability and reasonable price. The selection of devices has been thoroughly updated for the second edition, which has also been expanded to cover the latest ICs.The three sections of the book cover: Moder

  13. Mac OS X Snow Leopard pocket guide

    CERN Document Server

    Seiblod, Chris

    2009-01-01

    Whether you're new to the Mac or a longtime user, this handy book is the quickest way to get up to speed on Snow Leopard. Packed with concise information in an easy-to-read format, Mac OS X Snow Leopard Pocket Guide covers what you need to know and is an ideal resource for problem-solving on the fly. This book goes right to the heart of Snow Leopard, with details on system preferences, built-in applications, and utilities. You'll also find configuration tips, keyboard shortcuts, guides for troubleshooting, lots of step-by-step instructions, and more. Learn about new features and changes s

  14. Root Locus Algorithms for Programmable Pocket Calculators

    Science.gov (United States)

    Wechsler, E. R.

    1983-01-01

    Two algorithms are described which allow the plotting of individual points on a root locus diagram with or without time delay. The development was performed during the design of a continuous phase shifter used in the Baseband Antenna Combiner for the Deep Space Network (DSN). The algorithms, which are expected to be useful in similar DSN efforts, are simple enough to be implemented on a programmable pocket calculator. The coordinates of the open-loop zeros and poles, the gain constant K, and the time delay T are the data inputs.

  15. AIR for Javascript Developers Pocket Guide

    CERN Document Server

    Chambers, Mike; Hoyt, Kevin; Georgita, Dragos

    2009-01-01

    This book is the official guide to Adobe ® AIR[TM], written by members of the AIR team. With Adobe AIR, web developers can use technologies like HTML and JavaScript to build and deploy web applications to the desktop. Packed with examples, this book explains how AIR works and features recipes for performing common runtime tasks. Part of the Adobe Developer Library, this concise pocket guide explains: What Adobe AIR is, and the problems this runtime aims to solveHow to set up your development environmentThe HTML and JavaScript environments within AIRHow to create your first AIR application

  16. PoLi: A Virtual Screening Pipeline Based on Template Pocket and Ligand Similarity.

    Science.gov (United States)

    Roy, Ambrish; Srinivasan, Bharath; Skolnick, Jeffrey

    2015-08-24

    Often in pharmaceutical research the goal is to identify small molecules that can interact with and appropriately modify the biological behavior of a new protein target. Unfortunately, most proteins lack both known structures and small molecule binders, prerequisites of many virtual screening, VS, approaches. For such proteins, ligand homology modeling, LHM, that copies ligands from homologous and perhaps evolutionarily distant template proteins, has been shown to be a powerful VS approach to identify possible binding ligands. However, if we want to target a specific pocket for which there is no homologous holo template protein structure, then LHM will not work. To address this issue, in a new pocket-based approach, PoLi, we generalize LHM by exploiting the fact that the number of distinct small molecule ligand-binding pockets in proteins is small. PoLi identifies similar ligand-binding pockets in a holo template protein library, selectively copies relevant parts of template ligands, and uses them for VS. In practice, PoLi is a hybrid structure and ligand-based VS algorithm that integrates 2D fingerprint-based and 3D shape-based similarity metrics for improved virtual screening performance. On standard DUD and DUD-E benchmark databases, using modeled receptor structures, PoLi achieves an average enrichment factor of 13.4 and 9.6, respectively, in the top 1% of the screened library. In contrast, traditional docking-based VS using AutoDock Vina and homology-based VS using FINDSITE(filt) have an average enrichment of 1.6 (3.0) and 9.0 (7.9) on the DUD (DUD-E) sets, respectively. Experimental validation of PoLi predictions on dihydrofolate reductase, DHFR, using differential scanning fluorimetry, DSF, identifies multiple ligands with diverse molecular scaffolds, thus demonstrating the advantage of PoLi over current state-of-the-art VS methods.

  17. Nucleotide Selectivity at a Preinsertion Checkpoint of T7 RNA Polymerase Transcription Elongation.

    Science.gov (United States)

    E, Chao; Duan, Baogen; Yu, Jin

    2017-04-20

    Nucleotide selection is crucial for transcription fidelity control, in particular, for viral T7 RNA polymerase (RNAP) lack of proofreading activity. It has been recognized that multiple kinetic checkpoints exist prior to full nucleotide incorporation. In this work, we implemented intensive atomistic molecular dynamics (MD) simulations to quantify how strong the nucleotide selection is at the initial checkpoint of an elongation cycle of T7 RNAP. The incoming nucleotides bind into a preinsertion site where a critical tyrosine residue locates nearby to assist the nucleotide selection. We calculated the relative binding free energy between a noncognate nucleotide and a cognate one at a preinsertion configuration via alchemical simulations, showing that a small selection free energy or the binding free energy difference (∼3 k B T) exists between the two nucleotides. Indeed, another preinsertion configuration favored by the noncognate nucleotides was identified, which appears to be off path for further nucleotide insertion and additionally assists the nucleotide selection. By chemical master equation (CME) approach, we show that the small selection free energy at the preinsertion site along with the off-path noncognate nucleotide filtering can help substantially to reduce the error rate and to maintain the elongation rate high in the T7 RNAP transcription.

  18. Molecular mechanism of ATP binding and ion channel activation in P2X receptors

    Energy Technology Data Exchange (ETDEWEB)

    Hattori, Motoyuki; Gouaux, Eric (Oregon HSU)

    2012-10-24

    P2X receptors are trimeric ATP-activated ion channels permeable to Na{sup +}, K{sup +} and Ca{sup 2+}. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body {beta}-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.

  19. Dissecting functions of the retinoblastoma tumor suppressor and the related pocket proteins by integrating genetic, cell biology, and electrophoretic techniques

    DEFF Research Database (Denmark)

    Hansen, Klaus; Lukas, J; Holm, K

    1999-01-01

    The members of the 'pocket protein' family, comprising the retinoblastoma tumor suppressor (pRB) and its relatives, p107 and p130, negatively regulate cell proliferation and modulate fundamental biological processes including embryonic development, differentiation, homeostatic tissue renewal......, and defense against cancer. The large, multidomain pocket proteins act by binding a plethora of cell fate-determining and growth-stimulatory proteins, the most prominent of which are the E2F/DP transcription factors. These protein-protein interactions are in turn regulated by carefully orchestrated...

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

    OpenAIRE

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

    2009-01-01

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

  1. Single Nucleotide Polymorphism

    DEFF Research Database (Denmark)

    Børsting, Claus; Pereira, Vania; Andersen, Jeppe Dyrberg

    2014-01-01

    and briefly describe the methods that are preferred for SNP typing in forensic genetics. In addition, we will illustrate how SNPs can be used as investigative leads in the police investigation by discussing the use of ancestry informative markers and forensic DNA phenotyping. Modern DNA sequencing......Single nucleotide polymorphisms (SNPs) are the most frequent DNA sequence variations in the genome. They have been studied extensively in the last decade with various purposes in mind. In this chapter, we will discuss the advantages and disadvantages of using SNPs for human identification...... technologies (also called next generation sequencing or NGS) have the potential to completely transform forensic genetic investigations as we know them today. Here, we will make a short introduction to NGS and explain how NGS may combine analysis of the traditional forensic genetic markers with analysis...

  2. Oracle PL/SQL Language Pocket Reference

    CERN Document Server

    Feuerstein, Steven; Dawes, Chip

    2004-01-01

    While it's good to have a book with all the answers--like your trusty copy of Oracle PL/SQL Programming-- how often do you need all the answers? More likely, you just need a reminder, a quick answer to a problem you're up against. For these times, nothing's handier than the new edition of the Oracle PL/SQL Language Pocket Reference by PL/SQL experts Stephen Feuerstein, Bill Pribyl, and Chip Dawes. Newly updated for Oracle10g, this little book is always at the ready for the quick problem solving you need. The 3rd edition of this popular mini-reference boils down the most vital information fr

  3. Computational learning on specificity-determining residue-nucleotide interactions

    KAUST Repository

    Wong, Ka-Chun

    2015-11-02

    The protein–DNA interactions between transcription factors and transcription factor binding sites are essential activities in gene regulation. To decipher the binding codes, it is a long-standing challenge to understand the binding mechanism across different transcription factor DNA binding families. Past computational learning studies usually focus on learning and predicting the DNA binding residues on protein side. Taking into account both sides (protein and DNA), we propose and describe a computational study for learning the specificity-determining residue-nucleotide interactions of different known DNA-binding domain families. The proposed learning models are compared to state-of-the-art models comprehensively, demonstrating its competitive learning performance. In addition, we describe and propose two applications which demonstrate how the learnt models can provide meaningful insights into protein–DNA interactions across different DNA binding families.

  4. Nucleotide-Induced Conformational Changes in Escherichia coli DnaA Protein Are Required for Bacterial ORC to Pre-RC Conversion at the Chromosomal Origin.

    Science.gov (United States)

    Saxena, Rahul; Vasudevan, Sona; Patil, Digvijay; Ashoura, Norah; Grimwade, Julia E; Crooke, Elliott

    2015-11-24

    DnaA oligomerizes when bound to origins of chromosomal replication. Structural analysis of a truncated form of DnaA from Aquifex aeolicus has provided insight into crucial conformational differences within the AAA+ domain that are specific to the ATP- versus ADP- bound form of DnaA. In this study molecular docking of ATP and ADP onto Escherichia coli DnaA, modeled on the crystal structure of Aquifex aeolicus DnaA, reveals changes in the orientation of amino acid residues within or near the vicinity of the nucleotide-binding pocket. Upon limited proteolysis with trypsin or chymotrypsin ADP-DnaA, but not ATP-DnaA generated relatively stable proteolytic fragments of various sizes. Examined sites of limited protease susceptibility that differ between ATP-DnaA and ADP-DnaA largely reside in the amino terminal half of DnaA. The concentration of adenine nucleotide needed to induce conformational changes, as detected by these protease susceptibilities of DnaA, coincides with the conversion of an inactive bacterial origin recognition complex (bORC) to a replication efficient pre-replication complex (pre-RC) at the E. coli chromosomal origin of replication (oriC).

  5. Nucleotide-Induced Conformational Changes in Escherichia coli DnaA Protein Are Required for Bacterial ORC to Pre-RC Conversion at the Chromosomal Origin

    Directory of Open Access Journals (Sweden)

    Rahul Saxena

    2015-11-01

    Full Text Available DnaA oligomerizes when bound to origins of chromosomal replication. Structural analysis of a truncated form of DnaA from Aquifex aeolicus has provided insight into crucial conformational differences within the AAA+ domain that are specific to the ATP- versus ADP- bound form of DnaA. In this study molecular docking of ATP and ADP onto Escherichia coli DnaA, modeled on the crystal structure of Aquifex aeolicus DnaA, reveals changes in the orientation of amino acid residues within or near the vicinity of the nucleotide-binding pocket. Upon limited proteolysis with trypsin or chymotrypsin ADP-DnaA, but not ATP-DnaA generated relatively stable proteolytic fragments of various sizes. Examined sites of limited protease susceptibility that differ between ATP-DnaA and ADP-DnaA largely reside in the amino terminal half of DnaA. The concentration of adenine nucleotide needed to induce conformational changes, as detected by these protease susceptibilities of DnaA, coincides with the conversion of an inactive bacterial origin recognition complex (bORC to a replication efficient pre-replication complex (pre-RC at the E. coli chromosomal origin of replication (oriC.

  6. Effects of nucleotides and nucleotide analogue inhibitors of HIV-1 reverse transcriptase in a ratchet model of polymerase translocation.

    Science.gov (United States)

    Götte, Matthias

    2006-01-01

    A single cycle of nucleotide incorporation by the reverse transcriptase of the human immunodeficiency virus type 1 (HIV-1 RT) involves the initial binding of an incoming nucleotide, a conformational change that traps the substrate, the formation of a new phosphodiester bond, the release of pyrophosphate (PPi), and ultimately polymerase translocation, which clears the nucleotide binding site. This article reviews different mechanistic models for polymerase translocation with emphasis placed on HIV-1 RT. Structure-function analyses of stalled complexes of HIV-1 RT provide strong evidence to suggest that the enzyme can oscillate between pre- and post-translocational states. Nucleotide hydrolysis is not required for the movement of the polymerase in a stalled configuration; thermal energy is sufficient to allow random bidirectional sliding. The next complementary nucleotide, following the incorporated chain-terminator, acts like a pawl of a ratchet that traps the enzyme in the post-translocation state and prevents the reverse movement. Quantitative footprinting experiments have shown that the concentration of the templated nucleotide required to shift the translocational equilibrium forward depends crucially on the structure of the 3'end of the primer. Changes in the relative population of pre- and post-translocation complexes can influence rates of excision of incorporated NRTIs, which, in turn, affects drug susceptibility. The concept of a ratchet model of HIV-1 RT translocation and its implications for drug action and resistance, and the discovery and development of novel antiviral compounds is discussed.

  7. Molecular recognition of nucleotides in micelles and the development and expansion of a chemistry outreach program

    Science.gov (United States)

    Schechinger, Linda Sue

    I. To investigate the delivery of nucleotide-based drugs, we are studying molecular recognition of nucleotide derivatives in environments that are similar to cell membranes. The Nowick group previously discovered that membrane-like surfactant micelles tetradecyltrimethylammonium bromide (TTAB) micelle facilitate molecular of adenosine monophosphate (AMP) recognition. The micelles bind nucleotides by means of electrostatic interactions and hydrogen bonding. We observed binding by following 1H NMR chemical shift changes of unique hexylthymine protons upon addition of AMP. Cationic micelles are required for binding. In surfactant-free or sodium dodecylsulfate solutions, no hydrogen bonding is observed. These observations suggest that the cationic surfactant headgroups bind the nucleotide phosphate group, while the intramicellar base binds the nucleotide base. The micellar system was optimized to enhance binding and selectivity for adenosine nucleotides. The selectivity for adenosine and the number of phosphate groups attached to the adenosine were both investigated. Addition of cytidine, guanidine, or uridine monophosphates, results in no significant downfield shifting of the NH resonance. Selectivity for the phosphate is limited, since adenosine mono-, di-, and triphosphates all have similar binding constants. We successfully achieved molecular recognition of adenosine nucleotides in micellar environments. There is significant difference in the binding interactions between the adenosine nucleotides and three other natural nucleotides. II. The UCI Chemistry Outreach Program (UCICOP) addresses the declining interest of the nations youth for science. UCICOP brings fun and exciting chemistry experiments to local high schools, to remind students that science is fun and has many practical uses. Volunteer students and alumni of UCI perform the demonstrations using scripts and material provided by UCICOP. The preparation of scripts and materials is done by two coordinators

  8. Drug-like density: a method of quantifying the "bindability" of a protein target based on a very large set of pockets and drug-like ligands from the Protein Data Bank.

    Science.gov (United States)

    Sheridan, Robert P; Maiorov, Vladimir N; Holloway, M Katharine; Cornell, Wendy D; Gao, Ying-Duo

    2010-11-22

    One approach to estimating the "chemical tractability" of a candidate protein target where we know the atomic resolution structure is to examine the physical properties of potential binding sites. A number of other workers have addressed this issue. We characterize ~290,000 "pockets" from ~42,000 protein crystal structures in terms of a three parameter "pocket space": volume, buriedness, and hydrophobicity. A metric DLID (drug-like density) measures how likely a pocket is to bind a drug-like molecule. This is calculated from the count of other pockets in its local neighborhood in pocket space that contain drug-like cocrystallized ligands and the count of total pockets in the neighborhood. Surprisingly, despite being defined locally, a global trend in DLID can be predicted by a simple linear regression on log(volume), buriedness, and hydrophobicity. Two levels of simplification are necessary to relate the DLID of individual pockets to "targets": taking the best DLID per Protein Data Bank (PDB) entry (because any given crystal structure can have many pockets), and taking the median DLID over all PDB entries for the same target (because different crystal structures of the same protein can vary because of artifacts and real conformational changes). We can show that median DLIDs for targets that are detectably homologous in sequence are reasonably similar and that median DLIDs correlate with the "druggability" estimate of Cheng et al. (Nature Biotechnology 2007, 25, 71-75).

  9. Pocket book of integrals and mathematical formulas

    CERN Document Server

    Tallarida, Ronald J

    2008-01-01

    Convenient Organization of Essential Material so You Can Look up Formulas Fast Containing a careful selection of standard and timely topics, the Pocket Book of Integrals and Mathematical Formulas, Fourth Edition presents many numerical and statistical tables, scores of worked examples, and the most useful mathematical formulas for engineering and scientific applications. This fourth edition of a bestseller provides even more comprehensive coverage with the inclusion of several additional topics, all while maintaining its accessible, clear style and handy size. New to the Fourth Edition           An expanded chapter on series that covers many fascinating properties of the natural numbers that follow from number theory           New applications such as geostationary satellite orbits and drug kinetics           An expanded statistics section that discusses nonlinear regression as well as the normal approximation of the binomial distribution           Revised f...

  10. Improvement of personal dosimetry - Digital pocket dosemeter

    International Nuclear Information System (INIS)

    Radalj, Z.; Cerovac, Z.; Cerovac, H.; Brumen, V.; Prlic, I.

    1996-01-01

    Physical dosimetric surveillance of professional groups working with various radiation sources is a regular procedure in Croatia, established almost 40 years ago. Data available point out that the majority of professionals under surveillance are those employed in medical facilities, most of them working with X-ray sources. Depending on the nature of professional activities, personnel occupationally exposed to radiation sources are obliged to wear either film badge, TLD or film+TLD badge. Unfortunately, due to the line of data processing, all standard dosemeters have the same disadvantage i.e. up to 40 days delay in dose reporting, regarding the time of actual exposure. The significance of such a delay raises in cases when radiation dose was received within the short time or when technical failure on the operating unit(s) is suspected. Bearing this in mind, the additional dosimetric monitoring becomes an imperative. Therefore, we decided to introduce a palette of digital pocket dosemeters, meant to be used in different workplaces in the radiation zone, each of them being adjusted to the specificities of a particular workplace

  11. Targeting the Central Pocket in Human Transcription Factor TEAD as a Potential Cancer Therapeutic Strategy

    OpenAIRE

    Pobbati, Ajaybabu V.; Han, Xiao; Hung, Alvin W.; Weiguang, Seetoh; Huda, Nur; Chen, Guo-Ying; Kang, CongBao; Chia, Cheng San Brian; Luo, Xuelian; Hong, Wanjin; Poulsen, Anders

    2015-01-01

    The human TEAD family of transcription factors (TEAD1-4) is required for YAP-mediated transcription in the Hippo pathway. Hyperactivation of TEAD’s co-activator YAP contributes to tissue overgrowth and human cancers, suggesting that pharmacological interference of TEAD-YAP activity may be an effective strategy for anticancer therapy. Here we report the discovery of a central pocket in the YAP-binding domain (YBD) of TEAD that is targetable by small molecule inhibitors. Our X-ray crystallograp...

  12. Assessing the structural conservation of protein pockets to study functional and allosteric sites: implications for drug discovery

    Directory of Open Access Journals (Sweden)

    Daura Xavier

    2010-03-01

    Full Text Available Abstract Background With the classical, active-site oriented drug-development approach reaching its limits, protein ligand-binding sites in general and allosteric sites in particular are increasingly attracting the interest of medicinal chemists in the search for new types of targets and strategies to drug development. Given that allostery represents one of the most common and powerful means to regulate protein function, the traditional drug discovery approach of targeting active sites can be extended by targeting allosteric or regulatory protein pockets that may allow the discovery of not only novel drug-like inhibitors, but activators as well. The wealth of available protein structural data can be exploited to further increase our understanding of allosterism, which in turn may have therapeutic applications. A first step in this direction is to identify and characterize putative effector sites that may be present in already available structural data. Results We performed a large-scale study of protein cavities as potential allosteric and functional sites, by integrating publicly available information on protein sequences, structures and active sites for more than a thousand protein families. By identifying common pockets across different structures of the same protein family we developed a method to measure the pocket's structural conservation. The method was first parameterized using known active sites. We characterized the predicted pockets in terms of sequence and structural conservation, backbone flexibility and electrostatic potential. Although these different measures do not tend to correlate, their combination is useful in selecting functional and regulatory sites, as a detailed analysis of a handful of protein families shows. We finally estimated the numbers of potential allosteric or regulatory pockets that may be present in the data set, finding that pockets with putative functional and effector characteristics are widespread across

  13. Structure of a eukaryotic cyclic nucleotide-gated channel

    Science.gov (United States)

    Li, Minghui; Zhou, Xiaoyuan; Wang, Shu; Michailidis, Ioannis; Gong, Ye; Su, Deyuan; Li, Huan; Li, Xueming; Yang, Jian

    2018-01-01

    Summary Cyclic nucleotide-gated (CNG) channels are essential for vision and olfaction. They belong to the voltage-gated ion channel superfamily but their activities are controlled by intracellular cyclic nucleotides instead of transmembrane voltage. Here we report a 3.5 Å-resolution single-particle electron cryomicroscopy structure of a CNG channel from C. elegans in the cGMP-bound open state. The channel has an unusual voltage-sensor-like domain (VSLD), accounting for its deficient voltage dependence. A C-terminal linker connecting S6 and the cyclic nucleotide-binding domain interacts directly with both the VSLD and pore domain, forming a gating ring that couples conformational changes triggered by cyclic nucleotide binding to the gate. The selectivity filter is lined by the carboxylate side chains of a functionally important glutamate and three rings of backbone carbonyls. This structure provides a new framework for understanding mechanisms of ion permeation, gating and channelopathy of CNG channels and cyclic nucleotide modulation of related channels. PMID:28099415

  14. Calibrations of pocket dosemeters using a comparison method

    International Nuclear Information System (INIS)

    Somarriba V, I.

    1996-01-01

    This monograph is dedicated mainly to the calibration of pocket dosemeters. Various types of radiation sources used in hospitals and different radiation detectors with emphasis on ionization chambers are briefly presented. Calibration methods based on the use of a reference dosemeter were developed to calibrate all pocket dosemeters existing at the Radiation Physics and Metrology Laboratory. Some of these dosemeters were used in personnel dosimetry at hospitals. Moreover, a study was realized about factors that affect the measurements with pocket dosemeters in the long term, such as discharges due to cosmic radiation. A DBASE IV program was developed to store the information included in the hospital's registry

  15. Fluorone dyes have binding sites on both cytoplasmic and extracellular domains of Na,K-ATPase.

    Science.gov (United States)

    Havlíková, Marika; Huličiak, Miroslav; Bazgier, Václav; Berka, Karel; Kubala, Martin

    2013-02-01

    Combination of fluorescence techniques and molecular docking was used to monitor interaction of Na,K-ATPase and its large cytoplasmic loop connecting fourth and fifth transmembrane helices (C45) with fluorone dyes (i.e. eosin Y, 5(6)-carboxyeosin, rose bengal, fluorescein, and erythrosine B). Our data suggested that there are at least two binding sites for all used fluorone dyes, except of 5(6)-carboxyeosin. The first binding site is located on C45 loop, and it is sensitive to the presence of nucleotide. The other site is located on the extracellular part of the enzyme, and it is sensitive to the presence of Na(+) or K(+) ions. The molecular docking revealed that in the open conformation of C45 loop (which is obtained in the presence of ATP) all used fluorone dyes occupy position directly inside the ATP-binding pocket, while in the closed conformation (i.e. in the absence of any ligand) they are located only near the ATP-binding site depending on their different sizes. On the extracellular part of the protein, the molecular docking predicts two possible binding sites with similar binding energy near Asp897(α) or Gln69(β). The former was identified as a part of interaction site between α- and β-subunits, the latter is in contact with conserved FXYD sequence of the γ-subunit. Our findings provide structural explanation for numerous older studies, which were performed with fluorone dyes before the high-resolution structures were known. Further, fluorone dyes seem to be good probes for monitoring of intersubunit interactions influenced by Na(+) and K(+) binding. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Out of pocket spending for healthcare services: a study assessing ...

    African Journals Online (AJOL)

    Out of pocket spending for healthcare services: a study assessing the relationship between payment methods and perceived satisfaction with the quality of care in a tertiary health facility in Delta State, Nigeria.

  17. Pocket facts 2008 : Swedish Road Administration, roads and traffic

    Science.gov (United States)

    2008-05-01

    Pocket Facts offers an overall picture of the road : transport system. Here you will find a selection of : brief facts about roads, road transports, vehicles and : people in traffic. In addition, it presents the functions : and organisation at the SR...

  18. Nucleotide and Predicted Amino Acid Sequence-Based Analysis of the Avian Metapneumovirus Type C Cell Attachment Glycoprotein Gene: Phylogenetic Analysis and Molecular Epidemiology of U.S. Pneumoviruses

    Science.gov (United States)

    Alvarez, Rene; Lwamba, Humphrey M.; Kapczynski, Darrell R.; Njenga, M. Kariuki; Seal, Bruce S.

    2003-01-01

    A serologically distinct avian metapneumovirus (aMPV) was isolated in the United States after an outbreak of turkey rhinotracheitis (TRT) in February 1997. The newly recognized U.S. virus was subsequently demonstrated to be genetically distinct from European subtypes and was designated aMPV serotype C (aMPV/C). We have determined the nucleotide sequence of the gene encoding the cell attachment glycoprotein (G) of aMPV/C (Colorado strain and three Minnesota isolates) and predicted amino acid sequence by sequencing cloned cDNAs synthesized from intracellular RNA of aMPV/C-infected cells. The nucleotide sequence comprised 1,321 nucleotides with only one predicted open reading frame encoding a protein of 435 amino acids, with a predicted Mr of 48,840. The structural characteristics of the predicted G protein of aMPV/C were similar to those of the human respiratory syncytial virus (hRSV) attachment G protein, including two mucin-like regions (heparin-binding domains) flanking both sides of a CX3C chemokine motif present in a conserved hydrophobic pocket. Comparison of the deduced G-protein amino acid sequence of aMPV/C with those of aMPV serotypes A, B, and D, as well as hRSV revealed overall predicted amino acid sequence identities ranging from 4 to 16.5%, suggesting a distant relationship. However, G-protein sequence identities ranged from 72 to 97% when aMPV/C was compared to other members within the aMPV/C subtype or 21% for the recently identified human MPV (hMPV) G protein. Ratios of nonsynonymous to synonymous nucleotide changes were greater than one in the G gene when comparing the more recent Minnesota isolates to the original Colorado isolate. Epidemiologically, this indicates positive selection among U.S. isolates since the first outbreak of TRT in the United States. PMID:12682171

  19. A Series of Potent CREBBP Bromodomain Ligands Reveals an Induced-Fit Pocket Stabilized by a Cation–π Interaction**

    Science.gov (United States)

    Rooney, Timothy P C; Filippakopoulos, Panagis; Fedorov, Oleg; Picaud, Sarah; Cortopassi, Wilian A; Hay, Duncan A; Martin, Sarah; Tumber, Anthony; Rogers, Catherine M; Philpott, Martin; Wang, Minghua; Thompson, Amber L; Heightman, Tom D; Pryde, David C; Cook, Andrew; Paton, Robert S; Müller, Susanne; Knapp, Stefan; Brennan, Paul E; Conway, Stuart J

    2014-01-01

    The benzoxazinone and dihydroquinoxalinone fragments were employed as novel acetyl lysine mimics in the development of CREBBP bromodomain ligands. While the benzoxazinone series showed low affinity for the CREBBP bromodomain, expansion of the dihydroquinoxalinone series resulted in the first potent inhibitors of a bromodomain outside the BET family. Structural and computational studies reveal that an internal hydrogen bond stabilizes the protein-bound conformation of the dihydroquinoxalinone series. The side chain of this series binds in an induced-fit pocket forming a cation–π interaction with R1173 of CREBBP. The most potent compound inhibits binding of CREBBP to chromatin in U2OS cells. PMID:24821300

  20. Exploration of pH-dependent behavior of the anion receptor pocket of subdomain IIA of HSA: determination of effective pocket charge using the Debye-Hückel limiting law.

    Science.gov (United States)

    Bolel, Priyanka; Datta, Shubhashis; Mahapatra, Niharendu; Halder, Mintu

    2014-01-09

    Protein-ligand electrostatic interaction can be looked upon as ion receptor-ligand interaction, and the binding cavity of protein can be either an anion or cation receptor depending on the charge of the guest. Here we focus on the exploration of pH-modulated binding of a number of anionic ligands, specific to the subdomain IIA cavity of HSA, such as carmoisine, tartrazine, cochineal red, and warfarin. The logarithm of the binding constant is found to vary linearly with the square-root of ionic strength, indicating applicability of the Debye-Hückel limiting law to protein-ligand electrostatic binding equilibrium, and concludes that the subdomain IIA cavity is an anion receptor. The present approach is very unique that one can calculate the effective charge of the protein-based anion receptor pocket, and the calculated charge has been found to vary between +1 and +3 depending on the pH and ligand itself. The study also indicates that in such cases of specific ligand binding the pocket charge rather than the overall or surface charge of the macromolecule seems to have a paramount role in determining the strength of interaction. For the first time, it is demonstrated that the Debye-Hückel interionic interaction model can be successfully applied to understand the protein-based receptor-ligand electrostatic interaction in general.

  1. A single mutation in Taiwanese H6N1 influenza hemagglutinin switches binding to human-type receptors

    Energy Technology Data Exchange (ETDEWEB)

    de Vries, Robert P.; Tzarum, Netanel; Peng, Wenjie; Thompson, Andrew J.; Ambepitiya Wickramasinghe, Iresha N.; de la Pena, Alba T. Torrents; van Breemen, Marielle J.; Bouwman, Kim M.; Zhu, Xueyong; McBride, Ryan; Yu, Wenli; Sanders, Rogier W.; Verheije, Monique H.; Wilson, Ian A.; Paulson, James C.

    2017-07-10

    In June 2013, the first case of human infection with an avian H6N1 virus was reported in a Taiwanese woman. Although this was a single non-fatal case, the virus continues to circulate in Taiwanese poultry. As with any emerging avian virus that infects humans, there is concern that acquisition of human-type receptor specificity could enable transmission in the human population. Despite mutations in the receptor-binding pocket of the human H6N1 isolate, it has retained avian-type (NeuAcα2-3Gal) receptor specificity. However, we show here that a single nucleotide substitution, resulting in a change from Gly to Asp at position 225 (G225D), completely switches specificity to human-type (NeuAcα2-6Gal) receptors. Significantly, G225D H6 loses binding to chicken trachea epithelium and is now able to bind to human tracheal tissue. Structural analysis reveals that Asp225 directly interacts with the penultimate Gal of the human-type receptor, stabilizing human receptor binding.

  2. Diversity and evolutionary relationship of nucleotide binding site ...

    Indian Academy of Sciences (India)

    Degenerate primers based on the conserved motif (P-loop and GLPL) of the NBS domain from -genes were used to isolate RGAs from the genomic DNA of sweet potato cultivar Qingnong no. 2. Five distinct clusters of RGAs (22 sequences) with the characteristic NBS representing a highly diverse sample were identified in ...

  3. DNA polymerase having modified nucleotide binding site for DNA sequencing

    Science.gov (United States)

    Tabor, Stanley; Richardson, Charles

    1997-01-01

    Modified gene encoding a modified DNA polymerase wherein the modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase.

  4. Diversity and evolutionary relationship of nucleotide binding site ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    Sweet potato is China's fourth most important food crop after rice, wheat and maize. It produces a stable yield under a wide range of environmental conditions and is cultivated in more than 100 countries. China accounts for roughly 80% of the world's production. Sweet potato is widely used as food and animal feed, and is ...

  5. Identification of cyclic nucleotide gated channels using regular expressions

    KAUST Repository

    Zelman, Alice K.

    2013-09-03

    Cyclic nucleotide-gated channels (CNGCs) are nonselective cation channels found in plants, animals, and some bacteria. They have a six-transmembrane/one- pore structure, a cytosolic cyclic nucleotide-binding domain, and a cytosolic calmodulin-binding domain. Despite their functional similarities, the plant CNGC family members appear to have different conserved amino acid motifs within corresponding functional domains than animal and bacterial CNGCs do. Here we describe the development and application of methods employing plant CNGC-specific sequence motifs as diagnostic tools to identify novel candidate channels in different plants. These methods are used to evaluate the validity of annotations of putative orthologs of CNGCs from plant genomes. The methods detail how to employ regular expressions of conserved amino acids in functional domains of annotated CNGCs and together with Web tools such as PHI-BLAST and ScanProsite to identify novel candidate CNGCs in species including Physcomitrella patens. © Springer Science+Business Media New York 2013.

  6. Structural basis of substrate discrimination and integrin binding by autotaxin

    Energy Technology Data Exchange (ETDEWEB)

    Hausmann, Jens; Kamtekar, Satwik; Christodoulou, Evangelos; Day, Jacqueline E.; Wu, Tao; Fulkerson, Zachary; Albers, Harald M.H.G.; van Meeteren, Laurens A.; Houben, Anna J.S.; van Zeijl, Leonie; Jansen, Silvia; Andries, Maria; Hall, Troii; Pegg, Lyle E.; Benson, Timothy E.; Kasiem, Mobien; Harlos, Karl; Vander Kooi, Craig W.; Smyth, Susan S.; Ovaa, Huib; Bollen, Mathieu; Morris, Andrew J.; Moolenaar, Wouter H.; Perrakis, Anastassis (Pfizer); (Leuven); (Oxford); (NCI-Netherlands); (Kentucky)

    2013-09-25

    Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.

  7. Nucleotide excision repair in yeast

    NARCIS (Netherlands)

    Eijk, Patrick van

    2012-01-01

    Nucleotide Excision Repair (NER) is a conserved DNA repair pathway capable of removing a broad spectrum of DNA damage. In human cells a defect in NER leads to the disorder Xeroderma pigmentosum (XP). The yeast Saccharomyces cerevisiae is an excellent model organism to study the mechanism of NER. The

  8. Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain

    Science.gov (United States)

    Mistry, Pragnesh; Laird, Michelle H. W.; Schwarz, Ryan S.; Greene, Shannon; Dyson, Tristan; Snyder, Greg A.; Xiao, Tsan Sam; Chauhan, Jay; Fletcher, Steven; Toshchakov, Vladimir Y.; MacKerell, Alexander D.; Vogel, Stefanie N.

    2015-01-01

    Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein–protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C16H15NO4 (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors. PMID:25870276

  9. Unusual Treatment of Pacemaker Pocket Infection: A Case Report

    Directory of Open Access Journals (Sweden)

    Ali Kazemisaeed

    2010-12-01

    Full Text Available Pocket infection of a cardiac device is usually treated by removing the device and re-implanting it in a new site after complete treatment of the infection. This report illustrates a complicated case of pocket infection in the wake of the implantation of a permanent pacemaker (cardiac resynchronization therapy. The patient was treated conservatively through daily irrigation and dressing, broad-spectrum antibiotics, and debridement without the device being removed; the generator was kept out of the pocket for 5 weeks and then re-implanted in the same location successfully. The method of treatment presented herein can be of value, not least in the elderly population who might experience life-threatening events following the replacement of their cardiac devices.

  10. Crystal structure of the Candida albicans Kar3 kinesin motor domain fused to maltose-binding protein

    International Nuclear Information System (INIS)

    Delorme, Caroline; Joshi, Monika; Allingham, John S.

    2012-01-01

    Highlights: ► The Candida albicans Kar3 motor domain structure was solved as a maltose-binding protein fusion. ► The electrostatic surface and part of the ATPase pocket of the motor domain differs markedly from other kinesins. ► The MBP–Kar3 interface highlights a new site for intramolecular or intermolecular interactions. -- Abstract: In the human fungal pathogen Candida albicans, the Kinesin-14 motor protein Kar3 (CaKar3) is critical for normal mitotic division, nuclear fusion during mating, and morphogenic transition from the commensal yeast form to the virulent hyphal form. As a first step towards detailed characterization of this motor of potential medical significance, we have crystallized and determined the X-ray structure of the motor domain of CaKar3 as a maltose-binding protein (MBP) fusion. The structure shows strong conservation of overall motor domain topology to other Kar3 kinesins, but with some prominent differences in one of the motifs that compose the nucleotide-binding pocket and the surface charge distribution. The MBP and Kar3 modules are arranged such that MBP interacts with the Kar3 motor domain core at the same site where the neck linker of conventional kinesins docks during the “ATP state” of the mechanochemical cycle. This site differs from the Kar3 neck–core interface in the recent structure of the ScKar3Vik1 heterodimer. The position of MBP is also completely distinct from the Vik1 subunit in this complex. This may suggest that the site of MBP interaction on the CaKar3 motor domain provides an interface for the neck, or perhaps a partner subunit, at an intermediate state of its motile cycle that has not yet been observed for Kinesin-14 motors.

  11. Out-of-Pocket Spending for Hospitalizations Among Nonelderly Adults.

    Science.gov (United States)

    Adrion, Emily R; Ryan, Andrew M; Seltzer, Amanda C; Chen, Lena M; Ayanian, John Z; Nallamothu, Brahmajee K

    2016-09-01

    Patients' out-of-pocket spending for major health care expenses, such as inpatient care, may result in substantial financial distress. Limited contemporary data exist on out-of-pocket spending among nonelderly adults. To evaluate out-of-pocket spending associated with hospitalizations and to assess how this spending varied over time and by patient characteristics, region, and type of insurance. A retrospective analysis of medical claims for 7.3 million hospitalizations using 2009-2013 data from Aetna, UnitedHealthcare, and Humana insurance companies representing approximately 50 million members was performed. Out-of-pocket spending was evaluated by age, sex, type of insurance, region, and principal diagnosis or procedure for hospitalized adults aged 18 to 64 years who were enrolled in employer-sponsored and individual-market health insurance plans from January 1, 2009, to December 31, 2013. The study was conducted between July 1, 2015, and March 1, 2016. Primary outcomes were total out-of-pocket spending and spending attributed to deductibles, copayments, and coinsurance for all hospitalizations. Other outcomes included out-of-pocket spending associated with 7 commonly occurring inpatient diagnoses and procedures: acute myocardial infarction, live birth, pneumonia, appendicitis, coronary artery bypass graft, total knee arthroplasty, and spinal fusion. From 2009 to 2013, total cost sharing per inpatient hospitalization increased by 37%, from $738 in 2009 (95% CI, $736-$740) to $1013 in 2013 (95% CI, $1011-$1016), after adjusting for inflation and case-mix differences. This rise was driven primarily by increases in the amount applied to deductibles, which grew by 86% from $145 in 2009 (95% CI, $144-$146) to $270 in 2013 (95% CI, $269-$271), and by increases in coinsurance, which grew by 33% over the study period from $518 in 2009 (95% CI, $516-$520) to $688 in 2013 (95% CI, $686-$690). In 2013, total cost sharing was highest for enrollees in individual market plans

  12. CIED infection with either pocket or systemic infection presentation

    DEFF Research Database (Denmark)

    Ihlemann, Nikolaj; Møller-Hansen, Michael; Salado-Rasmussen, Kirsten

    2016-01-01

    -up no relapses and two cases of new infections were noted (2.8%). CONCLUSIONS: CIED infection with systemic or pocket infection was difficult to distinguish in clinical presentation and outcome. Complete device removal and antibiotic treatment of long duration was safe and without relapses....... infection during the period from 2005 to 2012 were retrospectively reviewed. CIED infections were categorized as systemic or pocket infections. Treatment included complete removal of the device, followed by antibiotic treatment of six weeks. RESULTS: Seventy-one device removals due to infection (32 systemic...

  13. Apache 2 Pocket Reference For Apache Programmers & Administrators

    CERN Document Server

    Ford, Andrew

    2008-01-01

    Even if you know the Apache web server inside and out, you still need an occasional on-the-job reminder -- especially if you're moving to the newer Apache 2.x. Apache 2 Pocket Reference gives you exactly what you need to get the job done without forcing you to plow through a cumbersome, doorstop-sized reference. This Book provides essential information to help you configure and maintain the server quickly, with brief explanations that get directly to the point. It covers Apache 2.x, giving web masters, web administrators, and programmers a quick and easy reference solution. This pocket r

  14. Why not to ''pocket shoot'': Radiology of intravenous drug abuse

    International Nuclear Information System (INIS)

    McCarroll, K.A.; Fisher, D.R.; Cawthon, L.A.; Donovan, K.R.; Roszler, M.H.; Kling, G.A.

    1987-01-01

    Our large population of intravenous drug abusers has increasingly resorted to supraclavicular central venous injection for vascular access. Few reports of complications associated with the practice of supraclavicular ''pocket'' injection have appeared in the radiologic literature. The authors describe the complications associated with this practice, including pneumothorax, mycotic aneurysm, arteriovenous fistula, jugular vein thrombosis, cellulitis, foreign body reaction, and neck abscess. In addition, the authors provide examples of sternoclavicular osteomyelitis. The anatomy of the ''pocket,'' and the pathophysiology and radiographic manifestations of these complications, are reviewed

  15. Canvas Pocket Reference Scripted Graphics for HTML5

    CERN Document Server

    Flanagan, David

    2010-01-01

    The Canvas element is a revolutionary feature of HTML5 that enables powerful graphics for rich Internet applications, and this pocket reference provides the essentials you need to put this element to work. If you have working knowledge of JavaScript, this book will help you create detailed, interactive, and animated graphics -- from charts to animations to video games -- whether you're a web designer or a programmer interested in graphics. Canvas Pocket Reference provides both a tutorial that covers all of the element's features with plenty of examples and a definitive reference to each of t

  16. Vibration Characteristics of Hydrodynamic Fluid Film Pocket Journal Bearings

    Directory of Open Access Journals (Sweden)

    N. S. Feng

    2010-01-01

    Full Text Available Theoretical analyses of hydrodynamic fluid film bearings with different bearing profiles rely on solutions of the Reynolds equation. This paper presents an approach used for analysing the so-called pocket bearings formed from a combination of offset circular bearing profiles. The results show that the variation of the dynamic bearing characteristics with different load inclinations for the pocket bearings is less than that for the elliptic bearing counterpart. It is shown that the natural frequencies as well as the critical speeds, and hence the vibrational behaviour, can also be significantly different for an industrial rotor supported by the different bearings.

  17. Tissue expander infections in children: look beyond the expander pocket.

    Science.gov (United States)

    Mason, A C; Davison, S P; Manders, E K

    1999-11-01

    Infection of the expander pocket is the most common complication encountered with soft-tissue expansion. It is usually due to direct inoculation with skin flora either at the time of expander insertion or from extrusion of the device. The authors report two cases of infection of tissue expanders in which the children had concomitant infected sites distant from the prosthesis. Etiological bacteria of common pediatric infections like otitis media and pharyngitis were cultured from the infected expander pocket, raising suspicion that translocation of the organism to the expander had occurred. Aggressive antibiotic treatment, removal of the prosthesis, and flap advancement is advocated.

  18. Pocket radar guide key facts, equations, and data

    CERN Document Server

    Curry, G Richard

    2010-01-01

    ThePocket Radar Guideis a concise collection of key radar facts and important radar data that provides you with necessary radar information when you are away from your office or references. It includes statements and comments on radar design, operation, and performance; equations describing the characteristics and performance of radar systems and their components; and tables with data on radar characteristics and key performance issues.It is intended to supplement other radar information sources by providing a pocket companion to refresh memory and provide details whenever you need them such a

  19. UML 2.0 Pocket Reference UML Syntax and Usage

    CERN Document Server

    Pilone, Dan

    2006-01-01

    Globe-trotting travelers have long resorted to handy, pocket-size dictionaries as an aid to communicating across the language barrier. Dan Pilone's UML 2.0 Pocket Reference is just such an aid for on-the-go developers who need to converse in the Unified Modeling Language (UML). Use this book to decipher the many UML diagrams you'll encounter on the path to delivering a modern software system. Updated to cover the very latest in UML, you'll find coverage of the following UML 2.0 diagram types: Class diagramsComponent diagrams*Sequence diagrams*Communication diagrams*Timing diagrams*Interactio

  20. A Hydrophobic Pocket in the Active Site of Glycolytic Aldolase Mediates Interactions with Wiskott-Aldrich Syndrome Protein

    Energy Technology Data Exchange (ETDEWEB)

    St-Jean,M.; Izard, T.; Sygusch, J.

    2007-01-01

    Aldolase plays essential catalytic roles in glycolysis and gluconeogenesis. However, aldolase is a highly abundant protein that is remarkably promiscuous in its interactions with other cellular proteins. In particular, aldolase binds to highly acidic amino acid sequences, including the C-terminus of the Wiskott-Aldrich syndrome protein, an actin nucleation promoting factor. Here we report the crystal structure of tetrameric rabbit muscle aldolase in complex with a C-terminal peptide of Wiskott-Aldrich syndrome protein. Aldolase recognizes a short, 4-residue DEWD motif (residues 498-501), which adopts a loose hairpin turn that folds about the central aromatic residue, enabling its tryptophan side chain to fit into a hydrophobic pocket in the active site of aldolase. The flanking acidic residues in this binding motif provide further interactions with conserved aldolase active site residues, Arg-42 and Arg-303, aligning their side chains and forming the sides of the hydrophobic pocket. The binding of Wiskott-Aldrich syndrome protein to aldolase precludes intramolecular interactions of its C-terminus with its active site, and is competitive with substrate as well as with binding by actin and cortactin. Finally, based on this structure a novel naphthol phosphate-based inhibitor of aldolase was identified and its structure in complex with aldolase demonstrated mimicry of the Wiskott-Aldrich syndrome protein-aldolase interaction. The data support a model whereby aldolase exists in distinct forms that regulate glycolysis or actin dynamics.

  1. Interactions of fluoride and guanine nucleotides with thyroid adenylate cyclase.

    Science.gov (United States)

    Goldhammer, A; Wolff, J

    1982-02-18

    The activation of bovine thyroid adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) by Gpp(NH)p has been studied using steady-state kinetic methods. This activation is complex and may be characterized by two Gpp(NH)p binding sites of different affinities with measured constants: Ka1 = 0.1 micro M and Ka2 = 2.9 micro M. GDP beta S does not completely inhibit the Gpp(NH)p activation: analysis of the data is consistent with a single GDP beta S inhibitory site which is competitive with the weaker Gpp(NH)p site. Guanine nucleotide effects upon F- activation of adenylate cyclase have been studied. When App(NH)p is the substrate, 10 micro M GTP along with 10 mM NaF gives higher activity than NaF alone, while GDP together with NaF inhibits the activity by 50% relative to NaF. These features are not observed when the complex is assayed with ATP in the presence of a nucleotide regenerating system or when analogs Gpp)NH)p or GDP beta S are used along with NaF. These effects were studied in three other membrane systems using App(NH)p as substrate: rat liver, rat ovary and turkey erythrocyte. No consistent pattern of guanine nucleotide effects upon fluoride activation could be observed in the different membrane preparations. Previous experiments showed that the size of soluble thyroid adenylate cyclase changed whether membranes were preincubated with Gpp(NH)p or NaF. This size change roughly corresponded to the molecular weight of the nucleotide regulatory protein. This finding, coupled with the present data, suggests that two guanine nucleotide binding sites may be involved in regulating thyroid cyclase and that these sites may be on different protein chains.

  2. Nucleotides increase the internal flexibility of filaments of dephosphorylated Acanthamoeba myosin II.

    Science.gov (United States)

    Redowicz, M J; Korn, E D; Rau, D C

    1996-05-24

    The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosin II minifilaments is dependent both on Mg2+ concentration and on the state of phosphorylation of three serine sites at the C-terminal end of the heavy chains. Previous electric birefringence experiments on minifilaments showed a large dependence of signal amplitude on the phosphorylation state and Mg2+ concentration, consistent with large changes in filament flexibility. These observations suggested that minifilament stiffness was important for function. We now report that the binding of nucleotides to dephosphorylated minifilaments at Mg2+ concentrations needed for optimal activity increases the flexibility by about 10-fold, as inferred from the birefringence signal amplitude increase. An increase in flexibility with nucleotide binding is not observed for dephosphorylated minifilaments at lower Mg2+ concentrations or for phosphorylated minifilaments at any Mg2+ concentrations examined. The relaxation times for minifilament rotations that are sensitive to the conformation myosin heads are also observed to depend on phosphorylation, Mg2+ concentration, and nucleotide binding. These latter experiments indicate that the actin-activated Mg2+ concentration, and nucleotide binding. These latter experiments indicate that the actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosin II correlates with both changes in myosin head conformation and the ability of minifilaments to cycle between stiff and flexible conformations coupled to nucleotide binding and release.

  3. Screening Analogs of β-OG Pocket Binder as Fusion Inhibitor of Dengue Virus 2.

    Science.gov (United States)

    Tambunan, Usman Sf; Zahroh, Hilyatuz; Parikesit, Arli A; Idrus, Syarifuddin; Kerami, Djati

    2015-01-01

    Dengue is an infectious disease caused by dengue virus (DENV) and transmitted between human hosts by mosquitoes. Recently, Indonesia was listed as a country with the highest cases of dengue by the Association of Southeast Asian Nations. The current treatment for dengue disease is supportive therapy; there is no antiviral drug available in the market against dengue. Therefore, a research on antiviral drug against dengue is very important, especially to prevent outbreak explosion. In this research, the development of dengue antiviral is performed through the inhibition of n-octyl-β-D-glucoside (β-OG) binding pocket on envelope protein of DENV by using analogs of β-OG pocket binder. There are 828 compounds used in this study, and all of them were screened based on the analysis of molecular docking, pharmacological character prediction of the compounds, and molecular dynamics simulation. The result of these analyses revealed that the compound that can be used as an antiviral candidate against DENV is 5-(3,4-dichlorophenyl)-N-[2-(p-tolyl) benzotriazol-5-yl]furan-2-carboxamide.

  4. Out-of-pocket payment for health services: constraints and ...

    African Journals Online (AJOL)

    Conclusion: This study brings to the fore the fact that most government employees and their dependants in Abakaliki have difficulties in accessing quality health care services via paying for them out-of-pocket. Keywords: Health services, payment, constraints, government employees. African Health Sciences 2011; 11(3): 481 ...

  5. Adhesion of Porphyromonas gingivalis serotypes to pocket epithelium

    NARCIS (Netherlands)

    Dierickx, K; Pauwels, M; Laine, ML; Van Eldere, J; Cassiman, JJ; van Winkelhoff, AJ; van Steenberghe, D; Quirynen, M

    Background: Porphyromonas gingivalis, a key pathogen in periodontitis, is able to adhere to and invade the pocket epithelium. Different capsular antigens of P gingivalis have been identified (K-serotyping). These P gingivalis capsular types show differences in adhesion capacity to human cell lines

  6. Book Review: Nelson Mandela: A Jacana Pocket Biography ...

    African Journals Online (AJOL)

    Book Title: Nelson Mandela: A Jacana Pocket Biography. Author: Colin Bundy. Jacana: Auckland Park, 2015. 159 pp. Full Text: EMAIL FULL TEXT EMAIL FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT · AJOL African Journals Online. HOW TO USE AJOL... for Researchers · for Librarians · for Authors ...

  7. Book Review: Chris Hani: A Jacana Pocket Biography | Smith | New ...

    African Journals Online (AJOL)

    Book Title: Chris Hani: A Jacana Pocket Biography. Author: Hugh Macmillan. Jacana: Auckland Park, 2014. 152 pp. Full Text: EMAIL FULL TEXT EMAIL FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT · AJOL African Journals Online. HOW TO USE AJOL... for Researchers · for Librarians · for Authors · FAQ's ...

  8. Reynolds number scaling of pocket events in the viscous sublayer

    Science.gov (United States)

    Metzger, M.; Fershtut, A.; Kunkel, C.; Klewicki, J.

    2017-12-01

    Recent findings [X. Wu et al., Proc. Natl. Acad. Sci. USA 114, E5292 (2017), 10.1073/pnas.1704671114] reinforce earlier assertions [e.g., R. Falco, Philos. Trans. R. Soc. London A 336, 103 (1991), 10.1098/rsta.1991.0069] that the sublayer pocket motions play a distinctly important role in near-wall dynamics. In the present study, smoke visualization and axial velocity measurements are combined in order to establish the scaling behavior of pocket events in the viscous sublayer of the turbulent boundary layer. In doing so, an identical analysis methodology is employed over an extensive range of friction Reynolds numbers 388 ≤δ+≤2.2 ×105 . Both the pocket width W and time interval between pocket events T increase logarithmically with Reynolds number when normalized by viscous units. Normalization of W and T by the Taylor microscales evaluated at a wall-normal location of about 100 viscous units, however, appears to successfully remove this Reynolds-number dependence. The present results are discussed in the context of motion formation owing to the three dimensionalization of the near-wall vorticity field and, concomitantly, the recurring perturbation of the viscous sublayer.

  9. The Use of Pocket Electronic Dictionaries by Thai University Students

    African Journals Online (AJOL)

    This article reports on a small-scale study of Thai-speaking learners using pocket electronic dictionaries (PEDs) to read an English news article. It investigates how the subjects use their PEDs for reading comprehension. Thirty-nine undergraduate students completed a questionnaire survey. Of these, four were chosen to ...

  10. Equity in out-of-pocket payment in Chile.

    Science.gov (United States)

    Mondaca, Alicia Lorena Núñez; Chi, Chunhuei

    2017-05-04

    To assess the distribution of financial burden in Chile, with a focus on the burden and progressivity of out-of-pocket payment. Based on the principle of ability to pay, we explore factors that contribute to inequities in the health system finance and issues about the burden of out-of-pocket payment, as well as the progressivity and redistributive effect of out-of-pocket payment in Chile. Our analysis is based on data from the 2006 National Survey on Satisfaction and Out-of-Pocket Payments. Results from this study indicate evidence of inequity, in spite of the progressivity of the healthcare system. Our analysis also identifies relevant policy variables such as education, insurance system, and method of payment that should be taken into consideration in the ongoing debates and research in improving the Chilean system. In order to reduce the detected disparities among income groups, healthcare priorities should target low-income groups. Furthermore, policies should explore changes in the access to education and its impact on equity.

  11. Nucleotide patterns aiding in prediction of eukaryotic promoters

    Science.gov (United States)

    Triska, Martin; Solovyev, Victor; Baranova, Ancha; Kel, Alexander

    2017-01-01

    Computational analysis of promoters is hindered by the complexity of their architecture. In less studied genomes with complex organization, false positive promoter predictions are common. Accurate identification of transcription start sites and core promoter regions remains an unsolved problem. In this paper, we present a comprehensive analysis of genomic features associated with promoters and show that probabilistic integrative algorithms-driven models allow accurate classification of DNA sequence into “promoters” and “non-promoters” even in absence of the full-length cDNA sequences. These models may be built upon the maps of the distributions of sequence polymorphisms, RNA sequencing reads on genomic DNA, methylated nucleotides, transcription factor binding sites, as well as relative frequencies of nucleotides and their combinations. Positional clustering of binding sites shows that the cells of Oryza sativa utilize three distinct classes of transcription factors: those that bind preferentially to the [-500,0] region (188 “promoter-specific” transcription factors), those that bind preferentially to the [0,500] region (282 “5′ UTR-specific” TFs), and 207 of the “promiscuous” transcription factors with little or no location preference with respect to TSS. For the most informative motifs, their positional preferences are conserved between dicots and monocots. PMID:29141011

  12. Nucleotide-mediated conformational changes of monomeric actin and Arp3 studied by molecular dynamics simulations.

    Science.gov (United States)

    Dalhaimer, Paul; Pollard, Thomas D; Nolen, Brad J

    2008-02-08

    Members of the actin family of proteins exhibit different biochemical properties when ATP, ADP-P(i), ADP, or no nucleotide is bound. We used molecular dynamics simulations to study the effect of nucleotides on the behavior of actin and actin-related protein 3 (Arp3). In all of the actin simulations, the nucleotide cleft stayed closed, as in most crystal structures. ADP was much more mobile within the cleft than ATP, despite the fact that both nucleotides adopt identical conformations in actin crystal structures. The nucleotide cleft of Arp3 opened in most simulations with ATP, ADP, and no bound nucleotide. Deletion of a C-terminal region of Arp3 that extends beyond the conserved actin sequence reduced the tendency of the Arp3 cleft to open. When the Arp3 cleft opened, we observed multiple instances of partial release of the nucleotide. Cleft opening in Arp3 also allowed us to observe correlated movements of the phosphate clamp, cleft mouth, and barbed-end groove, providing a way for changes in the nucleotide state to be relayed to other parts of Arp3. The DNase binding loop of actin was highly flexible regardless of the nucleotide state. The conformation of Ser14/Thr14 in the P1 loop was sensitive to the presence of the gamma-phosphate, but other changes observed in crystal structures were not correlated with the nucleotide state on nanosecond timescales. The divalent cation occupied three positions in the nucleotide cleft, one of which was not previously observed in actin or Arp2/3 complex structures. In sum, these simulations show that subtle differences in structures of actin family proteins have profound effects on their nucleotide-driven behavior.

  13. Coupling of guanine nucleotide inhibitory protein to somatostatin receptors on pancreatic acinar membranes

    International Nuclear Information System (INIS)

    Sakamoto, C.; Matozaki, T.; Nagao, M.; Baba, S.

    1987-01-01

    Guanine nucleotides and pertussis toxin were used to investigate whether somatostatin receptors interact with the guanine nucleotide inhibitory protein (NI) on pancreatic acinar membranes in the rat. Guanine nucleotides reduced 125 I-[Tyr 1 ]somatostatin binding to acinar membranes up to 80%, with rank order of potency being 5'-guanylyl imidodiphosphate [Gpp(NH)p]>GTP>TDP>GMP. Scatchard analysis revealed that the decrease in somatostatin binding caused by Gpp(NH)p was due to the decrease in the maximum binding capacity without a significant change in the binding affinity. The inhibitory effect of Gpp(NH)p was partially abolished in the absence of Mg 2+ . When pancreatic acini were treated with 1 μg/ml pertussis toxin for 4 h, subsequent 125 I-[Tyr 1 ]somatostatin binding to acinar membranes was reduced. Pertussis toxin treatment also abolished the inhibitory effect of somatostatin on vasoactive intestinal peptide-stimulated increase in cellular content of adenosine 3',5'-cyclic monophosphate (cAMP) in the acini. The present results suggest that 1) somatostatin probably functions in the pancreas to regulate adenylate cyclase enzyme system via Ni, 2) the extent of modification of Ni is correlated with the ability of somatostatin to inhibit cAMP accumulation in acini, and 3) guanine nucleotides also inhibit somatostatin binding to its receptor

  14. Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP Binding: Requirement for Establishing Chronic Persistent Infection

    Energy Technology Data Exchange (ETDEWEB)

    Drumm, J.; Mi, K; Bilder, P; Sun, M; Lim, J; Bielefeldt-Ohmann, H; Basaraba, R; So, M; Zhu, G; et. al.

    2009-01-01

    Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii Rv2623 binds ATP; and iv the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

  15. Nucleotide effects on the structure and dynamics of actin.

    Science.gov (United States)

    Zheng, Xiange; Diraviyam, Karthikeyan; Sept, David

    2007-08-15

    Adenosine 5'-triphosphate or ATP is the primary energy source within the cell, releasing its energy via hydrolysis into adenosine 5'-diphosphate or ADP. Actin is an important ATPase involved in many aspects of cellular function, and the binding and hydrolysis of ATP regulates its polymerization into actin filaments as well as its interaction with a host of actin-associated proteins. Here we study the dynamics of monomeric actin in ATP, ADP-Pi, and ADP states via molecular dynamics simulations. As observed in some crystal structures we see that the DNase-I loop is an alpha-helix in the ADP state but forms an unstructured coil domain in the ADP-Pi and ATP states. We also find that this secondary structure change is reversible, and by mimicking nucleotide exchange we can observe the transition between the helical and coil states. Apart from the DNase-I loop, we also see several key structural differences in the nucleotide binding cleft as well as in the hydrophobic cleft between subdomains 1 and 3 where WH2-containing proteins have been shown to interact. These differences provide a structural basis for understanding the observed differences between the various nucleotide states of actin and provide some insight into how ATP regulates the interaction of actin with itself and other proteins.

  16. Relating the shape of protein binding sites to binding affinity profiles: is there an association?

    Directory of Open Access Journals (Sweden)

    Bitter István

    2010-10-01

    Full Text Available Abstract Background Various pattern-based methods exist that use in vitro or in silico affinity profiles for classification and functional examination of proteins. Nevertheless, the connection between the protein affinity profiles and the structural characteristics of the binding sites is still unclear. Our aim was to investigate the association between virtual drug screening results (calculated binding free energy values and the geometry of protein binding sites. Molecular Affinity Fingerprints (MAFs were determined for 154 proteins based on their molecular docking energy results for 1,255 FDA-approved drugs. Protein binding site geometries were characterized by 420 PocketPicker descriptors. The basic underlying component structure of MAFs and binding site geometries, respectively, were examined by principal component analysis; association between principal components extracted from these two sets of variables was then investigated by canonical correlation and redundancy analyses. Results PCA analysis of the MAF variables provided 30 factors which explained 71.4% of the total variance of the energy values while 13 factors were obtained from the PocketPicker descriptors which cumulatively explained 94.1% of the total variance. Canonical correlation analysis resulted in 3 statistically significant canonical factor pairs with correlation values of 0.87, 0.84 and 0.77, respectively. Redundancy analysis indicated that PocketPicker descriptor factors explain 6.9% of the variance of the MAF factor set while MAF factors explain 15.9% of the total variance of PocketPicker descriptor factors. Based on the salient structures of the factor pairs, we identified a clear-cut association between the shape and bulkiness of the drug molecules and the protein binding site descriptors. Conclusions This is the first study to investigate complex multivariate associations between affinity profiles and the geometric properties of protein binding sites. We found that

  17. F pocket flexibility influences the tapasin dependence of two differentially disease-associated MHC Class I proteins.

    Science.gov (United States)

    Abualrous, Esam T; Fritzsche, Susanne; Hein, Zeynep; Al-Balushi, Mohammed S; Reinink, Peter; Boyle, Louise H; Wellbrock, Ursula; Antoniou, Antony N; Springer, Sebastian

    2015-04-01

    The human MHC class I protein HLA-B*27:05 is statistically associated with ankylosing spondylitis, unlike HLA-B*27:09, which differs in a single amino acid in the F pocket of the peptide-binding groove. To understand how this unique amino acid difference leads to a different behavior of the proteins in the cell, we have investigated the conformational stability of both proteins using a combination of in silico and experimental approaches. Here, we show that the binding site of B*27:05 is conformationally disordered in the absence of peptide due to a charge repulsion at the bottom of the F pocket. In agreement with this, B*27:05 requires the chaperone protein tapasin to a greater extent than the conformationally stable B*27:09 in order to remain structured and to bind peptide. Taken together, our data demonstrate a method to predict tapasin dependence and physiological behavior from the sequence and crystal structure of a particular class I allotype. Also watch the Video Abstract. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. 24 CFR 570.466 - Additional application submission requirements for Pockets of Poverty-employment opportunities.

    Science.gov (United States)

    2010-04-01

    ... requirements for Pockets of Poverty-employment opportunities. 570.466 Section 570.466 Housing and Urban... application submission requirements for Pockets of Poverty—employment opportunities. Applicants for Action Grants under the Pockets of Poverty provision must describe the number and, to the extent possible, the...

  19. Health Promoting Pocket Parks in a Landscape Architectural Perspective

    DEFF Research Database (Denmark)

    Peschardt, Karin Kragsig

    shown to have a positive influence on preventing lifestyle related diseases, although only limited research suggests how the various green spaces in the urban green infrastructure (UGI) can benefit health. Especially knowledge about the role of pocket parks is lacking. The study evaluates the health...... cities have become detached from nature and live most of their daily life indoors where sedentary work and physical inactivity characterise everyday life. It has been suggested that this inactivity has resulted in a rapid increase in a number of lifestyle diseases. Urban green spaces (UGS) have been......This thesis presents how the health potential of pocket parks can be improved through design from a landscape architectural perspective. In developed countries, the densification of cities is a wide-spread tendency which often results in a compact city planning structure. People who live in dense...

  20. Pocket PC-based portable gamma-ray spectrometer

    Directory of Open Access Journals (Sweden)

    Kamontip Ploykrachang

    2011-04-01

    Full Text Available A portable gamma-ray spectrometer based on a Pocket PC has been developed. A 12-bit pipeline analog-to-digitalconverter (ADC associated with an implemented pulse height histogram function on field programmable gate array (FPGAoperating at 15 MHz is employed for pulse height analysis from built-in pulse amplifier. The system, which interfaces withthe Pocket PC via an enhanced RS-232 serial port under the microcontroller facilitation, is utilized for spectrum acquisition,display and analysis. The pulse height analysis capability of the system was tested and it was found that the ADC integralnonlinearity of ±0.45% was obtained with the throughput rate at 160 kcps. The overall system performance was tested usinga PIN photodiode-CsI(Tl crystal coupled scintillation detector and gamma standard radioactive sources of Cs-137 andCo-60. Low cost and the compact system size as a result of the implemented logical function are also discussed.

  1. NMR studies on DNA binding specificity of the lac repressor

    NARCIS (Netherlands)

    Kopke Salinas, Roberto

    2005-01-01

    The thesis describes NMR structures of two protein-DNA complexes. The first structure shows how the protein, the DNA binding domain of lac repressor, recognizes its natural DNA binding site, by adaptation and read out of the nucleotide sequence. The second one shows how the DNA binding specificity

  2. Toothbrushing Behaviour and Periodontal Pocketing: An 11-year longitudinal study

    OpenAIRE

    Joshi S; Suominen AL; Knuuttila M; Bernabé E

    2018-01-01

    Aim To explore the association between toothbrushing behaviour and change in periodontal pocketing among adults. Methods We pooled data from 1,025 adults, aged 30–89 years, who participated in two national surveys in Finland (Health 2000 and Health 2011, BRIF8901) and reported their toothbrushing frequency. A cumulative measure of regular toothbrushing was created by counting the number of times participants reported brushing twice or more daily across the two surveys (ranging from ...

  3. Newnes circuit calculations pocket book with computer programs

    CERN Document Server

    Davies, Thomas J

    2013-01-01

    Newnes Circuit Calculations Pocket Book: With Computer Programs presents equations, examples, and problems in circuit calculations. The text includes 300 computer programs that help solve the problems presented. The book is comprised of 20 chapters that tackle different aspects of circuit calculation. The coverage of the text includes dc voltage, dc circuits, and network theorems. The book also covers oscillators, phasors, and transformers. The text will be useful to electrical engineers and other professionals whose work involves electronic circuitry.

  4. Toothbrushing behaviour and periodontal pocketing: An 11-year longitudinal study.

    Science.gov (United States)

    Joshi, Shriraksha; Suominen, Anna L; Knuuttila, Matti; Bernabé, Eduardo

    2018-02-01

    To explore the association between toothbrushing behaviour and change in periodontal pocketing among adults. We pooled data from 1,025 adults, aged 30-89 years, who participated in two national surveys in Finland (Health 2000 and Health 2011, BRIF8901) and reported their toothbrushing frequency. A cumulative measure of regular toothbrushing was created by counting the number of times participants reported brushing twice or more daily across the two surveys (ranging from 0 to 2). The association between toothbrushing behaviour and the number of teeth with periodontal pocket depth (PPD) ≥4 mm over 11 years was assessed in linear regression models adjusting for confounders. There was a clear dose-response relationship between toothbrushing frequency (either at baseline or follow-up) and change in number of teeth with PPD ≥ 4 mm. There was also evidence of a cumulative effect of regular toothbrushing on change in number of teeth with PPD ≥ 4 mm. Participants who reported brushing twice or more a day in both surveys developed 1.99 (95% CI: 1.02-2.95) fewer teeth with PPD ≥ 4 mm than those who did not report this behaviour in any survey. This 11-year prospective study showed that toothbrushing behaviour was associated with smaller increments in the number of teeth with periodontal pocketing. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Electrophysiological analysis of the mutated Na,K-ATPase cation binding pocket.

    NARCIS (Netherlands)

    Koenderink, J.B.; Geibel, S.; Grabsch, E.; Pont, J.J.H.H.M. de; Bamberg, E.; Friedrich, T.

    2003-01-01

    Na,K-ATPase mediates net electrogenic transport by extruding three Na+ ions and importing two K+ ions across the plasma membrane during each reaction cycle. We mutated putative cation coordinating amino acids in transmembrane hairpin M5-M6 of rat Na,K-ATPase: Asp776 (Gln, Asp, Ala), Glu779 (Asp,

  6. MeCP2 recognizes cytosine methylated tri-nucleotide and di-nucleotide sequences to tune transcription in the mammalian brain.

    Directory of Open Access Journals (Sweden)

    Sabine Lagger

    2017-05-01

    Full Text Available Mutations in the gene encoding the methyl-CG binding protein MeCP2 cause several neurological disorders including Rett syndrome. The di-nucleotide methyl-CG (mCG is the classical MeCP2 DNA recognition sequence, but additional methylated sequence targets have been reported. Here we show by in vitro and in vivo analyses that MeCP2 binding to non-CG methylated sites in brain is largely confined to the tri-nucleotide sequence mCAC. MeCP2 binding to chromosomal DNA in mouse brain is proportional to mCAC + mCG density and unexpectedly defines large genomic domains within which transcription is sensitive to MeCP2 occupancy. Our results suggest that MeCP2 integrates patterns of mCAC and mCG in the brain to restrain transcription of genes critical for neuronal function.

  7. Extracellular modulation of the silkmoth sex pheromone receptor activity by cyclic nucleotides.

    Directory of Open Access Journals (Sweden)

    Tatsuro Nakagawa

    Full Text Available Odorants and pheromones are essential to insects as chemical cues for finding food or an appropriate mating partner. These volatile compounds bind to olfactory receptors (Ors expressed by olfactory sensory neurons. Each insect Or functions as a ligand-gated ion channel and is a heteromeric complex that comprises one type of canonical Or and a highly conserved Orco subunit. Because there are many Or types, insect Ors can recognize with high specificity a myriad of chemical cues. Cyclic nucleotides can modulate the activity of insect Or-Orco complexes; however, the mechanism of action of these nucleotides is under debate. Here, we show that cyclic nucleotides, including cAMP and cGMP, interact with the silkmoth sex pheromone receptor complex, BmOr-1-BmOrco, from the outside of the cell and that these nucleotides act as antagonists at low concentrations and weak agonists at high concentrations. These cyclic nucleotides do not compete with the sex pheromone, bombykol, for binding to the BmOr-1 subunit. ATP and GTP also weakly inhibited BmOr-1-BmOrco activity, but D-ribose had no effect; these findings indicated that the purine moiety was crucial for the inhibition. Only the bombykol receptors have been so far shown to be subject to modulation by nucleotide-related compounds, indicating that this responsiveness to these compounds is not common for all insect Or-Orco complexes.

  8. Small organic compounds enhance antigen loading of class II major histocompatibility complex proteins by targeting the polymorphic P1 pocket

    DEFF Research Database (Denmark)

    Höpner, Sabine; Dickhaut, Katharina; Hofstätter, Maria

    2006-01-01

    immune responses by catalyzing the peptide loading of human class II MHC molecules HLA-DR. Here we show now that they achieve this by interacting with a defined binding site of the HLA-DR peptide receptor. Screening of a compound library revealed a set of adamantane derivatives that strongly accelerated...... the peptide loading rate. The effect was evident only for an allelic subset and strictly correlated with the presence of glycine at the dimorphic position beta86 of the HLA-DR molecule. The residue forms the floor of the conserved pocket P1, located in the peptide binding site of MHC molecule. Apparently......-susceptible" MHC molecules. As catalysts of antigen loading, compounds targeting P1 may be useful molecular tools to amplify the immune response. The observation, however, that the ligand repertoire can be affected through polymorphic sites form the outside may also imply that environmental factors could induce...

  9. A series of potent CREBBP bromodomain ligands reveals an induced-fit pocket stabilized by a cation-π interaction.

    Science.gov (United States)

    Rooney, Timothy P C; Filippakopoulos, Panagis; Fedorov, Oleg; Picaud, Sarah; Cortopassi, Wilian A; Hay, Duncan A; Martin, Sarah; Tumber, Anthony; Rogers, Catherine M; Philpott, Martin; Wang, Minghua; Thompson, Amber L; Heightman, Tom D; Pryde, David C; Cook, Andrew; Paton, Robert S; Müller, Susanne; Knapp, Stefan; Brennan, Paul E; Conway, Stuart J

    2014-06-10

    The benzoxazinone and dihydroquinoxalinone fragments were employed as novel acetyl lysine mimics in the development of CREBBP bromodomain ligands. While the benzoxazinone series showed low affinity for the CREBBP bromodomain, expansion of the dihydroquinoxalinone series resulted in the first potent inhibitors of a bromodomain outside the BET family. Structural and computational studies reveal that an internal hydrogen bond stabilizes the protein-bound conformation of the dihydroquinoxalinone series. The side chain of this series binds in an induced-fit pocket forming a cation-π interaction with R1173 of CREBBP. The most potent compound inhibits binding of CREBBP to chromatin in U2OS cells. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Loss of Nucleotide Excision Repair as a Source of Genomic Instability in Breast Cancer

    Science.gov (United States)

    2005-06-01

    sequence-specific mechanism of nucleotide excision repair. Genes Dev., 13, 768-785. DNA binding by short single strands of DNA requires the p53 C... Arabidopsis thaliana . The advantage signal (27). CPD-3 cells were further subcloned by single cell dilution, of using XP-A cells completely deficient...developed and optimized a novel technique for the detection of localized DNA damage and damage binding proteins in individual cells, using targeted

  11. Kinetics of the interactions between yeast elongation factors 1A and 1Balpha, guanine nucleotides, and aminoacyl-tRNA

    DEFF Research Database (Denmark)

    Gromadski, Kirill B; Schümmer, Tobias; Strømgaard, Anne

    2007-01-01

    of guanine nucleotides. At the concentrations of nucleotides and factors prevailing in the cell, the overall exchange rate is expected to be in the range of 6 s(-1), which is compatible with the rate of protein synthesis in the cell. eEF1A.GTP binds Phe-tRNA(Phe) with a K(d) of 3 nm, whereas eEF1A.GDP shows...... no significant binding, indicating that eEF1A has similar tRNA binding properties as its prokaryotic homolog, EF-Tu. Udgivelsesdato: 2007-Dec-7...

  12. The nucleotide exchange factor Ric-8A is a chaperone for the conformationally dynamic nucleotide-free state of Gαi1.

    Directory of Open Access Journals (Sweden)

    Celestine J Thomas

    Full Text Available Heterotrimeric G protein α subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of Gα, catalyzed by guanine nucleotide exchange factors (GEFs. In addition to transmembrane G protein-coupled receptors (GPCRs, which act on G protein heterotrimers, members of the family cytosolic proteins typified by mammalian Ric-8A are GEFs for Gi/q/12/13-class Gα subunits. Ric-8A binds to Gα•GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free Gαi1 is stable, but dissociates upon binding of GTP to Gαi1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from Gαi1, experiments were conducted to characterize the physical state of nucleotide-free Gαi1 (hereafter referred to as Gαi1[ ] in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free Gαi1 is more accessible to trypsinolysis than Gαi1•GDP, but less so than Gαi1[ ] alone. The TROSY-HSQC spectrum of [(15N]Gαi1[ ] bound to Ric-8A shows considerable loss of peak intensity relative to that of [(15N]Gαi1•GDP. Hydrogen-deuterium exchange in Gαi1[ ] bound to Ric-8A is 1.5-fold more extensive than in Gαi1•GDP. Differential scanning calorimetry shows that both Ric-8A and Gαi1•GDP undergo cooperative, irreversible unfolding transitions at 47° and 52°, respectively, while nucleotide-free Gαi1 shows a broad, weak transition near 35°. The unfolding transition for Ric-8A:Gαi1[ ] is complex, with a broad transition that peaks at 50°, suggesting that both Ric-8A and Gαi1[ ] are stabilized within the complex, relative to their respective free states. The C-terminus of Gαi1 is shown to be a critical binding element for Ric-8A, as is also the case for GPCRs, suggesting that the two types of GEF might promote nucleotide exchange by similar mechanisms, by acting as chaperones for the unstable and dynamic nucleotide-free state of Gα.

  13. Practical Pocket PC Application w/Biometric Security

    Science.gov (United States)

    Logan, Julian

    2004-01-01

    I work in the Flight Software Engineering Branch, where we provide design and development of embedded real-time software applications for flight and supporting ground systems to support the NASA Aeronautics and Space Programs. In addition, this branch evaluates, develops and implements new technologies for embedded real-time systems, and maintains a laboratory for applications of embedded technology. The majority of microchips that are used in modern society have been programmed using embedded technology. These small chips can be found in microwaves, calculators, home security systems, cell phones and more. My assignment this summer entails working with an iPAQ HP 5500 Pocket PC. This top-of-the-line hand-held device is one of the first mobile PC's to introduce biometric security capabilities. Biometric security, in this case a fingerprint authentication system, is on the edge of technology as far as securing information. The benefits of fingerprint authentication are enormous. The most significant of them are that it is extremely difficult to reproduce someone else's fingerprint, and it is equally difficult to lose or forget your own fingerprint as opposed to a password or pin number. One of my goals for this summer is to integrate this technology with another Pocket PC application. The second task for the summer is to develop a simple application that provides an Astronaut EVA (Extravehicular Activity) Log Book capability. The Astronaut EVA Log Book is what an astronaut would use to report the status of field missions, crew physical health, successes, future plans, etc. My goal is to develop a user interface into which these data fields can be entered and stored. The applications that I am developing are created using eMbedded Visual C++ 4.0 with the Pocket PC 2003 Software Development Kit provided by Microsoft.

  14. Electronic properties of carbon nanotubes complexed with a DNA nucleotide.

    Science.gov (United States)

    Chehelamirani, Morteza; da Silva, Maurício C; Salahub, Dennis R

    2017-03-08

    Electronic properties of carbon nanotubes (CNTs) play an important role in their interactions with nano-structured materials. In this work, interactions of adenosine monophosphate (AMP), a DNA nucleotide, with metallic and semi-conducting CNTs are studied using the density functional tight binding (DFTB) method. The electronic structure of semi-conducting CNTs was found to be changed as they turned to metallic CNTs in a vacuum upon interaction with the nucleotide while metallic CNTs remain metallic. Specifically, the band gap of semi-conducting CNTs was decreased by 0.79 eV on average while nearly no change was found in the metallic tubes. However, our investigations showed that the presence of explicit water molecules prevents the metallicity change and only small changes in the CNT band gap occur. According to our charge analysis, the average negative charge accumulated on CNTs upon interaction with the AMP was determined to be 0.77 e in a vacuum while it was 0.03 e in solution. Therefore, it is essential to include explicit water molecules in simulating complexes formed by DNA nucleotides and CNTs which were ignored in several past studies performed using quantum mechanical approaches.

  15. Newnes audio and Hi-Fi engineer's pocket book

    CERN Document Server

    Capel, Vivian

    2013-01-01

    Newnes Audio and Hi-Fi Engineer's Pocket Book, Second Edition provides concise discussion of several audio topics. The book is comprised of 10 chapters that cover different audio equipment. The coverage of the text includes microphones, gramophones, compact discs, and tape recorders. The book also covers high-quality radio, amplifiers, and loudspeakers. The book then reviews the concepts of sound and acoustics, and presents some facts and formulas relevant to audio. The text will be useful to sound engineers and other professionals whose work involves sound systems.

  16. Windows® Small Business Server 2008 Administrator's Pocket Consultant

    CERN Document Server

    Zacker, Craig

    2009-01-01

    Portable and precise, this pocket-sized guide delivers ready answers for administering Windows Small Business Server 2008. Zero in on core support tasks and tools using quick-reference tables, instructions, and lists. You'll get the focused information you need to solve problems and get the job done-whether at your desk or in the field. Get fast facts to: Plan, install, and configure a small business network Navigate the Windows SBS Console toolCreate and administer user and group accounts Manage automatic updates, disk storage, and shared printersConfigure mail settings and customize inte

  17. Environment protection and occupational safety - pocket book 1993/94

    International Nuclear Information System (INIS)

    Kroll, J.M.

    1993-01-01

    The pocket book, which formerly bore the title ''Ecology and environmental engineering'', contains dates and data and above all addresses of journalists publicists, photographers, including those living in the new Laender, of publications and documentations such as daily and weekly newspapers, of the electronic media, business and industry, the parliaments, governments and ministries of the Federation and the Laender, institutions of science and education and of associations and organisations it serves as a guide for all those who have committed themselves either privately or professionally to the cause of ecology/environment/occupational safety. (orig./HP) [de

  18. Pocket atlas of head and neck MRI anatomy

    Energy Technology Data Exchange (ETDEWEB)

    Lufkin, R.B.; Hanafee, W.N.

    1989-01-01

    This pocket atlas depicts the anatomy of the head and neck as seen in magnetic resonance (MR) images. The collection of 140 high-resolution images covers all major areas - neck, larynx, oropharynx, tongue, nasopharynx, skull base, sinuses, and temporal bone - displayed in sagittal, axial, and coronal MR image planes. The images show maximum fat/muscle contrast for better visualization of fascial planes. In certain areas of the anatomy, such as the neck and temporal bone, surface coils were used to achieve significant advantages in image quality over standard head or body coils.

  19. Windows® Group Policy Administrators Pocket Consultant

    CERN Document Server

    Stanek, William

    2009-01-01

    Portable and precise, this pocket-sized guide delivers ready answers for the day-to-day administration of Group Policy. Zero in on core support and maintenance tasks using quick-reference tables, instructions, and lists. You'll get the focused information you need to solve problems and get the job done-whether at your desk or in the field! Get fast facts to: Configure Local GPOs and Active Directory®-based GPOsManage policy preferences and settingsModel policy changes through the consoleMigrate and maintain the SYSVOLDiagnose and troubleshoot replication issuesKnow when to enforce, block,

  20. Contralateral Abdominal Pocketing in Salvation of Replanted Fingertips with Compromised Circulation

    Directory of Open Access Journals (Sweden)

    Hyung-Sup Shim

    2014-01-01

    Full Text Available Abdominal pocketing is one of the most useful methods in salvation of compromised replanted fingertips. Abdominal pocketing has generally been performed in the ipsilateral lower abdominal quadrant, but we have also performed contralateral pocketing at our institute. To determine which approach is more beneficial, a total of 40 patients underwent an abdominal pocketing procedure in either the ipsilateral or contralateral lower abdominal quadrant after fingertip replantation. Dates of abdominal pocketing after initial replantation, detachment after abdominal pocketing, range of motion (ROM before abdominal pocketing, and sequential ROM after the detachment operation and date of full ROM recovery and Disabilities of Arm, Shoulder, and Hand questionnaire (DASH score were recorded through medical chart review. Mean detachment date, mean abduction of shoulder after the detachment operation, and mean days to return to full ROM were not significantly different between the ipsilateral and contralateral pocketing groups. However, the mean DASH score was significantly lower in the contralateral group than the ipsilateral group. There were also fewer postoperative wound complications in the contralateral group than in the ipsilateral group. We, therefore, recommend contralateral abdominal pocketing rather than ipsilateral abdominal pocketing to increase patient comfort and reduce pain and complications.

  1. Contralateral Abdominal Pocketing in Salvation of Replanted Fingertips with Compromised Circulation

    Science.gov (United States)

    Shim, Hyung-Sup; Kim, Dong-Hwi; Kwon, Ho; Jung, Sung-No

    2014-01-01

    Abdominal pocketing is one of the most useful methods in salvation of compromised replanted fingertips. Abdominal pocketing has generally been performed in the ipsilateral lower abdominal quadrant, but we have also performed contralateral pocketing at our institute. To determine which approach is more beneficial, a total of 40 patients underwent an abdominal pocketing procedure in either the ipsilateral or contralateral lower abdominal quadrant after fingertip replantation. Dates of abdominal pocketing after initial replantation, detachment after abdominal pocketing, range of motion (ROM) before abdominal pocketing, and sequential ROM after the detachment operation and date of full ROM recovery and Disabilities of Arm, Shoulder, and Hand questionnaire (DASH) score were recorded through medical chart review. Mean detachment date, mean abduction of shoulder after the detachment operation, and mean days to return to full ROM were not significantly different between the ipsilateral and contralateral pocketing groups. However, the mean DASH score was significantly lower in the contralateral group than the ipsilateral group. There were also fewer postoperative wound complications in the contralateral group than in the ipsilateral group. We, therefore, recommend contralateral abdominal pocketing rather than ipsilateral abdominal pocketing to increase patient comfort and reduce pain and complications. PMID:25379539

  2. Association between intensity of smoking and periodontal pockets among young university students.

    Science.gov (United States)

    Salem, A; Hilow, H; Khraisat, A; Smadi, L; Ryalat, S

    2008-06-01

    Smoking is known to be a risk factor for the progression of periodontal disease. The aim of this study was to examine the association between intensity and duration of cigarette smoking with the presence of periodontal pockets at a young adult age in relation to oral hygiene practices. A comparative study was carried out on a systematically selected random sample of 357 Jordanian university students aged between 18-28 years. Subjects were interviewed about their smoking habits for the number of cigarettes smoked per day and the duration. Clinical examination was performed by a qualified periodontist for the detection of periodontal pockets (probing depth > or = 5 mm). Subjects were divided into four groups: Group A (smoker with pockets at many sites), Group B (smoker with no pockets), Group C (non-smoker with pockets), and group D (non-smoker with no pockets). Data analysis included descriptive statistics, Chi-square test of association, One-Way ANOVA, and Logistic Regression were performed (P periodontal pockets and smoking (P smoking, the more periodontal pockets were present (P periodontal pockets and patient's own oral hygiene practices was found among the four groups (p smoking and presence of periodontal pockets, especially with increased intensity and longer duration of smoking.

  3. Nucleotide diversity and phylogenetic relationships among ...

    Indian Academy of Sciences (India)

    The plastid genome regions of two intergenic spacers, psbA–trnH and trnL–trnF, were sequenced to study the nucleotide diversity and phylogenetic relationships among Gladiolus cultivars. Nucleotide diversity of psbA–trnH region was higher than trnL–trnF region of chloroplast. We employed Bayesian, maximum ...

  4. Assay of cyclic nucleotide phosphodiesterase using radiolabeled and fluorescent substrates

    International Nuclear Information System (INIS)

    Kincaid, R.L.; Manganiello, V.C.

    1988-01-01

    There are four major classes of phosphodiesterase with different specificities for cAMP and cGMP and different allosteric regulators. Type I phosphodiesterase is activated by calmodulin plus Ca/sup 2+/ and has a higher affinity for cGMP than cAMP. Type II phosphodiesterase likewise has a higher affinity for cGMP than cAMP, but the activity toward one substrate is markedly stimulated by low (micromolar) concentrations of the other nucleotide. Type III phosphodiesterase has a higher affinity for cAMP than cGMP; its activity is increased in responsive cells by certain hormones, e.g., insulin, isoproterenol. Type IV phosphodiesterase is the cGMP-specific enzyme, which also has an allosteric binding site for cGMP. An example of this class of enzyme is the one from retinal rod outer segments, which is activated by light via rhodopsin and the guanine nucleotide-binding protein transducin. There appears to be little structural relatedness among these enzymes based on immunologic analysis, consistent with the possibility that divergent forms evolved from an ancestral enzyme. Determination of the amount of a specific form of phosphodiesterase in crude material is often difficult. Modification of assay conditions by judicious choice of substrate and/or inhibitor concentrations may selectively favor (or reduce) the activity of a particular form; in many instances, however, some fractionation of enzymes may be necessary. This is discussed more fully in the final section of this chapter

  5. Design and Implementation of Accurate and Efficient Pocket Dosimeter

    International Nuclear Information System (INIS)

    Shehata, S.A.; Abdelkhalek, K.L.

    2005-01-01

    It is so important in the field of radiation therapy and radiation protection to have dosimeters to determine the absorbed dose, which is transferred to human body by ionizing radiation. In this paper the design and implementation of a wide-range pocket dosimeter (PKD-1) with high accuracy to measure personal equivalent dose and dose rate of gamma radiation will be presented. This pocket dosimeter is micro controller-based and powered from 9 V rechargeable battery. The overall power consumption is significantly reduced by smart software and hardware design allowing longer time intervals between recharging. The integrated alphanumerical LCD displays not only of the accumulated dose and current dose rate, but also displays alarm messages such as low battery. For reasons of power saving the LCD is activated on demand by pressing the push button or automatically when an alarm occurs. Audible and visual alarms have been added to PKD-1 in order that they cannot be accidentally overlooked or ignored. PKD-1 can be connected to any PC through its serial port (RS232) and User Interface software has been developed for easy displaying and recording of radiation readings over any time period

  6. Neglected Esophageal Injury Presenting With Spontaneously Shrunken Retroesophageal Pocket

    Directory of Open Access Journals (Sweden)

    Chin-Chih Chang

    2008-09-01

    Full Text Available Cervical stab wounds with a thoracic-inlet esophageal injury are extremely rare. A 30-year-old man presented with dysphagia and stridor. He had attempted suicide by stabbing his neck with a screwdriver followed by jumping from a building 10 days previously, when a cervical tracheal injury was found and surgically repaired. Physical examination was unremarkable. Lateral cervical radiography revealed an air-fluid level within an extensive retropharyngeal pocket. Follow-up radiography showed that the retropharyngeal lesion had shrunk spontaneously. Contrast esophagography demonstrated an extravasation at the thoracic inlet. The patient underwent surgical exploration of the esophagus via a lower neck incision. A thoracic-inlet esophageal slit was found and primary repair was performed. He resumed oral intake uneventfully on the 8th postoperative day. This was a rare case of esophageal injury secondary to cervical stabbing wounds, presenting with delayed occurrence and spontaneously shrunken retroesophageal pocket. Esophageal perforation can be easily missed if tracheal lesion is found. Both bronchoscopy and esophagoscopy are mandatory. In patients highly suspected to have esophageal injury but with a negative esophagoscopy result, contrast esophagography is indicated and can decrease the incidence of false-negative results.

  7. Detection of the amoeba Entamoeba gingivalis in periodontal pockets

    Science.gov (United States)

    Bonner, Mark; Amard, Véronique; Bar-Pinatel, Charlotte; Charpentier, Frédéric; Chatard, Jean-Michel; Desmuyck, Yvan; Ihler, Serge; Rochet, Jean-Pierre; Roux de La Tribouille, Véronique; Saladin, Luc; Verdy, Marion; Gironès, Núria; Fresno, Manuel; Santi-Rocca, Julien

    2014-01-01

    Periodontitis is a public health issue, being one of the most prevalent diseases worldwide. However, the aetiology of the disease is still unclear: genetics of patients cannot explain the dispersed or isolated localisation of gingival pockets, while bacteria-based models are insufficient to distinguish gingivitis and periodontitis. The possible role of parasites in the establishment of periodontitis has been poorly studied until now. The aim of this project was to study a potential link between colonisation of gingival crevices by the amoeba Entamoeba gingivalis and periodontitis. In eight different dental clinics in France, samples were taken in periodontal pockets (72) or healthy sites (33), and submitted to microscopic observation and molecular identification by PCR with a new set of primers designed to specifically detect E. gingivalis. This blind sample analysis showed the strong sensitivity of PCR compared with clinical diagnosis (58/72 = 81%), and microscopy (51/65 = 78%). The results of this work show that the parasites detected by microscopy mainly – if not exclusively – belong to the species E. gingivalis and that the presence of the parasite is correlated with periodontitis. PMID:24983705

  8. Breakdown of air pockets in downwardly inclined sewerage pressure mains.

    Science.gov (United States)

    Lubbers, C L; Clemens, F H L R

    2006-01-01

    In the Netherlands, wastewater is collected in municipal areas and transported to centralised WWTPs by an extensive system of pressure mains. Over the last decades these pressure mains did not receive much attention in terms of monitoring of performance or maintenance. A recent inventory showed that half of the pressure mains show an increased pressure loss for no directly obvious reason. One of the many causes that account for the reduction of the flow capacity is the occurrence of free gas in the pipeline. During dry weather periods with low flow velocities, gas may accumulate at high points in the system. Once the velocity increases during storm weather flow, the air pockets may be broken down and transported to the end of the system. A research study is started focussing on the description of the gas-water phenomena in wastewater pressure mains with respect to transportation of gas. An experimental facility is constructed for the study of multi-phase flow. This paper describes the preliminary results of experiments on breakdown rates of gas pockets as a function of inclination angle and water flow rate. The results show an increasing breakdown rate with increasing inclination angle.

  9. Choleragen-stimulated release of guanyl nucleotides from turkey erythrocyte membranes.

    Science.gov (United States)

    Burns, D L; Moss, J; Vaughan, M

    1982-01-10

    Choleragen stimulates adenylate cyclase by ADP ribosylating a guanyl nucleotide-binding regulatory protein (G/F). beta-Adrenergic hormones also activate the adenylate cyclase of turkey erythrocytes, and it is currently believed that they do so in part by decreasing the affinity of G/F factor for GDP, an effect which is manifested by a hormone-stimulated release of guanyl nucleotides from the membranes. Since choleragen might also activate adenylate cyclase by a similar mechanism, the effect of toxin treatment on the release of guanyl nucleotides from turkey erythrocyte membranes was examined. In the presence of NAD, choleragen was found to stimulate release of guanyl nucleotides from membranes which had been preloaded with radiolabeled GTP. No stimulation of release was observed with cAMP or when NAD was replaced by NADP, which does not serve as a substrate for choleragen-catalyzed ADP ribosylation. While either isoproterenol or choleragen can stimulate release of guanyl nucleotides from the membranes, the amount of guanyl nucleotide released in the presence of both isoproterenol and choleragen was no greater than that released by isoproterenol alone. Furthermore, when membranes were first treated with choleragen and NAD, the subsequent release of guanyl nucleotides induced by isoproterenol was reduced to approximately 15% of that observed with membranes not treated with the toxin. Therefore, choleragen may enhance release of guanine nucleotides from sites on the membranes that are also affected by beta-adrenergic agonists, sites which are thought to correspond to G/F. These data are consistent with the hypothesis that choleragen may stimulate adenylate cyclase, in part, by enhancing release of guanyl nucleotides, a mechanism similar to that of beta-adrenergic agonists.

  10. A comparison of different parametrized pocket formulae to calculate fusion barriers

    Science.gov (United States)

    Dutt, Ishwar

    2013-04-01

    Using twelve different parametrized pocket formulae fusion barrier heights and positions are calculated and confronted with available experimental data. All different parametrized pocket formulae are able to reproduce fusion barrier heights and positions within ±10% and ±20%, respectively. Our detailed study investigate that the parametrized pocket formulae based on models such as Bass 1980, Winther 1995, Prox 1988, and Royer 2001 reproduce the barrier heights nicely as compared to other formulae available in the literature.

  11. Conserved residues in the coiled-coil pocket of human immunodeficiency virus type 1 gp41 are essential for viral replication and interhelical interaction

    International Nuclear Information System (INIS)

    Mo Hongmei; Konstantinidis, Alex K.; Stewart, Kent D.; Dekhtyar, Tatyana; Ng, Teresa; Swift, Kerry; Matayoshi, Edmund D.; Kati, Warren; Kohlbrenner, William; Molla, Akhteruzzaman

    2004-01-01

    The human immunodeficiency virus type 1 (HIV-1) gp41 plays an important role in mediating the fusion of HIV with host cells. During the fusion process, three N-terminal helices and three C-terminal helices pack in an anti-parallel direction to form a six-helix bundle. X-ray crystallographic analysis of the gp41 core demonstrated that within each coiled-coil interface, there is a deep and large pocket, formed by a cluster of residues in the N-helix coiled-coil. In this report, we systematically analyzed the role of seven conserved residues that are either lining or packing this pocket on the infectivity and interhelical interaction using novel approaches. Our results show that residues L568, V570, W571, and K574 of the N-helix that are lining the side chain and right wall of the pocket are important for establishing a productive infection. Mutations V570A and W571A completely abolished replication, while replication of the L568A and K574A mutants was significantly attenuated relative to wild type. Similarly, residues W628, W631, and I635 of the C-helix that insert into the pocket are essential for infectivity. The impaired infectivity of these seven mutants is in part attributed to the loss in binding affinity of the interhelical interaction. Molecular modeling of the crystal structure of the coiled-coil further shows that alanine substitution of those residues disrupts the hydrophobic interaction between the N- and C-helix. These results suggest that the conserved residues in the coiled-coil domain play a key role in HIV infection and this coiled-coil pocket is a good target for development of inhibitors against HIV. In addition, our data indicate that the novel fluorescence polarization assay described in this study could be valuable in screening for inhibitors that block the interhelical interaction and HIV entry

  12. A Novel Regulator of Activation-Induced Cytidine Deaminase/APOBECs in Immunity and Cancer: Schrödinger’s CATalytic Pocket

    Directory of Open Access Journals (Sweden)

    Mani Larijani

    2017-04-01

    Full Text Available Activation-induced cytidine deaminase (AID and its relative APOBEC3 cytidine deaminases boost immune response by mutating immune or viral genes. Because of their genome-mutating activities, AID/APOBECs are also drivers of tumorigenesis. Due to highly charged surfaces, extensive non-specific protein–protein/nucleic acid interactions, formation of polydisperse oligomers, and general insolubility, structure elucidation of these proteins by X-ray crystallography and NMR has been challenging. Hence, almost all available AID/APOBEC structures are of mutated and/or truncated versions. In 2015, we reported a functional structure for AID using a combined computational–biochemical approach. In so doing, we described a new regulatory mechanism that is a first for human DNA/RNA-editing enzymes. This mechanism involves dynamic closure of the catalytic pocket. Subsequent X-ray and NMR studies confirmed our discovery by showing that other APOBEC3s also close their catalytic pockets. Here, we highlight catalytic pocket closure as an emerging and important regulatory mechanism of AID/APOBEC3s. We focus on three sub-topics: first, we propose that variable pocket closure rates across AID/APOBEC3s underlie differential activity in immunity and cancer and review supporting evidence. Second, we discuss dynamic pocket closure as an ever-present internal regulator, in contrast to other proposed regulatory mechanisms that involve extrinsic binding partners. Third, we compare the merits of classical approaches of X-ray and NMR, with that of emerging computational–biochemical approaches, for structural elucidation specifically for AID/APOBEC3s.

  13. Single-molecule microscopy reveals new insights into nucleotide selection by DNA polymerase I

    Science.gov (United States)

    Markiewicz, Radoslaw P.; Vrtis, Kyle B.; Rueda, David; Romano, Louis J.

    2012-01-01

    The mechanism by which DNA polymerases achieve their extraordinary accuracy has been intensely studied because of the linkage between this process and mutagenesis and carcinogenesis. Here, we have used single-molecule fluorescence microscopy to study the process of nucleotide selection and exonuclease action. Our results show that the binding of Escherichia coli DNA polymerase I (Klenow fragment) to a primer-template is stabilized by the presence of the next correct dNTP, even in the presence of a large excess of the other dNTPs and rNTPs. These results are consistent with a model where nucleotide selection occurs in the open complex prior to the formation of a closed ternary complex. Our assay can also distinguish between primer binding to the polymerase or exonuclease domain and, contrary to ensemble-averaged studies, we find that stable exonuclease binding only occurs with a mismatched primer terminus. PMID:22669904

  14. Structure of a Viral Cap-independent Translation Element That Functions via High Affinity Binding to the eIF4E Subunit of eIF4F*S⃞

    Science.gov (United States)

    Wang, Zhaohui; Treder, Krzysztof; Miller, W. Allen

    2009-01-01

    RNAs of many positive strand RNA viruses lack a 5′ cap structure and instead rely on cap-independent translation elements (CITEs) to facilitate efficient translation initiation. The mechanisms by which these RNAs recruit ribosomes are poorly understood, and for many viruses the CITE is unknown. Here we identify the first CITE of an umbravirus in the 3′-untranslated region of pea enation mosaic virus RNA 2. Chemical and enzymatic probing of the ∼100-nucleotide PEMV RNA 2 CITE (PTE), and mutagenesis revealed that it forms a long, bulged helix that branches into two short stem-loops, with a possible pseudoknot interaction between a C-rich bulge at the branch point and a G-rich bulge in the main helix. The PTE inhibited translation in trans, and addition of eIF4F, but not eIFiso4F, restored translation. Filter binding assays revealed that the PTE binds eIF4F and its eIF4E subunit with high affinity. Tight binding required an intact cap-binding pocket in eIF4E. Among many PTE mutants, there was a strong correlation between PTE-eIF4E binding affinity and ability to stimulate cap-independent translation. We conclude that the PTE recruits eIF4F by binding eIF4E. The PTE represents a different class of translation enhancer element, as defined by its structure and ability to bind eIF4E in the absence of an m7G cap. PMID:19276085

  15. Structure of a viral cap-independent translation element that functions via high affinity binding to the eIF4E subunit of eIF4F.

    Science.gov (United States)

    Wang, Zhaohui; Treder, Krzysztof; Miller, W Allen

    2009-05-22

    RNAs of many positive strand RNA viruses lack a 5' cap structure and instead rely on cap-independent translation elements (CITEs) to facilitate efficient translation initiation. The mechanisms by which these RNAs recruit ribosomes are poorly understood, and for many viruses the CITE is unknown. Here we identify the first CITE of an umbravirus in the 3'-untranslated region of pea enation mosaic virus RNA 2. Chemical and enzymatic probing of the approximately 100-nucleotide PEMV RNA 2 CITE (PTE), and mutagenesis revealed that it forms a long, bulged helix that branches into two short stem-loops, with a possible pseudoknot interaction between a C-rich bulge at the branch point and a G-rich bulge in the main helix. The PTE inhibited translation in trans, and addition of eIF4F, but not eIFiso4F, restored translation. Filter binding assays revealed that the PTE binds eIF4F and its eIF4E subunit with high affinity. Tight binding required an intact cap-binding pocket in eIF4E. Among many PTE mutants, there was a strong correlation between PTE-eIF4E binding affinity and ability to stimulate cap-independent translation. We conclude that the PTE recruits eIF4F by binding eIF4E. The PTE represents a different class of translation enhancer element, as defined by its structure and ability to bind eIF4E in the absence of an m(7)G cap.

  16. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    International Nuclear Information System (INIS)

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-01-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

  17. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    Energy Technology Data Exchange (ETDEWEB)

    Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  18. Polymorphism between HLA-A*0301 and A*0302 located outside the pocket F alters the PΩ peptide motif.

    Science.gov (United States)

    Elamin, N E; Bade-Doeding, C; Blasczyk, R; Eiz-Vesper, B

    2010-12-01

    The human leukocyte antigen (HLA)-A*03 group has more than 90 known members and is one of the largest families of HLA class I alleles, with the most common variant being HLA-A*0301. In this study, we determined the peptide-binding motif of the highly frequent Sudanese allele A*0302 and compared it with the previously published peptide-binding motif of A*0301. The two alleles differ only at two distinct residues Glu152Val and Leu156Gln, which are predicted to be part of specificity pockets D, C and E and thus in contact with the peptide. Soluble recombinant A*0302 was expressed, affinity purified and the bound peptides were then eluted and analysed by mass spectrometry. The peptide-binding motif of A*0302 differs significantly from the previously published HLA-A*0301 and the Glu152Val/Leu156Gln mismatches appear to have a significant impact on the peptide-binding features of A*0302 and A*0301. © 2010 John Wiley & Sons A/S.

  19. Non-thiolate ligation of nickel by nucleotide-free UreG of Klebsiella aerogenes

    Energy Technology Data Exchange (ETDEWEB)

    Martin-Diaconescu, Vlad; Joseph, Crisjoe A.; Boer, Jodi L.; Mulrooney, Scott B.; Hausinger, Robert P.; Maroney, Michael J.

    2016-12-21

    Nickel-dependent ureases are activated by a multiprotein complex that includes the GTPase UreG. Prior studies showed that nucleotide-free UreG from Klebsiella aerogenes is monomeric and binds one nickel or zinc ion with near-equivalent affinity using an undefined binding site, whereas nucleotide-free UreG from Helicobacter pylori selectively binds one zinc ion per dimer via a universally conserved Cys-Pro-His motif in each protomer. Iodoacetamide-treated K. aerogenes UreG was nearly unaffected in nickel binding compared to non-treated sample, suggesting the absence of thiolate ligands to the metal. X-ray absorption spectroscopy of nickel-bound UreG showed the metal possessed four-coordinate geometry with all O/N donor ligands including one imidazole, thus confirming the absence of thiolate ligation. The nickel site in Strep-tag II-modified protein possessed six-coordinate geometry, again with all O/N donor ligands, but now including two or three imidazoles. An identical site was noted for the Strep-tag II-modified H74A variant, substituted in the Cys-Pro-His motif, ruling out coordination by this His residue. These results are consistent with metal binding to both His6 and a His residue of the fusion peptide in Strep-tagged K. aerogenes UreG. We conclude that the nickel- and zinc-binding site in nucleotide-free K. aerogenes UreG is distinct from that of nucleotide-free H. pylori UreG and does not involve the Cys-Pro-His motif. Further, we show the Strep-tag II can perturb metal coordination of this protein.

  20. tPA-binding RNA Aptamers

    DEFF Research Database (Denmark)

    Bjerregaard, Nils

    2015-01-01

    -density lipoprotein receptor Related Protein-1 (LRP-1). Here, we describe the selection and characterisation of structured RNA ligands (“RNA aptamers”) to tPA, K18 and K32. Both aptamers were truncated to minimal 32-nucleotide constructs (v2) with improved or unchanged activities, and were shown to bind tPA with low...

  1. The Six-Inch Lunar Atlas A Pocket Field Guide

    CERN Document Server

    Spain, Don

    2009-01-01

    The Six-Inch Lunar Atlas has been designed specifically for use in the field by lunar observers so it’s perfect for fitting into an observer’s pocket! The author’s own lunar photographs were taken with a 6-inch (150mm) telescope and CCD camera, and closely match the visual appearance of the Moon when viewed through 3-inch to 8-inch telescopes. Each picture is shown oriented "as the Moon really is" when viewed from the northern hemisphere, and is supplemented by exquisite computer sketches that list the main features. Two separate computer sketches are provided to go with each photograph, one oriented to appear as seen through an SCT telescope (e.g. the Meade and Celestron ranges), the other oriented for Newtonian and refracting telescopes. Observers using the various types telescopes will find it extremely helpful to identify lunar features as the human brain is very poor at making "mirror-image" visual translations.

  2. Psychologist in a pocket: towards depression screening on mobile phones.

    Science.gov (United States)

    Bitsch, Jó Ágila; Ramos, Roann; Ix, Tim; Ferrer-Cheng, Paula Glenda; Wehrle, Klaus

    2015-01-01

    Depression is the most prevalent clinical disorder and one of the main causes of disability. This makes early detection of depressive symptoms critical in its prevention and management. This paper presents and discusses the development of Psychologist in a Pocket (PiaP), a mental mHealth application for Android which screens and monitors for these symptoms, and-given the explicit permission of the user-alerts a trusted contact such as the mental health professional or a close friend, if it detects symptoms. All text inputted electronically-such as short message services, emails, social network posts-is analyzed based on keywords related to depression based on DSM-5 and ICD criteria as well as Beck's Cognitive Theory of Depression and the Self-Focus Model. Data evaluation and collection happen in the background, on-device, without requiring any user involvement. Currently, the application is in an early prototype phase entering initial clinical validation.

  3. A pocket warning γ-dosimeter with numerical display

    International Nuclear Information System (INIS)

    Jones, A.R.

    1980-09-01

    A pocket warning dosimeter is described. It provides alarms (continuous tone and a flashing red light) when a presettable dose has been accumulated in the range .064 - 16.4 rads (0.64 - 164 μGy). This warning level can be selected in nine steps of 2 with a switch inside the dosimeter. The dose rate is indicated by a series of sound pulses whose repetition rate is proportional to the dose rate. At 1 rad/h (10 mGy/h) about 17 pluses/minute are emitted. The accumulated dose up to 20 rads (0.2 Gy) is displayed in steps of 1 mrad (10 μGy) with a liquid crystal display. A red LED lights before battery failure occurs. The effects of changes in temperature, battery voltage, dose rate and photon energy upon dosimeter sensitivity are presented. Finally, the applications of the dosimeter are discussed. (auth)

  4. A versatile pocket PIG ion source with permanent magnet

    International Nuclear Information System (INIS)

    Yu Jinxiang; Ren Xiaotang; Song Zhizhong

    1996-01-01

    A versatile pocket PIG ion source with permanent magnet is described in this paper. The source can produce both gas or metal and some negative ion beams. Normally 1-2 mA gas ion beams can be extracted at 40 W discharge power. Using Ar as auxiliary gas, tens of μA of solid or metal ions from cathode material are produced by cathode sputtering. The negative ions of some elements with larger electron affinity can be extracted from the source directly, so far using LaB 6 as cathode, more than 50 μA of H - , 1-2 mA of O - and F - ion beams have been extracted at 15-20 kV extraction voltage, the power consumption are normally less than 40, 50 and 100 W, respectively. (orig.)

  5. MARTINDALE'S DRUGS RESTRICTED IN SPORT POCKET COMPANION 2009

    Directory of Open Access Journals (Sweden)

    Sean C. Sweetman

    2009-06-01

    Full Text Available Over 500 drugs restricted in sport presented in alphabetical order. To inform and alert the athlete about the potential problem of drug taking for any kind of reasons on and off during training and competition.A comprehensive index of drug names, synonyms, medical usage, single and multi-ingredient preparations and trade (on occasion street names of drugs from 40 countries worldwide (Martindale data. The classification of World Anti-Doping Agency (WADA is added to the explanation of drugs limitation in sport in and out of competition. A glossary of common medical terms is also included.This pocket publication is a must-have list of restricted drugs for athletes, trainers, sports medicine professionals, in short for anyone in exercise physiology and human performance fields.

  6. Altered nucleotide-microtubule coupling and increased mechanical output by a kinesin mutant.

    Directory of Open Access Journals (Sweden)

    Hong-Lei Liu

    Full Text Available Kinesin motors hydrolyze ATP to produce force and do work in the cell--how the motors do this is not fully understood, but is thought to depend on the coupling of ATP hydrolysis to microtubule binding by the motor. Transmittal of conformational changes from the microtubule- to the nucleotide-binding site has been proposed to involve the central β-sheet, which could undergo large structural changes important for force production. We show here that mutation of an invariant residue in loop L7 of the central β-sheet of the Drosophila kinesin-14 Ncd motor alters both nucleotide and microtubule binding, although the mutated residue is not present in either site. Mutants show weak-ADP/tight-microtubule binding, instead of tight-ADP/weak-microtubule binding like wild type--they hydrolyze ATP faster than wild type, move faster in motility assays, and assemble long spindles with greatly elongated poles, which are also produced by simulations of assembly with tighter microtubule binding and faster sliding. The mutated residue acts like a mechanochemical coupling element--it transmits changes between the microtubule-binding and active sites, and can switch the state of the motor, increasing mechanical output by the motor. One possibility, based on our findings, is that movements by the residue and the loop that contains it could bend or distort the central β-sheet, mediating free energy changes that lead to force production.

  7. Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide–protein complexes

    Science.gov (United States)

    Kondo, Jiro; Westhof, Eric

    2011-01-01

    Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide–protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson–Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson–Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues. PMID:21737431

  8. The international nucleotide sequence database collaboration.

    Science.gov (United States)

    Karsch-Mizrachi, Ilene; Takagi, Toshihisa; Cochrane, Guy

    2018-01-04

    For more than 30 years, the International Nucleotide Sequence Database Collaboration (INSDC; http://www.insdc.org/) has been committed to capturing, preserving and providing access to comprehensive public domain nucleotide sequence and associated metadata which enables discovery in biomedicine, biodiversity and biological sciences. Since 1987, the DNA Data Bank of Japan (DDBJ) at the National Institute for Genetics in Mishima, Japan; the European Nucleotide Archive (ENA) at the European Molecular Biology Laboratory's European Bioinformatics Institute (EMBL-EBI) in Hinxton, UK; and GenBank at National Center for Biotechnology Information (NCBI), National Library of Medicine, National Institutes of Health in Bethesda, Maryland, USA have worked collaboratively to enable access to nucleotide sequence data in standardized formats for the worldwide scientific community. In this article, we reiterate the principles of the INSDC collaboration and briefly summarize the trends of the archival content. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

  9. Nucleotide excision repair in the test tube.

    NARCIS (Netherlands)

    N.G.J. Jaspers (Nicolaas); J.H.J. Hoeijmakers (Jan)

    1995-01-01

    textabstractThe eukaryotic nucleotide excision-repair pathway has been reconstituted in vitro, an achievement that should hasten the full enzymological characterization of this highly complex DNA-repair pathway.

  10. Nucleotide diversity and linkage disequilibrium in 11 expressed resistance candidate genes in Lolium perenne

    Directory of Open Access Journals (Sweden)

    Asp Torben

    2007-08-01

    Full Text Available Abstract Background Association analysis is an alternative way for QTL mapping in ryegrass. So far, knowledge on nucleotide diversity and linkage disequilibrium in ryegrass is lacking, which is essential for the efficiency of association analyses. Results 11 expressed disease resistance candidate (R genes including 6 nucleotide binding site and leucine rich repeat (NBS-LRR like genes and 5 non-NBS-LRR genes were analyzed for nucleotide diversity. For each of the genes about 1 kb genomic fragments were isolated from 20 heterozygous genotypes in ryegrass. The number of haplotypes per gene ranged from 9 to 27. On average, one single nucleotide polymorphism (SNP was present per 33 bp between two randomly sampled sequences for the 11 genes. NBS-LRR like gene fragments showed a high degree of nucleotide diversity, with one SNP every 22 bp between two randomly sampled sequences. NBS-LRR like gene fragments showed very high non-synonymous mutation rates, leading to altered amino acid sequences. Particularly LRR regions showed very high diversity with on average one SNP every 10 bp between two sequences. In contrast, non-NBS LRR resistance candidate genes showed a lower degree of nucleotide diversity, with one SNP every 112 bp. 78% of haplotypes occurred at low frequency ( Conclusion Substantial LD decay was found within a distance of 500 bp for most resistance candidate genes in this study. Hence, LD based association analysis is feasible and promising for QTL fine mapping of resistance traits in ryegrass.

  11. Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping

    Science.gov (United States)

    Hendershot, Jenna M.; O'Brien, Patrick J.

    2014-01-01

    Nucleotide flipping is a common feature of DNA-modifying enzymes that allows access to target sites within duplex DNA. Structural studies have identified many intercalating amino acid side chains in a wide variety of enzymes, but the functional contribution of these intercalating residues is poorly understood. We used site-directed mutagenesis and transient kinetic approaches to dissect the energetic contribution of intercalation for human alkyladenine DNA glycosylase, an enzyme that initiates repair of alkylation damage. When AAG flips out a damaged nucleotide, the void in the duplex is filled by a conserved tyrosine (Y162). We find that tyrosine intercalation confers 140-fold stabilization of the extrahelical specific recognition complex, and that Y162 functions as a plug to slow the rate of unflipping by 6000-fold relative to the Y162A mutant. Surprisingly, mutation to the smaller alanine side chain increases the rate of nucleotide flipping by 50-fold relative to the wild-type enzyme. This provides evidence against the popular model that DNA intercalation accelerates nucleotide flipping. In the case of AAG, DNA intercalation contributes to the specific binding of a damaged nucleotide, but this enhanced specificity comes at the cost of reduced speed of nucleotide flipping. PMID:25324304

  12. Structures of Asymmetric ClpX Hexamers Reveal Nucleotide-Dependent Motions in a AAA+ Protein-Unfolding Machine

    Energy Technology Data Exchange (ETDEWEB)

    Glynn, Steven E.; Martin, Andreas; Nager, Andrew R.; Baker, Tania A.; Sauer, Robert T.; MIT

    2010-02-02

    ClpX is a AAA+ machine that uses the energy of ATP binding and hydrolysis to unfold native proteins and translocate unfolded polypeptides into the ClpP peptidase. The crystal structures presented here reveal striking asymmetry in ring hexamers of nucleotide-free and nucleotide-bound ClpX. Asymmetry arises from large changes in rotation between the large and small AAA+ domains of individual subunits. These differences prevent nucleotide binding to two subunits, generate a staggered arrangement of ClpX subunits and pore loops around the hexameric ring, and provide a mechanism for coupling conformational changes caused by ATP binding or hydrolysis in one subunit to flexing motions of the entire ring. Our structures explain numerous solution studies of ClpX function, predict mechanisms for pore elasticity during translocation of irregular polypeptides, and suggest how repetitive conformational changes might be coupled to mechanical work during the ATPase cycle of ClpX and related molecular machines.

  13. Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.

  14. Effects of nucleotides and nucleosides on coagulation

    DEFF Research Database (Denmark)

    Bune, Laurids; Thaning, Pia; Johansson, Pär I

    2010-01-01

    Nucleotides, including ADP, ATP and uridine triphosphate (UTP), are discharged profusely in the circulation during many pathological conditions including sepsis. Sepsis can cause hypotension and systemic activation of the coagulation and fibrinolytic systems in humans, which may cause disseminated...... intravascular coagulation. We investigated whether nucleotide-induced cardiovascular collapse as provoked by systemic infusion of adenosine, ADP, ATP, UTP and nitric oxide affected the haemostatic system as assessed by whole blood thromboelastography (TEG) analysis. Ten pigs received a randomized infusion...

  15. Structural Insights into the Mechanisms of Action of Short-Peptide HIV-1 Fusion Inhibitors Targeting the Gp41 Pocket

    Directory of Open Access Journals (Sweden)

    Xiujuan Zhang

    2018-02-01

    Full Text Available The deep hydrophobic pocket of HIV-1 gp41 has been considered a drug target, but short-peptides targeting this site usually lack potent antiviral activity. By applying the M-T hook structure, we previously generated highly potent short-peptide fusion inhibitors that specifically targeted the pocket site, such as MT-SC22EK, HP23L, and LP-11. Here, the crystal structures of HP23L and LP-11 bound to the target mimic peptide N36 demonstrated the critical intrahelical and interhelical interactions, especially verifying that the hook-like conformation was finely adopted while the methionine residue was replaced by the oxidation-less prone residue leucine, and that addition of an extra glutamic acid significantly enhanced the binding and inhibitory activities. The structure of HP23L bound to N36 with two mutations (E49K and L57R revealed the critical residues and motifs mediating drug resistance and provided new insights into the mechanism of action of inhibitors. Therefore, the present data help our understanding for the structure-activity relationship (SAR of HIV-1 fusion inhibitors and facilitate the development of novel antiviral drugs.

  16. Iodide Binding in Sodium-Coupled Cotransporters.

    Science.gov (United States)

    Vergara-Jaque, Ariela; Fong, Peying; Comer, Jeffrey

    2017-12-26

    Several apical iodide translocation pathways have been proposed for iodide efflux out of thyroid follicular cells, including a pathway mediated by the sodium-coupled monocarboxylate transporter 1 (SMCT1), which remains controversial. Herein, we evaluate structural and functional similarities between SMCT1 and the well-studied sodium-iodide symporter (NIS) that mediates the first step of iodide entry into the thyroid. Free-energy calculations using a force field with electronic polarizability verify the presence of a conserved iodide-binding pocket between the TM2, TM3, and TM7 segments in hNIS, where iodide is coordinated by Phe67, Gln72, Cys91, and Gln94. We demonstrate the mutation of residue Gly93 of hNIS to a larger amino acid expels the side chain of a critical tryptophan residue (Trp255) into the interior of the binding pocket, partially occluding the iodide binding site and reducing iodide affinity, which is consistent with previous reports associating mutation of this residue with iodide uptake deficiency and hypothyroidism. Furthermore, we find that the position of Trp255 in this hNIS mutant mirrors that of Trp253 in wild-type hSMCT1, where a threonine (Thr91) occupies the position homologous to that occupied by glycine in wild-type hNIS (Gly93). Correspondingly, mutation of Thr91 to glycine in hSMCT1 makes the pocket structure more like that of wild-type hNIS, increasing its iodide affinity. These results suggest that wild-type hSMCT1 in the inward-facing conformation may bind iodide only very weakly, which may have implications for its ability to transport iodide.

  17. The role of nucleotides in the neuron--glia communication responsible for the brain functions.

    Science.gov (United States)

    Inoue, Kazuhide; Koizumi, Schuichi; Tsuda, Makoto

    2007-09-01

    Accumulating findings indicate that nucleotides play an important role in cell-to-cell communication through P2 purinoceptors, even though ATP is recognized primarily to be a source of free energy and nucleotides are key molecules in cells. P2 purinoceptors are divided into two families, ionotropic receptors (P2X) and metabotropic receptors (P2Y). P2X receptors (7 types; P2X(1)-P2X(7)) contain intrinsic pores that open by binding with ATP. P2Y (8 types; P2Y(1, 2, 4, 6, 11, 12, 13,) and (14)) are activated by nucleotides and couple to intracellular second-messenger systems through heteromeric G-proteins. Nucleotides are released or leaked from non-excitable cells as well as neurons in physiological and pathophysiological conditions. One of the most exciting cells in non-excitable cells is the glia cells, which are classified into astrocytes, oligodendrocytes, and microglia. Astrocytes express many types of P2 purinoceptors and release the 'gliotransmitter' ATP to communicate with neurons, microglia and the vascular walls of capillaries. Microglia also express many types of P2 purinoceptors and are known as resident macrophages in the CNS. ATP and other nucleotides work as 'warning molecules' especially through activating microglia in pathophysiological conditions. Microglia play a key role in neuropathic pain and show phagocytosis through nucleotide-evoked activation of P2X(4) and P2Y(6) receptors, respectively. Such strong molecular, cellular and system-level evidence for extracellular nucleotide signaling places nucleotides in the central stage of cell communications in glia/CNS.

  18. Dutch GPs' perceptions: the influence of out-of-pocket costs on prescribing

    NARCIS (Netherlands)

    Kasje, W.N.; Timmer, J.W.; Boendermaker, P.M.; Haaijer-Ruskamp, F.M.

    2002-01-01

    The aim of this study was to explore the impact of out-of-pocket costs on Dutch general practitioners' prescribing. A qualitative study using focus groups was conducted. An open-ended topic guide was used to elucidate the influence of out-of-pocket costs on decision making for the treatment of

  19. Denaturing gradient gel electrophoresis analysis to study bacterial community structure in pockets of periodontitis patients

    NARCIS (Netherlands)

    Zijnge, V.; Harmsen, H.J.M.; Kleinfelder, J.W.; Rest, M.E. van der; Degener, J.E.; Welling, G.W.

    Bacteria are involved in the onset and progression of periodontitis. A promising molecular technique, denaturing gradient gel electrophoresis (DGGE), to study microbial population dynamics in the subgingival pocket is presented. Twenty-three samples were taken from the subgingival pockets of nine

  20. Characteristics and tolerances of the pocket mouse and incidence of disease. [CNS lesions during space flights

    Science.gov (United States)

    Lindberg, R. G.; Kraft, L. M.; Simmonds, R. C.; Bailey, O. T.; Dunlap, W. A.; Haymaker, W.

    1975-01-01

    Studies carried out on the pocket mouse colony on Apollo XVII are reported. They revealed no serological evidence of viral disease, no pathogenic enterobacteria or respiratory Mycoplasma on culture, a 25% incidence of sarcosporidiosis, and a 2% incidence of chronic meningitis or meningoencephalitis. It is concluded that the pocket mouse is a highly adaptive animal and very well-suited to space flight.

  1. Towards Autonomous Navigation of Multiple Pocket-Drones in Real-World Environments

    NARCIS (Netherlands)

    McGuire, K.N.; Coppola, M.; de Wagter, C.; de Croon, G.C.H.E.

    2017-01-01

    Pocket-drones are inherently safe for flight near humans, and their small size allows maneuvering through narrow indoor environments. However, achieving autonomous flight of pocket-drones is challenging because of strict on-board hardware limitations. Further challenges arise when multiple

  2. The Role of Electronic Pocket Dictionaries as an English Learning Tool among Chinese Students

    Science.gov (United States)

    Jian, Hua-Li; Sandnes, Frode Eika; Law, Kris M. Y.; Huang, Yo-Ping; Huang, Yueh-Min

    2009-01-01

    This study addressed the role of electronic pocket dictionaries as a language learning tool among university students in Hong Kong and Taiwan. The target groups included engineering and humanities students at both undergraduate and graduate level. Speed of reference was found to be the main motivator for using an electronic pocket dictionary.…

  3. Effect of azithromycin on acid reflux, hiatus hernia and proximal acid pocket in the postprandial period

    NARCIS (Netherlands)

    Rohof, W. O.; Bennink, R. J.; de Ruigh, A. A.; Hirsch, D. P.; Zwinderman, A. H.; Boeckxstaens, G. E.

    2012-01-01

    Background The risk for acidic reflux is mainly determined by the position of the gastric acid pocket. It was hypothesised that compounds affecting proximal stomach tone might reduce gastro-oesophageal reflux by changing the acid pocket position. Objective To study the effect of azithromycin (Azi)

  4. Proton pump inhibitors reduce the size and acidity of the acid pocket in the stomach

    NARCIS (Netherlands)

    Rohof, Wout O.; Bennink, Roelof J.; Boeckxstaens, Guy E.

    2014-01-01

    The gastric acid pocket is believed to be the reservoir from which acid reflux events originate. Little is known about how changes in position, size, and acidity of the acid pocket contribute to the therapeutic effect of proton pump inhibitors (PPIs) in patients with gastroesophageal reflux disease

  5. PatchSurfers: Two methods for local molecular property-based binding ligand prediction.

    Science.gov (United States)

    Shin, Woong-Hee; Bures, Mark Gregory; Kihara, Daisuke

    2016-01-15

    Protein function prediction is an active area of research in computational biology. Function prediction can help biologists make hypotheses for characterization of genes and help interpret biological assays, and thus is a productive area for collaboration between experimental and computational biologists. Among various function prediction methods, predicting binding ligand molecules for a target protein is an important class because ligand binding events for a protein are usually closely intertwined with the proteins' biological function, and also because predicted binding ligands can often be directly tested by biochemical assays. Binding ligand prediction methods can be classified into two types: those which are based on protein-protein (or pocket-pocket) comparison, and those that compare a target pocket directly to ligands. Recently, our group proposed two computational binding ligand prediction methods, Patch-Surfer, which is a pocket-pocket comparison method, and PL-PatchSurfer, which compares a pocket to ligand molecules. The two programs apply surface patch-based descriptions to calculate similarity or complementarity between molecules. A surface patch is characterized by physicochemical properties such as shape, hydrophobicity, and electrostatic potentials. These properties on the surface are represented using three-dimensional Zernike descriptors (3DZD), which are based on a series expansion of a 3 dimensional function. Utilizing 3DZD for describing the physicochemical properties has two main advantages: (1) rotational invariance and (2) fast comparison. Here, we introduce Patch-Surfer and PL-PatchSurfer with an emphasis on PL-PatchSurfer, which is more recently developed. Illustrative examples of PL-PatchSurfer performance on binding ligand prediction as well as virtual drug screening are also provided. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. The limited value of three pathogen species in predicting healing of periodontal pockets.

    Science.gov (United States)

    Sewón, L; Karjalainen, S; Söderling, E; Hyyppä, T; Luukkala-Wardi, E; Mäkelä, M; Paunio, K; Varrela, T

    1999-10-01

    Baseline level of Actinobacillus actinomycetemcomitans has been suggested as being predictive of periodontal treatment outcome. We analyzed the presence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia in 55 deep periodontal pockets of 29 patients (18 men, 11 women, 37-75 years) before and after periodontal treatment. At baseline and after treatment, 62% and 33%, respectively, of the subjects presented with 1, 2, or a combination of all 3 pathogens. The mean pocket depth of 6.6 mm (0.4 mm) before treatment decreased to 2.2 mm (0.4 mm) in response to treatment (Ptreatment plan of non-surgical or surgical treatment was based on pocket depths and tooth morphology only. No antimicrobial medications were used during the treatment. Eighty-two percent of the deep pockets healed satisfactorily to periodontal therapy. In conclusion, we found that the presence of the 3 periodontopathogen species had little or no value in predicting healing of periodontal pockets.

  7. Modeling of arylamide helix mimetics in the p53 peptide binding site of hDM2 suggests parallel and anti-parallel conformations are both stable.

    Directory of Open Access Journals (Sweden)

    Jonathan C Fuller

    Full Text Available The design of novel α-helix mimetic inhibitors of protein-protein interactions is of interest to pharmaceuticals and chemical genetics researchers as these inhibitors provide a chemical scaffold presenting side chains in the same geometry as an α-helix. This conformational arrangement allows the design of high affinity inhibitors mimicking known peptide sequences binding specific protein substrates. We show that GAFF and AutoDock potentials do not properly capture the conformational preferences of α-helix mimetics based on arylamide oligomers and identify alternate parameters matching solution NMR data and suitable for molecular dynamics simulation of arylamide compounds. Results from both docking and molecular dynamics simulations are consistent with the arylamides binding in the p53 peptide binding pocket. Simulations of arylamides in the p53 binding pocket of hDM2 are consistent with binding, exhibiting similar structural dynamics in the pocket as simulations of known hDM2 binders Nutlin-2 and a benzodiazepinedione compound. Arylamide conformations converge towards the same region of the binding pocket on the 20 ns time scale, and most, though not all dihedrals in the binding pocket are well sampled on this timescale. We show that there are two putative classes of binding modes for arylamide compounds supported equally by the modeling evidence. In the first, the arylamide compound lies parallel to the observed p53 helix. In the second class, not previously identified or proposed, the arylamide compound lies anti-parallel to the p53 helix.

  8. Retinal Cyclic Nucleotide-Gated Channels: From Pathophysiology to Therapy

    Directory of Open Access Journals (Sweden)

    Stylianos Michalakis

    2018-03-01

    Full Text Available The first step in vision is the absorption of photons by the photopigments in cone and rod photoreceptors. After initial amplification within the phototransduction cascade the signal is translated into an electrical signal by the action of cyclic nucleotide-gated (CNG channels. CNG channels are ligand-gated ion channels that are activated by the binding of cyclic guanosine monophosphate (cGMP or cyclic adenosine monophosphate (cAMP. Retinal CNG channels transduce changes in intracellular concentrations of cGMP into changes of the membrane potential and the Ca2+ concentration. Structurally, the CNG channels belong to the superfamily of pore-loop cation channels and share a common gross structure with hyperpolarization-activated cyclic nucleotide-gated (HCN channels and voltage-gated potassium channels (KCN. In this review, we provide an overview on the molecular properties of CNG channels and describe their physiological role in the phototransduction pathways. We also discuss insights into the pathophysiological role of CNG channel proteins that have emerged from the analysis of CNG channel-deficient animal models and human CNG channelopathies. Finally, we summarize recent gene therapy activities and provide an outlook for future clinical application.

  9. Kinetic mechanism and nucleotide specificity of NADH peroxidase

    Energy Technology Data Exchange (ETDEWEB)

    Stoll, V.S.; Blanchard, J.S.

    1988-02-01

    NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between (/sup 14/C)NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilic attack by enzymatic dithiols.

  10. Kinetic mechanism and nucleotide specificity of NADH peroxidase

    International Nuclear Information System (INIS)

    Stoll, V.S.; Blanchard, J.S.

    1988-01-01

    NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between [ 14 C]NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilic attack by enzymatic dithiols

  11. Cyclic Nucleotide Monophosphates and Their Cyclases in Plant Signaling

    KAUST Repository

    Gehring, Christoph A.

    2017-10-04

    The cyclic nucleotide monophosphates (cNMPs), and notably 3′,5′-cyclic guanosine monophosphate (cGMP) and 3′,5′-cyclic adenosine monophosphate (cAMP) are now accepted as key signaling molecules in many processes in plants including growth and differentiation, photosynthesis, and biotic and abiotic defense. At the single molecule level, we are now beginning to understand how cNMPs modify specific target molecules such as cyclic nucleotide-gated channels, while at the systems level, a recent study of the Arabidopsis cNMP interactome has identified novel target molecules with specific cNMP-binding domains. A major advance came with the discovery and characterization of a steadily increasing number of guanylate cyclases (GCs) and adenylate cyclases (ACs). Several of the GCs are receptor kinases and include the brassinosteroid receptor, the phytosulfokine receptor, the Pep receptor, the plant natriuretic peptide receptor as well as a nitric oxide sensor. We foresee that in the near future many more molecular mechanisms and biological roles of GCs and ACs and their catalytic products will be discovered and further establish cNMPs as a key component of plant responses to the environment.

  12. Proofreading of misincorporated nucleotides in DNA transcription

    International Nuclear Information System (INIS)

    Voliotis, Margaritis; Liverpool, Tanniemola B; Cohen, Netta; Molina-París, Carmen

    2012-01-01

    The accuracy of DNA transcription is crucial for the proper functioning of the cell. Although RNA polymerases demonstrate selectivity for correct nucleotides, additional active mechanisms of transcriptional error correction are required to achieve observed levels of fidelity. Recent experimental findings have shed light on a particular mechanism of transcriptional error correction involving: (i) diffusive translocation of the RNA polymerase along the DNA (backtracking) and (ii) irreversible RNA cleavage. This mechanism achieves preferential cleavage of misincorporated nucleotides by biasing the local rates of translocation. Here, we study how misincorporated nucleotides affect backtracking dynamics and how this effect determines the level of transcriptional fidelity. We consider backtracking as a diffusive process in a periodic, one-dimensional energy landscape, which at a coarse-grained level gives rise to a hopping process between neighbouring local minima. We propose a model for how misincorporated nucleotides deform this energy landscape and hence affect the hopping rates. In particular, we show that this model can be used to derive both the theoretical limit on the fidelity (i.e. the minimum fraction of misincorporated nucleotides) and the actual fidelity relative to this optimum, achieved for specific combinations of the cleavage and polymerization rates. Finally, we study how external factors influencing backtracking dynamics affect transcriptional fidelity. We show that biologically relevant loads, similar to those exerted by nucleosomes or other transcriptional barriers, increase error correction. (paper)

  13. Positive versus negative modulation of different endogenous chemokines for CC-chemokine receptor 1 by small molecule agonists through allosteric versus orthosteric binding

    DEFF Research Database (Denmark)

    Jensen, Pia C; Thiele, Stefanie; Ulven, Trond

    2008-01-01

    and not CCL3 activation is affected by substitutions in the main ligand binding pocket including the conserved GluVII:06 anchor point. A series of metal-ion chelator complexes were found to act as full agonists on CCR1 and to be critically affected by the same substitutions in the main ligand binding pocket...... as CCL5 but not by mutations in the extracellular domain. In agreement with the overlapping binding sites, the small non-peptide agonists displaced radiolabeled CCL5 with high affinity. Interestingly, the same compounds acted as allosteric enhancers of the binding of CCL3 - with which they did...

  14. Inertial Pocket Navigation System: Unaided 3D Positioning.

    Science.gov (United States)

    Diaz, Estefania Munoz

    2015-04-17

    Inertial navigation systems use dead-reckoning to estimate the pedestrian's position. There are two types of pedestrian dead-reckoning, the strapdown algorithm and the step-and-heading approach. Unlike the strapdown algorithm, which consists of the double integration of the three orthogonal accelerometer readings, the step-and-heading approach lacks the vertical displacement estimation. We propose the first step-and-heading approach based on unaided inertial data solving 3D positioning. We present a step detector for steps up and down and a novel vertical displacement estimator. Our navigation system uses the sensor introduced in the front pocket of the trousers, a likely location of a smartphone. The proposed algorithms are based on the opening angle of the leg or pitch angle. We analyzed our step detector and compared it with the state-of-the-art, as well as our already proposed step length estimator. Lastly, we assessed our vertical displacement estimator in a real-world scenario. We found that our algorithms outperform the literature step and heading algorithms and solve 3D positioning using unaided inertial data. Additionally, we found that with the pitch angle, five activities are distinguishable: standing, sitting, walking, walking up stairs and walking down stairs. This information complements the pedestrian location and is of interest for applications, such as elderly care.

  15. The Use of Pocket Electronic Dictionaries by Thai University Students

    Directory of Open Access Journals (Sweden)

    Atipat Boonmoh

    2011-10-01

    Full Text Available

    ABSTRACT: This article reports on a small-scale study of Thai-speaking learners using pocket electronic dictionaries (PEDs to read an English news article. It investigates how the subjects use their PEDs for reading comprehension. Thirty-nine undergraduate students completed a questionnaire survey. Of these, four were chosen to participate in the experiment. Observations and interviews were utilized to ascertain how the subjects used their PEDs. The findings showed superficial and partial reading of the dictionary entries. It also revealed several factors that may hinder dictionary look-up success. On the basis of this research, guidelines for buying PEDs and for teaching PED skills are proposed.

    OPSOMMING: Die gebruik van sak- elektroniese woordeboeke deur Thaise universiteitstudente. Hierdie artikel doen verslag van 'n kleinskaalse studie van Thaisprekende aanleerders wat sak- elektroniese woordeboeke (SEW's gebruik om 'n Engelse nuusartikel te lees. Dit ondersoek hoe die proefpersone hul SAW's gebruik vir leesbegrip. Nege-en-dertig voorgraadse studente het 'n vraelysopname voltooi. Uit hulle is vier gekies om aan die eksperiment deel te neem. Waarnemings en onderhoude is aangewend om te bepaal hoe die proefpersone hul SEW's gebruik het. Die bevindings het oppervlakkige en gedeeltelike lees van die woordeboekinskrywings getoon. Dit het ook verskeie faktore uitgewys wat woordeboekopsoeksukses mag belemmer. Op grond van hierdie navorsing word riglyne vir die koop van SEW's en vir die onderrig van SEWvaardighede voorgestel.

    Sleutelwoorde: WOORDEBOEKGEBRUIK, SAK- ELEKTRONIESE WOORDEBOEK, WOORDEBOEKRAADPLEGING, EENTALIGE WOORDEBOEK, TWEETALIGE WOORDEBOEK

  16. Inertial Pocket Navigation System: Unaided 3D Positioning

    Directory of Open Access Journals (Sweden)

    Estefania Munoz Diaz

    2015-04-01

    Full Text Available Inertial navigation systems use dead-reckoning to estimate the pedestrian’s position. There are two types of pedestrian dead-reckoning, the strapdown algorithm and the step-and-heading approach. Unlike the strapdown algorithm, which consists of the double integration of the three orthogonal accelerometer readings, the step-and-heading approach lacks the vertical displacement estimation. We propose the first step-and-heading approach based on unaided inertial data solving 3D positioning. We present a step detector for steps up and down and a novel vertical displacement estimator. Our navigation system uses the sensor introduced in the front pocket of the trousers, a likely location of a smartphone. The proposed algorithms are based on the opening angle of the leg or pitch angle. We analyzed our step detector and compared it with the state-of-the-art, as well as our already proposed step length estimator. Lastly, we assessed our vertical displacement estimator in a real-world scenario. We found that our algorithms outperform the literature step and heading algorithms and solve 3D positioning using unaided inertial data. Additionally, we found that with the pitch angle, five activities are distinguishable: standing, sitting, walking, walking up stairs and walking down stairs. This information complements the pedestrian location and is of interest for applications, such as elderly care.

  17. Sonographic assessment of predictors of depth of the corner pocket for ultrasound-guided supraclavicular brachial plexus block

    Directory of Open Access Journals (Sweden)

    Naveen Yadav

    2016-01-01

    Conclusion: Prescanning of supraclavicular region for estimating depth of corner pocket should be done before choosing an appropriate size needle. Furthermore, the needle should not be advanced more than the predicted corner pocket depth.

  18. Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A.

    Science.gov (United States)

    Strotmeier, Jasmin; Mahrhold, Stefan; Krez, Nadja; Janzen, Constantin; Lou, Jianlong; Marks, James D; Binz, Thomas; Rummel, Andreas

    2014-04-02

    Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins. The most important serotype BoNT/A employs the synaptic vesicle glycoprotein 2 (SV2) isoforms A-C as neuronal receptors. Here, we identified their binding site by blocking SV2 interaction using monoclonal antibodies with characterised epitopes within the cell binding domain (HC). The site is located on the backside of the conserved ganglioside binding pocket at the interface of the HCC and HCN subdomains. The dimension of the binding pocket was characterised in detail by site directed mutagenesis allowing the development of potent inhibitors as well as modifying receptor binding properties. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  19. Pyrrolidine analogues of nucleosides and nucleotides

    Czech Academy of Sciences Publication Activity Database

    Rejman, Dominik; Pohl, Radek; Kovačková, Soňa; Kočalka, Petr; Švenková, Alžběta; Šanderová, Hana; Krásný, Libor; Rosenberg, Ivan

    -, č. 52 (2008), s. 577-578 ISSN 0261-3166. [Joint Symposium of the International Roundtable on Nucleosides, Nucleotides and Nucleic Acids /18./ and the International Symposium on Nucleic Acid Chemistry /35./. Kyoto, 08.09.2008-12.09.2008] R&D Projects: GA MŠk(CZ) LC06077; GA MŠk 2B06065; GA MZd NR9138 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50200510; CEZ:AV0Z50520514 Keywords : pyrrolidine * nucleoside * nucleotide Subject RIV: CC - Organic Chemistry

  20. PENGEMBANGAN ECONOMICS POCKET BOOK BERBASIS QUANTUM LEARNING UNTUK SISWA SEKOLAH MENENGAH ATAS

    Directory of Open Access Journals (Sweden)

    Ni Wayan Ayu Santi

    2017-04-01

    Full Text Available The purpose of this study is to produce and know the quality of quantum learning based economics pocket book. In developing economics pocket book the research uses design based research. The result of study show that the matter experts, education and education practtioners provide ratings that economics is a very decent to be used without revision. Besides design experts provide decent ratings with the revition. The result of the field test showed positive responses from students with the acquistion of a persentage of 90% in addition to the field tes results II also earn a percentage of 87,4%.  Based on the result above, economics pocket book is very decend for be used without revision.  Tujuan penelitian ini adalah untuk menghasilkan dan mengetahui kualitas economics pocket book berbasis quantum learning. Peneliti dalam mengembangkan economics pocket book menggunakan model pengembangan Design Based Research. Hasil penelitian menunjukkan ahli materi, pendidikan dan praktisi pendidikan memberikan penilaian bahwa economics pocket book sangat layak digunakan tanpa revisi, selain itu ahli desain memberikan penilaian bahwa layak digunakan dengan revisi. Hasil dari uji lapangan I menunjukkan tanggapan positif dari siswa dengan perolehan persentase 90%, selain itu hasil uji lapangan II juga memperoleh persentase sebesar 87,4%. Berdasarkan hasil tersebut maka economics pocket book berbasis quantum learning termasuk kualifikasi sangat layak digunakan tanpa revisi.

  1. Placement Of Cardiac PacemaKEr Trial (POCKET) - rationale and design: a randomized controlled trial.

    Science.gov (United States)

    Magnusson, Peter; Wennström, Leo; Kastberg, Robert; Liv, Per

    2017-01-01

    A pacemaker system consists of one or two leads connected to a device that is implanted into a pocket formed just below the collarbone. This pocket is typically subcutaneous, that is, located just above the pectoral fascia. Even though the size of pacemakers has decreased markedly, complications due to superficial implants do occur. An alternative technique would be intramuscular placement of the pacemaker device, but there are no randomized controlled trials (RCTs) to support this approach, which is the rationale for the Placement Of Cardiac PacemaKEr Trial (POCKET). The aim is to study if intramuscular is superior to subcutaneous placement of a pacemaker pocket. In October 2016, we started to enroll 200 consecutive patients with an indication for bradycardia pacemaker implantation. Patients are randomized to random block sizes, stratified by age group (cut-off: 65 years) and sex, and then randomized to either subcutaneous or intramuscular implant. A concealed allocation procedure is employed, using sequentially numbered, sealed envelopes. Pocket site is blinded to the patient and in all subsequent care. The primary endpoint is patient overall satisfaction with the pocket location at 24 months as measured using a visual analog scale (VAS) 0-10. Secondary endpoints are: complications, patient-reported satisfaction at 1, 12, and 24 months (overall satisfaction, pain, discomfort, degree of unsightly appearance, movement problems, and sleep problems due to device). POCKET is a prospective interventional RCT designed to evaluate if intramuscular is superior to subcutaneous placement of a bradycardia pacemaker during a two-year follow-up.

  2. Heterodimerization of Hox proteins with Pbx1 and oncoprotein E2a-Pbx1 generates unique DNA-binding specifities at nucleotides predicted to contact the N-terminal arm of the Hox homeodomain--demonstration of Hox-dependent targeting of E2a-Pbx1 in vivo.

    Science.gov (United States)

    Lu, Q; Kamps, M P

    1997-01-09

    Hox proteins control genetic programs that orchestrate development, and a large subset of Hox proteins can bind DNA elements as heterodimers with the Pbx family of homeodomain proteins. A transcriptionally activated version of Pbx1, E2a-Pbx1, is an oncoprotein in human pre-B cell leukemia that strongly suppresses differentiation and retains its ability to heterodimerize with Hox proteins. Because monomeric Hox proteins bind very similar DNA motifs, it is unclear how they activate diverse developmental programs. Here we demonstrate that heterodimers containing different Hox proteins and a common Pbx1 or E2a-Pbx1 partner bind different DNA motifs. Structural models suggest that the specificity of the Hox protein is altered by a conformation change involving residues in the N-terminal arm of the Hox homeodomain. Mutational analysis also supported the hypothesis that unique sequences in the N-terminal arm of the Hox homeodomain are at least partially responsible for mediating this specificity. In vivo, Hox proteins directed E2a-Pbx1-mediated transactivation with moderate specificity to cognate Hox-Pbx motifs. Thus, the development specificity of individual Hox proteins may be mediated, in part, by differential targeting of cellular genes by Pbx1-Hox complexes. Likewise, through its function as a common heterodimer partner, oncoprotein E2a-Pbx1 may be able to interfere with multiple programs of development that are induced by the sequential or simultaneous expression of Hox proteins during hematopoiesis.

  3. Periodontal Pocket Depth, Hyperglycemia, and Progression of Chronic Kidney Disease: A Population-Based Longitudinal Study.

    Science.gov (United States)

    Chang, Jia-Feng; Yeh, Jih-Chen; Chiu, Ya-Lin; Liou, Jian-Chiun; Hsiung, Jing-Ru; Tung, Tao-Hsin

    2017-01-01

    No large epidemiological study has been conducted to investigate the interaction and joint effects of periodontal pocket depth and hyperglycemia on progression of chronic kidney disease in patients with periodontal diseases. Periodontal pocket depth was utilized for the grading severity of periodontal disease in 2831 patients from January 2002 to June 2013. Progression of chronic kidney disease was defined as progression of color intensity in glomerular filtration rate and albuminuria grid of updated Kidney Disease-Improving Global Outcomes guidelines. Multivariable-adjusted hazard ratios (aHR) in various models were presented across different levels of periodontal pocket depth and hemoglobin A1c (HbA1c) in forest plots and 3-dimensional histograms. During 7621 person-years of follow-up, periodontal pocket depth and HbA1C levels were robustly associated with incremental risks for progression of chronic kidney disease (aHR 3.1; 95% confidence interval [CI], 2.0-4.6 for periodontal pocket depth >4.5 mm, and 2.5; 95% CI, 1.1-5.4 for HbA1C >6.5%, respectively). The interaction between periodontal pocket depth and HbA1C on progression of chronic kidney disease was strong (P periodontal pocket depth (>4.5 mm) and higher HbA1C (>6.5%) had the greatest risk (aHR 4.2; 95% CI, 1.7-6.8) compared with the lowest aHR group (periodontal pocket depth ≤3.8 mm and HbA1C ≤6%). Our study identified combined periodontal pocket depth and HbA1C as a valuable predictor of progression of chronic kidney disease in patients with periodontal diseases. While considering the interaction between periodontal diseases and hyperglycemia, periodontal survey and optimizing glycemic control are warranted to minimize the risk of worsening renal function. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Pharmacophore screening of the protein data bank for specific binding site chemistry.

    Science.gov (United States)

    Campagna-Slater, Valérie; Arrowsmith, Andrew G; Zhao, Yong; Schapira, Matthieu

    2010-03-22

    A simple computational approach was developed to screen the Protein Data Bank (PDB) for putative pockets possessing a specific binding site chemistry and geometry. The method employs two commonly used 3D screening technologies, namely identification of cavities in protein structures and pharmacophore screening of chemical libraries. For each protein structure, a pocket finding algorithm is used to extract potential binding sites containing the correct types of residues, which are then stored in a large SDF-formatted virtual library; pharmacophore filters describing the desired binding site chemistry and geometry are then applied to screen this virtual library and identify pockets matching the specified structural chemistry. As an example, this approach was used to screen all human protein structures in the PDB and identify sites having chemistry similar to that of known methyl-lysine binding domains that recognize chromatin methylation marks. The selected genes include known readers of the histone code as well as novel binding pockets that may be involved in epigenetic signaling. Putative allosteric sites were identified on the structures of TP53BP1, L3MBTL3, CHEK1, KDM4A, and CREBBP.

  5. Pocket money, eating behaviors, and weight status among Chinese children : The Childhood Obesity Study in China mega-cities

    NARCIS (Netherlands)

    Li, Miao; Xue, Hong; Jia, Peng; Zhao, Yaling; Wang, Zhiyong; Xu, Fei; Wang, Youfa

    2017-01-01

    Both the obesity rate and pocket money are rising among children in China. This study examined family correlates of children's pocket money, associations of pocket money with eating behaviors and weight status, and how the associations may be modified by schools' unhealthy food restrictions in urban

  6. Exploring the binding sites and binding mechanism for hydrotrope encapsulated griseofulvin drug on γ-tubulin protein.

    Science.gov (United States)

    Das, Shubhadip; Paul, Sandip

    2018-01-01

    The protein γ-tubulin plays an important role in centrosomal clustering and this makes it an attractive therapeutic target for treating cancers. Griseofulvin, an antifungal drug, has recently been used to inhibit proliferation of various types of cancer cells. It can also affect the microtubule dynamics by targeting the γ-tubulin protein. So far, the binding pockets of γ-tubulin protein are not properly identified and the exact mechanism by which the drug binds to it is an area of intense speculation and research. The aim of the present study is to investigate the binding mechanism and binding affinity of griseofulvin on γ-tubulin protein using classical molecular dynamics simulations. Since the drug griseofulvin is sparingly soluble in water, here we also present a promising approach for formulating and achieving delivery of hydrophobic griseofulvin drug via hydrotrope sodium cumene sulfonate (SCS) cluster. We observe that the binding pockets of γ-tubulin protein are mainly formed by the H8, H9 helices and S7, S8, S14 strands and the hydrophobic interactions between the drug and γ-tubulin protein drive the binding process. The release of the drug griseofulvin from the SCS cluster is confirmed by the coordination number analysis. We also find hydrotrope-induced alteration of the binding sites of γ-tubulin protein and the weakening of the drug-protein interactions.

  7. Exploring the binding sites and binding mechanism for hydrotrope encapsulated griseofulvin drug on γ-tubulin protein.

    Directory of Open Access Journals (Sweden)

    Shubhadip Das

    Full Text Available The protein γ-tubulin plays an important role in centrosomal clustering and this makes it an attractive therapeutic target for treating cancers. Griseofulvin, an antifungal drug, has recently been used to inhibit proliferation of various types of cancer cells. It can also affect the microtubule dynamics by targeting the γ-tubulin protein. So far, the binding pockets of γ-tubulin protein are not properly identified and the exact mechanism by which the drug binds to it is an area of intense speculation and research. The aim of the present study is to investigate the binding mechanism and binding affinity of griseofulvin on γ-tubulin protein using classical molecular dynamics simulations. Since the drug griseofulvin is sparingly soluble in water, here we also present a promising approach for formulating and achieving delivery of hydrophobic griseofulvin drug via hydrotrope sodium cumene sulfonate (SCS cluster. We observe that the binding pockets of γ-tubulin protein are mainly formed by the H8, H9 helices and S7, S8, S14 strands and the hydrophobic interactions between the drug and γ-tubulin protein drive the binding process. The release of the drug griseofulvin from the SCS cluster is confirmed by the coordination number analysis. We also find hydrotrope-induced alteration of the binding sites of γ-tubulin protein and the weakening of the drug-protein interactions.

  8. Crystal Structures and Binding Dynamics of Odorant-Binding Protein 3 from two aphid species Megoura viciae and Nasonovia ribisnigri.

    Science.gov (United States)

    Northey, Tom; Venthur, Herbert; De Biasio, Filomena; Chauviac, Francois-Xavier; Cole, Ambrose; Ribeiro, Karlos Antonio Lisboa; Grossi, Gerarda; Falabella, Patrizia; Field, Linda M; Keep, Nicholas H; Zhou, Jing-Jiang

    2016-04-22

    Aphids use chemical cues to locate hosts and find mates. The vetch aphid Megoura viciae feeds exclusively on the Fabaceae, whereas the currant-lettuce aphid Nasonovia ribisnigri alternates hosts between the Grossulariaceae and Asteraceae. Both species use alarm pheromones to warn of dangers. For N. ribisnigri this pheromone is a single component (E)-β-farnesene but M. viciae uses a mixture of (E)-β-farnesene, (-)-α-pinene, β-pinene, and limonene. Odorant-binding proteins (OBP) are believed to capture and transport such semiochemicals to their receptors. Here, we report the first aphid OBP crystal structures and examine their molecular interactions with the alarm pheromone components. Our study reveals some unique structural features: 1) the lack of an internal ligand binding site; 2) a striking groove in the surface of the proteins as a putative binding site; 3) the N-terminus rather than the C-terminus occupies the site closing off the conventional OBP pocket. The results from fluorescent binding assays, molecular docking and dynamics demonstrate that OBP3 from M. viciae can bind to all four alarm pheromone components and the differential ligand binding between these very similar OBP3s from the two aphid species is determined mainly by the direct π-π interactions between ligands and the aromatic residues of OBP3s in the binding pocket.

  9. EspM2 is a RhoA guanine nucleotide exchange factor.

    Science.gov (United States)

    Arbeloa, Ana; Garnett, James; Lillington, James; Bulgin, Richard R; Berger, Cedric N; Lea, Susan M; Matthews, Steve; Frankel, Gad

    2010-05-01

    We investigated how the type III secretion system WxxxE effectors EspM2 of enterohaemorrhagic Escherichia coli, which triggers stress fibre formation, and SifA of Salmonella enterica serovar Typhimurium, which is involved in intracellular survival, modulate Rho GTPases. We identified a direct interaction between EspM2 or SifA and nucleotide-free RhoA. Nuclear Magnetic Resonance Spectroscopy revealed that EspM2 has a similar fold to SifA and the guanine nucleotide exchange factor (GEF) effector SopE. EspM2 induced nucleotide exchange in RhoA but not in Rac1 or H-Ras, while SifA induced nucleotide exchange in none of them. Mutating W70 of the WxxxE motif or L118 and I127 residues, which surround the catalytic loop, affected the stability of EspM2. Substitution of Q124, located within the catalytic loop of EspM2, with alanine, greatly attenuated the RhoA GEF activity in vitro and the ability of EspM2 to induce stress fibres upon ectopic expression. These results suggest that binding of SifA to RhoA does not trigger nucleotide exchange while EspM2 is a unique Rho GTPase GEF.

  10. A strategy using NMR peptide structures of thromboxane A2 receptor as templates to construct ligand-recognition pocket of prostacyclin receptor

    Directory of Open Access Journals (Sweden)

    Ruan Ke-He

    2005-11-01

    Full Text Available Abstract Background: Prostacyclin receptor (IP and thromboxane A2 receptor (TP belong to rhodopsin-type G protein-coupling receptors and respectively bind to prostacyclin and thromboxane A2 derived from arachidonic acid. Recently, we have determined the extracellular loop (eLP structures of the human TP receptor by 2-D 1H NMR spectroscopy using constrained peptides mimicking the individual eLP segments. The studies have identified the segment along with several residues in the eLP domains important to ligand recognition, as well as proposed a ligand recognition pocket for the TP receptor. Results: The IP receptor shares a similar primary structure in the eLPs with those of the TP receptor. Forty percent residues in the second eLPs of the receptors are identical, which is the major region involved in forming the ligand recognition pocket in the TP receptor. Based on the high homology score, the eLP domains of the IP receptor were constructed by the homology modeling approach using the NMR structures of the TP eLPs as templates, and then configured to the seven transmembrane (TM domains model constructed using the crystal structure of the bovine rhodopsin as a template. A NMR structure of iloprost was docked into the modeled IP ligand recognition pocket. After dynamic studies, the segments and residues involved in the IP ligand recognition were proposed. A key residue, Arg173 involved in the ligand recognition for the IP receptor, as predicted from the modeling, was confirmed by site-directed mutagenesis. Conclusion: A 3-D model of the human IP receptor was constructed by homology modeling using the crystal structure of bovine rhodopsin TM domains and the NMR structures of the synthetic constrained peptides of the eLP domains of the TP receptor as templates. This strategy can be applied to molecular modeling and the prediction of ligand recognition pockets for other prostanoid receptors.

  11. A single nucleotide change affects fur-dependent regulation of sodB in H. pylori.

    Directory of Open Access Journals (Sweden)

    Beth M Carpenter

    Full Text Available Helicobacter pylori is a significant human pathogen that has adapted to survive the many stresses found within the gastric environment. Superoxide Dismutase (SodB is an important factor that helps H. pylori combat oxidative stress. sodB was previously shown to be repressed by the Ferric Uptake Regulator (Fur in the absence of iron (apo-Fur regulation [1]. Herein, we show that apo regulation is not fully conserved among all strains of H. pylori. apo-Fur dependent changes in sodB expression are not observed under iron deplete conditions in H. pylori strains G27, HPAG1, or J99. However, Fur regulation of pfr and amiE occurs as expected. Comparative analysis of the Fur coding sequence between G27 and 26695 revealed a single amino acid difference, which was not responsible for the altered sodB regulation. Comparison of the sodB promoters from G27 and 26695 also revealed a single nucleotide difference within the predicted Fur binding site. Alteration of this nucleotide in G27 to that of 26695 restored apo-Fur dependent sodB regulation, indicating that a single base difference is at least partially responsible for the difference in sodB regulation observed among these H. pylori strains. Fur binding studies revealed that alteration of this single nucleotide in G27 increased the affinity of Fur for the sodB promoter. Additionally, the single base change in G27 enabled the sodB promoter to bind to apo-Fur with affinities similar to the 26695 sodB promoter. Taken together these data indicate that this nucleotide residue is important for direct apo-Fur binding to the sodB promoter.

  12. Achieving single-nucleotide resolution of 5-methylcytosine detection with TALEs.

    Science.gov (United States)

    Kubik, Grzegorz; Summerer, Daniel

    2015-01-19

    We report engineered transcription-activator-like effectors (TALEs) as the first DNA-binding molecules that detect 5-methylcytosine (mC) at single-nucleotide resolution with fully programmable sequence selectivity. This is achieved by a design strategy such that a single cytosine (C) in a DNA sequence is selectively interrogated for its mC-modification level by targeting with a discriminatory TALE repeat; other Cs are ignored by targeting with universal-binding TALE repeats. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Out-of-pocket expenditure on institutional delivery in India.

    Science.gov (United States)

    Mohanty, Sanjay K; Srivastava, Akanksha

    2013-05-01

    Though promotion of institutional delivery is used as a strategy to reduce maternal and neonatal mortality, about half of the deliveries in India are conducted at home without any medical care. Among women who deliver at home, one in four cites cost as barrier to facility-based care. The relative share of deliveries in private health centres has increased over time and the associated costs are often catastrophic for poor households. Though research has identified socio-economic, demographic and geographic barriers to the utilization of maternal care, little is known on the cost differentials in delivery care in India. The objective of this paper is to understand the regional pattern and socio-economic differentials in out-of-pocket (OOP) expenditure on institutional delivery by source of provider in India. The study utilizes unit data from the District Level Household and Facility Survey (DLHS-3), conducted in India during 2007-08. Descriptive statistics, principal component analyses and a two-part model are used in the analyses. During 2004-08, the mean OOP expenditure for a delivery in a public health centre in India was US$39 compared with US$139 in a private health centre. The predicted expenditure for a caesarean delivery was six times higher than for a normal delivery. With an increase in the economic status and educational attainment of mothers, the propensity and rate of OOP expenditure increases, linking higher OOP expenditure to quality of care. The OOP expenditure in public health centres, adjusting for inflation, has declined over time, possibly due to increased spending under the National Rural Health Mission. Based on these findings, we recommend that facilities in public health centres of poorly performing states are improved and that public-private partnership models are developed to reduce the economic burden for households of maternal care in India.

  14. A pocket device for high-throughput optofluidic holographic microscopy

    Science.gov (United States)

    Mandracchia, B.; Bianco, V.; Wang, Z.; Paturzo, M.; Bramanti, A.; Pioggia, G.; Ferraro, P.

    2017-06-01

    Here we introduce a compact holographic microscope embedded onboard a Lab-on-a-Chip (LoC) platform. A wavefront division interferometer is realized by writing a polymer grating onto the channel to extract a reference wave from the object wave impinging the LoC. A portion of the beam reaches the samples flowing along the channel path, carrying their information content to the recording device, while one of the diffraction orders from the grating acts as an off-axis reference wave. Polymeric micro-lenses are delivered forward the chip by Pyro-ElectroHydroDynamic (Pyro-EHD) inkjet printing techniques. Thus, all the required optical components are embedded onboard a pocket device, and fast, non-iterative, reconstruction algorithms can be used. We use our device in combination with a novel high-throughput technique, named Space-Time Digital Holography (STDH). STDH exploits the samples motion inside microfluidic channels to obtain a synthetic hologram, mapped in a hybrid space-time domain, and with intrinsic useful features. Indeed, a single Linear Sensor Array (LSA) is sufficient to build up a synthetic representation of the entire experiment (i.e. the STDH) with unlimited Field of View (FoV) along the scanning direction, independently from the magnification factor. The throughput of the imaging system is dramatically increased as STDH provides unlimited FoV, refocusable imaging of samples inside the liquid volume with no need for hologram stitching. To test our embedded STDH microscopy module, we counted, imaged and tracked in 3D with high-throughput red blood cells moving inside the channel volume under non ideal flow conditions.

  15. Expressed sequence tags (ESTs) and single nucleotide ...

    African Journals Online (AJOL)

    Expressed Sequence Tags (ESTs) and Single Nucleotide Polymorphisms (SNPs) are providing in depth knowledge in plant biology, breeding and biotechnology. The emergence of many novel molecular marker techniques are changing and accelerating the process of producing mutations in plant molecular biology ...

  16. Retrieval and Representation of Nucleotide Sequence of ...

    African Journals Online (AJOL)

    Nigerian Journal of Basic and Applied Science (March, 2013), 21(1): 27-32. DOI: http://dx.doi.org/10.4314/njbas.v21i1.4. ISSN 0794-5698. Retrieval and Representation of Nucleotide Sequence of Saccharomyces cerevisiae Cystathionine. Gamma-Lyase (CYS3) Gene in Five Formats. *R. A. Umar, H. Abdullahi and N. Lawal.

  17. Incision Coordination in Nucleotide Excision Repair

    NARCIS (Netherlands)

    W.L. de Laat (Wouter)

    1998-01-01

    textabstractThis thesis aims to contribute to the understanding of the molecular mechanism that underlies one of the main DNA repair pathways in mammals, nucleotide excision rcpair. In chapter 1 the relevance of DNA repair in general is outlined. An overview of mammalian strategies to counteract DNA

  18. Single-nucleotide polymorphisms in peroxisome proliferator ...

    Indian Academy of Sciences (India)

    Prakash

    the metabolic syndrome (MS) and type 2 diabetes. We also investigated the correlation of these two single-nucleotide polymorphisms (SNPs) with plasma resistin levels. The C1431T SNP was associated with higher levels of plasma resistin (P = 0.017). Furthermore, C1431T was associated with resistin in different tertiles.

  19. Role of a Hydrophobic Pocket in Polyamine Interactions with the Polyspecific Organic Cation Transporter OCT3*

    Science.gov (United States)

    Li, Dan C.; Nichols, Colin G.; Sala-Rabanal, Monica

    2015-01-01

    Organic cation transporter 3 (OCT3, SLC22A3) is a polyspecific, facilitative transporter expressed in astrocytes and in placental, intestinal, and blood-brain barrier epithelia, and thus elucidating the molecular mechanisms underlying OCT3 substrate recognition is critical for the rational design of drugs targeting these tissues. The pharmacology of OCT3 is distinct from that of other OCTs, and here we investigated the role of a hydrophobic cavity tucked within the translocation pathway in OCT3 transport properties. Replacement of an absolutely conserved Asp by charge reversal (D478E), neutralization (D478N), or even exchange (D478E) abolished MPP+ uptake, demonstrating this residue to be obligatory for OCT3-mediated transport. Mutations at non-conserved residues lining the putative binding pocket of OCT3 to the corresponding residue in OCT1 (L166F, F450L, and E451Q) reduced the rate of MPP+ transport, but recapitulated the higher sensitivity pharmacological profile of OCT1. Thus, interactions of natural polyamines (putrescine, spermidine, spermine) and polyamine-like potent OCT1 blockers (1,10-diaminodecane, decamethonium, bistriethylaminodecane, and 1,10-bisquinuclidinedecane) with wild-type OCT3 were weak, but were significantly potentiated in the mutant OCT3s. Conversely, a reciprocal mutation in OCT1 (F161L) shifted the polyamine-sensitivity phenotype toward that of OCT3. Further analysis indicated that OCT1 and OCT3 can recognize essentially the same substrates, but the strength of substrate-transporter interactions is weaker in OCT3, as informed by the distinct makeup of the hydrophobic cleft. The residues identified here are key contributors to both the observed differences between OCT3 and OCT1 and to the mechanisms of substrate recognition by OCTs in general. PMID:26405039

  20. Role of a Hydrophobic Pocket in Polyamine Interactions with the Polyspecific Organic Cation Transporter OCT3.

    Science.gov (United States)

    Li, Dan C; Nichols, Colin G; Sala-Rabanal, Monica

    2015-11-13

    Organic cation transporter 3 (OCT3, SLC22A3) is a polyspecific, facilitative transporter expressed in astrocytes and in placental, intestinal, and blood-brain barrier epithelia, and thus elucidating the molecular mechanisms underlying OCT3 substrate recognition is critical for the rational design of drugs targeting these tissues. The pharmacology of OCT3 is distinct from that of other OCTs, and here we investigated the role of a hydrophobic cavity tucked within the translocation pathway in OCT3 transport properties. Replacement of an absolutely conserved Asp by charge reversal (D478E), neutralization (D478N), or even exchange (D478E) abolished MPP(+) uptake, demonstrating this residue to be obligatory for OCT3-mediated transport. Mutations at non-conserved residues lining the putative binding pocket of OCT3 to the corresponding residue in OCT1 (L166F, F450L, and E451Q) reduced the rate of MPP(+) transport, but recapitulated the higher sensitivity pharmacological profile of OCT1. Thus, interactions of natural polyamines (putrescine, spermidine, spermine) and polyamine-like potent OCT1 blockers (1,10-diaminodecane, decamethonium, bistriethylaminodecane, and 1,10-bisquinuclidinedecane) with wild-type OCT3 were weak, but were significantly potentiated in the mutant OCT3s. Conversely, a reciprocal mutation in OCT1 (F161L) shifted the polyamine-sensitivity phenotype toward that of OCT3. Further analysis indicated that OCT1 and OCT3 can recognize essentially the same substrates, but the strength of substrate-transporter interactions is weaker in OCT3, as informed by the distinct makeup of the hydrophobic cleft. The residues identified here are key contributors to both the observed differences between OCT3 and OCT1 and to the mechanisms of substrate recognition by OCTs in general. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.