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Sample records for active site position

  1. Nucleosome positioning and kinetics near transcription-start-site barriers are controlled by interplay between active remodeling and DNA sequence

    PubMed Central

    Parmar, Jyotsana J.; Marko, John F.; Padinhateeri, Ranjith

    2014-01-01

    We investigate how DNA sequence, ATP-dependent chromatin remodeling and nucleosome-depleted ‘barriers’ co-operate to determine the kinetics of nucleosome organization, in a stochastic model of nucleosome positioning and dynamics. We find that ‘statistical’ positioning of nucleosomes against ‘barriers’, hypothesized to control chromatin structure near transcription start sites, requires active remodeling and therefore cannot be described using equilibrium statistical mechanics. We show that, unlike steady-state occupancy, DNA site exposure kinetics near a barrier is dominated by DNA sequence rather than by proximity to the barrier itself. The timescale for formation of positioning patterns near barriers is proportional to the timescale for active nucleosome eviction. We also show that there are strong gene-to-gene variations in nucleosome positioning near barriers, which are eliminated by averaging over many genes. Our results suggest that measurement of nucleosome kinetics can reveal information about sequence-dependent regulation that is not apparent in steady-state nucleosome occupancy. PMID:24068556

  2. A Conserved Surface Loop in Type I Dehydroquinate Dehydratases Positions an Active Site Arginine and Functions in Substrate Binding

    SciTech Connect

    Light, Samuel H.; Minasov, George; Shuvalova, Ludmilla; Peterson, Scott N.; Caffrey, Michael; Anderson, Wayne F.; Lavie, Arnon

    2012-04-18

    Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD that address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild-type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of involvement of the loop in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, closure of the loop necessitates a conformational change of a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria make the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.

  3. Effects of protonation state of Asp181 and position of active site water molecules on the conformation of PTP1B.

    PubMed

    Ozcan, Ahmet; Olmez, Elif Ozkirimli; Alakent, Burak

    2013-05-01

    In protein tyrosine phosphatase 1B (PTP1B), the flexible WPD loop adopts a closed conformation (WPDclosed ) in the active state of PTP1B, bringing the catalytic Asp181 close to the active site pocket, while WPD loop is in an open conformation (WPDopen ) in the inactive state. Previous studies showed that Asp181 may be protonated at physiological pH, and ordered water molecules exist in the active site. In the current study, molecular dynamics simulations are employed at different Asp181 protonation states and initial positions of active site water molecules, and compared with the existing crystallographic data of PTP1B. In WPDclosed conformation, the active site is found to maintain its conformation only in the protonated state of Asp181 in both free and liganded states, while Asp181 is likely to be deprotonated in WPDopen conformation. When the active site water molecule network that is a part of the free WPDclosed crystal structure is disrupted, intermediate WPD loop conformations, similar to that in the PTPRR crystal structure, are sampled in the MD simulations. In liganded PTP1B, one active site water molecule is found to be important for facilitating the orientation of Cys215 and the phosphate ion, thus may play a role in the reaction. In conclusion, conformational stability of WPD loop, and possibly catalytic activity of PTP1B, is significantly affected by the protonation state of Asp181 and position of active site water molecules, showing that these aspects should be taken into consideration both in MD simulations and inhibitor design. PMID:23239271

  4. Unveiling hidden catalytic contributions of the conserved His/Trp-III in tyrosine recombinases: assembly of a novel active site in Flp recombinase harboring alanine at this position

    PubMed Central

    Ma, Chien-Hui; Kwiatek, Agnieszka; Bolusani, Swetha; Voziyanov, Yuri; Jayaram, Makkuni

    2007-01-01

    Summary The catalytic pentad of tyrosine recombinases, that assists the tyrosine nucleophile, includes a conserved histidine/tryptophan (His/Trp-III). Flp and Cre harbor tryptophan at this position; most of their kin recombinases display histidine. Contrary to the conservation rule, Flp(W330F) is a much stronger recombinase than Flp(W330H). The hydrophobicity of Trp-330 or Phe-330 is utilized in correctly positioning Tyr-343 during the strand cleavage step of recombination. Why then is phenylalanine almost never encountered in the recombinase family at this conserved position? Using exogenous nucleophiles and synthetic methylphosphonate or 5'-thiolate substrates, we decipher that Trp-330 also assists in the activation of the scissile phosphate and the departure of the 5'-hydroxyl leaving group. These two functions are consistent with the hydrogen bonding property of Trp-330 as well as its location in structures of the Flp recombination complexes. However, van der Waals contact between Trp-330 and Arg-308 may also be important for the phosphate activation step. A structure based suppression strategy permits the inactive variant Flp(W330A) to be rescued by a second site mutation A339M. Modeling alanine and methionine at positions 330 and 339, respectively, in the Flp crystal structure suggests a plausible mechanism for active site restoration. Successful suppression suggests the possibility of evolving, by design, new active site configurations for tyrosine recombination. PMID:17367810

  5. Experimental and Computational Mutagenesis To Investigate the Positioning of a General Base within an Enzyme Active Site

    PubMed Central

    2015-01-01

    The positioning of catalytic groups within proteins plays an important role in enzyme catalysis, and here we investigate the positioning of the general base in the enzyme ketosteroid isomerase (KSI). The oxygen atoms of Asp38, the general base in KSI, were previously shown to be involved in anion–aromatic interactions with two neighboring Phe residues. Here we ask whether those interactions are sufficient, within the overall protein architecture, to position Asp38 for catalysis or whether the side chains that pack against Asp38 and/or the residues of the structured loop that is capped by Asp38 are necessary to achieve optimal positioning for catalysis. To test positioning, we mutated each of the aforementioned residues, alone and in combinations, in a background with the native Asp general base and in a D38E mutant background, as Glu at position 38 was previously shown to be mispositioned for general base catalysis. These double-mutant cycles reveal positioning effects as large as 103-fold, indicating that structural features in addition to the overall protein architecture and the Phe residues neighboring the carboxylate oxygen atoms play roles in positioning. X-ray crystallography and molecular dynamics simulations suggest that the functional effects arise from both restricting dynamic fluctuations and disfavoring potential mispositioned states. Whereas it may have been anticipated that multiple interactions would be necessary for optimal general base positioning, the energetic contributions from positioning and the nonadditive nature of these interactions are not revealed by structural inspection and require functional dissection. Recognizing the extent, type, and energetic interconnectivity of interactions that contribute to positioning catalytic groups has implications for enzyme evolution and may help reveal the nature and extent of interactions required to design enzymes that rival those found in biology. PMID:24597914

  6. Identification of the conserved spatial position of key active-site atoms in glycoside hydrolase 13 family members.

    PubMed

    Kumar, Vikash

    2010-07-19

    A computational study on the glycoside hydrolase 13 (GH13) family of the CAZy database has been carried out at the atomic level in order to identify the conserved positions that may be responsible for recognition of the substrate. Analysis with substrate analog-, inhibitor-, or product-bound 3D structures was carried out to find the atomic spatial arrangement of the amino acids that make -2, -1, +1, and +2 subsites and water oxygen atoms around the ligand. The identified conserved positions of subsites were independent from the nature of the amino acid. The -1 and +1 subsites have more conserved positions than the -2 and +2 subsites. Some of the clusters of the -1 and +1 subsites have atoms of the same chemical nature. A spatially conserved position for water, which is stabilized by a hydrogen bond with the carboxyl group of a proton donor (Glu) and Asp of the catalytic triad, was found in the -1 subsite of 75% of the enzymes subjected to analysis. This position could be the region of hydrolytic water. PMID:20557875

  7. The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site

    PubMed Central

    Aurelius, Oskar; Johansson, Renzo; Bågenholm, Viktoria; Lundin, Daniel; Tholander, Fredrik; Balhuizen, Alexander; Beck, Tobias; Sahlin, Margareta; Sjöberg, Britt-Marie; Mulliez, Etienne; Logan, Derek T.

    2015-01-01

    Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), alone and in several complexes, including with the allosteric effector dATP and its cognate substrate CTP. In the crystal structure of the enzyme as purified, tmNrdD lacks a cysteine for radical transfer to the substrate pre-positioned in the active site. Nevertheless activity assays using anaerobic cell extracts from T. maritima demonstrate that the class III RNR is enzymatically active. Other genetic and microbiological evidence is summarized indicating that the enzyme is important for T. maritima. Mutation of either of two cysteine residues in a disordered loop far from the active site results in inactive enzyme. We discuss the possible mechanisms for radical initiation of substrate reduction given the collected evidence from the crystal structure, our activity assays and other published work. Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist. Finally, we present a phylogenetic analysis showing that the structure of tmNrdD is representative of a new RNR subclass IIIh, present in all Thermotoga

  8. Enhancer-like activity of A1gR1-binding site in alginate gene activation: positional, orientational, and sequence specificity.

    PubMed Central

    Fujiwara, S; Zielinski, N A; Chakrabarty, A M

    1993-01-01

    Significant activation of promoters of alginate genes such as algD or algC occurs in mucoid Pseudomonas aeruginosa during its proliferation in the lungs of cystic fibrosis patients. These promoters have been shown to be responsive to environmental signals such as high osmolarity. The signaling is mediated by a so-called two-component signal transduction system, in which a soluble protein, AlgR2, undergoes autophosphorylation and transfers the phosphate to a DNA-binding response regulator protein, AlgR1. The phosphorylated form of AlgR1 has a high affinity for binding at upstream sequences of both the algC and algD promoters. Two AlgR1-binding sites (ABS) have been reported upstream of the algC gene. One of the two ABSs (algC-ABS1, located at -94 to -81) is critical for the algC activation process, while the second ABS (algC-ABS2, located at +161 to +174) is only weakly active. We now report the presence of a third ABS within the structural gene of algC, and this ABS (algC-ABS3) is also important for algC promoter activation. algC-ABS1 can be replaced functionally by algC-ABS2, algD-ABS1, or algD-ABS2 and somewhat weakly by algD-ABS3. Introduction of a half-integral turn in the DNA helix between the algC site of transcription initiation and algC-ABS1 allowed only slight reduction of promoter activity, suggesting that the binding site could be appreciably functional even when present in the opposite face of the helix. Activation of the algC promoter is independent of the relative location (upstream or downstream of the mRNA start site), the number of copies, or the orientation of algC-ABS1, suggesting that it behaves like a eukaryotic enhancer element in promoting transcription from the algC promoter. Images PMID:8366031

  9. Optical positions of active galaxies

    NASA Astrophysics Data System (ADS)

    Meurs, E. J. A.

    1984-04-01

    Optical positions are calculated for 26 active galaxies (mainly Markarian dn Arakelian objects), using the plate-measuring apparatus at Leiden Observatory on the O plates of the Palomar Sky Survey and applying AGK-3 data in the reductions. The results are presented in a table and have accuracy 0.5 arcsec; a comparison with the positions determined by Clements (1981, 1983) for 19 objects reveals a possible offset of -0.28 arcsec in the right-ascension determinations.

  10. Site directed immobilization of glucose-6-phosphate dehydrogenase via thiol-disulfide interchange: influence on catalytic activity of cysteines introduced at different positions.

    PubMed

    Simons, J R; Mosisch, M; Torda, A E; Hilterhaus, L

    2013-08-10

    This study shows the effect of site-directed enzyme immobilization upon the enzyme activity of covalently bound glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Immobilization points were introduced at sterically accessible sites in order to control the protein's orientation and twice as much activity was recovered in comparison to conventionally immobilized enzyme. Immobilization of G6PDH via genetically engineered cysteine provided a simple, but effective method to control the immobilization process. G6PDH variants with cysteine close to the active center (L218C), close to the dimer interface (D205C) as well as far from the active center (D453C) showed changes in activity and the efficacy of immobilization. PMID:23770076

  11. Positive Surface over Positive Scattered Data Sites.

    PubMed

    Ibraheem, Farheen; Hussain, Malik Zawwar; Bhatti, Akhlaq Ahmad

    2015-01-01

    The aim of this paper is to develop a local positivity preserving scheme when the data amassed from different sources is positioned at sparse points. The proposed algorithm first triangulates the irregular data using Delauny triangulation method, therewith interpolates each boundary and radial curve of the triangle by C¹ rational trigonometric cubic function. Half of the parameters in the description of the interpolant are constrained to keep up the positive shape of data while the remaining half are set free for users' requirement. Orthogonality of trigonometric function assures much smoother surface as compared to polynomial functions. The proposed scheme can be of great use in areas of surface reconstruction and deformation, signal processing, CAD/CAM design, solving differential equations, and image restoration. PMID:26057122

  12. Positive Surface over Positive Scattered Data Sites

    PubMed Central

    Ibraheem, Farheen; Hussain, Malik Zawwar; Bhatti, Akhlaq Ahmad

    2015-01-01

    The aim of this paper is to develop a local positivity preserving scheme when the data amassed from different sources is positioned at sparse points. The proposed algorithm first triangulates the irregular data using Delauny triangulation method, therewith interpolates each boundary and radial curve of the triangle by C¹ rational trigonometric cubic function. Half of the parameters in the description of the interpolant are constrained to keep up the positive shape of data while the remaining half are set free for users’ requirement. Orthogonality of trigonometric function assures much smoother surface as compared to polynomial functions. The proposed scheme can be of great use in areas of surface reconstruction and deformation, signal processing, CAD/CAM design, solving differential equations, and image restoration. PMID:26057122

  13. Positive-Unlabeled Learning for Pupylation Sites Prediction

    PubMed Central

    Cao, Jun-Zhe

    2016-01-01

    Pupylation plays a key role in regulating various protein functions as a crucial posttranslational modification of prokaryotes. In order to understand the molecular mechanism of pupylation, it is important to identify pupylation substrates and sites accurately. Several computational methods have been developed to identify pupylation sites because the traditional experimental methods are time-consuming and labor-sensitive. With the existing computational methods, the experimentally annotated pupylation sites are used as the positive training set and the remaining nonannotated lysine residues as the negative training set to build classifiers to predict new pupylation sites from the unknown proteins. However, the remaining nonannotated lysine residues may contain pupylation sites which have not been experimentally validated yet. Unlike previous methods, in this study, the experimentally annotated pupylation sites were used as the positive training set whereas the remaining nonannotated lysine residues were used as the unlabeled training set. A novel method named PUL-PUP was proposed to predict pupylation sites by using positive-unlabeled learning technique. Our experimental results indicated that PUL-PUP outperforms the other methods significantly for the prediction of pupylation sites. As an application, PUL-PUP was also used to predict the most likely pupylation sites in nonannotated lysine sites. PMID:27579315

  14. Positive-Unlabeled Learning for Pupylation Sites Prediction.

    PubMed

    Jiang, Ming; Cao, Jun-Zhe

    2016-01-01

    Pupylation plays a key role in regulating various protein functions as a crucial posttranslational modification of prokaryotes. In order to understand the molecular mechanism of pupylation, it is important to identify pupylation substrates and sites accurately. Several computational methods have been developed to identify pupylation sites because the traditional experimental methods are time-consuming and labor-sensitive. With the existing computational methods, the experimentally annotated pupylation sites are used as the positive training set and the remaining nonannotated lysine residues as the negative training set to build classifiers to predict new pupylation sites from the unknown proteins. However, the remaining nonannotated lysine residues may contain pupylation sites which have not been experimentally validated yet. Unlike previous methods, in this study, the experimentally annotated pupylation sites were used as the positive training set whereas the remaining nonannotated lysine residues were used as the unlabeled training set. A novel method named PUL-PUP was proposed to predict pupylation sites by using positive-unlabeled learning technique. Our experimental results indicated that PUL-PUP outperforms the other methods significantly for the prediction of pupylation sites. As an application, PUL-PUP was also used to predict the most likely pupylation sites in nonannotated lysine sites. PMID:27579315

  15. Salt site performance assessment activities

    SciTech Connect

    Kircher, J.F.; Gupta, S.K.

    1983-01-01

    During this year the first selection of the tools (codes) for performance assessments of potential salt sites have been tentatively selected and documented; the emphasis has shifted from code development to applications. During this period prior to detailed characterization of a salt site, the focus is on bounding calculations, sensitivity and with the data available. The development and application of improved methods for sensitivity and uncertainty analysis is a focus for the coming years activities and the subject of a following paper in these proceedings. Although the assessments to date are preliminary and based on admittedly scant data, the results indicate that suitable salt sites can be identified and repository subsystems designed which will meet the established criteria for protecting the health and safety of the public. 36 references, 5 figures, 2 tables.

  16. Effect of the piperazine unit and metal-binding site position on the solubility and anti-proliferative activity of ruthenium(II)- and osmium(II)- arene complexes of isomeric indolo[3,2-c]quinoline-piperazine hybrids.

    PubMed

    Filak, Lukas K; Kalinowski, Danuta S; Bauer, Theresa J; Richardson, Des R; Arion, Vladimir B

    2014-07-01

    In this study, the indoloquinoline backbone and piperazine were combined to prepare indoloquinoline-piperazine hybrids and their ruthenium- and osmium-arene complexes in an effort to generate novel antitumor agents with improved aqueous solubility. In addition, the position of the metal-binding unit was varied, and the effect of these structural alterations on the aqueous solubility and antiproliferative activity of their ruthenium- and osmium-arene complexes was studied. The indoloquinoline-piperazine hybrids L(1-3) were prepared in situ and isolated as six ruthenium and osmium complexes [(η(6)-p-cymene)M(L(1-3))Cl]Cl, where L(1) = 6-(4-methylpiperazin-1-yl)-N-(pyridin-2-yl-methylene)-11H-indolo[3,2-c]quinolin-2-N-amine, M = Ru ([1a]Cl), Os ([1b]Cl), L(2) = 6-(4-methylpiperazin-1-yl)-N-(pyridin-2-yl-methylene)-11H-indolo[3,2-c]quinolin-4-N-amine, M = Ru ([2a]Cl), Os ([2b]Cl), L(3) = 6-(4-methylpiperazin-1-yl)-N-(pyridin-2-yl-methylene)-11H-indolo[3,2-c]quinolin-8-N-amine, M = Ru ([3a]Cl), Os ([3b]Cl). The compounds were characterized by elemental analysis, one- and two-dimensional NMR spectroscopy, ESI mass spectrometry, IR and UV-vis spectroscopy, and single-crystal X-ray diffraction. The antiproliferative activity of the isomeric ruthenium and osmium complexes [1a,b]Cl-[3a,b]Cl was examined in vitro and showed the importance of the position of the metal-binding site for their cytotoxicity. Those complexes containing the metal-binding site located at the position 4 of the indoloquinoline scaffold ([2a]Cl and [2b]Cl) demonstrated the most potent antiproliferative activity. The results provide important insight into the structure-activity relationships of ruthenium- and osmium-arene complexes with indoloquinoline-piperazine hybrid ligands. These studies can be further utilized for the design and development of more potent chemotherapeutic agents. PMID:24927493

  17. Effect of the Piperazine Unit and Metal-Binding Site Position on the Solubility and Anti-Proliferative Activity of Ruthenium(II)- and Osmium(II)- Arene Complexes of Isomeric Indolo[3,2-c]quinoline—Piperazine Hybrids

    PubMed Central

    2014-01-01

    In this study, the indoloquinoline backbone and piperazine were combined to prepare indoloquinoline–piperazine hybrids and their ruthenium- and osmium-arene complexes in an effort to generate novel antitumor agents with improved aqueous solubility. In addition, the position of the metal-binding unit was varied, and the effect of these structural alterations on the aqueous solubility and antiproliferative activity of their ruthenium- and osmium-arene complexes was studied. The indoloquinoline–piperazine hybrids L1–3 were prepared in situ and isolated as six ruthenium and osmium complexes [(η6-p-cymene)M(L1–3)Cl]Cl, where L1 = 6-(4-methylpiperazin-1-yl)-N-(pyridin-2-yl-methylene)-11H-indolo[3,2-c]quinolin-2-N-amine, M = Ru ([1a]Cl), Os ([1b]Cl), L2 = 6-(4-methylpiperazin-1-yl)-N-(pyridin-2-yl-methylene)-11H-indolo[3,2-c]quinolin-4-N-amine, M = Ru ([2a]Cl), Os ([2b]Cl), L3 = 6-(4-methylpiperazin-1-yl)-N-(pyridin-2-yl-methylene)-11H-indolo[3,2-c]quinolin-8-N-amine, M = Ru ([3a]Cl), Os ([3b]Cl). The compounds were characterized by elemental analysis, one- and two-dimensional NMR spectroscopy, ESI mass spectrometry, IR and UV–vis spectroscopy, and single-crystal X-ray diffraction. The antiproliferative activity of the isomeric ruthenium and osmium complexes [1a,b]Cl–[3a,b]Cl was examined in vitro and showed the importance of the position of the metal-binding site for their cytotoxicity. Those complexes containing the metal-binding site located at the position 4 of the indoloquinoline scaffold ([2a]Cl and [2b]Cl) demonstrated the most potent antiproliferative activity. The results provide important insight into the structure–activity relationships of ruthenium- and osmium-arene complexes with indoloquinoline–piperazine hybrid ligands. These studies can be further utilized for the design and development of more potent chemotherapeutic agents. PMID:24927493

  18. Position Statement on Active Outdoor Play

    PubMed Central

    Tremblay, Mark S.; Gray, Casey; Babcock, Shawna; Barnes, Joel; Costas Bradstreet, Christa; Carr, Dawn; Chabot, Guylaine; Choquette, Louise; Chorney, David; Collyer, Cam; Herrington, Susan; Janson, Katherine; Janssen, Ian; Larouche, Richard; Pickett, William; Power, Marlene; Sandseter, Ellen Beate Hansen; Simon, Brenda; Brussoni, Mariana

    2015-01-01

    A diverse, cross-sectorial group of partners, stakeholders and researchers, collaborated to develop an evidence-informed Position Statement on active outdoor play for children aged 3–12 years. The Position Statement was created in response to practitioner, academic, legal, insurance and public debate, dialogue and disagreement on the relative benefits and harms of active (including risky) outdoor play. The Position Statement development process was informed by two systematic reviews, a critical appraisal of the current literature and existing position statements, engagement of research experts (N = 9) and cross-sectorial individuals/organizations (N = 17), and an extensive stakeholder consultation process (N = 1908). More than 95% of the stakeholders consulted strongly agreed or somewhat agreed with the Position Statement; 14/17 participating individuals/organizations endorsed it; and over 1000 additional individuals and organizations requested their name be listed as a supporter. The final Position Statement on Active Outdoor Play states: “Access to active play in nature and outdoors—with its risks— is essential for healthy child development. We recommend increasing children’s opportunities for self-directed play outdoors in all settings—at home, at school, in child care, the community and nature.” The full Position Statement provides context for the statement, evidence supporting it, and a series of recommendations to increase active outdoor play opportunities to promote healthy child development. PMID:26062040

  19. Position Statement on Active Outdoor Play.

    PubMed

    Tremblay, Mark S; Gray, Casey; Babcock, Shawna; Barnes, Joel; Bradstreet, Christa Costas; Carr, Dawn; Chabot, Guylaine; Choquette, Louise; Chorney, David; Collyer, Cam; Herrington, Susan; Janson, Katherine; Janssen, Ian; Larouche, Richard; Pickett, William; Power, Marlene; Sandseter, Ellen Beate Hansen; Simon, Brenda; Brussoni, Mariana

    2015-06-01

    A diverse, cross-sectorial group of partners, stakeholders and researchers, collaborated to develop an evidence-informed Position Statement on active outdoor play for children aged 3-12 years. The Position Statement was created in response to practitioner, academic, legal, insurance and public debate, dialogue and disagreement on the relative benefits and harms of active (including risky) outdoor play. The Position Statement development process was informed by two systematic reviews, a critical appraisal of the current literature and existing position statements, engagement of research experts (N=9) and cross-sectorial individuals/organizations (N=17), and an extensive stakeholder consultation process (N=1908). More than 95% of the stakeholders consulted strongly agreed or somewhat agreed with the Position Statement; 14/17 participating individuals/organizations endorsed it; and over 1000 additional individuals and organizations requested their name be listed as a supporter. The final Position Statement on Active Outdoor Play states: "Access to active play in nature and outdoors--with its risks--is essential for healthy child development. We recommend increasing children's opportunities for self-directed play outdoors in all settings--at home, at school, in child care, the community and nature." The full Position Statement provides context for the statement, evidence supporting it, and a series of recommendations to increase active outdoor play opportunities to promote healthy child development. PMID:26062040

  20. Discursive Positionings and Emotions in Modelling Activities

    ERIC Educational Resources Information Center

    Daher, Wajeeh

    2015-01-01

    Mathematical modelling is suggested as an activity through which students engage in meaningful mathematics. In the current research, the modelling activity of a group of four seventh-grade students was analysed using the discursive analysis framework. The research findings show that the positionings and emotions of the group members during their…

  1. Discursive positionings and emotions in modelling activities

    NASA Astrophysics Data System (ADS)

    Daher, Wajeeh

    2015-11-01

    Mathematical modelling is suggested as an activity through which students engage in meaningful mathematics. In the current research, the modelling activity of a group of four seventh-grade students was analysed using the discursive analysis framework. The research findings show that the positionings and emotions of the group members during their participation in the modelling activity changed as the activity proceeded. Overall, it can be said that three of the four group members acted as insiders, while the fourth acted as an outsider, and only, towards the end of the group's work on the activity, he acted as an insider. Moreover, the research findings point at four factors that affected the group members' positionings and emotions during the modelling activity: the member's characteristics, the member's history of learning experiences, the activity characteristics and the modelling phases. Furthermore, the different positionings of the group members in the different modelling phases were accompanied by different emotions experienced by them, where being an insider and a collaborator resulted in positive emotions, while being an outsider resulted in negative emotions.

  2. Private well/spring position paper, Rifle, Colorado, sites

    SciTech Connect

    1995-05-01

    This report describes the hydrogeochemical relationship between the New and Old Rifle processing sites and 15 domestic wells in their vicinity. The relationship of the domestic wells to the Old and New Rifle tailings sites requires clarification due to recent advances in understanding of Rifle site conceptual models. In order to form a bridge from the Rifle remedial action plan (RAP) and the recent baseline risk assessment to this position paper, several issues require discussion. First, through analysis of long-term ground water level data, the hydraulic gradient between the former tailings and private wells and springs was assessed. Second, in the Rifle RAP there was not a strong emphasis placed on describing regional influences on water quality in the vicinity of the processing sites. This document uses available information coupled with theory of regional ground water flow to describe regional flow systems north of Rifle. Third, the definition of background water quality from the RAP has been refined in several ways. Also, for the recent baseline risk assessment, all alluvial wells used to define background for the sites were located east of Old Rifle. In the RAP, alluvial background wells were also placed between the sites (downgradient of Old Rifle). Two additional wells were installed for the recent baseline risk assessment upgradient of Old Rifle which verified that several of the older wells (RFO-01-0597 and -0598) were in locations representative of background.

  3. Positional distribution of transcription factor binding sites in Arabidopsis thaliana

    PubMed Central

    Yu, Chun-Ping; Lin, Jinn-Jy; Li, Wen-Hsiung

    2016-01-01

    Binding of a transcription factor (TF) to its DNA binding sites (TFBSs) is a critical step to initiate the transcription of its target genes. It is therefore interesting to know where the TFBSs of a gene are likely to locate in the promoter region. Here we studied the positional distribution of TFBSs in Arabidopsis thaliana, for which many known TFBSs are now available. We developed a method to identify the locations of TFBSs in the promoter sequences of genes in A. thaliana. We found that the distribution is nearly bell-shaped with a peak at 50 base pairs (bp) upstream of the transcription start site (TSS) and 86% of the TFBSs are in the region from −1,000 bp to +200 bp with respect to the TSS. Our distribution was supported by chromatin immunoprecipitation sequencing and microarray data and DNase I hypersensitive site sequencing data. When TF families were considered separately, differences in positional preference were observed between TF families. Our study of the positional distribution of TFBSs seems to be the first in a plant. PMID:27117388

  4. Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals

    PubMed Central

    Denisov, Stepan; Bazykin, Georgii; Favorov, Alexander; Mironov, Andrey; Gelfand, Mikhail

    2015-01-01

    Splice sites (SSs)—short nucleotide sequences flanking introns—are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints. PMID:26642327

  5. Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals.

    PubMed

    Denisov, Stepan; Bazykin, Georgii; Favorov, Alexander; Mironov, Andrey; Gelfand, Mikhail

    2015-01-01

    Splice sites (SSs)--short nucleotide sequences flanking introns--are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints. PMID:26642327

  6. Active site specificity of plasmepsin II.

    PubMed Central

    Westling, J.; Cipullo, P.; Hung, S. H.; Saft, H.; Dame, J. B.; Dunn, B. M.

    1999-01-01

    Members of the aspartic proteinase family of enzymes have very similar three-dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site-directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues. PMID:10548045

  7. Autonomous site selection and instrument positioning for sample acquisition

    NASA Astrophysics Data System (ADS)

    Shaw, A.; Barnes, D.; Pugh, S.

    The European Space Agency Aurora Exploration Program aims to establish a European long-term programme for the exploration of Space, culminating in a human mission to space in the 2030 timeframe. Two flagship missions, namely Mars Sample Return and ExoMars, have been proposed as recognised steps along the way. The Exomars Rover is the first of these flagship missions and includes a rover carrying the Pasteur Payload, a mobile exobiology instrumentation package, and the Beagle 2 arm. The primary objective is the search for evidence of past or present life on mars, but the payload will also study the evolution of the planet and the atmosphere, look for evidence of seismological activity and survey the environment in preparation for future missions. The operation of rovers in unknown environments is complicated, and requires large resources not only on the planet but also in ground based operations. Currently, this can be very labour intensive, and costly, if large teams of scientists and engineers are required to assess mission progress, plan mission scenarios, and construct a sequence of events or goals for uplink. Furthermore, the constraints in communication imposed by the time delay involved over such large distances, and line-of-sight required, make autonomy paramount to mission success, affording the ability to operate in the event of communications outages and be opportunistic with respect to scientific discovery. As part of this drive to reduce mission costs and increase autonomy the Space Robotics group at the University of Wales, Aberystwyth is researching methods of autonomous site selection and instrument positioning, directly applicable to the ExoMars mission. The site selection technique used builds on the geometric reasoning algorithms used previously for localisation and navigation [Shaw 03]. It is proposed that a digital elevation model (DEM) of the local surface, generated during traverse and without interaction from ground based operators, can be

  8. Active site of ribulosebisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.; Stringer, C.D.; Milanez, S.; Lee, E.H.

    1985-01-01

    Previous affinity labeling studies and comparative sequence analyses have identified two different lysines at the active site of ribulosebisphosphate carboxylase/oxygenase and have suggested their essentiality to function. The essential lysines occupy positions 166 and 329 in the Rhodospirillum rubrum enzyme and positions 175 and 334 in the spinach enzyme. Based on the pH-dependencies of inactivations of the two enzymes by trinitrobenzene sulfonate, Lys-166 (R. rubrum enzyme) exhibits a pK/sub a/ of 7.9 and Lys-334 (spinach enzyme) exhibits a pK/sub a/ of 9.0. These low pK/sub a/ values as well as the enhanced nucleophilicities of the lysyl residues argue that both are important to catalysis rather than to substrate binding. Lys-166 may correspond to the essential base that initiates catalysis and that displays a pK/sub a/ of 7.5 in the pH-curve for V/sub max//K/sub m/. Cross-linking experiments with 4,4'-diisothiocyano-2,2'-disulfonate stilbene demonstrate that the two active-site lysines are within 12 A. 50 refs., 7 figs., 1 tab.

  9. Distinct tyrosine autophosphorylation sites negatively and positively modulate neu-mediated transformation.

    PubMed Central

    Dankort, D L; Wang, Z; Blackmore, V; Moran, M F; Muller, W J

    1997-01-01

    A number of cytoplasmic signaling molecules are thought to mediate mitogenic signaling from the activated Neu receptor tyrosine kinase through binding specific phosphotyrosine residues located within the intracellular portion of Neu/c-ErbB-2. An activated neu oncogene containing tyrosine-to-phenylalanine substitutions at each of the known autophosphorylation sites was generated and assessed for its specific transforming potential in Rat1 and NIH 3T3 fibroblasts. Mutation of these sites resulted in a dramatic impairment of the transforming potential of neu. To assess the role of these tyrosine phosphorylation sites in cellular transformation, the transforming potential of a series of mutants in which individual tyrosine residues were restored to this transformation-debilitated neu mutant was evaluated. Reversion of any one of four mutated sites to tyrosine residues restored wild-type transforming activity. While each of these transforming mutants displayed Ras-dependent signaling, the transforming activity of two of these mutants was correlated with their ability to bind either the GRB2 or SHC adapter molecules that couple receptor tyrosine kinases to the Ras signaling pathway. By contrast, restoration of a tyrosine residue located at position 1028 completely suppressed the basal transforming activity of this mutated neu molecule or other transforming neu molecules which possessed single tyrosine residues. These data argue that the transforming potential of activated neu is mediated both by positive and negative regulatory tyrosine phosphorylation sites. PMID:9271418

  10. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1990-10-01

    DOE Order 5820.2A requires that low-level waste (LLW) disposal sites active on or after September 1988 and all transuranic (TRU) waste storage sites be monitored periodically to assure that radioactive contamination does not escape from the waste sites and pose a threat to the public or to the environment. This plan describes such a monitoring program for the active LLW disposal sites in SWSA 6 and the TRU waste storage sites in SWSA 5 North. 14 refs., 8 figs.

  11. Toward Understanding the Catalytic Mechanism of Human Paraoxonase 1: Site-Specific Mutagenesis at Position 192

    PubMed Central

    Aggarwal, Geetika; Prajapati, Rameshwar; Tripathy, Rajan K.; Bajaj, Priyanka; Iyengar, A. R. Satvik; Sangamwar, Abhay T.; Pande, Abhay H.

    2016-01-01

    Human paraoxonase 1 (h-PON1) is a serum enzyme that can hydrolyze a variety of substrates. The enzyme exhibits anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial and organophosphate-hydrolyzing activities. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against a variety conditions in human. However, the crystal structure of h-PON1 is not solved and the molecular details of how the enzyme hydrolyzes different substrates are not clear yet. Understanding the catalytic mechanism(s) of h-PON1 is important in developing the enzyme for therapeutic use. Literature suggests that R/Q polymorphism at position 192 in h-PON1 dramatically modulates the substrate specificity of the enzyme. In order to understand the role of the amino acid residue at position 192 of h-PON1 in its various hydrolytic activities, site-specific mutagenesis at position 192 was done in this study. The mutant enzymes were produced using Escherichia coli expression system and their hydrolytic activities were compared against a panel of substrates. Molecular dynamics simulation studies were employed on selected recombinant h-PON1 (rh-PON1) mutants to understand the effect of amino acid substitutions at position 192 on the structural features of the active site of the enzyme. Our results suggest that, depending on the type of substrate, presence of a particular amino acid residue at position 192 differentially alters the micro-environment of the active site of the enzyme resulting in the engagement of different subsets of amino acid residues in the binding and the processing of substrates. The result advances our understanding of the catalytic mechanism of h-PON1. PMID:26829396

  12. Mutation of Glycosylation Sites in BST-2 Leads to Its Accumulation at Intracellular CD63-Positive Vesicles without Affecting Its Antiviral Activity against Multivesicular Body-Targeted HIV-1 and Hepatitis B Virus

    PubMed Central

    Han, Zhu; Lv, Mingyu; Shi, Ying; Yu, Jinghua; Niu, Junqi; Yu, Xiao-Fang; Zhang, Wenyan

    2016-01-01

    BST-2/tetherin blocks the release of various enveloped viruses including HIV-1 with a “physical tethering” model. The detailed contribution of N-linked glycosylation to this model is controversial. Here, we confirmed that mutation of glycosylation sites exerted an effect of post-translational mis-trafficking, leading to an accumulation of BST-2 at intracellular CD63-positive vesicles. BST-2 with this phenotype potently inhibited the release of multivesicular body-targeted HIV-1 and hepatitis B virus, without affecting the co-localization of BST-2 with EEA1 and LAMP1. These results suggest that N-linked glycosylation of human BST-2 is dispensable for intracellular virion retention and imply that this recently discovered intracellular tethering function may be evolutionarily distinguished from the canonical antiviral function of BST-2 by tethering nascent virions at the cell surface. PMID:26938549

  13. Educational Activity Sites for High School Students

    ERIC Educational Resources Information Center

    Troutner, Joanne

    2005-01-01

    Finding quality Internet resources for high school students is a continuing challenge. Several high-quality web sites are presented for educators and students. These sites offer activities to learn how an art conservator looks at paintings, create a newspaper, research and develop an end product, build geometry and physics skills, explore science…

  14. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

    The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

  15. Comprehensive School Physical Activity Programs. Position Statement

    ERIC Educational Resources Information Center

    National Association for Sport and Physical Education, 2008

    2008-01-01

    The National Association for Sport and Physical Education (NASPE) recommends that all PK-12 schools implement a Comprehensive School Physical Activity Program. Schools play an important role in public health, and the physical, mental, and social benefits of regular physical activity for youth are well documented. Leading public health, medical,…

  16. Overlapping binding sites drive allosteric agonism and positive cooperativity in type 4 metabotropic glutamate receptors.

    PubMed

    Rovira, Xavier; Malhaire, Fanny; Scholler, Pauline; Rodrigo, Jordi; Gonzalez-Bulnes, Patricia; Llebaria, Amadeu; Pin, Jean-Philippe; Giraldo, Jesús; Goudet, Cyril

    2015-01-01

    Type 4 metabotropic glutamate (mGlu4) receptors are emerging targets for the treatment of various disorders. Accordingly, numerous mGlu4-positive allosteric modulators (PAMs) have been identified, some of which also display agonist activity. To identify the structural bases for their allosteric action, we explored the relationship between the binding pockets of mGlu4 PAMs with different chemical scaffolds and their functional properties. By use of innovative mGlu4 biosensors and second-messenger assays, we show that all PAMs enhance agonist action on the receptor through different degrees of allosteric agonism and positive cooperativity. For example, whereas VU0155041 and VU0415374 display equivalent efficacies [log(τ(B)) = 1.15 ± 0.38 and 1.25 ± 0.44, respectively], they increase the ability of L-AP4 to stabilize the active conformation of the receptor by 4 and 39 times, respectively. Modeling and docking studies identify 2 overlapping binding pockets as follows: a first site homologous to the pocket of natural agonists of class A GPCRs linked to allosteric agonism and a second one pointing toward a site topographically homologous to the Na(+) binding pocket of class A GPCRs, occupied by PAMs exhibiting the strongest cooperativity. These results reveal that intrinsic efficacy and cooperativity of mGlu4 PAMs are correlated with their binding mode, and vice versa, integrating structural and functional knowledge from different GPCR classes. PMID:25342125

  17. Associations among positive mood, brain, and cardiovascular activities in an affectively positive situation.

    PubMed

    Matsunaga, Masahiro; Isowa, Tokiko; Kimura, Kenta; Miyakoshi, Makoto; Kanayama, Noriaki; Murakami, Hiroki; Fukuyama, Seisuke; Shinoda, Jun; Yamada, Jitsuhiro; Konagaya, Toshihiro; Kaneko, Hiroshi; Ohira, Hideki

    2009-03-31

    It is hypothesized that experiencing positive emotions such as pleasure leads to a perception of the body being in a positive state. This study demonstrated associations among positive mood, brain, and cardiovascular activities by simultaneously recording these activities when positive emotions were evoked in participants watching films revolving around a love story. Heart rate variability analysis revealed increased parasympathetic nervous activity while watching the film. The following brain regions were significantly activated in the positive condition relative to the control condition: medial prefrontal cortex, thalamus, superior temporal gyrus, inferior frontal gyrus, and cerebellum. Further, covariate analyses indicated that these brain regions were temporally associated with subjective positive mood. Activities of brain regions considered to be related to interoceptive awareness, such as the insular cortex, anterior cingulate cortex, amygdala, and orbitofrontal cortex, were also temporally associated with the cardiovascular change. These results suggest that while an individual experiences positive emotions, activities of the central nervous system and cardiovascular system may be interrelated, and the brain may perceive the body to be in a positive state. PMID:19368841

  18. Corrosion Research And Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2001-01-01

    This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

  19. Corrosion Research and Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2002-01-01

    This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

  20. Phylogenetic analysis and positive-selection site detecting of vascular endothelial growth factor family in vertebrates.

    PubMed

    He, Wenwu; Tang, Yanyan; Qi, Bin; Lu, Chuansen; Qin, Chao; Wei, Yunfei; Yi, Jiachao; Chen, Mingwu

    2014-02-10

    Vascular endothelial growth factor (VEGF), known to play an important role in vascular homeostasis, vascular integrity and angiogenesis, is little known about the evolutionary relationship of its five members especially the role of gene duplication and natural selection in the evolution of the VEGF family. In this study, seventy-five full-length cDNA sequences from 33 vertebrate species were extracted from the NCBI's GenBank, UniProt protein database and the Ensembl database. By phylogenetic analyses, we investigated the origin, conservation, and evolution of the VEGFs. Five VEGF family members in vertebrates might be formed by gene duplication. The inferred evolutionary transitions that separate members which belong to different gene clusters correlated with changes in functional properties. Selection analysis and protein structure analysis were combined to explain the relationship of the site-specific evolution in the vertebrate VEGF family. Eleven positive selection sites, one transmembrane region and the active sites were detected in this process. PMID:24200960

  1. ON-LINE TOOLS FOR PROPER VERTICAL POSITIONING OF VERTICAL SAMPLING INTERVALS DURING SITE ASSESSMENT

    EPA Science Inventory

    This presentation presents on-line tools for proper vertical positioning of vertical sampling intervals during site assessment. Proper vertical sample interval selection is critical for generate data on the vertical distribution of contamination. Without vertical delineation, th...

  2. Potential sites of CFTR activation by tyrosine kinases.

    PubMed

    Billet, Arnaud; Jia, Yanlin; Jensen, Timothy J; Hou, Yue-Xian; Chang, Xiu-Bao; Riordan, John R; Hanrahan, John W

    2016-05-01

    The CFTR chloride channel is tightly regulated by phosphorylation at multiple serine residues. Recently it has been proposed that its activity is also regulated by tyrosine kinases, however the tyrosine phosphorylation sites remain to be identified. In this study we examined 2 candidate tyrosine residues near the boundary between the first nucleotide binding domain and the R domain, a region which is important for channel function but devoid of PKA consensus sequences. Mutating tyrosines at positions 625 and 627 dramatically reduced responses to Src or Pyk2 without altering the activation by PKA, suggesting they may contribute to CFTR regulation. PMID:26645934

  3. Position specific variation in the rate of evolution intranscription factor binding sites

    SciTech Connect

    Moses, Alan M.; Chiang, Derek Y.; Kellis, Manolis; Lander, EricS.; Eisen, Michael B.

    2003-08-28

    The binding sites of sequence specific transcription factors are an important and relatively well-understood class of functional non-coding DNAs. Although a wide variety of experimental and computational methods have been developed to characterize transcription factor binding sites, they remain difficult to identify. Comparison of non-coding DNA from related species has shown considerable promise in identifying these functional non-coding sequences, even though relatively little is known about their evolution. Here we analyze the genome sequences of the budding yeasts Saccharomyces cerevisiae, S. bayanus, S. paradoxus and S. mikataeto study the evolution of transcription factor binding sites. As expected, we find that both experimentally characterized and computationally predicted binding sites evolve slower than surrounding sequence, consistent with the hypothesis that they are under purifying selection. We also observe position-specific variation in the rate of evolution within binding sites. We find that the position-specific rate of evolution is positively correlated with degeneracy among binding sites within S. cerevisiae. We test theoretical predictions for the rate of evolution at positions where the base frequencies deviate from background due to purifying selection and find reasonable agreement with the observed rates of evolution. Finally, we show how the evolutionary characteristics of real binding motifs can be used to distinguish them from artifacts of computational motif finding algorithms. As has been observed for protein sequences, the rate of evolution in transcription factor binding sites varies with position, suggesting that some regions are under stronger functional constraint than others. This variation likely reflects the varying importance of different positions in the formation of the protein-DNA complex. The characterization of the pattern of evolution in known binding sites will likely contribute to the effective use of comparative

  4. Activities for Teaching Positive Psychology: A Guide for Instructors

    ERIC Educational Resources Information Center

    Froh, Jeffrey J., Ed.; Parks, Acacia C., Ed.

    2012-01-01

    Positive psychology is a rapidly expanding area of study that is of great interest to students at the graduate, undergraduate, and high school levels. But the field is so broad that teachers who want to cover all the bases when designing a positive psychology course may have difficulty locating and selecting materials. "Activities for Teaching…

  5. Differential Active Site Loop Conformations Mediate Promiscuous Activities in the Lactonase SsoPox

    PubMed Central

    Elias, Mikael; Chabriere, Eric

    2013-01-01

    Enzymes are proficient catalysts that enable fast rates of Michaelis-complex formation, the chemical step and products release. These different steps may require different conformational states of the active site that have distinct binding properties. Moreover, the conformational flexibility of the active site mediates alternative, promiscuous functions. Here we focused on the lactonase SsoPox from Sulfolobus solfataricus. SsoPox is a native lactonase endowed with promiscuous phosphotriesterase activity. We identified a position in the active site loop (W263) that governs its flexibility, and thereby affects the substrate specificity of the enzyme. We isolated two different sets of substitutions at position 263 that induce two distinct conformational sampling of the active loop and characterized the structural and kinetic effects of these substitutions. These sets of mutations selectively and distinctly mediate the improvement of the promiscuous phosphotriesterase and oxo-lactonase activities of SsoPox by increasing active-site loop flexibility. These observations corroborate the idea that conformational diversity governs enzymatic promiscuity and is a key feature of protein evolvability. PMID:24086491

  6. Profile comparison revealed deviation from structural constraint at the positively selected sites.

    PubMed

    Oda, Hiroyuki; Ota, Motonori; Toh, Hiroyuki

    2016-09-01

    The amino acid substitutions at a site are affected by mixture of various constraints. It is also known that the amino acid substitutions are accelerated at sites under positive selection. However, the relationship between the substitutions at positively selected sites and the constraints has not been thoroughly examined. The advances in computational biology have enabled us to divide the mixture of the constraints into the structural constraint and the remainings by using the amino acid sequences and the tertiary structures, which is expressed as the deviation of the mixture of constraints from the structural constraint. Here, two types of profiles, or matrices with the size of 20 x (site length), are compared. One of the profiles represents the mixture of constraints, and is generated from a multiple amino acid sequence alignment, whereas the other is designed to represent the structural constraints. We applied the profile comparison method to proteins under positive selection to examine the relationship between the positive selection and constraints. The results suggested that the constraint at a site under positive selection tends to be deviated from the structural constraint at the site. PMID:27443483

  7. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  8. Probing the promiscuous active site of myo-inositol dehydrogenase using synthetic substrates, homology modeling, and active site modification.

    PubMed

    Daniellou, Richard; Zheng, Hongyan; Langill, David M; Sanders, David A R; Palmer, David R J

    2007-06-26

    The active site of myo-inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis recognizes a variety of mono- and disaccharides, as well as 1l-4-O-substituted inositol derivatives. It catalyzes the NAD+-dependent oxidation of the axial alcohol of these substrates with comparable kinetic constants. We have found that 4-O-p-toluenesulfonyl-myo-inositol does not act as a substrate for IDH, in contrast to structurally similar compounds such as those bearing substituted benzyl substituents in the same position. X-ray crystallographic analysis of 4-O-p-toluenesulfonyl-myo-inositol and 4-O-(2-naphthyl)methyl-myo-inositol, which is a substrate for IDH, shows a distinct difference in the preferred conformation of the aryl substituent. Conformational analysis of known substrates of IDH suggests that this conformational difference may account for the difference in reactivity of 4-O-p-toluenesulfonyl-myo-inositol in the presence of IDH. A sequence alignment of IDH with the homologous glucose-fructose oxidoreductase allowed the construction of an homology model of inositol dehydrogenase, to which NADH and 4-O-benzyl-scyllo-inosose were docked and the active site energy minimized. The active site model is consistent with all experimental results and suggests that a conserved tyrosine-glycine-tyrosine motif forms the hydrophobic pocket adjoining the site of inositol recognition. Y233F and Y235F retain activity, while Y233R and Y235R do not. A histidine-aspartate pair, H176 and D172, are proposed to act as a dyad in which H176 is the active site acid/base. The enzyme is inactivated by diethyl pyrocarbonate, and the mutants H176A and D172N show a marked loss of activity. Kinetic isotope effect experiments with D172N indicate that chemistry is rate-determining for this mutant. PMID:17539607

  9. Active control of a flexible structure using a modal positive position feedback controller

    NASA Technical Reports Server (NTRS)

    Poh, S.; Baz, A.

    1990-01-01

    The feasibility of a new Modal Positive Position Feedback (MPPF) strategy in controlling the vibration of a complex flexible structure using a single piezo-electric active structural member is demonstrated. The control strategy generates its control forces by manipulating only the modal position signals of the structure to provide a damping action to undamped modes. This is in contrast to conventional modal controllers that rely in their operation on negative feedback of both the modal position and velocity. The proposed strategy is very simple to design and implement as it designs the controller at the uncoupled modal level and utilizes simple first order filters to achieve the Positive Position Feedback effect. The performance of the new strategy is enhanced by augmenting it with a time sharing strategy to share a small number of actuators between larger number of modes. The effectiveness of the new strategy is validated experimentally on a flexible box-type structure that has four bays and its first two bending modes are 2.015 and 6.535 Hz respectively. A single piezo-electric actuator is utilized as an active structural member to control several transverse bending modes of the structure. The performance of the active control system is determined in the time and the frequency domains. The results are compared with those obtained when using the Independent Modal Space Control (IMSC) of Meirovitch. The experimental results suggest the potential of the proposed strategy as a viable means for controlling the vibration of large flexible structures in real time.

  10. Active control of a flexible structure using a modal positive position feedback controller

    NASA Technical Reports Server (NTRS)

    Poh, S.; Baz, A.

    1990-01-01

    The feasibility of a new Modal Positive Position Feedback (MPPF) strategy in controlling the vibration of a complex flexible structure using a single piezo-electric active structural member is demonstrated. The control strategy generates its control forces by manipulating only the modal position signals of the structure to provide a damping action to undamped modes. This is in contrast to conventional modal controllers that rely in their operation on negative feedback of both the modal position and velocity. The proposed strategy is very simple to design and implement as it designs the controller at the uncoupled modal level and utilizes simple first order filters to achieve the Positive Position Feedback effect. The performance of the new strategy is enhanced by augmenting it with a time sharing strategy to share a small number of actuators between larger number of modes. The effectiveness of the new strategy is validated experimentally on a flexible box-type structure that has four bays and its first two bending modes are 2.015 and 6.535 Hz, respectively. A single piezo-electric actuator is utilized as an active structural member to control several transverse bending modes of the structure. The performance of the active control system is determined in the time and the frequency domains. The results are compared with those obtained when using the Independent Modal Space Control (IMSC) of Meirovitch. The experimental results suggest the potential of the proposed strategy as a viable means for controlling the vibration of large flexible structures in real time.

  11. Optimized position weight matrices in prediction of novel putative binding sites for transcription factors in the Drosophila melanogaster genome.

    PubMed

    Morozov, Vyacheslav Y; Ioshikhes, Ilya P

    2013-01-01

    Position weight matrices (PWMs) have become a tool of choice for the identification of transcription factor binding sites in DNA sequences. DNA-binding proteins often show degeneracy in their binding requirement and thus the overall binding specificity of many proteins is unknown and remains an active area of research. Although existing PWMs are more reliable predictors than consensus string matching, they generally result in a high number of false positive hits. Our previous study introduced a promising approach to PWM refinement in which known motifs are used to computationally mine putative binding sites directly from aligned promoter regions using composition of similar sites. In the present study, we extended this technique originally tested on single examples of transcription factors (TFs) and showed its capability to optimize PWM performance to predict new binding sites in the fruit fly genome. We propose refined PWMs in mono- and dinucleotide versions similarly computed for a large variety of transcription factors of Drosophila melanogaster. Along with the addition of many auxiliary sites the optimization includes variation of the PWM motif length, the binding sites location on the promoters and the PWM score threshold. To assess the predictive performance of the refined PWMs we compared them to conventional TRANSFAC and JASPAR sources. The results have been verified using performed tests and literature review. Overall, the refined PWMs containing putative sites derived from real promoter content processed using optimized parameters had better general accuracy than conventional PWMs. PMID:23936309

  12. Position dependent rate dampening in any active hand controller

    NASA Technical Reports Server (NTRS)

    Gregory, William W. (Inventor); Kauffman, James W. (Inventor)

    1994-01-01

    A control system for an active hand controller, for example, uses a control stick connected to and controlled by a motor. Electronics are provided to control the motor to eliminate oscillations due to motor torque and high gain due to breakout at the control stick when the control stick is at about its null position. Both hardware as well as software implementations can provide position dependent dampening to the control sticks such that when the control stick is located about a null position, a higher rate of dampening is provided than when the control stick is located outside the null position, when a lower rate of dampening is provided. The system provides a stable active hand controller control stick without degraded force and feel characteristics of the system.

  13. Identification of Ice Nucleation Active Sites on Silicate Dust Particles

    NASA Astrophysics Data System (ADS)

    Zolles, Tobias; Burkart, Julia; Häusler, Thomas; Pummer, Bernhard; Hitzenberger, Regina; Grothe, Hinrich

    2015-04-01

    Mineral dusts originating from Earth's crust are known to be important atmospheric ice nuclei. In agreement with earlier studies, feldspar was found as the most active of the tested natural mineral dusts [1-3]. Nevertheless, among those structures K-feldspar showed by far the highest ice nucleation activity. In this study, the reasons for its activity and the difference in the activity of the different feldspars were investigated in closer details. Conclusions are drawn from scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, and oil-immersion freezing experiments. We give a potential explanation of the increased ice nucleation activity of K-feldspar. The ice nucleating sites are very much dependent on the alkali ion present by altering the water structure and the feldspar surface. The higher activity of K-feldspar can be attributed to the presence of potassium ions on the surface and surface bilayer. The alkali-ions have different hydration shells and thus an influence on the ice nucleation activity of feldspar. Chaotropic behavior of Calcium and Sodium ions are lowering the ice nucleation potential of the other feldspars, while kosmotropic Potassium has a neutral or even positive effect. Furthermore we investigated the influence of milling onto the ice nucleation of quartz particles. The ice nucleation activity can be increased by mechanical milling, by introducing more molecular, nucleation active defects to the particle surface. This effect is larger than expected by plane surface increase. [1] Atkinson et al. The Importance of Feldspar for Ice Nucleation by Mineral Dust in Mixed-Phase Clouds. Nature 2013, 498, 355-358. [2] Yakobi-Hancock et al.. Feldspar Minerals as Efficient Deposition Ice Nuclei. Atmos. Chem. Phys. 2013, 13, 11175-11185. [3] Zolles et al. Identification of Ice Nucleation Active Sites on Feldspar Dust Particles. J. Phys. Chem. A 2015 accepted.

  14. Transcription Factor Binding Site Positioning in Yeast: Proximal Promoter Motifs Characterize TATA-Less Promoters

    PubMed Central

    Erb, Ionas; van Nimwegen, Erik

    2011-01-01

    The availability of sequence specificities for a substantial fraction of yeast's transcription factors and comparative genomic algorithms for binding site prediction has made it possible to comprehensively annotate transcription factor binding sites genome-wide. Here we use such a genome-wide annotation for comprehensively studying promoter architecture in yeast, focusing on the distribution of transcription factor binding sites relative to transcription start sites, and the architecture of TATA and TATA-less promoters. For most transcription factors, binding sites are positioned further upstream and vary over a wider range in TATA promoters than in TATA-less promoters. In contrast, a group of ‘proximal promoter motifs’ (GAT1/GLN3/DAL80, FKH1/2, PBF1/2, RPN4, NDT80, and ROX1) occur preferentially in TATA-less promoters and show a strong preference for binding close to the transcription start site in these promoters. We provide evidence that suggests that pre-initiation complexes are recruited at TATA sites in TATA promoters and at the sites of the other proximal promoter motifs in TATA-less promoters. TATA-less promoters can generally be classified by the proximal promoter motif they contain, with different classes of TATA-less promoters showing different patterns of transcription factor binding site positioning and nucleosome coverage. These observations suggest that different modes of regulation of transcription initiation may be operating in the different promoter classes. In addition we show that, across all promoter classes, there is a close match between nucleosome free regions and regions of highest transcription factor binding site density. This close agreement between transcription factor binding site density and nucleosome depletion suggests a direct and general competition between transcription factors and nucleosomes for binding to promoters. PMID:21931670

  15. A position-dependent transcription-activating domain in TFIIIA.

    PubMed

    Mao, X; Darby, M K

    1993-12-01

    Transcription of the Xenopus 5S RNA gene by RNA polymerase III requires the gene-specific factor TFIIIA. To identify domains within TFIIIA that are essential for transcriptional activation, we have expressed C-terminal deletion, substitution, and insertion mutants of TFIIIA in bacteria as fusions with maltose-binding protein (MBP). The MBP-TFIIIA fusion protein specifically binds to the 5S RNA gene internal control region and complements transcription in a TFIIIA-depleted oocyte nuclear extract. Random, cassette-mediated mutagenesis of the carboxyl region of TFIIIA, which is not required for promoter binding, has defined a 14-amino-acid region that is critical for transcriptional activation. In contrast to activators of RNA polymerase II, the activity of the TFIIIA activation domain is strikingly sensitive to its position relative to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are deleted, transcriptional activity is lost. Surprisingly, diverse amino acids can replace these eight amino acids with restoration of full transcriptional activity, suggesting that the length and not the sequence of this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reduction in transcription proportional to the number of amino acids introduced. We propose that to function, the transcription-activating domain of TFIIIA must be correctly positioned at a minimum distance from the DNA-binding domain. PMID:8246967

  16. Hydrologic applications of GPS site-position observations in the Western U.S

    NASA Astrophysics Data System (ADS)

    Ouellette, Karli J.

    Permanent Global Positioning System (GPS) networks have been established around the globe for a variety of uses, most notably to monitor the activity of fault lines and tectonic plate motion. A model for utilizing GPS as a tool for hydrologic monitoring is also developed. First, observations of the recent movement of the land surface throughout California by the Scripps Orbit and Permanent Array Center (SOPAC) GPS network are explored. Significant seasonal cycles and long term trends are related to historical observations of land subsidence. The pattern of deformation throughout the state appears to be caused by the occurrence of poroelastic deformation of the aquifer in the Central Valley, and elastic crustal loading by surface water and the winter snowpack in the Sierra Nevada Mountains. The result is a sort of teeter-totter motion between the Valley and the mountains where the Valley sinks in the dry season while the mountains lift, and the mountains sink in the wet season while the Valley lifts. Next, the elastic crustal deformation caused by the winter snowpack is explored more thoroughly at 6 high elevations throughout the Western United States. Expected annual deformation as a result of thermoelastic and snow water equivalent are calculated using SNOTEL observations and an elastic half-space model. The results demonstrate the dominance of snow loading on the seasonal vertical land surface deformation at all 6 GPS stations. The model is then reversed and applied to the GPS vertical site-position observations in order to predict snow water equivalent. The results are compared to SNOTEL observations of snow water equivalent and soil moisture. The study concludes that GPS site-position observations are able to predict variations in snow water equivalent and soil moisture with good accuracy. Then a model which incorporates both elastic crustal loading and poroelastic deformation was used to predict groundwater storage variations at 54 GPS stations throughout the

  17. Position-dependent splicing activation and repression by SR and hnRNP proteins rely on common mechanisms

    PubMed Central

    Erkelenz, Steffen; Mueller, William F.; Evans, Melanie S.; Busch, Anke; Schöneweis, Katrin; Hertel, Klemens J.; Schaal, Heiner

    2013-01-01

    Alternative splicing is regulated by splicing factors that modulate splice site selection. In some cases, however, splicing factors show antagonistic activities by either activating or repressing splicing. Here, we show that these opposing outcomes are based on their binding location relative to regulated 5′ splice sites. SR proteins enhance splicing only when they are recruited to the exon. However, they interfere with splicing by simply relocating them to the opposite intronic side of the splice site. hnRNP splicing factors display analogous opposing activities, but in a reversed position dependence. Activation by SR or hnRNP proteins increases splice site recognition at the earliest steps of exon definition, whereas splicing repression promotes the assembly of nonproductive complexes that arrest spliceosome assembly prior to splice site pairing. Thus, SR and hnRNP splicing factors exploit similar mechanisms to positively or negatively influence splice site selection. PMID:23175589

  18. Physical Activity Is Positively Associated with Episodic Memory in Aging

    PubMed Central

    Hayes, Scott M.; Alosco, Michael L.; Hayes, Jasmeet P.; Cadden, Margaret; Peterson, Kristina M.; Allsup, Kelly; Forman, Daniel E.; Sperling, Reisa A.; Verfaellie, Mieke

    2016-01-01

    Aging is associated with performance reductions in executive function and episodic memory, although there is substantial individual variability in cognition among older adults. One factor that may be positively associated with cognition in aging is physical activity. To date, few studies have objectively assessed physical activity in young and older adults, and examined whether physical activity is differentially associated with cognition in aging. Young (n = 29, age 18–31 years) and older adults (n = 31, ages 55–82 years) completed standardized neuropsychological testing to assess executive function and episodic memory capacities. An experimental face-name relational memory task was administered to augment assessment of episodic memory. Physical activity (total step count and step rate) was objectively assessed using an accelerometer, and hierarchical regressions were used to evaluate relationships between cognition and physical activity. Older adults performed more poorly on tasks of executive function and episodic memory. Physical activity was positively associated with a composite measure of visual episodic memory and face-name memory accuracy in older adults. Physical activity associations with cognition were independent of sedentary behavior, which was negatively correlated with memory performance. Physical activity was not associated with cognitive performance in younger adults. Physical activity is positively associated with episodic memory performance in aging. The relationship appears to be strongest for face-name relational memory and visual episodic memory, likely attributable to the fact that these tasks make strong demands on the hippocampus. The results suggest that physical activity relates to cognition in older, but not younger adults. PMID:26581790

  19. Active diffusion positions the nucleus in mouse oocytes.

    PubMed

    Almonacid, Maria; Ahmed, Wylie W; Bussonnier, Matthias; Mailly, Philippe; Betz, Timo; Voituriez, Raphaël; Gov, Nir S; Verlhac, Marie-Hélène

    2015-04-01

    In somatic cells, the position of the cell centroid is dictated by the centrosome. The centrosome is instrumental in nucleus positioning, the two structures being physically connected. Mouse oocytes have no centrosomes, yet harbour centrally located nuclei. We demonstrate how oocytes define their geometric centre in the absence of centrosomes. Using live imaging of oocytes, knockout for the formin 2 actin nucleator, with off-centred nuclei, together with optical trapping and modelling, we discover an unprecedented mode of nucleus positioning. We document how active diffusion of actin-coated vesicles, driven by myosin Vb, generates a pressure gradient and a propulsion force sufficient to move the oocyte nucleus. It promotes fluidization of the cytoplasm, contributing to nucleus directional movement towards the centre. Our results highlight the potential of active diffusion, a prominent source of intracellular transport, able to move large organelles such as nuclei, providing in vivo evidence of its biological function. PMID:25774831

  20. Draft position paper on knowledge management in space activities

    NASA Technical Reports Server (NTRS)

    Holm, Jeanne; Moura, Denis

    2003-01-01

    As other fields of industry, space activities are facing the challenge of Knowledge Management and the International Academy of Astronautics decided to settle in 2002 a Study Group to analyse the problem and issue general guidelines. This communication presents the draft position paper of this group in view to be discussed during the 2003 IAF Congress.

  1. Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426

    PubMed Central

    Zhang, Yu; An, Jiao; Yang, Guang-Yu; Bai, Aixi; Zheng, Baisong; Lou, Zhiyong; Wu, Geng; Ye, Wei; Chen, Hai-Feng; Feng, Yan; Manco, Giuseppe

    2015-01-01

    Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity. PMID:25706379

  2. Association between Positional Dependency and Obstruction Site in Obstructive Sleep Apnea Syndrome

    PubMed Central

    Sunwoo, Woong Sang; Hong, Sung-Lyong; Kim, Sang-Wook; Park, Sung Joon; Han, Doo Hee; Kim, Jeong-Whun; Lee, Chul Hee

    2012-01-01

    Objectives The purpose of this study is to find out associations between positional dependency and obstructive levels based on sleep videofluoroscopy (SVF) in patients with obstructive sleep apnea syndrome (OSAS). Methods Retrospective review was made of 91 OSAS patients who underwent polysomnography and SVF from August 2009 through June 2010. Polysomnography variables including apnea-hypopnea index (AHI), supine AHI, non-supine AHI, time spent in supine sleep position of the total sleep time and positional dependency (PD) were analyzed. Obstruction sites were evaluated as SVF variables. Results Of 91 patients, 65 (71.4%) were positional patients (PP) and 26 (28.6%) were non-positional patients (NPP). An analysis of polysomnography variables according to PD revealed that overall AHI, non-supine AHI and supine AHI in PP was significantly lower than that in NPP. The patients with soft palate obstruction (SP type) were more likely to have PD than the patients with tongue base obstruction (TB type; P=0.046). PD was inversely related to OSAS severity significantly (P=0.001). Conclusion These results provide evidence that positional dependent patients may have higher success rate of soft palate OSA surgery alone than non-positional dependent patients. Although PD may be associated with obstruction site, PD only itself may not be useful in planning surgical treatment for OSAS. PMID:23205227

  3. Positive selection neighboring functionally essential sites and disease-implicated regions of mammalian reproductive proteins

    PubMed Central

    2010-01-01

    Background Reproductive proteins are central to the continuation of all mammalian species. The evolution of these proteins has been greatly influenced by environmental pressures induced by pathogens, rival sperm, sexual selection and sexual conflict. Positive selection has been demonstrated in many of these proteins with particular focus on primate lineages. However, the mammalia are a diverse group in terms of mating habits, population sizes and germ line generation times. We have examined the selective pressures at work on a number of novel reproductive proteins across a wide variety of mammalia. Results We show that selective pressures on reproductive proteins are highly varied. Of the 10 genes analyzed in detail, all contain signatures of positive selection either across specific sites or in specific lineages or a combination of both. Our analysis of SP56 and Col1a1 are entirely novel and the results show positively selected sites present in each gene. Our findings for the Col1a1 gene are suggestive of a link between positive selection and severe disease type. We find evidence in our dataset to suggest that interacting proteins are evolving in symphony: most likely to maintain interacting functionality. Conclusion Our in silico analyses show positively selected sites are occurring near catalytically important regions suggesting selective pressure to maximize efficient fertilization. In those cases where a mechanism of protein function is not fully understood, the sites presented here represent ideal candidates for mutational study. This work has highlighted the widespread rate heterogeneity in mutational rates across the mammalia and specifically has shown that the evolution of reproductive proteins is highly varied depending on the species and interacting partners. We have shown that positive selection and disease are closely linked in the Col1a1 gene. PMID:20149245

  4. Positioning.

    ERIC Educational Resources Information Center

    Conone, Ruth M.

    The key to positioning is the creation of a clear benefit image in the consumer's mind. One positioning strategy is creating in the prospect's mind a position that takes into consideration the company's or agency's strengths and weaknesses as well as those of its competitors. Another strategy is to gain entry into a position ladder owned by…

  5. Generalized site occupancy models allowing for false positive and false negative errors

    USGS Publications Warehouse

    Royle, J. Andrew; Link, W.A.

    2006-01-01

    Site occupancy models have been developed that allow for imperfect species detection or ?false negative? observations. Such models have become widely adopted in surveys of many taxa. The most fundamental assumption underlying these models is that ?false positive? errors are not possible. That is, one cannot detect a species where it does not occur. However, such errors are possible in many sampling situations for a number of reasons, and even low false positive error rates can induce extreme bias in estimates of site occupancy when they are not accounted for. In this paper, we develop a model for site occupancy that allows for both false negative and false positive error rates. This model can be represented as a two-component finite mixture model and can be easily fitted using freely available software. We provide an analysis of avian survey data using the proposed model and present results of a brief simulation study evaluating the performance of the maximum-likelihood estimator and the naive estimator in the presence of false positive errors.

  6. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

    While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

  7. Positive Active Material For Alkaline Electrolyte Storage Battert Nickel Electrodes

    DOEpatents

    Bernard, Patrick; Baudry, Michelle

    2000-12-05

    A method of manufacturing a positive active material for nickel electrodes of alkaline storage batteries which consists of particles of hydroxide containing mainly nickel and covered with a layer of a hydroxide phase based on nickel and yttrium is disclosed. The proportion of the hydroxide phase is in the range 0.15% to 3% by weight of yttrium expressed as yttrium hydroxide relative to the total weight of particles.

  8. Novel positive allosteric modulators of GABAA receptors with anesthetic activity

    PubMed Central

    Maldifassi, Maria C.; Baur, Roland; Pierce, David; Nourmahnad, Anahita; Forman, Stuart A.; Sigel, Erwin

    2016-01-01

    GABAA receptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics and anesthetics. We previously identified novel ligands of the classical benzodiazepine binding pocket in α1β2γ2 GABAA receptors using an experiment-guided virtual screening (EGVS) method. This screen also identified novel ligands for intramembrane low affinity diazepam site(s). In the current study we have further characterized compounds 31 and 132 identified with EGVS as well as 4-O-methylhonokiol. We investigated the site of action of these compounds in α1β2γ2 GABAA receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology combined with a benzodiazepine site antagonist and transmembrane domain mutations. All three compounds act mainly through the two β+/α− subunit transmembrane interfaces of the GABAA receptors. We then used concatenated receptors to dissect the involvement of individual β+/α− interfaces. We further demonstrated that these compounds have anesthetic activity in a small aquatic animal model, Xenopus laevis tadpoles. The newly identified compounds may serve as scaffolds for the development of novel anesthetics. PMID:27198062

  9. Differences among Job Positions Related to Communication Errors at Construction Sites

    NASA Astrophysics Data System (ADS)

    Takahashi, Akiko; Ishida, Toshiro

    In a previous study, we classified the communicatio n errors at construction sites as faulty intention and message pattern, inadequate channel pattern, and faulty comprehension pattern. This study seeks to evaluate the degree of risk of communication errors and to investigate differences among people in various job positions in perception of communication error risk . Questionnaires based on the previous study were a dministered to construction workers (n=811; 149 adminis trators, 208 foremen and 454 workers). Administrators evaluated all patterns of communication error risk equally. However, foremen and workers evaluated communication error risk differently in each pattern. The common contributing factors to all patterns wer e inadequate arrangements before work and inadequate confirmation. Some factors were common among patterns but other factors were particular to a specific pattern. To help prevent future accidents at construction sites, administrators should understand how people in various job positions perceive communication errors and propose human factors measures to prevent such errors.

  10. Site-specific aflatoxin B sub 1 adduction of sequence-positioned nucleosome core particles

    SciTech Connect

    Moyer, R.A.

    1988-01-01

    The question of how the presence of nucleosomal packing of DNA modifies carcinogen interaction at specific sites cannot be answered by studies on whole chromatin or bulk nucleosomes because of the heterogeneity of DNA sequences in the particles. This problem was circumvented by constructing nucleosomes that are homogenous in DNA-histone contact points. A cloned DNA fragment, containing a sea urchin 5S gene which precisely positions a histone octamer was employed. By using {sup 32}P end-labeled DNA and genotoxins that allow cleavage at sites of attack, the frequency of adduction at every susceptible nucleotide can be determined on sequencing gels. The small methylating agent dimethyl sulfate (DMS) and the bulky alkylating agent afatoxin B{sub 1}-dichloride (AFB{sub 1}-Cl{sub 2}) were used to probe the influence of DNA-histone interactions on DNA alkylation patterns in sequence-positioned core particles.

  11. ER-endosome contact sites in endosome positioning and protrusion outgrowth.

    PubMed

    Raiborg, Camilla; Wenzel, Eva M; Pedersen, Nina M; Stenmark, Harald

    2016-04-15

    The endoplasmic reticulum (ER) makes abundant contacts with endosomes, and the numbers of contact sites increase as endosomes mature. It is already clear that such contact sites have diverse compositions and functions, but in this mini-review we will focus on two particular types of ER-endosome contact sites that regulate endosome positioning. Formation of ER-endosome contact sites that contain the cholesterol-binding protein oxysterol-binding protein-related protein 1L (ORP1L) is coordinated with loss of the minus-end-directed microtubule motor Dynein from endosomes. Conversely, formation of ER-endosome contact sites that contain the Kinesin-1-binding protein Protrudin results in transfer of the plus-end-directed microtubule motor Kinesin-1 from ER to endosomes. We discuss the possibility that formation of these two types of contact sites is coordinated as a 'gear-shift' mechanism for endosome motility, and we review evidence that Kinesin-1-mediated motility of late endosomes (LEs) to the cell periphery promotes outgrowth of neurites and other protrusions. PMID:27068952

  12. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, Virginia C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program --now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history The missions will develop technology and acquire data necessary for eventual human Exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines be opportunities for the Mars community to provide input into the landing site selection process.

  13. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, V. C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program -- now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history. The missions will develop technology and acquire data necessary for eventual human exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines the opportunities for the Mars community to provide input into the landing site selection process.

  14. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

    PubMed

    Youngblut, Matthew D; Tsai, Chi-Lin; Clark, Iain C; Carlson, Hans K; Maglaqui, Adrian P; Gau-Pan, Phonchien S; Redford, Steven A; Wong, Alan; Tainer, John A; Coates, John D

    2016-04-22

    Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth. PMID:26940877

  15. Promoting positive psychology using social networking sites: a study of new college entrants on Facebook.

    PubMed

    Chang, Shu-Man; Lin, Yung-Hsiu; Lin, Chi-Wei; Chang, Her-Kun; Chong, Ping Pete

    2014-05-01

    This study explores the potential of promoting college students' positive psychological development using popular online social networks. Online social networks have dramatically changed the ways college students manage their social relationships. Social network activities, such as checking Facebook posts dominates students' Internet time and has the potential to assist students' positive development. Positive psychology is a scientific study of how ordinary individuals can apply their strength effectively when facing objective difficulties and how this capability can be cultivated with certain approaches. A positive message delivery approach was designed for a group of new college entrants. A series of positive messages was edited by university counselors and delivered by students to their Facebook social groups. Responses from each posted positive messages were collected and analyzed by researchers. The responses indicated that: (1) relationships of individual engagement and social influence in this study can partially explain the observed student behavior; (2) using class-based social groups can promote a positive atmosphere to enhance strong-tie relationships in both the physical and virtual environments, and (3) promoting student's positive attitudes can substantially impact adolescents' future developments, and many positive attitudes can be cultivated by emotional events and social influence. PMID:24785540

  16. Promoting Positive Psychology Using Social Networking Sites: A Study of New College Entrants on Facebook

    PubMed Central

    Chang, Shu-Man; Lin, Yung-Hsiu; Lin, Chi-Wei; Chang, Her-Kun; Chong, Ping Pete

    2014-01-01

    This study explores the potential of promoting college students’ positive psychological development using popular online social networks. Online social networks have dramatically changed the ways college students manage their social relationships. Social network activities, such as checking Facebook posts dominates students’ Internet time and has the potential to assist students’ positive development. Positive psychology is a scientific study of how ordinary individuals can apply their strength effectively when facing objective difficulties and how this capability can be cultivated with certain approaches. A positive message delivery approach was designed for a group of new college entrants. A series of positive messages was edited by university counselors and delivered by students to their Facebook social groups. Responses from each posted positive messages were collected and analyzed by researchers. The responses indicated that: (1) relationships of individual engagement and social influence in this study can partially explain the observed student behavior; (2) using class-based social groups can promote a positive atmosphere to enhance strong-tie relationships in both the physical and virtual environments, and (3) promoting student’s positive attitudes can substantially impact adolescents’ future developments, and many positive attitudes can be cultivated by emotional events and social influence. PMID:24785540

  17. Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites.

    PubMed

    Zhang, Jiantao; Zhang, Zhenlu; Chukkapalli, Vineela; Nchoutmboube, Jules A; Li, Jianhui; Randall, Glenn; Belov, George A; Wang, Xiaofeng

    2016-02-23

    All positive-strand RNA viruses reorganize host intracellular membranes to assemble their viral replication complexes (VRCs); however, how these viruses modulate host lipid metabolism to accommodate such membrane proliferation and rearrangements is not well defined. We show that a significantly increased phosphatidylcholine (PC) content is associated with brome mosaic virus (BMV) replication in both natural host barley and alternate host yeast based on a lipidomic analysis. Enhanced PC levels are primarily associated with the perinuclear ER membrane, where BMV replication takes place. More specifically, BMV replication protein 1a interacts with and recruits Cho2p (choline requiring 2), a host enzyme involved in PC synthesis, to the site of viral replication. These results suggest that PC synthesized at the site of VRC assembly, not the transport of existing PC, is responsible for the enhanced accumulation. Blocking PC synthesis by deleting the CHO2 gene resulted in VRCs with wider diameters than those in wild-type cells; however, BMV replication was significantly inhibited, highlighting the critical role of PC in VRC formation and viral replication. We further show that enhanced PC levels also accumulate at the replication sites of hepatitis C virus and poliovirus, revealing a conserved feature among a group of positive-strand RNA viruses. Our work also highlights a potential broad-spectrum antiviral strategy that would disrupt PC synthesis at the sites of viral replication but would not alter cellular processes. PMID:26858414

  18. Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites

    PubMed Central

    Zhang, Jiantao; Zhang, Zhenlu; Chukkapalli, Vineela; Nchoutmboube, Jules A.; Li, Jianhui; Randall, Glenn; Belov, George A.; Wang, Xiaofeng

    2016-01-01

    All positive-strand RNA viruses reorganize host intracellular membranes to assemble their viral replication complexes (VRCs); however, how these viruses modulate host lipid metabolism to accommodate such membrane proliferation and rearrangements is not well defined. We show that a significantly increased phosphatidylcholine (PC) content is associated with brome mosaic virus (BMV) replication in both natural host barley and alternate host yeast based on a lipidomic analysis. Enhanced PC levels are primarily associated with the perinuclear ER membrane, where BMV replication takes place. More specifically, BMV replication protein 1a interacts with and recruits Cho2p (choline requiring 2), a host enzyme involved in PC synthesis, to the site of viral replication. These results suggest that PC synthesized at the site of VRC assembly, not the transport of existing PC, is responsible for the enhanced accumulation. Blocking PC synthesis by deleting the CHO2 gene resulted in VRCs with wider diameters than those in wild-type cells; however, BMV replication was significantly inhibited, highlighting the critical role of PC in VRC formation and viral replication. We further show that enhanced PC levels also accumulate at the replication sites of hepatitis C virus and poliovirus, revealing a conserved feature among a group of positive-strand RNA viruses. Our work also highlights a potential broad-spectrum antiviral strategy that would disrupt PC synthesis at the sites of viral replication but would not alter cellular processes. PMID:26858414

  19. The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.

    PubMed

    Taylor, J C; Markham, G D

    1999-11-12

    S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the

  20. Active vibration control of a sandwich plate by non-collocated positive position feedback

    NASA Astrophysics Data System (ADS)

    Ferrari, Giovanni; Amabili, Marco

    2015-04-01

    The active vibration control of a free rectangular sandwich plate by using the Positive Position Feedback (PPF) algorithm was experimentally investigated in a previous study. Four normal modes were controlled by four nearly collocated couples of piezoelectric sensors and actuators. The experimental results of the control showed some limitation, especially in the Multi-Input Multi-Output (MIMO) configuration. This was attributed to the specific type of sensors and their conditioning, as well as to the phase shifts present in the vibration at different points of the structure. An alternative approach is here undertaken by abandoning the configuration of quasi-perfect collocation between sensor and actuator. The positioning of the piezoelectric patches is still led by the strain energy value distribution on the plate; each couple of sensor and actuator is now placed on the same face of the plate but in two distinct positions, opposed and symmetrical with respect to the geometric center of the plate. Single-Input Single-Output (SISO) PPF is tested and the transfer function parameters of the controller are tuned according to the measured values of modal damping. Then the participation matrices necessary for the MIMO control algorithm are determined by means of a completely experimental procedure. PPF is able to mitigate the vibration of the first four natural modes, in spite of the rigid body motions due to the free boundary conditions. The amplitude reduction achieved with the non-collocated configuration is much larger than the one obtained with the nearby collocated one. The phase lags were addressed in the MIMO algorithm by correction phase delays, further increasing the performance of the controller.

  1. Dynamic positioning system based on active disturbance rejection technology

    NASA Astrophysics Data System (ADS)

    Lei, Zhengling; Guo, Chen; Fan, Yunsheng

    2015-08-01

    A dynamically positioned vessel, by the International Maritime Organization (IMO) and the certifying class societies (DNV, ABS, LR, etc.), is defined as a vessel that maintains its position and heading (fixed location or pre-determined track) exclusively by means of active thrusters. The development of control technology promotes the upgrading of dynamic positioning (DP) systems. Today there are two different DP systems solutions available on the market: DP system based on PID regulator and that based on model-based control. Both systems have limited disturbance rejection capability due to their design principle. In this paper, a new DP system solution is proposed based on Active Disturbance Rejection Control (ADRC) technology. This technology is composed of Tracking-Differentiator (TD), Extended State Observer (ESO) and Nonlinear Feedback Combination. On one hand, both TD and ESO can act as filters and can be used in place of conventional filters; on the other hand, the total disturbance of the system can be estimated and compensated by ESO, which therefore enhances the system's disturbance rejection capability. This technology's advantages over other methods lie in two aspects: 1) This method itself can not only achieve control objectives but also filter noisy measurements without other specialized filters; 2) This method offers a new useful approach to suppress the ocean disturbance. The simulation results demonstrate the effectiveness of the proposed method.

  2. The active site of ribulose-bisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.

    1991-01-01

    The active site of ribulose-bisphosphate carboxylase/oxygenase requires interacting domains of adjacent, identical subunits. Most active-site residues are located within the loop regions of an eight-stranded {beta}/{alpha}-barrel which constitutes the larger C-terminal domain; additional key residues are located within a segment of the smaller N-terminal domain which partially covers the mouth of the barrel. Site-directed mutagenesis of the gene encoding the enzyme from Rhodospirillum rubrum has been used to delineate functions of active-site residues. 6 refs., 2 figs.

  3. Global Positioning System Synchronized Active Light Autonomous Docking System

    NASA Technical Reports Server (NTRS)

    Howard, Richard T. (Inventor); Book, Michael L. (Inventor); Bryan, Thomas C. (Inventor); Bell, Joseph L. (Inventor)

    1996-01-01

    A Global Positioning System Synchronized Active Light Autonomous Docking System (GPSSALADS) for automatically docking a chase vehicle with a target vehicle comprising at least one active light emitting target which is operatively attached to the target vehicle. The target includes a three-dimensional array of concomitantly flashing lights which flash at a controlled common frequency. The GPSSALADS further comprises a visual tracking sensor operatively attached to the chase vehicle for detecting and tracking the target vehicle. Its performance is synchronized with the flash frequency of the lights by a synchronization means which is comprised of first and second internal clocks operatively connected to the active light target and visual tracking sensor, respectively, for providing timing control signals thereto, respectively. The synchronization means further includes first and second Global Positioning System receivers operatively connected to the first and second internal clocks, respectively, for repeatedly providing simultaneous synchronization pulses to the internal clocks, respectively. In addition, the GPSSALADS includes a docking process controller means which is operatively attached to the chase vehicle and is responsive to the visual tracking sensor for producing commands for the guidance and propulsion system of the chase vehicle.

  4. Global Positioning System Synchronized Active Light Autonomous Docking System

    NASA Technical Reports Server (NTRS)

    Howard, Richard (Inventor)

    1994-01-01

    A Global Positioning System Synchronized Active Light Autonomous Docking System (GPSSALADS) for automatically docking a chase vehicle with a target vehicle comprises at least one active light emitting target which is operatively attached to the target vehicle. The target includes a three-dimensional array of concomitantly flashing lights which flash at a controlled common frequency. The GPSSALADS further comprises a visual tracking sensor operatively attached to the chase vehicle for detecting and tracking the target vehicle. Its performance is synchronized with the flash frequency of the lights by a synchronization means which is comprised of first and second internal clocks operatively connected to the active light target and visual tracking sensor, respectively, for providing timing control signals thereto, respectively. The synchronization means further includes first and second Global Positioning System receivers operatively connected to the first and second internal clocks, respectively, for repeatedly providing simultaneous synchronization pulses to the internal clocks, respectively. In addition, the GPSSALADS includes a docking process controller means which is operatively attached to the chase vehicle and is responsive to the visual tracking sensor for producing commands for the guidance and propulsion system of the chase vehicle.

  5. A study on the flexibility of enzyme active sites

    PubMed Central

    2011-01-01

    Background A common assumption about enzyme active sites is that their structures are highly conserved to specifically distinguish between closely similar compounds. However, with the discovery of distinct enzymes with similar reaction chemistries, more and more studies discussing the structural flexibility of the active site have been conducted. Results Most of the existing works on the flexibility of active sites focuses on a set of pre-selected active sites that were already known to be flexible. This study, on the other hand, proposes an analysis framework composed of a new data collecting strategy, a local structure alignment tool and several physicochemical measures derived from the alignments. The method proposed to identify flexible active sites is highly automated and robust so that more extensive studies will be feasible in the future. The experimental results show the proposed method is (a) consistent with previous works based on manually identified flexible active sites and (b) capable of identifying potentially new flexible active sites. Conclusions This proposed analysis framework and the former analyses on flexibility have their own advantages and disadvantage, depending on the cause of the flexibility. In this regard, this study proposes an alternative that complements previous studies and helps to construct a more comprehensive view of the flexibility of enzyme active sites. PMID:21342563

  6. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    SciTech Connect

    Zull, Lawrence M.; Yeniscavich, William

    2008-01-15

    The Defense Nuclear Facilities Safety Board (Board) is an independent federal agency established by Congress in 1988 to provide nuclear safety oversight of activities at U.S. Department of Energy (DOE) defense nuclear facilities. The activities under the Board's jurisdiction include the design, construction, startup, operation, and decommissioning of defense nuclear facilities at DOE sites. This paper reviews the Board's safety oversight of decommissioning activities at DOE sites, identifies the safety problems observed, and discusses Board initiatives to improve the safety of decommissioning activities at DOE sites. The decommissioning of former defense nuclear facilities has reduced the risk of radioactive material contamination and exposure to the public and site workers. In general, efforts to perform decommissioning work at DOE defense nuclear sites have been successful, and contractors performing decommissioning work have a good safety record. Decommissioning activities have recently been completed at sites identified for closure, including the Rocky Flats Environmental Technology Site, the Fernald Closure Project, and the Miamisburg Closure Project (the Mound site). The Rocky Flats and Fernald sites, which produced plutonium parts and uranium materials for defense needs (respectively), have been turned into wildlife refuges. The Mound site, which performed R and D activities on nuclear materials, has been converted into an industrial and technology park called the Mound Advanced Technology Center. The DOE Office of Legacy Management is responsible for the long term stewardship of these former EM sites. The Board has reviewed many decommissioning activities, and noted that there are valuable lessons learned that can benefit both DOE and the contractor. As part of its ongoing safety oversight responsibilities, the Board and its staff will continue to review the safety of DOE and contractor decommissioning activities at DOE defense nuclear sites.

  7. DOE site performance assessment activities. Radioactive Waste Technical Support Program

    SciTech Connect

    Not Available

    1990-07-01

    Information on performance assessment capabilities and activities was collected from eight DOE sites. All eight sites either currently dispose of low-level radioactive waste (LLW) or plan to dispose of LLW in the near future. A survey questionnaire was developed and sent to key individuals involved in DOE Order 5820.2A performance assessment activities at each site. The sites surveyed included: Hanford Site (Hanford), Idaho National Engineering Laboratory (INEL), Los Alamos National Laboratory (LANL), Nevada Test Site (NTS), Oak Ridge National Laboratory (ORNL), Paducah Gaseous Diffusion Plant (Paducah), Portsmouth Gaseous Diffusion Plant (Portsmouth), and Savannah River Site (SRS). The questionnaire addressed all aspects of the performance assessment process; from waste source term to dose conversion factors. This report presents the information developed from the site questionnaire and provides a comparison of site-specific performance assessment approaches, data needs, and ongoing and planned activities. All sites are engaged in completing the radioactive waste disposal facility performance assessment required by DOE Order 5820.2A. Each site has achieved various degrees of progress and have identified a set of critical needs. Within several areas, however, the sites identified common needs and questions.

  8. Savannah River Site prioritization of transition activities

    SciTech Connect

    Finley, R.H.

    1993-11-01

    Effective management of SRS conversion from primarily a production facility to other missions (or Decontamination and Decommissioning (D&D)) requires a systematic and consistent method of prioritizing the transition activities. This report discusses the design of a prioritizing method developed to achieve systematic and consistent methods of prioritizing these activities.

  9. The Distribution of Active Force Generators Controls Mitotic Spindle Position

    NASA Astrophysics Data System (ADS)

    Grill, Stephan W.; Howard, Jonathon; Schäffer, Erik; Stelzer, Ernst H. K.; Hyman, Anthony A.

    2003-07-01

    During unequal cell divisions a mitotic spindle is eccentrically positioned before cell cleavage. To determine the basis of the net force imbalance that causes spindle displacement in one-cell Caenorhabditis elegans embryos, we fragmented centrosomes with an ultraviolet laser. Analysis of the mean and variance of fragment speeds suggests that the force imbalance is due to a larger number of force generators pulling on astral microtubules of the posterior aster relative to the anterior aster. Moreover, activation of heterotrimeric guanine nucleotide-binding protein (G protein) α subunits is required to generate these astral forces.

  10. Ionizable Side Chains at Catalytic Active Sites of Enzymes

    PubMed Central

    Jimenez-Morales, David; Liang, Jie

    2012-01-01

    Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

  11. Dinucleotide Weight Matrices for Predicting Transcription Factor Binding Sites: Generalizing the Position Weight Matrix

    PubMed Central

    Siddharthan, Rahul

    2010-01-01

    Background Identifying transcription factor binding sites (TFBS) in silico is key in understanding gene regulation. TFBS are string patterns that exhibit some variability, commonly modelled as “position weight matrices” (PWMs). Though convenient, the PWM has significant limitations, in particular the assumed independence of positions within the binding motif; and predictions based on PWMs are usually not very specific to known functional sites. Analysis here on binding sites in yeast suggests that correlation of dinucleotides is not limited to near-neighbours, but can extend over considerable gaps. Methodology/Principal Findings I describe a straightforward generalization of the PWM model, that considers frequencies of dinucleotides instead of individual nucleotides. Unlike previous efforts, this method considers all dinucleotides within an extended binding region, and does not make an attempt to determine a priori the significance of particular dinucleotide correlations. I describe how to use a “dinucleotide weight matrix” (DWM) to predict binding sites, dealing in particular with the complication that its entries are not independent probabilities. Benchmarks show, for many factors, a dramatic improvement over PWMs in precision of predicting known targets. In most cases, significant further improvement arises by extending the commonly defined “core motifs” by about 10bp on either side. Though this flanking sequence shows no strong motif at the nucleotide level, the predictive power of the dinucleotide model suggests that the “signature” in DNA sequence of protein-binding affinity extends beyond the core protein-DNA contact region. Conclusion/Significance While computationally more demanding and slower than PWM-based approaches, this dinucleotide method is straightforward, both conceptually and in implementation, and can serve as a basis for future improvements. PMID:20339533

  12. Centrosome movement in the early divisions of Caenorhabditis elegans: A cortical site determining centrosome position

    SciTech Connect

    Hyman, A.A. )

    1989-09-01

    In Caenorhabditis elegans embryos, early blastomeres of the P cell lineage divide successively on the same axis. This axis is a consequence of the specific rotational movement of the pair of centrosomes and nucleus. A laser has been used to perturb the centrosome movements that determine the pattern of early embryonic divisions. The results support a previously proposed model in which a centrosome rotates towards its correct position by shortening of connections, possibly microtubules, between a centrosome and a defined site on the cortex of the embryo.

  13. Relative position and pose measurement approach of specific operation site of space non-cooperative target

    NASA Astrophysics Data System (ADS)

    Chen, Feng; Zhou, Ying; Li, Ronghua; Huang, Jianming

    2015-12-01

    In order to achieve the rendezvous and capture of the space non-cooperative target, the relative position and pose measurement of non-cooperative target must be resolved. Since the marker is not installed into the non-cooperative target and there is no inter satellite link to transfer the information, so it is very difficult to measure the relative position and pose measurement of non-cooperative target. The solar array connecting frame of non-cooperative targets have their characters and are easy to capture, so the position and pose measurement of specific operation site of non-cooperative target based on stereo vision has been studied in this paper. The method composed of image acquiring, image filtering, edge detection, feature extraction and relative pose measurement. Finally, the relative position and attitude parameters of the solar wing connection were obtained and provided to the control system. The results of simulation and ground verification show that the algorithm is accurate and effective, and can satisfy the technical requirements of the on orbit operation. The measurement approach can be used for engineering implementation.

  14. Active Sites Environmental Monitoring Program FY 1996 annual report

    SciTech Connect

    Morrissey, C.M.; Marshall, D.S.; Cunningham, G.R.

    1997-11-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1995 through September 1996. The Radioactive Solid Waste Operations Group (RSWOG) of the Waste Management and Remedial Action Division (WMRAD) and the Environmental Sciences Division (ESD) at Oak Ridge National Laboratory (ORNL) established ASEMP in 1989. The purpose of the program is to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 North as required by Chapters 2 and 3 of US Department of Energy Order 5820.2A.

  15. Active sites environmental monitoring Program - Program Plan: Revision 2

    SciTech Connect

    Morrissey, C.M.; Hicks, D.S.; Ashwood, T.L.; Cunningham, G.R.

    1994-05-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of active low-level-waste (LLW) and transuranic (TRU) waste facilities at Oak Ridge National Laboratory (ORNL). Several changes have recently occurred in regard to the sites that are currently used for waste storage and disposal. These changes require a second set of revisions to the ASEMP program plan. This document incorporates those revisions. This program plan presents the organization and procedures for monitoring the active sites. The program plan also provides internal reporting levels to guide the evaluation of monitoring results.

  16. Greater positive schizotypy relates to reduced N100 activity during rejection scenes.

    PubMed

    Premkumar, Preethi; Onwumere, Juliana; Wilson, Daniel; Sumich, Alexander; Castro, Antonio; Kumari, Veena; Kuipers, Elizabeth

    2014-08-01

    Social anxiety due to rejection sensitivity (RS) exacerbates psychosis-like experiences in the general population. While reduced dorsal anterior cingulate cortex (dACC) activity during social rejection in high schizotypy has suggested self-distancing from rejection, earlier stages of mental processing such as feature encoding could also contribute to psychosis-like experiences. This study aimed to determine the stage of mental processing of social rejection that relates to positive schizotypy. Forty-one healthy participants were assessed for schizotypy and RS. Event-related potential amplitudes (ERPs) were measured at frontal, temporal and parieto-occipital sites and their cortical sources (dACC, temporal pole and lingual gyrus) at early (N100) and late (P300 and late slow wave, LSW) timeframes during rejection, acceptance and neutral scenes. ERPs were compared between social interaction types. Correlations were performed between positive schizotypy (defined as the presence of perceptual aberrations, hallucinatory experiences and magical thinking), RS and ERPs during rejection. Amplitude was greater during rejection than acceptance or neutral conditions at the dACC-P300, parieto-occipital-P300, dACC-LSW and frontal-LSW. RS correlated positively with positive schizotypy. Reduced dACC N100 activity during rejection correlated with greater positive schizotypy and RS. Reduced dACC N100 activity and greater RS independently predicted positive schizotypy. An N100 deficit that indicates reduced feature encoding of rejection scenes increases with greater positive schizotypy and RS. Higher RS shows that a greater tendency to misattribute ambiguous social situations as rejecting also increases with positive schizotypy. These two processes, namely primary bottom-up sensory processing and secondary misattribution of rejection, combine to increase psychosis-like experiences. PMID:25010933

  17. The active site behaviour of electrochemically synthesised gold nanomaterials.

    PubMed

    Plowman, Blake J; O'Mullane, Anthony P; Bhargava, Suresh K

    2011-01-01

    Even though gold is the noblest of metals, a weak chemisorber and is regarded as being quite inert, it demonstrates significant electrocatalytic activity in its nanostructured form. It is demonstrated here that nanostructured and even evaporated thin films of gold are covered with active sites which are responsible for such activity. The identification of these sites is demonstrated with conventional electrochemical techniques such as cyclic voltammetry as well as a large amplitude Fourier transformed alternating current (FT-ac) method under acidic and alkaline conditions. The latter technique is beneficial in determining if an electrode process is either Faradaic or capacitive in nature. The observed behaviour is analogous to that observed for activated gold electrodes whose surfaces have been severely disrupted by cathodic polarisation in the hydrogen evolution region. It is shown that significant electrochemical oxidation responses occur at discrete potential values well below that for the formation of the compact monolayer oxide of bulk gold and are attributed to the facile oxidation of surface active sites. Several electrocatalytic reactions are explored in which the onset potential is determined by the presence of such sites on the surface. Significantly, the facile oxidation of active sites is used to drive the electroless deposition of metals such as platinum, palladium and silver from their aqueous salts on the surface of gold nanostructures. The resultant surface decoration of gold with secondary metal nanoparticles not only indicates regions on the surface which are rich in active sites but also provides a method to form interesting bimetallic surfaces. PMID:22455038

  18. Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

    PubMed

    Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

    2016-06-17

    Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes. PMID:26990764

  19. GITR Activation Positively Regulates Immune Responses against Toxoplasma gondii

    PubMed Central

    Costa, Frederico R. C.; Mota, Caroline M.; Santiago, Fernanda M.; Silva, Murilo V.; Ferreira, Marcela D.; Fonseca, Denise M.; Silva, João S.; Mineo, José R.; Mineo, Tiago W. P.

    2016-01-01

    Toxoplasma gondii is a widespread parasite responsible for causing clinical diseases especially in pregnant and immunosuppressed individuals. Glucocorticoid-induced TNF receptor (GITR), which is also known as TNFRS18 and belongs to the TNF receptor superfamily, is found to be expressed in various cell types of the immune system and provides an important costimulatory signal for T cells and myeloid cells. However, the precise role of this receptor in the context of T. gondii infection remains elusive. Therefore, the current study investigated the role of GITR activation in the immunoregulation mechanisms induced during the experimental infection of mice with T. gondii. Our data show that T. gondii infection slightly upregulates GITR expression in Treg cells and B cells, but the most robust increment in expression was observed in macrophages and dendritic cells. Interestingly, mice infected and treated with an agonistic antibody anti-GITR (DTA-1) presented a robust increase in pro-inflammatory cytokine production at preferential sites of parasite replication, which was associated with the decrease in latent brain parasitism of mice under treatment with DTA-1. Several in vivo and in vitro analysis were performed to identify the cellular mechanisms involved in GITR activation upon infection, however no clear alterations were detected in the phenotype/function of macrophages, Tregs and B cells under treatment with DTA-1. Therefore, GITR appears as a potential target for intervention during infection by the parasite Toxoplasma gondii, even though further studies are still necessary to better characterize the immune response triggered by GITR activation during T. gondii infection. PMID:27027302

  20. Ultrastructure of the replication sites of positive-strand RNA viruses

    SciTech Connect

    Harak, Christian; Lohmann, Volker

    2015-05-15

    Positive strand RNA viruses replicate in the cytoplasm of infected cells and induce intracellular membranous compartments harboring the sites of viral RNA synthesis. These replication factories are supposed to concentrate the components of the replicase and to shield replication intermediates from the host cell innate immune defense. Virus induced membrane alterations are often generated in coordination with host factors and can be grouped into different morphotypes. Recent advances in conventional and electron microscopy have contributed greatly to our understanding of their biogenesis, but still many questions remain how viral proteins capture membranes and subvert host factors for their need. In this review, we will discuss different representatives of positive strand RNA viruses and their ways of hijacking cellular membranes to establish replication complexes. We will further focus on host cell factors that are critically involved in formation of these membranes and how they contribute to viral replication. - Highlights: • Positive strand RNA viruses induce massive membrane alterations. • Despite the great diversity, replication complexes share many similarities. • Host factors play a pivotal role in replication complex biogenesis. • Use of the same host factors by several viruses hints to similar functions.

  1. Real-Time GNSS Positioning Along Canada's Active Coastal Margin

    NASA Astrophysics Data System (ADS)

    Henton, J. A.; Dragert, H.; Lu, Y.

    2014-12-01

    High-rate, low-latency Global Navigation Satellite System (GNSS) data are being refined for real-time applications to monitor and report motions related to large earthquakes in coastal British Columbia. Given the tectonic setting of Canada's west coast, specific goals for real-time regional geodetic monitoring are: (1) the collection of GNSS data with adequate station density to identify the deformation field for regional earthquakes with M>7.3; (2) the robust, continuous real-time analyses of GNSS data with a precision of 1-2 cm and a latency of less than 10s; and (3) the display of results with attending automated alarms and estimations of earthquake parameters. Megathrust earthquakes (M>8) are the primary targets for immediate identification, since the tsunamis they generate will strike the coast within 15 to 20 min. However, large (6.0positioning streams for regional sites received from the Canadian Geodetic Survey (CGS), the Jet Propulsion Laboratory (JPL), and the Plate Boundary Observatory (PBO). The comparison of these various real-time solutions allows a realistic evaluation of day-to-day software performance especially when faced with adverse conditions such as data gaps or poor satellite geometry. Forward models for scenario earthquakes in this region are used to "fingerprint" the coseismic displacements expected from various offshore events which allows an evaluation of the effectiveness of the current regional coverage. The present distribution and density of real-time sites is largely sufficient for aiding the timely estimation of size, location

  2. Active site - a site of binding of affinity inhibitors in baker's yeast inorganic pyrophosphatase

    SciTech Connect

    Svyato, I.E.; Sklyankina, V.A.; Avaeva, S.M.

    1986-03-20

    The interaction of the enzyme-substrate complex with methyl phosphate, O-phosphoethanolamine, O-phosphopropanolamine, N-acetylphosphoserine, and phosphoglyolic acid, as well as pyrophosphatase, modified by monoesters of phosphoric acid, with pyrophosphate and tripolyphosphate, was investigated. It was shown that the enzyme containing the substrate in the active site does not react with monophosphates, but modified pyrophosphatase entirely retains the ability to bind polyanions to the regulatory site. It is concluded that the inactivation of baker's yeast inorganic pyrophosphatase by monoesters of phosphoric acid, which are affinity inhibitors of it, is the result of modification of the active site of the enzyme.

  3. Bidirectional associations between valued activities and adolescent positive adjustment in a longitudinal study: positive mood as a mediator.

    PubMed

    DesRoches, Andrea; Willoughby, Teena

    2014-02-01

    Although activity involvement has been linked to positive youth development, the value that adolescents place on these activities (i.e., how much they enjoy the activities, find them important, and spend time on them) has received less attention. The purpose of the present study was to examine the bidirectional longitudinal association between engagement in valued activities and adolescent positive adjustment (optimism, purpose in life, and self-esteem), as well as investigate a possible underlying mechanism for this link. High school students (N = 2,270, 48.7% female) from Ontario, Canada completed questionnaires annually in grades 10, 11, and 12. Auto-regressive cross-lagged path analyses were conducted over time, controlling for gender, parental education, and academic grades. Greater engagement in valued activities predicted higher optimism, purpose, and self-esteem over time. Importantly, the results did not support an alternate hypothesis of selection effects, in that adolescents who were better adjusted were not more likely than their peers to engage in valued activities over time. We also found that the longitudinal associations between valued activities and positive adjustment may be due partly to an underlying effect of increased positive mood. Thus, engagement in valued activities appears to be important for adolescent positive adjustment, and may help to foster thriving. Communities, educators, and parents should actively support and encourage adolescents to develop valued activities, and seek to ensure that there are ample opportunities and resources available for them to do so. PMID:23625185

  4. Activity of quinolones against gram-positive cocci: clinical features.

    PubMed

    Giamarellou, H

    1995-01-01

    The potential role of the commercially available fluoroquinolones in the treatment of Gram-positive infections is discussed on the basis of data obtained from animal experiments and clinical trials. In respiratory tract infections, and particularly in community-acquired pneumonia, it is evident that the presently available quinolones cannot be prescribed empirically as first-line therapy because of their borderline activity against Streptococcus pneumoniae and anaerobes. Reports of pneumococcal seeding in other tissues during quinolone therapy render their administration a debatable issue. Experience in endocarditis is limited to the use of ciprofloxacin plus rifampicin in intravenous drug users with right-sided Staphylococcus aureus endocarditis. Patients with staphylococcal osteomyelitis are included among cases of other bone infections. In noncontrolled studies ciprofloxacin, ofloxacin and pefloxacin attained a staphylococcal eradication rate ranging from 70 to 100%, while the addition of rifampicin has been proven to reduce the emergence of resistant mutants during therapy. In soft tissue and skin structure infections that also involve Gram-negative bacteria, ciprofloxacin and ofloxacin eradicated 72.6 and 89% of staphylococci, respectively; however, the presence of diabetes or vascular disease compromised the success of treatment. In staphylococcal peritonitis complicating continuous ambulatory peritoneal dialysis, results with ciprofloxacin given intravenously or intraperitoneally were promising. In infections in neutropenic hosts, success of prophylaxis or therapy is still not clear, since colonisation and breakthrough bacteraemias with viridans streptococci and staphylococci have been reported. Furthermore, therapeutic results are compromised by the low response rate in Gram-positive infections. Despite the reported clinical efficacy of the newer fluoroquinolones, physicians should be alerted to the emergence of staphylococci resistant to fluoroquinolones

  5. Small-Activating RNA Can Change Nucleosome Positioning in Human Fibroblasts.

    PubMed

    Wang, Bin; Sun, Jing; Shi, Jiandong; Guo, Qing; Tong, Xiangrong; Zhang, Jin; Hu, Ningzhu; Hu, YunZhang

    2016-07-01

    RNA activation (RNAa) is a mechanism of positive gene expression regulation mediated by small-activating RNAs (saRNAs), which target gene promoters and have been used as tools to manipulate gene expression. Studies have shown that RNAa is associated with epigenetic modifications at promoter regions; however, it is unclear whether these modifications are the cause or a consequence of RNAa. In this study, we examined changes in nucleosome repositioning and the involvement of RNA polymerase II (RNAPII) in this process. We screened saRNAs for OCT4 (POU5F1), SOX2, and NANOG, and identified several novel saRNAs. We found that nucleosome positioning was altered after saRNA treatment and that the formation of nucleosome-depleted regions (NDRs) contributed to RNAa at sites of RNAPII binding, such as the TATA box, CpG islands (CGIs), proximal enhancers, and proximal promoters. Moreover, RNAPII appeared to be bound specifically to NDRs. These results suggested that changes in nucleosome positions resulted from RNAa. We thus propose a hypothesis that targeting promoter regions using exogenous saRNAs can induce the formation of NDRs, exposing regulatory binding sites to recruit RNAPII, a key component of preinitiation complex, and leading to increased initiation of transcription. PMID:26993320

  6. A small ribozyme with dual-site kinase activity

    PubMed Central

    Biondi, Elisa; Maxwell, Adam W.R.; Burke, Donald H.

    2012-01-01

    Phosphoryl transfer onto backbone hydroxyls is a recognized catalytic activity of nucleic acids. We find that kinase ribozyme K28 possesses an unusually complex active site that promotes (thio)phosphorylation of two residues widely separated in primary sequence. After allowing the ribozyme to radiolabel itself by phosphoryl transfer from [γ-32P]GTP, DNAzyme-mediated cleavage yielded two radiolabeled cleavage fragments, indicating phosphorylation sites within each of the two cleavage fragments. These sites were mapped by alkaline digestion and primer extension pausing. Enzymatic digestion and mutational analysis identified nucleotides important for activity and established the active structure as being a constrained pseudoknot with unusual connectivity that may juxtapose the two reactive sites. Nuclease sensitivities for nucleotides near the pseudoknot core were altered in the presence of GTPγS, indicating donor-induced folding. The 5′ target site was more strongly favored in full-length ribozyme K28 (128 nt) than in truncated RNAs (58 nt). Electrophoretic mobilities of self-thiophosphorylated products on organomercurial gels are distinct from the 5′ mono-thiophosphorylated product produced by reaction with polynucleotide kinase, potentially indicating simultaneous labeling of both sites within individual RNA strands. Our evidence supports a single, compact structure with local dynamics, rather than global rearrangement, as being responsible for dual-site phosphorylation. PMID:22618879

  7. N6-Methyldeoxyadenosine Marks Active Transcription Start Sites in Chlamydomonas

    PubMed Central

    Chen, Kai; Deng, Xin; Yu, Miao; Han, Dali; Hao, Ziyang; Liu, Jianzhao; Lu, Xingyu; Dore, Louis C; Weng, Xiaocheng; Ji, Quanjiang; Mets, Laurens; He, Chuan

    2015-01-01

    SUMMARY N6-methyldeoxyadenosine (6mA or m6A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria, and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution, and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms. PMID:25936837

  8. High physical activity in young children suggests positive effects by altering autoantigen-induced immune activity.

    PubMed

    Carlsson, E; Ludvigsson, J; Huus, K; Faresjö, M

    2016-04-01

    Physical activity in children is associated with several positive health outcomes such as decreased cardiovascular risk factors, improved lung function, enhanced motor skill development, healthier body composition, and also improved defense against inflammatory diseases. We examined how high physical activity vs a sedentary lifestyle in young children influences the immune response with focus on autoimmunity. Peripheral blood mononuclear cells, collected from 55 5-year-old children with either high physical activity (n = 14), average physical activity (n = 27), or low physical activity (n = 14), from the All Babies In Southeast Sweden (ABIS) cohort, were stimulated with antigens (tetanus toxoid and beta-lactoglobulin) and autoantigens (GAD65 , insulin, HSP60, and IA-2). Immune markers (cytokines and chemokines), C-peptide and proinsulin were analyzed. Children with high physical activity showed decreased immune activity toward the autoantigens GAD65 (IL-5, P < 0.05), HSP60 and IA-2 (IL-10, P < 0.05) and also low spontaneous pro-inflammatory immune activity (IL-6, IL-13, IFN-γ, TNF-α, and CCL2 (P < 0.05)) compared with children with an average or low physical activity. High physical activity in young children seems to have positive effects on the immune system by altering autoantigen-induced immune activity. PMID:25892449

  9. Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site

    SciTech Connect

    Carra,J.; McHugh, C.; Mulligan, S.; Machiesky, L.; Soares, A.; Millard, C.

    2007-01-01

    We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding to a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.

  10. Estimating snow water equivalent from GPS vertical site-position observations in the western United States

    PubMed Central

    Ouellette, Karli J; de Linage, Caroline; Famiglietti, James S

    2013-01-01

    [1] Accurate estimation of the characteristics of the winter snowpack is crucial for prediction of available water supply, flooding, and climate feedbacks. Remote sensing of snow has been most successful for quantifying the spatial extent of the snowpack, although satellite estimation of snow water equivalent (SWE), fractional snow covered area, and snow depth is improving. Here we show that GPS observations of vertical land surface loading reveal seasonal responses of the land surface to the total weight of snow, providing information about the stored SWE. We demonstrate that the seasonal signal in Scripps Orbit and Permanent Array Center (SOPAC) GPS vertical land surface position time series at six locations in the western United States is driven by elastic loading of the crust by the snowpack. GPS observations of land surface deformation are then used to predict the water load as a function of time at each location of interest and compared for validation to nearby Snowpack Telemetry observations of SWE. Estimates of soil moisture are included in the analysis and result in considerable improvement in the prediction of SWE. Citation: Ouellette, K. J., C. de Linage, and J. S. Famiglietti (2013), Estimating snow water equivalent from GPS vertical site-position observations in the western United States, Water Resour. Res., 49, 2508–2518, doi:10.1002/wrcr.20173. PMID:24223442

  11. Estimating snow water equivalent from GPS vertical site-position observations in the western United States.

    PubMed

    Ouellette, Karli J; de Linage, Caroline; Famiglietti, James S

    2013-05-01

    [1] Accurate estimation of the characteristics of the winter snowpack is crucial for prediction of available water supply, flooding, and climate feedbacks. Remote sensing of snow has been most successful for quantifying the spatial extent of the snowpack, although satellite estimation of snow water equivalent (SWE), fractional snow covered area, and snow depth is improving. Here we show that GPS observations of vertical land surface loading reveal seasonal responses of the land surface to the total weight of snow, providing information about the stored SWE. We demonstrate that the seasonal signal in Scripps Orbit and Permanent Array Center (SOPAC) GPS vertical land surface position time series at six locations in the western United States is driven by elastic loading of the crust by the snowpack. GPS observations of land surface deformation are then used to predict the water load as a function of time at each location of interest and compared for validation to nearby Snowpack Telemetry observations of SWE. Estimates of soil moisture are included in the analysis and result in considerable improvement in the prediction of SWE. Citation: Ouellette, K. J., C. de Linage, and J. S. Famiglietti (2013), Estimating snow water equivalent from GPS vertical site-position observations in the western United States, Water Resour. Res., 49, 2508-2518, doi:10.1002/wrcr.20173. PMID:24223442

  12. Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin.

    PubMed Central

    Gasymov, O. K.; Abduragimov, A. R.; Yusifov, T. N.; Glasgow, B. J.

    2000-01-01

    The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence. Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL. Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the apo-proteins. These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern-Volmer plots are inconsistent with a ligand that is

  13. Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin.

    PubMed

    Gasymov, O K; Abduragimov, A R; Yusifov, T N; Glasgow, B J

    2000-02-01

    The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence. Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL. Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the apo-proteins. These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern-Volmer plots are inconsistent with a ligand that is

  14. Dashboard applications to monitor experiment activities at sites

    NASA Astrophysics Data System (ADS)

    Andreeva, Julia; Belforte, Stefano; Boehm, Max; Casajus, Adrian; Flix, Josep; Gaidioz, Benjamin; Grigoras, Costin; Kokoszkiewicz, Lukasz; Lanciotti, Elisa; Rocha, Ricardo; Saiz, Pablo; Santinelli, Roberto; Sidorova, Irina; Sciabà, Andrea; Tsaregorodtsev, Andrei

    2010-04-01

    In the framework of a distributed computing environment, such as WLCG, monitoring has a key role in order to keep under control activities going on in sites located in different countries and involving people based in many different sites. To be able to cope with such a large scale heterogeneous infrastructure, it is necessary to have monitoring tools providing a complete and reliable view of the overall performance of the sites. Moreover, the structure of a monitoring system critically depends on the object to monitor and on the users it is addressed to. In this article we will describe two different monitoring systems both aimed to monitor activities and services provided in the WLCG framework, but designed in order to meet the requirements of different users: Site Status Board has an overall view of the services available in all the sites supporting an experiment, whereas Siteview provides a complete view of all the activities going on at a site, for all the experiments supported by the site.

  15. Architecture and active site of particulate methane monooxygenase

    PubMed Central

    Culpepper, Megen A.; Rosenzweig, Amy C.

    2012-01-01

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria, organisms that live on methane gas as their sole carbon source. Understanding pMMO function has important implications for bioremediation applications and for the development of new, environmentally friendly catalysts for the direct conversion of methane to methanol. Crystal structures of pMMOs from three different methanotrophs reveal a trimeric architecture, consisting of three copies each of the pmoB, pmoA, and pmoC subunits. There are three distinct metal centers in each protomer of the trimer, mononuclear and dinuclear copper sites in the periplasmic regions of pmoB and a mononuclear site within the membrane that can be occupied by copper or zinc. Various models for the pMMO active site have been proposed within these structural constraints, including dicopper, tricopper, and diiron centers. Biochemical and spectroscopic data on pMMO and recombinant soluble fragments, denoted spmoB proteins, indicate that the active site involves copper and is located at the site of the dicopper center in the pmoB subunit. Initial spectroscopic evidence for O2 binding at this site has been obtained. Despite these findings, questions remain about the active site identity and nuclearity and will be the focus of future studies. PMID:22725967

  16. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination

    PubMed Central

    Rowley, Paul A.; Kachroo, Aashiq H.; Ma, Chien-Hui; Maciaszek, Anna D.; Guga, Piotr; Jayaram, Makkuni

    2015-01-01

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5′ to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  17. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

    PubMed

    Rowley, Paul A; Kachroo, Aashiq H; Ma, Chien-Hui; Maciaszek, Anna D; Guga, Piotr; Jayaram, Makkuni

    2015-07-13

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  18. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site.

    PubMed

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2domains reveal that the (HhH)2domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  19. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site

    PubMed Central

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-01-01

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  20. On-site hospital helipads: resource document for the NAEMSP position paper on on-site hospital helipads.

    PubMed

    Thomas, Stephen H

    2009-01-01

    The purpose of this article is to address the question of helipad location for hospitals using helicopter emergency medical services (HEMS). A helipad is defined as off-site, or remote, if a ground ambulance is required for patient transport between the helicopter and the hospital's patient care area. On-site helipads are those for which no ground ambulance transport is required between the hospital and the helicopter. The article describes the attributes of on-site helipads, which include elimination of the inherent risks of additional patient transfers, enhanced availability of emergency medical services (EMS) resources that would otherwise be used for extra transfer, and decreased time to arrival at the receiving treatment site. It is acknowledged that helipad placement decisions are informed by non-patient care issues and a paucity of research. Nevertheless, when the choice is viewed from a patient care perspective, there is a clear preference for on-site helipad location. PMID:19499480

  1. ON-LINE TOOLS FOR PROPER VERTICAL POSITIONING OF VERTICAL SAMPLING INTERVALS DURING SITE ASSESSMENT: MEETING IN MONTGOMERY, ALABAMA

    EPA Science Inventory

    This presentation, On-Line Tools for Proper Vertical Positioning of Sampling Intervals During Site Assessment, describes an approach to locating monitoring wells that is based on application of ground water models. The ideal use of both the model and site assessment funds is to ...

  2. Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase

    PubMed Central

    Seibold, Steve A.; Singh, Badri Nath; Zhang, Chunfen; Kireeva, Maria; Domecq, Céline; Bouchard, Annie; Nazione, Anthony M.; Feig, Michael; Cukier, Robert I.; Coulombe, Benoit; Kashlev, Mikhail; Hampsey, Michael; Burton, Zachary F.

    2010-01-01

    Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant β R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge α-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in β R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site “latch” assembly that includes a key trigger helix residue Tt β’ H1242 and highly conserved active site residues β E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed. PMID:20478425

  3. Molecular Imprint of Enzyme Active Site by Camel Nanobodies

    PubMed Central

    Li, Jiang-Wei; Xia, Lijie; Su, Youhong; Liu, Hongchun; Xia, Xueqing; Lu, Qinxia; Yang, Chunjin; Reheman, Kalbinur

    2012-01-01

    Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach. PMID:22374998

  4. Altered Nucleosome Positioning at the Transcription Start Site and Deficient Transcriptional Initiation in Friedreich Ataxia*

    PubMed Central

    Chutake, Yogesh K.; Costello, Whitney N.; Lam, Christina; Bidichandani, Sanjay I.

    2014-01-01

    Most individuals with Friedreich ataxia (FRDA) are homozygous for an expanded GAA triplet repeat (GAA-TR) mutation in intron 1 of the FXN gene, which results in deficiency of FXN transcript. Consistent with the expanded GAA-TR sequence as a cause of variegated gene silencing, evidence for heterochromatin has been detected in intron 1 in the immediate vicinity of the expanded GAA-TR mutation in FRDA. Transcriptional deficiency in FRDA is thought to result from deficient elongation through the expanded GAA-TR sequence because of repeat-proximal heterochromatin and abnormal DNA structures adopted by the expanded repeat. There is also evidence for deficient transcriptional initiation in FRDA, but its relationship to the expanded GAA-TR mutation remains unclear. We show that repressive chromatin extends from the expanded GAA-TR in intron 1 to the upstream regions of the FXN gene, involving the FXN transcriptional start site. Using a chromatin accessibility assay and a high-resolution nucleosome occupancy assay, we found that the major FXN transcriptional start site, which is normally in a nucleosome-depleted region, is rendered inaccessible by altered nucleosome positioning in FRDA. Consistent with the altered epigenetic landscape the FXN gene promoter, a typical CpG island promoter, was found to be in a transcriptionally non-permissive state in FRDA. Both metabolic labeling of nascent transcripts and an unbiased whole transcriptome analysis revealed a severe deficiency of transcriptional initiation in FRDA. Deficient transcriptional initiation, and not elongation, is the major cause of FXN transcriptional deficiency in FRDA, and it is related to the spread of repressive chromatin from the expanded GAA-TR mutation. PMID:24737321

  5. Active Sites Environmental Monitoring Program: Mid-FY 1991 report

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1991-10-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1990 through March 1991. The ASEMP was established in 1989 by Solid Waste Operations and the Environmental Sciences Division to provide early detection and performance monitoring at active low-level radioactive waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. Monitoring results continue to demonstrate the no LLW is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II began during this reporting period and 115 vaults had been loaded by the end of March 1991.

  6. An active-site peptide from pepsin C

    PubMed Central

    Kay, J.; Ryle, A. P.

    1971-01-01

    Porcine pepsin C is inactivated rapidly and irreversibly by diazoacetyl-dl-norleucine methyl ester in the presence of cupric ions at pH values above 4.5. The inactivation is specific in that complete inactivation accompanies the incorporation of 1mol of inhibitor residue/mol of enzyme and evidence has been obtained to suggest that the reaction occurs with an active site residue. The site of reaction is the β-carboxyl group of an aspartic acid residue in the sequence Ile-Val-Asp-Thr. This sequence is identical with the active-site sequence in pepsin and the significance of this in terms of the different activities of the two enzymes is discussed. PMID:4942834

  7. Spindle assembly checkpoint proteins are positioned close to core microtubule attachment sites at kinetochores

    PubMed Central

    Wan, Xiaohu; Cheerambathur, Dhanya; Gassmann, Reto; Suzuki, Aussie; Lawrimore, Josh; Desai, Arshad; Salmon, E.D.

    2013-01-01

    Spindle assembly checkpoint proteins have been thought to reside in the peripheral corona region of the kinetochore, distal to microtubule attachment sites at the outer plate. However, recent biochemical evidence indicates that checkpoint proteins are closely linked to the core kinetochore microtubule attachment site comprised of the Knl1–Mis12–Ndc80 (KMN) complexes/KMN network. In this paper, we show that the Knl1–Zwint1 complex is required to recruit the Rod–Zwilch–Zw10 (RZZ) and Mad1–Mad2 complexes to the outer kinetochore. Consistent with this, nanometer-scale mapping indicates that RZZ, Mad1–Mad2, and the C terminus of the dynein recruitment factor Spindly are closely juxtaposed with the KMN network in metaphase cells when their dissociation is blocked and the checkpoint is active. In contrast, the N terminus of Spindly is ∼75 nm outside the calponin homology domain of the Ndc80 complex. These results reveal how checkpoint proteins are integrated within the substructure of the kinetochore and will aid in understanding the coordination of microtubule attachment and checkpoint signaling during chromosome segregation. PMID:23979716

  8. Active Healthy Kids Canada's Position on Active Video Games for Children and Youth.

    PubMed

    Chaput, Jean-Philippe; Leblanc, Allana G; McFarlane, Allison; Colley, Rachel C; Thivel, David; Biddle, Stuart Jh; Maddison, Ralph; Leatherdale, Scott T; Tremblay, Mark S

    2013-12-01

    The effect of active video games (AVGs) on acute energy expenditure has previously been reported; however, the influence of AVGs on other health-related lifestyle indicators remains unclear. To address this knowledge gap, Active Healthy Kids Canada (AHKC) convened an international group of researchers to conduct a systematic review to understand whether AVGs should be promoted to increase physical activity and improve health indicators in children and youth (zero to 17 years of age). The present article outlines the process and outcomes of the development of the AHKC's position on active video games for children and youth. In light of the available evidence, AHKC does not recommend AVGs as a strategy to help children be more physically active. However, AVGs may exchange some sedentary time for light- to moderate-intensity physical activity, and there may be specific situations in which AVGs provide benefit (eg, motor skill development in special populations and rehabilitation). PMID:24497779

  9. Active chemisorption sites in functionalized ionic liquids for carbon capture.

    PubMed

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

    Development of novel technologies for the efficient and reversible capture of CO2 is highly desired. In the last decade, CO2 capture using ionic liquids has attracted intensive attention from both academia and industry, and has been recognized as a very promising technology. Recently, a new approach has been developed for highly efficient capture of CO2 by site-containing ionic liquids through chemical interaction. This perspective review focuses on the recent advances in the chemical absorption of CO2 using site-containing ionic liquids, such as amino-based ionic liquids, azolate ionic liquids, phenolate ionic liquids, dual-functionalized ionic liquids, pyridine-containing ionic liquids and so on. Other site-containing liquid absorbents such as amine-based solutions, switchable solvents, and functionalized ionic liquid-amine blends are also investigated. Strategies have been discussed for how to activate the existent reactive sites and develop novel reactive sites by physical and chemical methods to enhance CO2 absorption capacity and reduce absorption enthalpy. The carbon capture mechanisms of these site-containing liquid absorbents are also presented. Particular attention has been paid to the latest progress in CO2 capture in multiple-site interactions by amino-free anion-functionalized ionic liquids. In the last section, future directions and prospects for carbon capture by site-containing ionic liquids are outlined. PMID:27243042

  10. Positional asphyxia without active restraint following an assault.

    PubMed

    Fernando, Tarini; Byard, Roger W

    2013-11-01

    Deaths due to positional asphyxia are most often accidental, associated with alcohol and/or drug intoxication. A 19-year-old male is reported who was assaulted and placed in a head-down position in the back of a car were he was later found dead. Brush abrasions indicated that he had been dragged to the vehicle. The head and right shoulder were wedged into the foot well with the body uppermost. At autopsy, there was marked congestion of the face, neck, and upper chest with conjunctival ecchymoses, bruising of the face and scalp, focal subarachnoid hemorrhage, minor cerebral contusion, and diffuse cerebral swelling with early hypoxic ischemic encephalopathy (HIE). Toxicology was negative. Death was attributed to HIE resulting from the unusual positioning of the body. Cases of positional asphyxia involving others may not always include restraint, and when encountered should initiate a careful evaluation of the possible events and lethal pathophysiological processes. PMID:23786332

  11. Roles of s3 site residues of nattokinase on its activity and substrate specificity.

    PubMed

    Wu, Shuming; Feng, Chi; Zhong, Jin; Huan, Liandong

    2007-09-01

    Nattokinase (Subtilisin NAT, NK) is a bacterial serine protease with high fibrinolytic activity. To probe their roles on protease activity and substrate specificity, three residues of S3 site (Gly(100), Ser(101) and Leu(126)) were mutated by site-directed mutagenesis. Kinetics parameters of 20 mutants were measured using tetrapeptides as substrates, and their fibrinolytic activities were determined by fibrin plate method. Results of mutation analysis showed that Gly(100) and Ser(101) had reverse steric and electrostatic effects. Residues with bulky or positively charged side chains at position 100 decreased the substrate binding and catalytic activity drastically, while residues with the same characters at position 101 could obviously enhance protease and fibrinolytic activity of NK. Mutation of Leu(126) might impair the structure of the active cleft and drastically decreased the activity of NK. Kinetics studies of the mutants showed that S3 residues were crucial to keep protease activity while they moderately affected substrate specificity of NK. The present study provided some original insight into the P3-S3 interaction in NK and other subtilisins, as well as showed successful protein engineering cases to improve NK as a potential therapeutic agent. PMID:17673485

  12. Rat intestinal trehalase. Studies of the active site.

    PubMed

    Chen, C C; Guo, W J; Isselbacher, K J

    1987-11-01

    Rat intestinal trehalase was solubilized, purified and reconstituted into proteoliposomes. With octyl glucoside as the solubilizing detergent, the purified protein appeared as a single band on SDS/polyacrylamide-gel electrophoresis with an apparent molecular mass of 67 kDa. Kinetic studies indicated that the active site of this enzyme can be functionally divided into two adjacent regions, namely a binding site (with pKa 4.8) and a catalytic site (with pKa 7.2). Other findings suggested that the catalytic site contains a functional thiol group, which is sensitive to inhibition by N-ethylmaleimide, Hg2+ and iodoacetate. Substrate protection and iodoacetate labelling of the thiol group demonstrated that only a protein of 67 kDa was labelled. Furthermore, sucrose and phlorizin protected the thiol group, but Tris-like inhibitors did not. Structure-inhibition analysis of Tris-like inhibitors, the pH effect of Tris inhibition and Tris protection of 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide inactivation permitted characterization and location of a separate site containing a carboxy group for Tris binding, which may also be the binding region. On the basis of these findings, a possible structure for the active site of trehalase is proposed. PMID:3426558

  13. Active Site and Remote Contributions to Catalysis in Methylthioadenosine Nucleosidases

    PubMed Central

    Thomas, Keisha; Cameron, Scott A.; Almo, Steven C.; Burgos, Emmanuel S.; Gulab, Shivali A.; Schramm, Vern L.

    2015-01-01

    5′-Methylthioadenosine/S-adenosyl-L-homocysteine nucleosidases (MTANs) catalyze the hydrolysis of 5′-methylthioadenosine to adenine and 5-methylthioribose. The amino acid sequences of the MTANs from Vibrio cholerae (VcMTAN) and Escherichia coli (EcMTAN) are 60% identical and 75% similar. Protein structure folds and kinetic properties are similar. However, binding of transition-state analogues is dominated by favorable entropy in VcMTAN and by enthalpy in EcMTAN. Catalytic sites of VcMTAN and EcMTAN in contact with reactants differ by two residues; Ala113 and Val153 in VcMTAN are Pro113 and Ile152, respectively, in EcMTAN. We mutated the VcMTAN catalytic site residues to match those of EcMTAN in anticipation of altering its properties toward EcMTAN. Inhibition of VcMTAN by transition-state analogues required filling both active sites of the homodimer. However, in the Val153Ile mutant or double mutants, transition-state analogue binding at one site caused complete inhibition. Therefore, a single amino acid, Val153, alters the catalytic site cooperativity in VcMTAN. The transition-state analogue affinity and thermodynamics in mutant VcMTAN became even more unlike those of EcMTAN, the opposite of expectations from catalytic site similarity; thus, catalytic site contacts in VcMTAN are unable to recapitulate the properties of EcMTAN. X-ray crystal structures of EcMTAN, VcMTAN, and a multiple-site mutant of VcMTAN most closely resembling EcMTAN in catalytic site contacts show no major protein conformational differences. The overall protein architectures of these closely related proteins are implicated in contributing to the catalytic site differences. PMID:25806409

  14. Resonant active sites in catalytic ammonia synthesis: A structural model

    NASA Astrophysics Data System (ADS)

    Cholach, Alexander R.; Bryliakova, Anna A.; Matveev, Andrey V.; Bulgakov, Nikolai N.

    2016-03-01

    Adsorption sites Mn consisted of n adjacent atoms M, each bound to the adsorbed species, are considered within a realistic model. The sum of bonds Σ lost by atoms in a site in comparison with the bulk atoms was used for evaluation of the local surface imperfection, while the reaction enthalpy at that site was used as a measure of activity. The comparative study of Mn sites (n = 1-5) at basal planes of Pt, Rh, Ir, Fe, Re and Ru with respect to heat of N2 dissociative adsorption QN and heat of Nad + Had → NHad reaction QNH was performed using semi-empirical calculations. Linear QN(Σ) increase and QNH(Σ) decrease allowed to specify the resonant Σ for each surface in catalytic ammonia synthesis at equilibrium Nad coverage. Optimal Σ are realizable for Ru2, Re2 and Ir4 only, whereas other centers meet steric inhibition or unreal crystal structure. Relative activity of the most active sites in proportion 5.0 × 10- 5: 4.5 × 10- 3: 1: 2.5: 3.0: 1080: 2270 for a sequence of Pt4, Rh4, Fe4(fcc), Ir4, Fe2-5(bcc), Ru2, Re2, respectively, is in agreement with relevant experimental data. Similar approach can be applied to other adsorption or catalytic processes exhibiting structure sensitivity.

  15. Water in the Active Site of Ketosteroid Isomerase

    PubMed Central

    Hanoian, Philip; Hammes-Schiffer, Sharon

    2011-01-01

    Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two waters in the Y16S mutant, one water in the Y16F and FFF mutants, and intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of 1H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less

  16. Crystallographic Analysis of Active Site Contributions to Regiospecificity in the Diiron Enzyme Toluene 4-Monooxygenase

    SciTech Connect

    Bailey, Lucas J.; Acheson, Justin F.; McCoy, Jason G.; Elsen, Nathaniel L.; Phillips, Jr., George N.; Fox, Brian G.

    2014-10-02

    Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric {mu}-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors p-NH{sub 2}-benzoate and p-Br-benzoate showed a {mu}-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 and A107, while F196 positioned the aromatic ring against this surface via a {pi}-stacking interaction. The proximity of G103 and F176 to the para substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (<1 {angstrom}) were produced in F176, F196, and other interior residues of the active site. Likewise, products bound to the diiron center in either the presence or absence of effector protein did not significantly shift the position of the interior residues, suggesting that positioning of the cognate substrates will not be strongly influenced by effector protein binding. Thus, changes in product distributions in the absence of the effector protein are proposed to arise from differences in rates of chemical steps of the reaction relative to motion of substrates within the active site channel of the uncomplexed, less efficient enzyme, while structural changes in diiron ligand geometry associated with cycling between diferrous and diferric states are discussed for their potential

  17. Rapid deamination of cyclobutane pyrimidine dimer photoproducts at TCG sites in a translationally and rotationally positioned nucleosome in vivo.

    PubMed

    Cannistraro, Vincent J; Pondugula, Santhi; Song, Qian; Taylor, John-Stephen

    2015-10-30

    Sunlight-induced C to T mutation hot spots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C and 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by the DNA damage bypass polymerase η, thereby defining a probable mechanism for the origin of UV-induced C to T mutations. Deamination rates for T(m)CG CPDs have been found to vary 12-fold with rotational position in a nucleosome in vitro. To determine the influence of nucleosome structure on deamination rates in vivo, we determined the deamination rates of CPDs at TCG sites in a stably positioned nucleosome within the FOS promoter in HeLa cells. A procedure for in vivo hydroxyl radical footprinting with Fe-EDTA was developed, and, together with results from a cytosine methylation protection assay, we determined the translational and rotational positions of the TCG sites. Consistent with the in vitro observations, deamination was slower for one CPD located at an intermediate rotational position compared with two other sites located at outside positions, and all were much faster than for CPDs at non-TCG sites. Photoproduct formation was also highly suppressed at one site, possibly due to its interaction with a histone tail. Thus, it was shown that CPDs of TCG sites deaminate the fastest in vivo and that nucleosomes can modulate both their formation and deamination, which could contribute to the UV mutation hot spots and cold spots. PMID:26354431

  18. Energy transfer at the active sites of heme proteins

    SciTech Connect

    Dlott, D.D.; Hill, J.R.

    1995-12-31

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes.

  19. Chemical Modification of Papain and Subtilisin: An Active Site Comparison

    ERIC Educational Resources Information Center

    St-Vincent, Mireille; Dickman, Michael

    2004-01-01

    An experiment using methyle methanethiosulfonate (MMTS) and phenylmethylsulfonyl flouride (PMSF) to specifically modify the cysteine and serine residues in the active sites of papain and subtilism respectively is demonstrated. The covalent modification of these enzymes and subsequent rescue of papain shows the beginning biochemist that proteins…

  20. Changes in active site histidine hydrogen bonding trigger cryptochrome activation.

    PubMed

    Ganguly, Abir; Manahan, Craig C; Top, Deniz; Yee, Estella F; Lin, Changfan; Young, Michael W; Thiel, Walter; Crane, Brian R

    2016-09-01

    Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions. PMID:27551082

  1. Process evaluation of "Girls on the Run": exploring implementation in a physical activity-based positive youth development program.

    PubMed

    Iachini, Aidyn L; Beets, Michael W; Ball, Annahita; Lohman, Mary

    2014-10-01

    Many positive youth development programs rely on physical activity as a primary program component. Referred to as physical activity-based youth development programs, these program designs have great potential for promoting healthy youth development. This study examined how one such physical activity-based positive youth development program was implemented in order to identify design features critical to maximizing positive youth outcomes. This mixed method, multi-site process evaluation of Girls on the Run (GOTR) utilized focus groups, site visits, and self-report implementation checklists. Implementation scores were calculated to assess implementation fidelity across twenty-nine sites, and qualitative data were inductively analyzed to identify factors influential for implementation. Results reveal variability in how GOTR was implemented. Five themes emerged from the data that represented factors serving as facilitators or barriers to programmatic implementation. These included contextual/environmental factors (e.g., parental involvement, relationships with school personnel), organizational factors (e.g., implementation support and responsiveness of staff), program-specific factors (e.g., curriculum design), coach factors (e.g., existing relationships with participants, responsiveness to participant's needs), and youth factors (e.g., behavioral and discipline issues). Study findings have implications for improving the design of physical activity-based and other positive youth development programs, with relevance to evaluators, program planners, youth development leaders, and others working with children and youth. PMID:24858574

  2. Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor

    PubMed Central

    Spatzal, Thomas; Perez, Kathryn A; Howard, James B; Rees, Douglas C

    2015-01-01

    Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32–1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis. DOI: http://dx.doi.org/10.7554/eLife.11620.001 PMID:26673079

  3. Active Women: Perspectives on Their Structural Position in the Community.

    ERIC Educational Resources Information Center

    Moyer, Harriett; And Others

    An exploratory pilot study to determine the personal characteristics of women community leaders and their position in the power structure was conducted in Chippewa and Eau Claire Counties, Wisconsin. The research design involved a comparison of three samples: the traditional power structure identified through reputational techniques; the active…

  4. Positive Youth Development through Physical Activity: Opportunities for Physical Educators

    ERIC Educational Resources Information Center

    Hemphill, Michael A.

    2014-01-01

    As physical educators continue to advocate for school-based PE, they should also consider ways to extend their work into community settings in an effort to ensure that all kids have an opportunity to develop physical literacy. This article describes how positive youth development programs can provide an opportunity for physical educators to engage…

  5. Collecting "Total" Vocational Education and Training Activity. Position Paper

    ERIC Educational Resources Information Center

    Karmel, Tom

    2011-01-01

    In this position paper, NCVER's Managing Director, Dr Tom Karmel, argues that the submission of vocational education and training student data should be mandated as a condition of registration for all registered training organisations, including private providers. This will ensure a comprehensive data collection that gives a realistic view of…

  6. Active sites environmental monitoring program. Annual report FY 1992

    SciTech Connect

    Morrissey, C.M.; Ashwood, T.L.; Hicks, D.S.

    1994-04-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) at ORNL from October 1991 through September 1992. Solid Waste Operations and the Environmental Sciences Division established ASEMP in 1989 to provide early detection and performance monitoring at active low-level waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by Chapter 2 and 3 of US Department of Energy Order 5820.2A. The Interim Waste Management Facility (IWMF) began operation in December 1991. Monitoring results from the tumulus and IWMF disposal pads continue to indicate that no LLW is leaching from the storage vaults. Storm water falling on the IWMF active pad was collected and transported to the Process Waste Treatment Plant while operators awaited approval of the National Pollutant Discharge Elimination System (NPDES) permit. Several of the recent samples collected from the active IWMF pad had pH levels above the NPDES limit of 9.0 because of alkali leached from the concrete. The increase in gross beta activity has been slight; only 1 of the 21 samples collected contained activity above the 5.0 Bq/L action level. Automated sample-collection and flow-measurement equipment has been installed at IWMF and is being tested. The flume designed to electronically measure flow from the IWMF pads and underpads is too large to be of practical value for measuring most flows at this site. Modification of this system will be necessary. A CO{sub 2} bubbler system designed to reduce the pH of water from the pads is being tested at IWMF.

  7. Positive feedback of protein kinase C proteolytic activation during apoptosis.

    PubMed Central

    Leverrier, Sabrina; Vallentin, Alice; Joubert, Dominique

    2002-01-01

    In contrast with protein kinase Calpha (PKCalpha) and PKCepsilon, which are better known for promoting cell survival, PKCdelta is known for its pro-apoptotic function, which is exerted mainly through a caspase-3-dependent proteolytic activation pathway. In the present study, we used the rat GH3B6 pituitary adenoma cell line to show that PKCalpha and PKCepsilon are activated and relocalized together with PKCdelta when apoptosis is induced by a genotoxic stress. Proteolytic activation is a crucial step used by the three isoforms since: (1) the catalytic domains of the PKCalpha, PKCepsilon or PKCdelta isoforms (CDalpha, CDepsilon and CDdelta respectively) accumulated, and this accumulation was dependent on the activity of both calpain and caspase; and (2) transient expression of CDalpha, CDepsilon or CDdelta sufficed to induce apoptosis. However, following this initial step of proteolytic activation, the pathways diverge; cytochrome c release and caspase-3 activation are induced by CDepsilon and CDdelta, but not by CDalpha. Another interesting finding of the present study is the proteolysis of PKCdelta induced by CDepsilon expression that revealed the existence of a cross-talk between PKC isoforms during apoptosis. Hence the PKC family may participate in the apoptotic process of pituitary adenoma cells at two levels: downstream of caspase and calpain, and via retro-activation of caspase-3, resulting in the amplification of its own proteolytic activation. PMID:12238950

  8. Position paper on active countermeasures for computer networks.

    SciTech Connect

    Van Randwyk, Jamie A.

    2003-07-01

    Computer security professionals have used passive network countermeasures for several years in order to secure computer networks. Passive countermeasures such as firewalls and intrusion detection systems are effective but their use alone is not enough to protect a network. Active countermeasures offer new ways of protecting a computer network. Corporations and government entities should adopt active network countermeasures as a means of protecting their computer networks.

  9. Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation

    PubMed Central

    Zhang, Hua; Song, Lei; Cong, Haolong

    2015-01-01

    ABSTRACT Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5′ untranslated region (5′UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation. IMPORTANCE The nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection

  10. Active-Site-Accessible, Porphyrinic Metal;#8722;Organic Framework Materials

    SciTech Connect

    Farha, Omar K.; Shultz, Abraham M.; Sarjeant, Amy A.; Nguyen, SonBinh T.; Hupp, Joseph T.

    2012-02-06

    On account of their structural similarity to cofactors found in many metallo-enzymes, metalloporphyrins are obvious potential building blocks for catalytically active, metal-organic framework (MOF) materials. While numerous porphyrin-based MOFs have already been described, versions featuring highly accessible active sites and permanent microporosity are remarkably scarce. Indeed, of the more than 70 previously reported porphyrinic MOFs, only one has been shown to be both permanently microporous and contain internally accessible active sites for chemical catalysis. Attempts to generalize the design approach used in this single successful case have failed. Reported here, however, is the synthesis of an extended family of MOFs that directly incorporate a variety of metalloporphyrins (specifically Al{sup 3+}, Zn{sup 2+}, Pd{sup 2+}, Mn{sup 3+}, and Fe{sup 3+} complexes). These robust porphyrinic materials (RPMs) feature large channels and readily accessible active sites. As an illustrative example, one of the manganese-containing RPMs is shown to be catalytically competent for the oxidation of alkenes and alkanes.

  11. Nest predation increases with parental activity: Separating nest site and parental activity effects

    USGS Publications Warehouse

    Martin, T.E.; Scott, J.; Menge, C.

    2000-01-01

    Alexander Skutch hypothesized that increased parental activity can increase the risk of nest predation. We tested this hypothesis using ten open-nesting bird species in Arizona, USA. Parental activity was greater during the nestling than incubation stage because parents visited the nest frequently to feed their young during the nestling stage. However, nest predation did not generally increase with parental activity between nesting stages across the ten study species. Previous investigators have found similar results. We tested whether nest site effects might yield higher predation during incubation because the most obvious sites are depredated most rapidly. We conducted experiments using nest sites from the previous year to remove parental activity. Our results showed that nest sites have highly repeatable effects on nest predation risk; poor nest sites incurred rapid predation and caused predation rates to be greater during the incubation than nestling stage. This pattern also was exhibited in a bird species with similar (i.e. controlled) parental activity between nesting stages. Once nest site effects are taken into account, nest predation shows a strong proximate increase with parental activity during the nestling stage within and across species. Parental activity and nest sites exert antagonistic influences on current estimates of nest predation between nesting stages and both must be considered in order to understand current patterns of nest predation, which is an important source of natural selection.

  12. Nest predation increases with parental activity: separating nest site and parental activity effects.

    PubMed Central

    Martin, T E; Scott, J; Menge, C

    2000-01-01

    Alexander Skutch hypothesized that increased parental activity can increase the risk of nest predation. We tested this hypothesis using ten open-nesting bird species in Arizona, USA. Parental activity was greater during the nestling than incubation stage because parents visited the nest frequently to feed their young during the nestling stage. However, nest predation did not generally increase with parental activity between nesting stages across the ten study species. Previous investigators have found similar results. We tested whether nest site effects might yield higher predation during incubation because the most obvious sites are depredated most rapidly. We conducted experiments using nest sites from the previous year to remove parental activity. Our results showed that nest sites have highly repeatable effects on nest predation risk; poor nest sites incurred rapid predation and caused predation rates to be greater during the incubation than nestling stage. This pattern also was exhibited in a bird species with similar (i.e. controlled) parental activity between nesting stages. Once nest site effects are taken into account, nest predation shows a strong proximate increase with parental activity during the nestling stage within and across species. Parental activity and nest sites exert antagonistic influences on current estimates of nest predation between nesting stages and both must be considered in order to understand current patterns of nest predation, which is an important source of natural selection. PMID:11413645

  13. Involvement of I2-imidazoline binding sites in positive and negative morphine analgesia modulatory effects.

    PubMed

    Gentili, Francesco; Cardinaletti, Claudia; Carrieri, Antonio; Ghelfi, Francesca; Mattioli, Laura; Perfumi, Marina; Vesprini, Cristian; Pigini, Maria

    2006-12-28

    Some studies, suggesting the involvement of I(2)-imidazoline binding sites (I(2)-IBS) in morphine analgesia modulation, prompted us to examine on mice antinociceptive assays the effect produced by 1 (phenyzoline), that in view of its high I(2)-IBS affinity and high I(2)-IBS selectivity with regard to I(1)-IBS, alpha(2)-adrenoreceptors and mu-opioid receptors might be considered the first interesting I(2)-IBS ligand. The study was also applied to its ortho phenyl derivative 2 (diphenyzoline), designed and prepared in order to produce a possible modification of the biological profile of 1. Diphenyzoline (2) retains a significant I(2)-IBS selectivity with regard to I(1)-IBS, alpha(2)-adrenoreceptors and mu-opioid receptors. Moreover, by the functional assays 1 and 2 proved inactive at all alpha(2)-adrenoreceptors subtypes up to 10(-3) M. As expected, phenyzoline and diphenyzoline, which are structurally related, highlighted an interesting "positive" or "negative", respectively, morphine analgesia modulatory effect. In fact, 1 (s.c. 10 mg/kg) enhanced morphine analgesia (60% and 40% in mouse tail-flick and mouse hot-plate, respectively), while 2 (s.c. 10 mg/kg) decreased it (-41% and -20%, respectively). The ability to decrease morphine analgesia had never been observed before in I(2)-IBS ligands. These effects were not affected by i.p. treatment of animals with yohimbine (a selective alpha(2)-adrenoreceptor antagonist, 0.625 mg/kg) or efaroxan (an I(1)-IBS/alpha(2)-adrenoreceptor antagonist, 1.0 mg/kg). In contrast, they were completely reversed by i.p. treatment of animals with idazoxan (an I(2)-IBS/alpha(2)-adrenoreceptor antagonist, 2 mg/kg). Moreover, compound 2, in mouse tail-flick test, was able to potentiate by 23% the naloxone-induced decrease of morphine analgesia. Therefore, the results of this study indicate the crucial involvement of I(2)-IBS in the morphine analgesia modulatory effects of 1 and 2. PMID:17081513

  14. Localization of the active site of an enzyme, bacterial luciferase, using two-quantum affinity modification

    NASA Astrophysics Data System (ADS)

    Benimetskaya, L. Z.; Gitelzon, I. I.; Kozionov, Andrew L.; Novozhilov, S. Y.; Petushkov, V. N.; Rodionova, N. S.; Stockman, Mark I.

    1991-11-01

    For the first time the method of two-quantum affinity modification has been employed to probe the structure of an enzyme, bacterial luciferase. Position of the flavin-binding site of this enzyme, which was previously unknown, has been established. The obtained data indicate that the flavin site is positioned on the (alpha) -subunit. The closest contact of the protein chain of the enzyme with the chromophoric group of the flavin takes place near 80 +/- 10 and 120 +/- 10 amino acid residues; the regions 50 +/- 10 and 215 +/- 10 are also close to the flavin. The established localization does not contradict suggestions on positions of the flavin and phosphate sites of the bacterial luciferase, which had earlier been made from the data on evolutionary stability of various luciferases. The present method can, in principle, be applied to a great number of enzymes, including all flavin-dependent enzymes. Enzymatic catalysis has high speed and specificity. Creation of a method of determination of the elements of the primary structure of a protein, making up the active site (in which substratum conversion occurs), could be a significant advance in clearing up mechanisms of enzymatic catalysis. It was proposed to localize active sites of the enzymes, whose substrata are chromophores, using this method of two-quantum affinity modification. An enzyme- substratum complex is irradiated with laser light of sufficiently long wavelength ((lambda) 300 nm) which is not directly absorbed by the enzyme. Two-quantum quasiresonant excitation of the substratum activates it to the state with energy 5-7 eV, which is then radiativelessly transferred to neighboring protein groups. This energy exceeds the energy of activation of peptide bond breakage. Therefore, the enzyme will be disrupted in the vicinity of its active site. In the present paper the above approach has been implemented for the first time. Information has been obtained about the position of the flavin-binding site of bacterial

  15. Molecular Basis for Enzymatic Sulfite Oxidation -- HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY

    SciTech Connect

    Bailey, Susan; Rapson, Trevor; Johnson-Winters, Kayunta; Astashkin, Andrei; Enemark, John; Kappler, Ulrike

    2008-11-10

    Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (~;;60 and 200 s-1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

  16. Active Sites Environmental Monitoring Program. FY 1993: Annual report

    SciTech Connect

    Morrissey, C.M.; Ashwood, T.L.; Hicks, D.S.; Marsh, J.D.

    1994-08-01

    This report continues a series of annual and semiannual reports that present the results of the Active Sites Environmental Monitoring Program (ASEMP) monitoring activities. The report details monitoring data for fiscal year (FY) 1993 and is divided into three major areas: SWSA 6 [including tumulus pads, Interim Waste Management Facility (IWMF), and other sites], the low-level Liquid-Waste Solidification Project (LWSP), and TRU-waste storage facilities in SWSA 5 N. The detailed monitoring methodology is described in the second revision of the ASEMP program plan. This report also presents a summary of the methodology used to gather data for each major area along with the results obtained during FY 1993.

  17. Charged Nonclassical Antifolates with Activity Against Gram-Positive and Gram-Negative Pathogens.

    PubMed

    Scocchera, Eric; Reeve, Stephanie M; Keshipeddy, Santosh; Lombardo, Michael N; Hajian, Behnoush; Sochia, Adrienne E; Alverson, Jeremy B; Priestley, Nigel D; Anderson, Amy C; Wright, Dennis L

    2016-07-14

    Although classical, negatively charged antifolates such as methotrexate possess high affinity for the dihydrofolate reductase (DHFR) enzyme, they are unable to penetrate the bacterial cell wall, rendering them poor antibacterial agents. Herein, we report a new class of charged propargyl-linked antifolates that capture some of the key contacts common to the classical antifolates while maintaining the ability to passively diffuse across the bacterial cell wall. Eight synthesized compounds exhibit extraordinary potency against Gram-positive S. aureus with limited toxicity against mammalian cells and good metabolic profile. High resolution crystal structures of two of the compounds reveal extensive interactions between the carboxylate and active site residues through a highly organized water network. PMID:27437079

  18. Insight into the mechanism of phosphoenolpyruvate mutase catalysis derived from site-directed mutagenesis studies of active site residues.

    PubMed

    Jia, Y; Lu, Z; Huang, K; Herzberg, O; Dunaway-Mariano, D

    1999-10-26

    PEP mutase catalyzes the conversion of phosphoenolpyruvate (PEP) to phosphonopyruvate in biosynthetic pathways leading to phosphonate secondary metabolites. A recent X-ray structure [Huang, K., Li, Z., Jia, Y., Dunaway-Mariano, D., and Herzberg, O. (1999) Structure (in press)] of the Mytilus edulis enzyme complexed with the Mg(II) cofactor and oxalate inhibitor reveals an alpha/beta-barrel backbone-fold housing an active site in which Mg(II) is bound by the two carboxylate groups of the oxalate ligand and the side chain of D85 and, via bridging water molecules, by the side chains of D58, D85, D87, and E114. The oxalate ligand, in turn, interacts with the side chains of R159, W44, and S46 and the backbone amide NHs of G47 and L48. Modeling studies identified two feasible PEP binding modes: model A in which PEP replaces oxalate with its carboxylate group interacting with R159 and its phosphoryl group positioned close to D58 and Mg(II) shifting slightly from its original position in the crystal structure, and model B in which PEP replaces oxalate with its phosphoryl group interacting with R159 and Mg(II) retaining its original position. Site-directed mutagenesis studies of the key mutase active site residues (R159, D58, D85, D87, and E114) were carried out in order to evaluate the catalytic roles predicted by the two models. The observed retention of low catalytic activity in the mutants R159A, D85A, D87A, and E114A, coupled with the absence of detectable catalytic activity in D58A, was interpreted as evidence for model A in which D58 functions in nucleophilic catalysis (phosphoryl transfer), R159 functions in PEP carboxylate group binding, and the carboxylates of D85, D87 and E114 function in Mg(II) binding. These results also provide evidence against model B in which R159 serves to mediate the phosphoryl transfer. A catalytic motif, which could serve both the phosphoryl transfer and the C-C cleavage enzymes of the PEP mutase superfamily, is proposed. PMID:10571990

  19. Active sites in char gasification: Final technical report

    SciTech Connect

    Wojtowicz, M.; Lilly, W.D.; Perkins, M.T.; Hradil, G.; Calo, J.M.; Suuberg, E.M.

    1987-09-01

    Among the key variables in the design of gasifiers and combustors is the reactivity of the chars which must be gasified or combusted. Significant loss of unburned char is unacceptable in virtually any process; the provision of sufficient residence time for complete conversion is essential. A very wide range of reactivities are observed, depending upon the nature of the char in a process. The current work focuses on furthering the understanding of gasification reactivities of chars. It has been well established that the reactivity of char to gasification generally depends upon three principal factors: (1) the concentration of ''active sites'' in the char; (2) mass transfer within the char; and (3) the type and concentration of catalytic impurities in the char. The present study primarily addresses the first factor. The subject of this research is the origin, nature, and fate of active sites in chars derived from parent hydrocarbons with coal-like structure. The nature and number of the active sites and their reactivity towards oxygen are examined in ''model'' chars derived from phenol-formaldehyde type resins. How the active sites are lost by the process of thermal annealing during heat treatment of chars are studied, and actual rate for the annealing process is derived. Since intrinsic char reactivities are of primary interest in the present study, a fair amount of attention was given to the model char synthesis and handling so that the effect of catalytic impurities and oxygen-containing functional groups in the chemical structure of the material were minimized, if not completely eliminated. The project would not be considered complete without comparing characteristic features of synthetic chars with kinetic behavior exhibited by natural chars, including coal chars.

  20. Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase

    SciTech Connect

    Scott, A.I.; Roessner, C.A.; Stolowich, N.J.; Karuso, P.; Williams, H.J.; Grant, S.K.; Gonzalez, M.D.; Hoshino, T. )

    1988-10-18

    The active site of porphobilinogen (PBG){sup 1} deaminase from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-242, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA{sup {minus}} strain of E. coli the enzyme was enriched from (5-{sup 13}C)ALA and examined by {sup 1}H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked heat to tail and terminating in a CH{sub 2}-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-(2,11-{sup 13}C{sub 2})PBG reveals that the aminomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the {alpha}-free pyrrole. NMR spectroscopy of the ES{sub 2} complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the {alpha}-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.

  1. Brownian aggregation rate of colloid particles with several active sites

    SciTech Connect

    Nekrasov, Vyacheslav M.; Yurkin, Maxim A.; Chernyshev, Andrei V.; Polshchitsin, Alexey A.; Yakovleva, Galina E.; Maltsev, Valeri P.

    2014-08-14

    We theoretically analyze the aggregation kinetics of colloid particles with several active sites. Such particles (so-called “patchy particles”) are well known as chemically anisotropic reactants, but the corresponding rate constant of their aggregation has not yet been established in a convenient analytical form. Using kinematic approximation for the diffusion problem, we derived an analytical formula for the diffusion-controlled reaction rate constant between two colloid particles (or clusters) with several small active sites under the following assumptions: the relative translational motion is Brownian diffusion, and the isotropic stochastic reorientation of each particle is Markovian and arbitrarily correlated. This formula was shown to produce accurate results in comparison with more sophisticated approaches. Also, to account for the case of a low number of active sites per particle we used Monte Carlo stochastic algorithm based on Gillespie method. Simulations showed that such discrete model is required when this number is less than 10. Finally, we applied the developed approach to the simulation of immunoagglutination, assuming that the formed clusters have fractal structure.

  2. Systematic mutagenesis of the active site omega loop of TEM-1 beta-lactamase.

    PubMed Central

    Petrosino, J F; Palzkill, T

    1996-01-01

    Beta-Lactamase is a bacterial protein that provides resistance against beta-lactam antibiotics. TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Normally, this enzyme has high levels of hydrolytic activity for penicillins, but mutant beta-lactamases have evolved with activity toward a variety of beta-lactam antibiotics. It has been shown that active site substitutions are responsible for changes in the substrate specificity. Since mutant beta-lactamases pose a serious threat to antimicrobial therapy, the mechanisms by which mutations can alter the substrate specificity of TEM-1 beta-lactamase are of interest. Previously, screens of random libraries encompassing 31 of 55 active site amino acid positions enabled the identification of the residues responsible for maintaining the substrate specificity of TEM-1 beta-lactamase. In addition to substitutions found in clinical isolates, many other specificity-altering mutations were also identified. Interestingly, many nonspecific substitutions in the N-terminal half of the active site omega loop were found to increase ceftazidime hydrolytic activity and decrease ampicillin hydrolytic activity. To complete the active sight study, eight additional random libraries were constructed and screened for specificity-altering mutations. All additional substitutions found to alter the substrate specificity were located in the C-terminal half of the active site loop. These mutants, much like the N-terminal omega loop mutants, appear to be less stable than the wild-type enzyme. Further analysis of a 165-YYG-167 triple mutant, selected for high levels of ceftazidime hydrolytic activity, provides an example of the correlation which exists between enzyme instability and increased ceftazidime hydrolytic activity in the ceftazidime-selected omega loop mutants. PMID:8606154

  3. Computational and Biochemical Docking of the Irreversible Cocaine Analog RTI 82 Directly Demonstrates Ligand Positioning in the Dopamine Transporter Central Substrate-binding Site*

    PubMed Central

    Dahal, Rejwi Acharya; Pramod, Akula Bala; Sharma, Babita; Krout, Danielle; Foster, James D.; Cha, Joo Hwan; Cao, Jianjing; Newman, Amy Hauck; Lever, John R.; Vaughan, Roxanne A.; Henry, L. Keith

    2014-01-01

    The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3β-(p-chlorophenyl)tropane-2β-carboxylic acid, 4′-azido-3′-iodophenylethyl ester ([125I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [125I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors. PMID:25179220

  4. Computational and biochemical docking of the irreversible cocaine analog RTI 82 directly demonstrates ligand positioning in the dopamine transporter central substrate-binding site.

    PubMed

    Dahal, Rejwi Acharya; Pramod, Akula Bala; Sharma, Babita; Krout, Danielle; Foster, James D; Cha, Joo Hwan; Cao, Jianjing; Newman, Amy Hauck; Lever, John R; Vaughan, Roxanne A; Henry, L Keith

    2014-10-24

    The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3β-(p-chlorophenyl)tropane-2β-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([(125)I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [(125)I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors. PMID:25179220

  5. Plants Level of Chromium and Nickel at a Refuse Site, Any Positive Impact?

    NASA Astrophysics Data System (ADS)

    Ololade, I. A.; Ashoghon, A. O.; Adeyemi, O.

    Trace metals, including heavy metals can be dangerous to the biota and human beings. Consequently, a study of the accumulation of two unpopular heavy metals, Chromium (Cr) and Nickel (Ni), in four species of plants were carried out. At Ojota refuse sites (Old and New) in Lagos State, Nigeria, from where samples were taken; knowledge about these metals were scarce. The results obtained from the analysis of leaves and roots of plants showed that the sites were heavily polluted by chromium and nickel containing substances, which were indiscriminately dumped at the sites. Values were far above the background level with higher concentrations being recorded at the New Refuse Site (NRS). The concentrations obtained were also found to correlate strongly with the results of some soil physico-chemical properties, which were determined during the study. The plants used in the present research were observed to display a higher level of tolerance to metal concentration, an important characteristic of hyper-accumulator plants in phytoremediation study. Consequently, they are recommended for cultivation in non-grazing heavy metal polluted sites. However, livestock feedings and vegetable consumption at the present sites should be discouraged to avoid metal poisoning.

  6. Using the global positioning system in support of environmental characterization at the Hanford Site in Washington State

    SciTech Connect

    Peterson, L.B.; Tzemos, S.; Dietz, L.A.

    1993-10-01

    The US Department of Energy`s 1,450 km{sup 2} Hanford Site in southeastern Washington State accumulated hazardous wastes for more than 50 years. To support the Site`s mission of environmental restoration and cleanup, the Global Positioning System (GPS) is being used to verify waste site locations and provide location information for field samples. Collected GPS data are stored for use in the Hanford Geographic Information System (HGIS). The NAVSTAR GPS is a space-based electronic navigation and positioning system designed and operated by the US Department of Defense (DOD). The system consists of three major components: (1) the space segment, comprising 24 earth-orbiting satellites; (2) the control segment, made up of 5 control and monitoring stations placed around the globe; and (3) the user segment, which includes users worldwide. When declared fully operational by the DOD, the NAVSTAR GPS will allow users to identify their geographical position anywhere on earth at any time. There are no user fees for the service and anyone with a GPS receiver may use the system worldwide. The one major hindrance to the system is the DOD policy concerning a security option called Selective Availability (SA). Selective Availability affects the usability of the system by intentional manipulation of the GPS signals to degrade the accuracy of the user`s positions. The period and magnitude of degradation is solely a DOD privilege. The DOD policy on SA is to vary the error in position calculated from the Standard Positioning Service code to approximately 100 m root-mean squared (RMS). With SA on and other possible errors included, users may know their location to within a few hundred meters. While this accuracy is good for many applications, it is too inaccurate for others.

  7. Threshold occupancy and specific cation binding modes in the hammerhead ribozyme active site are required for active conformation

    PubMed Central

    Lee, Tai-Sung; Giambaşu, George M.; Sosa, Carlos P.; Martick, Monika; Scott, William G.; York, Darrin M.

    2009-01-01

    The relationship between formation of active in-line attack conformations and monovalent (Na+) and divalent (Mg2+) metal ion binding in the hammerhead ribozyme has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence or absence of a Mg2+ in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the hammerhead ribozyme folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of hammerhead ribozyme catalysis and support the supposition that Mg2+, in addition to stabilizing active conformations, plays a specific chemical role in catalysis. PMID:19265710

  8. Identification of active-site residues in protease 3C of hepatitis A virus by site-directed mutagenesis.

    PubMed Central

    Gosert, R; Dollenmaier, G; Weitz, M

    1997-01-01

    Picornavirus 3C proteases (3Cpro) are cysteine proteases related by amino acid sequence to trypsin-like serine proteases. Comparisons of 3Cpro of hepatitis A virus (HAV) to those of other picornaviruses have resulted in prediction of active-site residues: histidine at position 44 (H44), aspartic acid (D98), and cysteine (C172). To test whether these residues are key members of a putative catalytic triad, oligonucleotide-directed mutagenesis was targeted to 3Cpro in the context of natural polypeptide precursor P3. Autocatalytic processing of the polyprotein containing wild-type or variant 3Cpro was tested by in vivo expression of vaccinia virus-HAV chimeras in an animal cell-T7 hybrid system and by in vitro translation of corresponding RNAs. Comparison with proteins present in HAV-infected cells showed that both expression systems mimicked authentic polyprotein processing. Individual substitutions of H44 by tyrosine and of C172 by glycine or serine resulted in complete loss of the virus-specific proteolytic cascade. In contrast, a P3 polyprotein in which D98 was substituted by asparagine underwent only slightly delayed processing, while an additional substitution of valine (V47) by glycine within putative protein 3A caused a more pronounced loss of processing. Therefore, apparently H44 and C172 are active-site constituents whereas D98 is not. The results, furthermore, suggest that substitution of amino acid residues distant from polyprotein cleavage sites may reduce proteolytic activity, presumably by altering substrate conformation. PMID:9060667

  9. Wobble Pairs of the HDV Ribozyme Play Specific Roles in Stabilization of Active Site Dynamics

    PubMed Central

    Sripathi, Kamali N.; Banáš, Pavel; Reblova, Kamila; Šponer, Jiři; Otyepka, Michal

    2015-01-01

    The hepatitis delta virus (HDV) is the only known human pathogen whose genome contains a catalytic RNA motif (ribozyme). The overall architecture of the HDV ribozyme is that of a double-nested pseudoknot, with two GU pairs flanking the active site. Although extensive studies have shown that mutation of either wobble results in decreased catalytic activity, little work has focused on linking these mutations to specific structural effects on catalytic fitness. Here we use molecular dynamics simulations based on an activated structure to probe the active site dynamics as a result of wobble pair mutations. In both wild-type and mutant ribozymes, the in-line fitness of the active site (as a measure of catalytic proficiency) strongly depends on the presence of a C75(N3H3+)N1(O5′) hydrogen bond, which positions C75 as the general acid for the reaction. Our mutational analyses show that each GU wobble supports catalytically fit conformations in distinct ways; the reverse G25U20 wobble promotes high in-line fitness, high occupancy of the C75(N3H3+)G1(O5′) general-acid hydrogen bond and stabilization of the G1U37 wobble, while the G1U37 wobble acts more locally by stabilizing high in-line fitness and the C75(N3H3+)G1(O5′) hydrogen bond. We also find that stable type I A-minor and P1.1 hydrogen bonding above and below the active site, respectively, prevent local structural disorder from spreading and disrupting global conformation. Taken together, our results define specific, often redundant architectural roles for several structural motifs of the HDV ribozyme active site, expanding the known roles of these motifs within all HDV-like ribozymes and other structured RNAs. PMID:25631765

  10. Molecular Evolution and Circulation Patterns of Human Respiratory Syncytial Virus Subgroup A: Positively Selected Sites in the Attachment G Glycoprotein

    PubMed Central

    Zlateva, Kalina T.; Lemey, Philippe; Vandamme, Anne-Mieke; Van Ranst, Marc

    2004-01-01

    Human respiratory syncytial virus (HRSV) is the most common etiological agent of acute lower respiratory tract disease in infants and can cause repeated infections throughout life. In this study, we have analyzed nucleotide sequences encompassing 629 bp at the carboxy terminus of the G glycoprotein gene for HRSV subgroup A strains isolated over 47 years, including 112 Belgian strains isolated over 19 consecutive years (1984 to 2002). By using a maximum likelihood method, we have tested the presence of diversifying selection and identified 13 positively selected sites with a posterior probability above 0.5. The sites under positive selection correspond to sites of O glycosylation or to amino acids that were previously described as monoclonal antibody-induced in vitro escape mutants. Our findings suggest that the evolution of subgroup A HRSV G glycoprotein is driven by immune pressure operating in certain codon positions located mainly in the second hypervariable region of the ectodomain. Phylogenetic analysis revealed the prolonged cocirculation of two subgroup A lineages among the Belgian population and the possible extinction of three other lineages. The evolutionary rate of HRSV subgroup A isolates was estimated to be 1.83 × 10−3 nucleotide substitutions/site/year, projecting the most recent common ancestor back to the early 1940s. PMID:15078950

  11. Current activities handbook: formerly utilized sites remedial action program

    SciTech Connect

    1981-02-27

    This volume is one of a series produced under contract with the DOE, by Politech Corporation to develop a legislative and regulatory data base to assist the FUSRAP management in addressing the institutional and socioeconomic issues involved in carrying out the Formerly Utilized Sites Remedial Action Program. This Information Handbook series contains information about all relevant government agencies at the Federal and state levels, the pertinent programs they administer, each affected state legislature, and current Federal and state legislative and regulatory initiatives. This volume is a compilation of information about the activities each of the thirteen state legislatures potentially affected by the Formerly Utilized Sites Remedial Action Program. It contains a description of the state legislative procedural rules and a schedule of each legislative session; a summary of pending relevant legislation; the name and telephone number of legislative and state agency contacts; and the full text of all bills identified.

  12. Activity of T-DM1 in Her2-positive breast cancer brain metastases.

    PubMed

    Bartsch, Rupert; Berghoff, Anna S; Vogl, Ursula; Rudas, Margaretha; Bergen, Elisabeth; Dubsky, Peter; Dieckmann, Karin; Pinker, Katja; Bago-Horvath, Zsuzsanna; Galid, Arik; Oehler, Leopold; Zielinski, Christoph C; Gnant, Michael; Steger, Guenther G; Preusser, Matthias

    2015-10-01

    Brain metastases (BM) are frequently diagnosed in metastatic Her2-positive breast cancer. Local treatment remains the standard of care but lapatinib plus capecitabine was recently established as systemic therapy option. Due to a disruption of the blood-brain/tumour-barrier at metastatic sites, even large molecules may penetrate into the central nervous system (CNS). Here, we report on the activity of T-DM1 in Her2-positive breast cancer BM. T-DM1 was administered at a dose of 3.6 mg once every 3 weeks as primary systemic therapy for BM or upon documented CNS progression after initial local treatment. Thus, this study allowed for the appraisal of T-DM1 activity in BM. Restaging was conducted every 12 weeks with MRI or whenever symptoms of disease progression occurred. Ten patients were included; in two asymptomatic subjects, T-DM1 was administered as primary therapy, while eight had progressive BM. All patients had received prior treatment with trastuzumab, six had already received lapatinib, and three pertuzumab as well. Three patients had partial remission of BM, and two patient had stable disease lasting for ≥6 months; two further patients had stable disease for <6 months while three progressed despite treatment. At 8.5 months median follow-up, intracranial PFS was 5 months, and median OS from initiation of T-DM1 was not reached. Local treatment of BM remains the standard of care; lapatinib plus capecitabine is currently the best established systemic therapy option. Still, T-DM1 apparently offers relevant clinical activity in BM and further investigation is warranted. PMID:26303828

  13. Use of an Improved Matching Algorithm to Select Scaffolds for Enzyme Design Based on a Complex Active Site Model.

    PubMed

    Huang, Xiaoqiang; Xue, Jing; Lin, Min; Zhu, Yushan

    2016-01-01

    Active site preorganization helps native enzymes electrostatically stabilize the transition state better than the ground state for their primary substrates and achieve significant rate enhancement. In this report, we hypothesize that a complex active site model for active site preorganization modeling should help to create preorganized active site design and afford higher starting activities towards target reactions. Our matching algorithm ProdaMatch was improved by invoking effective pruning strategies and the native active sites for ten scaffolds in a benchmark test set were reproduced. The root-mean squared deviations between the matched transition states and those in the crystal structures were < 1.0 Å for the ten scaffolds, and the repacking calculation results showed that 91% of the hydrogen bonds within the active sites are recovered, indicating that the active sites can be preorganized based on the predicted positions of transition states. The application of the complex active site model for de novo enzyme design was evaluated by scaffold selection using a classic catalytic triad motif for the hydrolysis of p-nitrophenyl acetate. Eighty scaffolds were identified from a scaffold library with 1,491 proteins and four scaffolds were native esterase. Furthermore, enzyme design for complicated substrates was investigated for the hydrolysis of cephalexin using scaffold selection based on two different catalytic motifs. Only three scaffolds were identified from the scaffold library by virtue of the classic catalytic triad-based motif. In contrast, 40 scaffolds were identified using a more flexible, but still preorganized catalytic motif, where one scaffold corresponded to the α-amino acid ester hydrolase that catalyzes the hydrolysis and synthesis of cephalexin. Thus, the complex active site modeling approach for de novo enzyme design with the aid of the improved ProdaMatch program is a promising approach for the creation of active sites with high catalytic

  14. Use of an Improved Matching Algorithm to Select Scaffolds for Enzyme Design Based on a Complex Active Site Model

    PubMed Central

    Huang, Xiaoqiang; Xue, Jing; Lin, Min; Zhu, Yushan

    2016-01-01

    Active site preorganization helps native enzymes electrostatically stabilize the transition state better than the ground state for their primary substrates and achieve significant rate enhancement. In this report, we hypothesize that a complex active site model for active site preorganization modeling should help to create preorganized active site design and afford higher starting activities towards target reactions. Our matching algorithm ProdaMatch was improved by invoking effective pruning strategies and the native active sites for ten scaffolds in a benchmark test set were reproduced. The root-mean squared deviations between the matched transition states and those in the crystal structures were < 1.0 Å for the ten scaffolds, and the repacking calculation results showed that 91% of the hydrogen bonds within the active sites are recovered, indicating that the active sites can be preorganized based on the predicted positions of transition states. The application of the complex active site model for de novo enzyme design was evaluated by scaffold selection using a classic catalytic triad motif for the hydrolysis of p-nitrophenyl acetate. Eighty scaffolds were identified from a scaffold library with 1,491 proteins and four scaffolds were native esterase. Furthermore, enzyme design for complicated substrates was investigated for the hydrolysis of cephalexin using scaffold selection based on two different catalytic motifs. Only three scaffolds were identified from the scaffold library by virtue of the classic catalytic triad-based motif. In contrast, 40 scaffolds were identified using a more flexible, but still preorganized catalytic motif, where one scaffold corresponded to the α-amino acid ester hydrolase that catalyzes the hydrolysis and synthesis of cephalexin. Thus, the complex active site modeling approach for de novo enzyme design with the aid of the improved ProdaMatch program is a promising approach for the creation of active sites with high catalytic

  15. Detecting grizzly bear use of ungulate carcasses using global positioning system telemetry and activity data.

    PubMed

    Ebinger, Michael R; Haroldson, Mark A; van Manen, Frank T; Costello, Cecily M; Bjornlie, Daniel D; Thompson, Daniel J; Gunther, Kerry A; Fortin, Jennifer K; Teisberg, Justin E; Pils, Shannon R; White, P J; Cain, Steven L; Cross, Paul C

    2016-07-01

    Global positioning system (GPS) wildlife collars have revolutionized wildlife research. Studies of predation by free-ranging carnivores have particularly benefited from the application of location clustering algorithms to determine when and where predation events occur. These studies have changed our understanding of large carnivore behavior, but the gains have concentrated on obligate carnivores. Facultative carnivores, such as grizzly/brown bears (Ursus arctos), exhibit a variety of behaviors that can lead to the formation of GPS clusters. We combined clustering techniques with field site investigations of grizzly bear GPS locations (n = 732 site investigations; 2004-2011) to produce 174 GPS clusters where documented behavior was partitioned into five classes (large-biomass carcass, small-biomass carcass, old carcass, non-carcass activity, and resting). We used multinomial logistic regression to predict the probability of clusters belonging to each class. Two cross-validation methods-leaving out individual clusters, or leaving out individual bears-showed that correct prediction of bear visitation to large-biomass carcasses was 78-88 %, whereas the false-positive rate was 18-24 %. As a case study, we applied our predictive model to a GPS data set of 266 bear-years in the Greater Yellowstone Ecosystem (2002-2011) and examined trends in carcass visitation during fall hyperphagia (September-October). We identified 1997 spatial GPS clusters, of which 347 were predicted to be large-biomass carcasses. We used the clustered data to develop a carcass visitation index, which varied annually, but more than doubled during the study period. Our study demonstrates the effectiveness and utility of identifying GPS clusters associated with carcass visitation by a facultative carnivore. PMID:26971522

  16. Detecting grizzly bear use of ungulate carcasses using global positioning system telemetry and activity data

    USGS Publications Warehouse

    Ebinger, Michael R.; Haroldson, Mark A.; van Manen, Frank T.; Costello, Cecily M; Bjornlie, Daniel D; Thompson, Daniel J.; Gunther, Kerry A.; Fortin, Jennifer K.; Teisberg, Justin E.; Pils, Shannon R; White, P J; Cain, Steven L; Cross, Paul C.

    2016-01-01

    Global positioning system (GPS) wildlife collars have revolutionized wildlife research. Studies of predation by free-ranging carnivores have particularly benefited from the application of location clustering algorithms to determine when and where predation events occur. These studies have changed our understanding of large carnivore behavior, but the gains have concentrated on obligate carnivores. Facultative carnivores, such as grizzly/brown bears (Ursus arctos), exhibit a variety of behaviors that can lead to the formation of GPS clusters. We combined clustering techniques with field site investigations of grizzly bear GPS locations (n = 732 site investigations; 2004–2011) to produce 174 GPS clusters where documented behavior was partitioned into five classes (large-biomass carcass, small-biomass carcass, old carcass, non-carcass activity, and resting). We used multinomial logistic regression to predict the probability of clusters belonging to each class. Two cross-validation methods—leaving out individual clusters, or leaving out individual bears—showed that correct prediction of bear visitation to large-biomass carcasses was 78–88%, whereas the false-positive rate was 18–24%. As a case study, we applied our predictive model to a GPS data set of 266 bear-years in the Greater Yellowstone Ecosystem (2002–2011) and examined trends in carcass visitation during fall hyperphagia (September–October). We identified 1997 spatial GPS clusters, of which 347 were predicted to be large-biomass carcasses. We used the clustered data to develop a carcass visitation index, which varied annually, but more than doubled during the study period. Our study demonstrates the effectiveness and utility of identifying GPS clusters associated with carcass visitation by a facultative carnivore.

  17. Promoting Teachers' Positive Attitude towards Web Use: A Study in Web Site Development

    ERIC Educational Resources Information Center

    Akpinar, Yavuz; Bayramoglu, Yusuf

    2008-01-01

    The purpose of the study was to examine effects of a compact training for developing web sites on teachers' web attitude, as composed of: web self efficacy, perceived web enjoyment, perceived web usefulness and behavioral intention to use the web. To measure the related constructs, the Web Attitude Scale was adapted into Turkish and tested with a…

  18. Differential behavior of the sub-sites of cytochrome 450 active site in binding of substrates, and products (implications for coupling/uncoupling).

    PubMed

    Narasimhulu, Shakunthala

    2007-03-01

    The cytochrome P450 catalyzes hydroxylation of many substrates in the presence of O(2) and specific electron transport system. The ternary complex S-Fe(+)O(2) with substrate and O(2) bound to their respective sites on the reduced enzyme is an important intermediate in the formation of the hydroxylating species. Then the active site may be considered as having two sub-sites geared for entirely different types of functionally relevant interactions. The two sites are the substrate binding site, the specific protein residues (Site I), and the L(6) position of the iron (Site II) to which O(2) binds upon reduction. In the ferric enzyme, when substrate binds to Site I, the low spin six-coordinated P450 is converted to the readily reducible high spin five coordinated state. Certain amines and OH compounds, such as products of P450-catalyzed reactions, can bind to Site II resulting in six coordinated inhibited complexes. Then the substrate and product interactions with the two sub-sites can regulate the functional state of the enzyme during catalysis. Product interactions have received very little attention. CYP101 is the only P450 in which X-ray and spectroscopic data on all three structures, the substrate-free, camphor-bound and the 5-exo-OHcamphor-bound are available. The substrate-free CYP101 is low spin and six-coordinated with a water molecule ligated at the L(6) position of the iron. The substrate camphor binds to Site I, and releases the L(6) water despite its inability to bind to this site, indicating that Site I binding can inhibit Site II ligation. The product 5-exo-OHcamphor in addition to binding to Site I, binds to Site II through its -OH group forming Fe-O bond, resulting in the low spin six-coordinated complex. New temperature-jump relaxation kinetic data indicating that Site II ligation inhibits Site I binding are presented. It appears that the Site I and Site II function as interacting sub-sites. The inhibitory allosteric interactions between the two sub-sites

  19. Identification of covalent active site inhibitors of dengue virus protease

    PubMed Central

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

  20. Mutation at positively selected positions in the binding site for HLA-C shows that KIR2DL1 is a more refined but less adaptable NK cell receptor than KIR2DL3.

    PubMed

    Hilton, Hugo G; Vago, Luca; Older Aguilar, Anastazia M; Moesta, Achim K; Graef, Thorsten; Abi-Rached, Laurent; Norman, Paul J; Guethlein, Lisbeth A; Fleischhauer, Katharina; Parham, Peter

    2012-08-01

    Through recognition of HLA class I, killer cell Ig-like receptors (KIR) modulate NK cell functions in human immunity and reproduction. Although a minority of HLA-A and -B allotypes are KIR ligands, HLA-C allotypes dominate this regulation, because they all carry either the C1 epitope recognized by KIR2DL2/3 or the C2 epitope recognized by KIR2DL1. The C1 epitope and C1-specific KIR evolved first, followed several million years later by the C2 epitope and C2-specific KIR. Strong, varying selection pressure on NK cell functions drove the diversification and divergence of hominid KIR, with six positions in the HLA class I binding site of KIR being targets for positive diversifying selection. Introducing each naturally occurring residue at these positions into KIR2DL1 and KIR2DL3 produced 38 point mutants that were tested for binding to 95 HLA- A, -B, and -C allotypes. Modulating specificity for HLA-C is position 44, whereas positions 71 and 131 control cross-reactivity with HLA-A*11:02. Dominating avidity modulation is position 70, with lesser contributions from positions 68 and 182. KIR2DL3 has lower avidity and broader specificity than KIR2DL1. Mutation could increase the avidity and change the specificity of KIR2DL3, whereas KIR2DL1 specificity was resistant to mutation, and its avidity could only be lowered. The contrasting inflexibility of KIR2DL1 and adaptability of KIR2DL3 fit with C2-specific KIR having evolved from C1-specific KIR, and not vice versa. Substitutions restricted to activating KIR all reduced the avidity of KIR2DL1 and KIR2DL3, further evidence that activating KIR function often becomes subject to selective attenuation. PMID:22772445

  1. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer.

    PubMed

    Dinpajooh, Mohammadhasan; Martin, Daniel R; Matyushov, Dmitry V

    2016-01-01

    Enzymes in biology's energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work. PMID:27306204

  2. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer

    NASA Astrophysics Data System (ADS)

    Dinpajooh, Mohammadhasan; Martin, Daniel R.; Matyushov, Dmitry V.

    2016-06-01

    Enzymes in biology’s energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work.

  3. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer

    PubMed Central

    Dinpajooh, Mohammadhasan; Martin, Daniel R.; Matyushov, Dmitry V.

    2016-01-01

    Enzymes in biology’s energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work. PMID:27306204

  4. The copper active site of CBM33 polysaccharide oxygenases.

    PubMed

    Hemsworth, Glyn R; Taylor, Edward J; Kim, Robbert Q; Gregory, Rebecca C; Lewis, Sally J; Turkenburg, Johan P; Parkin, Alison; Davies, Gideon J; Walton, Paul H

    2013-04-24

    The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme's three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61. PMID:23540833

  5. The Copper Active Site of CBM33 Polysaccharide Oxygenases

    PubMed Central

    2013-01-01

    The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme’s three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61. PMID:23540833

  6. Alkyl isocyanates as active site-directed inactivators of guinea pig liver transglutaminase.

    PubMed

    Gross, M; Whetzel, N K; Folk, J E

    1975-10-10

    Alkyl isocyanates are effective inactivators of guinea pig liver transglutaminase. Based on the specificity of the reaction the protection against inactivation by glutamine substrate, and the essential nature of calcium for the inactivation reaction, it is concluded that these reagents act as amide substrate analogs and, thus function in an active site-specific manner. Support for the contention that inactivation results from alkyl thiocarbamate ester formation through the single active site sulfhydryl group of the enzyme is (a) the loss of one free--SH group and the incorporation of 1 mol of reagent/mol of enzyme in the reaction, (b) similarity in chemical properties of the inactive enzyme derivative formed to those previously reported for another alkyl thiocarbamoylenzyme and an alkyl thiocarbamoylcysteine derivative, and (c) the finding that labeled peptides from digests of [methyl-14C]thiocarbamoyltransglutaminase and those from digests of iodoacetamide-inactivated enzyme occupy similar positions on peptide maps. Transglutaminase was found to be inactivated neither by urethan anlogs of its active ester substrates nor by urea analogs of its amide substrates. It is concluded on the basis of these findings that inactive carbamoylenzyme derivatives are formed only by direct addition of the transglutaminase active--SH group to the isocyanate C--N double bond, and not, like several serine active site enzymes, by nucleophilic displacement with urethan analogs of substrate, or by nucleophilic displacement with urea analogs of substrate. PMID:240837

  7. Brain activations during judgments of positive self-conscious emotion and positive basic emotion: pride and joy.

    PubMed

    Takahashi, Hidehiko; Matsuura, Masato; Koeda, Michihiko; Yahata, Noriaki; Suhara, Tetsuya; Kato, Motoichiro; Okubo, Yoshiro

    2008-04-01

    We aimed to investigate the neural correlates associated with judgments of a positive self-conscious emotion, pride, and elucidate the difference between pride and a basic positive emotion, joy, at the neural basis level using functional magnetic resonance imaging. Study of the neural basis associated with pride might contribute to a better understanding of the pride-related behaviors observed in neuropsychiatric disorders. Sixteen healthy volunteers were studied. The participants read sentences expressing joy or pride contents during the scans. Pride conditions activated the right posterior superior temporal sulcus and left temporal pole, the regions implicated in the neural substrate of social cognition or theory of mind. However, against our prediction, we did not find brain activation in the medial prefrontal cortex, a region responsible for inferring others' intention or self-reflection. Joy condition produced activations in the ventral striatum and insula/operculum, the key nodes of processing of hedonic or appetitive stimuli. Our results support the idea that pride is a self-conscious emotion, requiring the ability to detect the intention of others. At the same time, judgment of pride might require less self-reflection compared with those of negative self-conscious emotions such as guilt or embarrassment. PMID:17638925

  8. An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis

    SciTech Connect

    Eren, Elif; Murphy, Megan; Goguen, Jon; van den Berg, Bert

    2010-08-13

    The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 {angstrom}. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.

  9. Target-classification approach applied to active UXO sites

    NASA Astrophysics Data System (ADS)

    Shubitidze, F.; Fernández, J. P.; Shamatava, Irma; Barrowes, B. E.; O'Neill, K.

    2013-06-01

    This study is designed to illustrate the discrimination performance at two UXO active sites (Oklahoma's Fort Sill and the Massachusetts Military Reservation) of a set of advanced electromagnetic induction (EMI) inversion/discrimination models which include the orthonormalized volume magnetic source (ONVMS), joint diagonalization (JD), and differential evolution (DE) approaches and whose power and flexibility greatly exceed those of the simple dipole model. The Fort Sill site is highly contaminated by a mix of the following types of munitions: 37-mm target practice tracers, 60-mm illumination mortars, 75-mm and 4.5'' projectiles, 3.5'', 2.36'', and LAAW rockets, antitank mine fuzes with and without hex nuts, practice MK2 and M67 grenades, 2.5'' ballistic windshields, M2A1-mines with/without bases, M19-14 time fuzes, and 40-mm practice grenades with/without cartridges. The site at the MMR site contains targets of yet different sizes. In this work we apply our models to EMI data collected using the MetalMapper (MM) and 2 × 2 TEMTADS sensors. The data for each anomaly are inverted to extract estimates of the extrinsic and intrinsic parameters associated with each buried target. (The latter include the total volume magnetic source or NVMS, which relates to size, shape, and material properties; the former includes location, depth, and orientation). The estimated intrinsic parameters are then used for classification performed via library matching and the use of statistical classification algorithms; this process yielded prioritized dig-lists that were submitted to the Institute for Defense Analyses (IDA) for independent scoring. The models' classification performance is illustrated and assessed based on these independent evaluations.

  10. Spectroscopic Definition of the Ferroxidase Site in M Ferritin: Comparison of Binuclear Substrate vs. Cofactor Active Sites

    PubMed Central

    Schwartz, Jennifer K.; Liu, Xiaofeng S.; Tosha, Takehiko; Theil, Elizabeth C.; Solomon, Edward I.

    2008-01-01

    Maxi ferritins, 24 subunit protein nanocages, are essential in humans, plants, bacteria, and other animals for the concentration and storage of iron as hydrated ferric oxide, while minimizing free radical generation or use by pathogens. Formation of the precursors to these ferric oxides is catalyzed at a non-heme biferrous substrate site, which has some parallels with the cofactor sites in other biferrous enzymes. A combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) has been used to probe Fe(II) binding to the substrate active site in frog M ferritin. These data determined that the active site within each subunit consists of two inequivalent five-coordinate (5C) ferrous centers that are weakly anti-ferromagnetically coupled, consistent with a μ-1,3 carboxylate bridge. The active site ligand set is unusual and likely includes a terminal water bound to each Fe(II) center. The Fe(II) ions bind to the active sites in a concerted manner, and cooperativity among the sites in each subunit is observed, potentially providing a mechanism for the control of ferritin iron loading. Differences in geometric and electronic structure – including a weak ligand field, availability of two water ligands at the biferrous substrate site, and the single carboxylate bridge in ferritin – coincide with the divergent reaction pathways observed between this substrate site and the previously studied cofactor active sites. PMID:18576633

  11. Metal active site elasticity linked to activation of homocysteine in methionine synthases

    SciTech Connect

    Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.; Matthews, Rowena G.; Smith, Janet L.; Ludwig, Martha L.

    2008-04-02

    Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.

  12. Evidence for segmental mobility in the active site of pepsin

    SciTech Connect

    Pohl, J.; Strop, P.; Senn, H.; Foundling, S.; Kostka, V.

    1986-05-01

    The low hydrolytic activity (k/sub cat/ < 0.001 s/sup -1/) of chicken pepsin (CP) towards tri- and tetrapeptides is enhanced at least 100 times by modification of its single sulfhydryl group of Cys-115, with little effect on K/sub m/-values. Modification thus simulates the effect of secondary substrate binding on pepsin catalysis. The rate of Cys-115 modification is substantially decreased in the presence of some competitive inhibitors, suggesting its active site location. Experiments with CP alkylated at Cys-115 with Acrylodan as a fluorescent probe or with N-iodoacetyl-(4-fluoro)-aniline as a /sup 19/F-nmr probe suggest conformation change around Cys-115 to occur on substrate or substrate analog binding. The difference /sup 1/H-nmr spectra (500 MHz) of unmodified free and inhibitor-complexed CP reveal chemical shifts almost exclusively in the aromatic region. The effects of Cu/sup + +/ on /sup 19/F- and /sup 1/H-nmr spectra have been studied. Examination of a computer graphics model of CP based on E. parasitica pepsin-inhibitor complex X-ray coordinates suggests that Cys-115 is located near the S/sub 3//S/sub 5/ binding site. The results are interpreted in favor of segmental mobility of this region important for pepsin substrate binding and catalysis.

  13. Eel calcitonin binding site distribution and antinociceptive activity in rats

    SciTech Connect

    Guidobono, F.; Netti, C.; Sibilia, V.; Villa, I.; Zamboni, A.; Pecile, A.

    1986-03-01

    The distribution of binding site for (/sup 125/I)-eel-calcitonin (ECT) to rat central nervous system, studied by an autoradiographic technique, showed concentrations of binding in the diencephalon, the brain stem and the spinal cord. Large accumulations of grains were seen in the hypothalamus, the amygdala, in the fasciculus medialis prosencephali, in the fasciculus longitudinalis medialis, in the ventrolateral part of the periventricular gray matter, in the lemniscus medialis and in the raphe nuclei. The density of grains in the reticular formation and in the nucleus tractus spinalis nervi trigemini was more moderate. In the spinal cord, grains were scattered throughout the dorsal horns. Binding of the ligand was displaced equally by cold ECT and by salmon CT(sCT), indicating that both peptides bind to the same receptors. Human CT was much weaker than sCT in displacing (/sup 125/I)-ECT binding. The administration of ECT into the brain ventricles of rats dose-dependently induced a significant and long-lasting enhancement of hot-plate latencies comparable with that obtained with sCT. The antinociceptive activity induced by ECT is compatible with the topographical distribution of binding sites for the peptide and is a further indication that fish CTs are active in the mammalian brain.

  14. Molecular Cloning, Characterization and Positively Selected Sites of the Glutathione S-Transferase Family from Locusta migratoria

    PubMed Central

    Zhang, Min; Qin, Guohua; Li, Daqi; Zhu, Kun Yan; Ma, Enbo; Zhang, Jianzhen

    2014-01-01

    Glutathione S-transferases (GSTs) are multifunctional enzymes that are involved in the metabolism of endogenous and exogenous compounds and are related to insecticide resistance. The purpose of this study was to provide new information on the molecular characteristics and the positive selection of locust GSTs. Based on the transcriptome database, we sequenced 28 cytosolic GSTs and 4 microsomal GSTs from the migratory locust (Locusta migratoria). We assigned the 28 cytosolic GSTs into 6 classes—sigma, epsilon, delta, theta, omega and zeta, and the 4 microsomal GSTs into 2 subclasses—insect and MGST3. The tissue- and stage-expression patterns of the GSTs differed at the mRNA level. Further, the substrate specificities and kinetic constants of the cytosolic GSTs differed markedly at the protein level. The results of likelihood ratio tests provided strong evidence for positive selection in the delta class. The result of Bayes Empirical Bayes analysis identified 4 amino acid sites in the delta class as positive selection sites. These sites were located on the protein surface. Our findings will facilitate the elucidation of the molecular characteristics and evolutionary aspects of insect GST superfamily. PMID:25486043

  15. Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity.

    PubMed

    Panayiotou, Richard; Miralles, Francesc; Pawlowski, Rafal; Diring, Jessica; Flynn, Helen R; Skehel, Mark; Treisman, Richard

    2016-01-01

    The myocardin-related transcription factors (MRTF-A and MRTF-B) regulate cytoskeletal genes through their partner transcription factor SRF. The MRTFs bind G-actin, and signal-regulated changes in cellular G-actin concentration control their nuclear accumulation. The MRTFs also undergo Rho- and ERK-dependent phosphorylation, but the function of MRTF phosphorylation, and the elements and signals involved in MRTF-A nuclear export are largely unexplored. We show that Rho-dependent MRTF-A phosphorylation reflects relief from an inhibitory function of nuclear actin. We map multiple sites of serum-induced phosphorylation, most of which are S/T-P motifs and show that S/T-P phosphorylation is required for transcriptional activation. ERK-mediated S98 phosphorylation inhibits assembly of G-actin complexes on the MRTF-A regulatory RPEL domain, promoting nuclear import. In contrast, S33 phosphorylation potentiates the activity of an autonomous Crm1-dependent N-terminal NES, which cooperates with five other NES elements to exclude MRTF-A from the nucleus. Phosphorylation thus plays positive and negative roles in the regulation of MRTF-A. PMID:27304076

  16. Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity

    PubMed Central

    Panayiotou, Richard; Miralles, Francesc; Pawlowski, Rafal; Diring, Jessica; Flynn, Helen R; Skehel, Mark; Treisman, Richard

    2016-01-01

    The myocardin-related transcription factors (MRTF-A and MRTF-B) regulate cytoskeletal genes through their partner transcription factor SRF. The MRTFs bind G-actin, and signal-regulated changes in cellular G-actin concentration control their nuclear accumulation. The MRTFs also undergo Rho- and ERK-dependent phosphorylation, but the function of MRTF phosphorylation, and the elements and signals involved in MRTF-A nuclear export are largely unexplored. We show that Rho-dependent MRTF-A phosphorylation reflects relief from an inhibitory function of nuclear actin. We map multiple sites of serum-induced phosphorylation, most of which are S/T-P motifs and show that S/T-P phosphorylation is required for transcriptional activation. ERK-mediated S98 phosphorylation inhibits assembly of G-actin complexes on the MRTF-A regulatory RPEL domain, promoting nuclear import. In contrast, S33 phosphorylation potentiates the activity of an autonomous Crm1-dependent N-terminal NES, which cooperates with five other NES elements to exclude MRTF-A from the nucleus. Phosphorylation thus plays positive and negative roles in the regulation of MRTF-A. DOI: http://dx.doi.org/10.7554/eLife.15460.001 PMID:27304076

  17. Effect of seat positions on discomfort, muscle activation, pressure distribution and pedal force during cycling.

    PubMed

    Verma, Rachita; Hansen, Ernst A; de Zee, Mark; Madeleine, Pascal

    2016-04-01

    The aim of this study was to measure and analyse discomfort and biomechanics of cycling, i.e., muscle activation, centre of pressure of seat pressure profiles and pedal forces as a function of seat position. Twenty-one recreationally active individuals cycled for 10min at 100W on an ergometer cycle using five different seat positions. The neutral position was considered as basic seat position and was compared with upward, downward, forward and backward seat positions. The initial bout was repeated at the end of the recording session. Discomfort increased for upward and backward condition compared with neutral (P<0.05). Normalized surface electromyography from gastrocnemius decreased in the downward and forward position but increased in the upward and backward position. The minimum force became less negative for forward position compared with neutral seat position (P<0.05). The degree of variability of centre of pressure increased in the upward and backward position and the entropy of the centre of pressure of sitting posture for backward position decreased compared with neutral seat position (P<0.05). The present study revealed that consecutive changes of seat position over time lead to increase in discomfort as well as alterations of the biomechanics of cycling. PMID:26938676

  18. Quantitative test of the barrier nucleosome model for statistical positioning of nucleosomes up- and downstream of transcription start sites.

    PubMed

    Möbius, Wolfram; Gerland, Ulrich

    2010-01-01

    The positions of nucleosomes in eukaryotic genomes determine which parts of the DNA sequence are readily accessible for regulatory proteins and which are not. Genome-wide maps of nucleosome positions have revealed a salient pattern around transcription start sites, involving a nucleosome-free region (NFR) flanked by a pronounced periodic pattern in the average nucleosome density. While the periodic pattern clearly reflects well-positioned nucleosomes, the positioning mechanism is less clear. A recent experimental study by Mavrich et al. argued that the pattern observed in Saccharomyces cerevisiae is qualitatively consistent with a "barrier nucleosome model," in which the oscillatory pattern is created by the statistical positioning mechanism of Kornberg and Stryer. On the other hand, there is clear evidence for intrinsic sequence preferences of nucleosomes, and it is unclear to what extent these sequence preferences affect the observed pattern. To test the barrier nucleosome model, we quantitatively analyze yeast nucleosome positioning data both up- and downstream from NFRs. Our analysis is based on the Tonks model of statistical physics which quantifies the interplay between the excluded-volume interaction of nucleosomes and their positional entropy. We find that although the typical patterns on the two sides of the NFR are different, they are both quantitatively described by the same physical model with the same parameters, but different boundary conditions. The inferred boundary conditions suggest that the first nucleosome downstream from the NFR (the +1 nucleosome) is typically directly positioned while the first nucleosome upstream is statistically positioned via a nucleosome-repelling DNA region. These boundary conditions, which can be locally encoded into the genome sequence, significantly shape the statistical distribution of nucleosomes over a range of up to approximately 1,000 bp to each side. PMID:20808881

  19. Active Sites Environmental Monitoring Program: Program plan. Revision 1

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  20. Physical properties of naked DNA influence nucleosome positioning and correlate with transcription start and termination sites in yeast

    PubMed Central

    2011-01-01

    Background In eukaryotic organisms, DNA is packaged into chromatin structure, where most of DNA is wrapped into nucleosomes. DNA compaction and nucleosome positioning have clear functional implications, since they modulate the accessibility of genomic regions to regulatory proteins. Despite the intensive research effort focused in this area, the rules defining nucleosome positioning and the location of DNA regulatory regions still remain elusive. Results Naked (histone-free) and nucleosomal DNA from yeast were digested by microccocal nuclease (MNase) and sequenced genome-wide. MNase cutting preferences were determined for both naked and nucleosomal DNAs. Integration of their sequencing profiles with DNA conformational descriptors derived from atomistic molecular dynamic simulations enabled us to extract the physical properties of DNA on a genomic scale and to correlate them with chromatin structure and gene regulation. The local structure of DNA around regulatory regions was found to be unusually flexible and to display a unique pattern of nucleosome positioning. Ab initio physical descriptors derived from molecular dynamics were used to develop a computational method that accurately predicts nucleosome enriched and depleted regions. Conclusions Our experimental and computational analyses jointly demonstrate a clear correlation between sequence-dependent physical properties of naked DNA and regulatory signals in the chromatin structure. These results demonstrate that nucleosome positioning around TSS (Transcription Start Site) and TTS (Transcription Termination Site) (at least in yeast) is strongly dependent on DNA physical properties, which can define a basal regulatory mechanism of gene expression. PMID:21981773

  1. Active Site and Laminarin Binding in Glycoside Hydrolase Family 55*

    PubMed Central

    Bianchetti, Christopher M.; Takasuka, Taichi E.; Deutsch, Sam; Udell, Hannah S.; Yik, Eric J.; Bergeman, Lai F.; Fox, Brian G.

    2015-01-01

    The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100–10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties. PMID:25752603

  2. Active site and laminarin binding in glycoside hydrolase family 55.

    PubMed

    Bianchetti, Christopher M; Takasuka, Taichi E; Deutsch, Sam; Udell, Hannah S; Yik, Eric J; Bergeman, Lai F; Fox, Brian G

    2015-05-01

    The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100-10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties. PMID:25752603

  3. Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions

    PubMed Central

    Thanaraj, T. A.; Clark, Francis

    2001-01-01

    It has been previously observed that the intrinsically weak variant GC donor sites, in order to be recognized by the U2-type spliceosome, possess strong consensus sequences maximized for base pair formation with U1 and U5/U6 snRNAs. However, variability in signal strength is a fundamental mechanism for splice site selection in alternative splicing. Here we report human alternative GC-AG introns (for the first time from any species), and show that while constitutive GC-AG introns do possess strong signals at their donor sites, a large subset of alternative GC-AG introns possess weak consensus sequences at their donor sites. Surprisingly, this subset of alternative isoforms shows strong consensus at acceptor exon positions 1 and 2. The improved consensus at the acceptor exon can facilitate a strong interaction with U5 snRNA, which tethers the two exons for ligation during the second step of splicing. Further, these isoforms nearly always possess alternative acceptor sites and exhibit particularly weak polypyrimidine tracts characteristic of AG-dependent introns. The acceptor exon nucleotides are part of the consensus required for the U2AF35-mediated recognition of AG in such introns. Such improved consensus at acceptor exons is not found in either normal or alternative GT-AG introns having weak donor sites or weak polypyrimidine tracts. The changes probably reflect mechanisms that allow GC-AG alternative intron isoforms to cope with two conflicting requirements, namely an apparent need for differential splice strength to direct the choice of alternative sites and a need for improved donor signals to compensate for the central mismatch base pair (C-A) in the RNA duplex of U1 snRNA and the pre-mRNA. The other important findings include (i) one in every twenty alternative introns is a GC-AG intron, and (ii) three of every five observed GC-AG introns are alternative isoforms. PMID:11410667

  4. Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site.

    PubMed

    Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

    2015-01-01

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called 'catalytic residues' are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes. PMID:25902402

  5. Probing Oxygen Activation Sites in Two Flavoprotein Oxidases Using Chloride as an Oxygen Surrogate

    SciTech Connect

    Kommoju, Phaneeswara-Rao; Chen, Zhi-wei; Bruckner, Robert C.; Mathews, F. Scott; Jorns, Marilyn Schuman

    2011-08-16

    A single basic residue above the si-face of the flavin ring is the site of oxygen activation in glucose oxidase (GOX) (His516) and monomeric sarcosine oxidase (MSOX) (Lys265). Crystal structures of both flavoenzymes exhibit a small pocket at the oxygen activation site that might provide a preorganized binding site for superoxide anion, an obligatory intermediate in the two-electron reduction of oxygen. Chloride binds at these polar oxygen activation sites, as judged by solution and structural studies. First, chloride forms spectrally detectable complexes with GOX and MSOX. The protonated form of His516 is required for tight binding of chloride to oxidized GOX and for rapid reaction of reduced GOX with oxygen. Formation of a binary MSOX-chloride complex requires Lys265 and is not observed with Lys265Met. Binding of chloride to MSOX does not affect the binding of a sarcosine analogue (MTA, methylthioactetate) above the re-face of the flavin ring. Definitive evidence is provided by crystal structures determined for a binary MSOX-chloride complex and a ternary MSOX-chloride-MTA complex. Chloride binds in the small pocket at a position otherwise occupied by a water molecule and forms hydrogen bonds to four ligands that are arranged in approximate tetrahedral geometry: Lys265:NZ, Arg49:NH1, and two water molecules, one of which is hydrogen bonded to FAD:N5. The results show that chloride (i) acts as an oxygen surrogate, (ii) is an effective probe of polar oxygen activation sites, and (iii) provides a valuable complementary tool to the xenon gas method that is used to map nonpolar oxygen-binding cavities.

  6. Kinetic model of ethopropazine interaction with horse serum butyrylcholinesterase and its docking into the active site.

    PubMed

    Golicnik, Marko; Sinko, Goran; Simeon-Rudolf, Vera; Grubic, Zoran; Stojan, Jure

    2002-02-01

    The action of a potent tricyclic cholinesterase inhibitor ethopropazine on the hydrolysis of acetylthiocholine and butyrylthiocholine by purified horse serum butyrylcholinesterase (EC 3.1.1.8) was investigated at 25 and 37 degrees C. The enzyme activities were measured on a stopped-flow apparatus and the analysis of experimental data was done by applying a six-parameter model for substrate hydrolysis. The model, which was introduced to explain the kinetics of Drosophila melanogaster acetylcholinesterase [Stojan et al. (1998) FEBS Lett. 440, 85-88], is defined with two dissociation constants and four rate constants and can describe both cooperative phenomena, apparent activation at low substrate concentrations and substrate inhibition by excess of substrate. For the analysis of the data in the presence of ethopropazine at two temperatures, we have enlarged the reaction scheme to allow primarily its competition with the substrate at the peripheral site, but the competition at the acylation site was not excluded. The proposed reaction scheme revealed, upon analysis, competitive effects of ethopropazine at both sites; at 25 degrees C, three enzyme-inhibitor dissociation constants could be evaluated; at 37 degrees C, only two constants could be evaluated. Although the model considers both cooperative phenomena, it appears that decreased enzyme sensitivity at higher temperature, predominantly for the ligands at the peripheral binding site, makes the determination of some expected enzyme substrate and/or inhibitor complexes technically impossible. The same reason might also account for one of the paradoxes in cholinesterases: activities at 25 degrees C at low substrate concentrations are higher than at 37 degrees C. Positioning of ethopropazine in the active-site gorge by molecular dynamics simulations shows that A328, W82, D70, and Y332 amino acid residues stabilize binding of the inhibitor. PMID:11811945

  7. Active Site Detection by Spatial Conformity and Electrostatic Analysis—Unravelling a Proteolytic Function in Shrimp Alkaline Phosphatase

    PubMed Central

    Chakraborty, Sandeep; Minda, Renu; Salaye, Lipika; Bhattacharjee, Swapan K.; Rao, Basuthkar J.

    2011-01-01

    Computational methods are increasingly gaining importance as an aid in identifying active sites. Mostly these methods tend to have structural information that supplement sequence conservation based analyses. Development of tools that compute electrostatic potentials has further improved our ability to better characterize the active site residues in proteins. We have described a computational methodology for detecting active sites based on structural and electrostatic conformity - CataLytic Active Site Prediction (CLASP). In our pipelined model, physical 3D signature of any particular enzymatic function as defined by its active sites is used to obtain spatially congruent matches. While previous work has revealed that catalytic residues have large pKa deviations from standard values, we show that for a given enzymatic activity, electrostatic potential difference (PD) between analogous residue pairs in an active site taken from different proteins of the same family are similar. False positives in spatially congruent matches are further pruned by PD analysis where cognate pairs with large deviations are rejected. We first present the results of active site prediction by CLASP for two enzymatic activities - β-lactamases and serine proteases, two of the most extensively investigated enzymes. The results of CLASP analysis on motifs extracted from Catalytic Site Atlas (CSA) are also presented in order to demonstrate its ability to accurately classify any protein, putative or otherwise, with known structure. The source code and database is made available at www.sanchak.com/clasp/. Subsequently, we probed alkaline phosphatases (AP), one of the well known promiscuous enzymes, for additional activities. Such a search has led us to predict a hitherto unknown function of shrimp alkaline phosphatase (SAP), where the protein acts as a protease. Finally, we present experimental evidence of the prediction by CLASP by showing that SAP indeed has protease activity in vitro. PMID

  8. Global positioning system reoccupation of early triangulation sites - Tectonic deformation of the Southern Coast Ranges

    NASA Technical Reports Server (NTRS)

    Shen, Zheng-Kang; Jackson, David D.

    1993-01-01

    We study tectonic deformation in the Southern Coast Range, California. We use triangulation and astronomic azimuth data collected since 1875, trilateration since 1970, and global positioning system data collected from 1986 to 1987. Two modeling techniques have been used. An elastic block-fault model is applied to study the tectonic motion of the San Andreas Fault and the San Gregorio-Hosgri Fault. Station velocities are modeled to study regional deformations. Results show that the regional deformation is predominantly controlled by deep strike-slip motion along the San Andreas Fault, at a rate of 33 +/- 2 mm/yr. Deep slip along the San Gregorio-Hosgri Fault is about 0-4 mm/yr, assuming a locked suit to a depth of 20 km. Convergence normal to the San Andreas Fault in the Southern Coast Ranges is not greater than 0.02 microrad/yr.

  9. Metavanadate at the active site of the phosphatase VHZ.

    PubMed

    Kuznetsov, Vyacheslav I; Alexandrova, Anastassia N; Hengge, Alvan C

    2012-09-01

    Vanadate is a potent modulator of a number of biological processes and has been shown by crystal structures and NMR spectroscopy to interact with numerous enzymes. Although these effects often occur under conditions where oligomeric forms dominate, the crystal structures and NMR data suggest that the inhibitory form is usually monomeric orthovanadate, a particularly good inhibitor of phosphatases because of its ability to form stable trigonal-bipyramidal complexes. We performed a computational analysis of a 1.14 Å structure of the phosphatase VHZ in complex with an unusual metavanadate species and compared it with two classical trigonal-bipyramidal vanadate-phosphatase complexes. The results support extensive delocalized bonding to the apical ligands in the classical structures. In contrast, in the VHZ metavanadate complex, the central, planar VO(3)(-) moiety has only one apical ligand, the nucleophilic Cys95, and a gap in electron density between V and S. A computational analysis showed that the V-S interaction is primarily ionic. A mechanism is proposed to explain the formation of metavanadate in the active site from a dimeric vanadate species that previous crystallographic evidence has shown to be able to bind to the active sites of phosphatases related to VHZ. Together, the results show that the interaction of vanadate with biological systems is not solely reliant upon the prior formation of a particular inhibitory form in solution. The catalytic properties of an enzyme may act upon the oligomeric forms primarily present in solution to generate species such as the metavanadate ion observed in the VHZ structure. PMID:22876963

  10. Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1. Homology guided site-directed mutagenesis.

    PubMed

    Schoch, Guillaume A; Attias, Roger; Le Ret, Monique; Werck-Reichhart, Danièle

    2003-09-01

    CYP73 enzymes are highly conserved cytochromes P450 in plant species that catalyse the regiospecific 4-hydroxylation of cinnamic acid to form precursors of lignin and many other phenolic compounds. A CYP73A1 homology model based on P450 experimentally solved structures was used to identify active site residues likely to govern substrate binding and regio-specific catalysis. The functional significance of these residues was assessed using site-directed mutagenesis. Active site modelling predicted that N302 and I371 form a hydrogen bond and hydrophobic contacts with the anionic site or aromatic ring of the substrate. Modification of these residues led to a drastic decrease in substrate binding and metabolism without major perturbation of protein structure. Changes to residue K484, which is located too far in the active site model to form a direct contact with cinnamic acid in the oxidized enzyme, did not influence initial substrate binding. However, the K484M substitution led to a 50% loss in catalytic activity. K484 may affect positioning of the substrate in the reduced enzyme during the catalytic cycle, or product release. Catalytic analysis of the mutants with structural analogues of cinnamic acid, in particular indole-2-carboxylic acid that can be hydroxylated with different regioselectivities, supports the involvement of N302, I371 and K484 in substrate docking and orientation. PMID:12950252

  11. Structure/function correlations over binuclear non-heme iron active sites.

    PubMed

    Solomon, Edward I; Park, Kiyoung

    2016-09-01

    Binuclear non-heme iron enzymes activate O2 to perform diverse chemistries. Three different structural mechanisms of O2 binding to a coupled binuclear iron site have been identified utilizing variable-temperature, variable-field magnetic circular dichroism spectroscopy (VTVH MCD). For the μ-OH-bridged Fe(II)2 site in hemerythrin, O2 binds terminally to a five-coordinate Fe(II) center as hydroperoxide with the proton deriving from the μ-OH bridge and the second electron transferring through the resulting μ-oxo superexchange pathway from the second coordinatively saturated Fe(II) center in a proton-coupled electron transfer process. For carboxylate-only-bridged Fe(II)2 sites, O2 binding as a bridged peroxide requires both Fe(II) centers to be coordinatively unsaturated and has good frontier orbital overlap with the two orthogonal O2 π* orbitals to form peroxo-bridged Fe(III)2 intermediates. Alternatively, carboxylate-only-bridged Fe(II)2 sites with only a single open coordination position on an Fe(II) enable the one-electron formation of Fe(III)-O2 (-) or Fe(III)-NO(-) species. Finally, for the peroxo-bridged Fe(III)2 intermediates, further activation is necessary for their reactivities in one-electron reduction and electrophilic aromatic substitution, and a strategy consistent with existing spectral data is discussed. PMID:27369780

  12. Upregulation of RNase E activity by mutation of a site that uncompetitively interferes with RNA binding

    PubMed Central

    Lee, Minho; Shin, Eunkyoung; Jeon, Che Ok; Cha, Chang-Jun; Han, Seung Hyun; Kim, Su-Jin; Lee, Sang-Won; Lee, Younghoon; Ha, Nam-Chul

    2011-01-01

    Escherichia coli RNase E contains a site that selectively binds to RNAs containing 5′-monophosphate termini, increasing the efficiency of endonucleolytic cleavage of these RNAs. Random mutagenesis of N-Rne, the N-terminal catalytic region of RNase E, identified a hyperactive variant that remains preferentially responsive to phosphorylation at 5′ termini. Biochemical analyses showed that the mutation (Q36R), which replaces glutamine with arginine at a position distant from the catalytic site, increases formation of stable RNA-protein complexes without detectably affecting the enzyme's secondary or tertiary structure. Studies of cleavage of fluorogenic substrate and EMSA experiments indicated that the Q36R mutation increases catalytic activity and RNA binding. however, UV crosslinking and mass spectrometry studies suggested that the mutant enzyme lacks an RNA binding site present in its wild-type counterpart. Two substrate-bound tryptic peptides, 65HGFLPLK71—which includes amino acids previously implicated in substrate binding and catalysis—and 24LYDLDIESPGHEQK37—which includes the Q36 locus—were identified in wild-type enzyme complexes, whereas only the shorter peptide was observed for complexes containing Q36R. Our results identify a novel RNase E locus that disparately affects the number of substrate binding sites and catalytic activity of the enzyme. We propose a model that may account for these surprising effects. PMID:22186084

  13. Structure–function analysis of the extracellular domain of the pneumococcal cell division site positioning protein MapZ

    PubMed Central

    Manuse, Sylvie; Jean, Nicolas L.; Guinot, Mégane; Lavergne, Jean-Pierre; Laguri, Cédric; Bougault, Catherine M.; VanNieuwenhze, Michael S.; Grangeasse, Christophe; Simorre, Jean-Pierre

    2016-01-01

    Accurate placement of the bacterial division site is a prerequisite for the generation of two viable and identical daughter cells. In Streptococcus pneumoniae, the positive regulatory mechanism involving the membrane protein MapZ positions precisely the conserved cell division protein FtsZ at the cell centre. Here we characterize the structure of the extracellular domain of MapZ and show that it displays a bi-modular structure composed of two subdomains separated by a flexible serine-rich linker. We further demonstrate in vivo that the N-terminal subdomain serves as a pedestal for the C-terminal subdomain, which determines the ability of MapZ to mark the division site. The C-terminal subdomain displays a patch of conserved amino acids and we show that this patch defines a structural motif crucial for MapZ function. Altogether, this structure–function analysis of MapZ provides the first molecular characterization of a positive regulatory process of bacterial cell division. PMID:27346279

  14. Structure-function analysis of the extracellular domain of the pneumococcal cell division site positioning protein MapZ.

    PubMed

    Manuse, Sylvie; Jean, Nicolas L; Guinot, Mégane; Lavergne, Jean-Pierre; Laguri, Cédric; Bougault, Catherine M; VanNieuwenhze, Michael S; Grangeasse, Christophe; Simorre, Jean-Pierre

    2016-01-01

    Accurate placement of the bacterial division site is a prerequisite for the generation of two viable and identical daughter cells. In Streptococcus pneumoniae, the positive regulatory mechanism involving the membrane protein MapZ positions precisely the conserved cell division protein FtsZ at the cell centre. Here we characterize the structure of the extracellular domain of MapZ and show that it displays a bi-modular structure composed of two subdomains separated by a flexible serine-rich linker. We further demonstrate in vivo that the N-terminal subdomain serves as a pedestal for the C-terminal subdomain, which determines the ability of MapZ to mark the division site. The C-terminal subdomain displays a patch of conserved amino acids and we show that this patch defines a structural motif crucial for MapZ function. Altogether, this structure-function analysis of MapZ provides the first molecular characterization of a positive regulatory process of bacterial cell division. PMID:27346279

  15. Structure-function analysis of the extracellular domain of the pneumococcal cell division site positioning protein MapZ

    NASA Astrophysics Data System (ADS)

    Manuse, Sylvie; Jean, Nicolas L.; Guinot, Mégane; Lavergne, Jean-Pierre; Laguri, Cédric; Bougault, Catherine M.; Vannieuwenhze, Michael S.; Grangeasse, Christophe; Simorre, Jean-Pierre

    2016-06-01

    Accurate placement of the bacterial division site is a prerequisite for the generation of two viable and identical daughter cells. In Streptococcus pneumoniae, the positive regulatory mechanism involving the membrane protein MapZ positions precisely the conserved cell division protein FtsZ at the cell centre. Here we characterize the structure of the extracellular domain of MapZ and show that it displays a bi-modular structure composed of two subdomains separated by a flexible serine-rich linker. We further demonstrate in vivo that the N-terminal subdomain serves as a pedestal for the C-terminal subdomain, which determines the ability of MapZ to mark the division site. The C-terminal subdomain displays a patch of conserved amino acids and we show that this patch defines a structural motif crucial for MapZ function. Altogether, this structure-function analysis of MapZ provides the first molecular characterization of a positive regulatory process of bacterial cell division.

  16. Mimicking enzymatic active sites on surfaces for energy conversion chemistry.

    PubMed

    Gutzler, Rico; Stepanow, Sebastian; Grumelli, Doris; Lingenfelder, Magalí; Kern, Klaus

    2015-07-21

    Metal-organic supramolecular chemistry on surfaces has matured to a point where its underlying growth mechanisms are well understood and structures of defined coordination environments of metal atoms can be synthesized in a controlled and reproducible procedure. With surface-confined molecular self-assembly, scientists have a tool box at hand which can be used to prepare structures with desired properties, as for example a defined oxidation number and spin state of the transition metal atoms within the organic matrix. From a structural point of view, these coordination sites in the supramolecular structure resemble the catalytically active sites of metallo-enzymes, both characterized by metal centers coordinated to organic ligands. Several chemical reactions take place at these embedded metal ions in enzymes and the question arises whether these reactions also take place using metal-organic networks as catalysts. Mimicking the active site of metal atoms and organic ligands of enzymes in artificial systems is the key to understanding the selectivity and efficiency of enzymatic reactions. Their catalytic activity depends on various parameters including the charge and spin configuration in the metal ion, but also on the organic environment, which can stabilize intermediate reaction products, inhibits catalytic deactivation, and serves mostly as a transport channel for the reactants and products and therefore ensures the selectivity of the enzyme. Charge and spin on the transition metal in enzymes depend on the one hand on the specific metal element, and on the other hand on its organic coordination environment. These two parameters can carefully be adjusted in surface confined metal-organic networks, which can be synthesized by virtue of combinatorial mixing of building synthons. Different organic ligands with varying functional groups can be combined with several transition metals and spontaneously assemble into ordered networks. The catalytically active metal

  17. Positive affect modulates activity in the visual cortex to images of high calorie foods.

    PubMed

    Killgore, William D S; Yurgelun-Todd, Deborah A

    2007-05-01

    Activity within the visual cortex can be influenced by the emotional salience of a stimulus, but it is not clear whether such cortical activity is modulated by the affective status of the individual. This study used functional magnetic resonance imaging (fMRI) to examine the relationship between affect ratings on the Positive and Negative Affect Schedule and activity within the occipital cortex of 13 normal-weight women while viewing images of high calorie and low calorie foods. Regression analyses revealed that when participants viewed high calorie foods, Positive Affect correlated significantly with activity within the lingual gyrus and calcarine cortex, whereas Negative Affect was unrelated to visual cortex activity. In contrast, during presentations of low calorie foods, affect ratings, regardless of valence, were unrelated to occipital cortex activity. These findings suggest a mechanism whereby positive affective state may affect the early stages of sensory processing, possibly influencing subsequent perceptual experience of a stimulus. PMID:17464782

  18. Positive Control Mutations in the MyoD Basic Region Fail to Show Cooperative DNA Binding and Transcriptional Activation in vitro

    NASA Astrophysics Data System (ADS)

    Bengal, Eyal; Flores, Osvaldo; Rangarajan, Pundi N.; Chen, Amy; Weintraub, Harold; Verma, Inder M.

    1994-06-01

    An in vitro transcription system from HeLa cells has been established in which MyoD and E47 proteins activate transcription both as homodimers and heterodimers. However, heterodimers activate transcription more efficiently than homodimers, and function synergistically from multiple binding sites. Positive control mutants in the basic region of MyoD that have previously been shown to be defective in initiating the myogenic program, can bind DNA but have lost their ability to function as transcriptional activators in vitro. Additionally, positive control mutants, unlike wild-type MyoD, fail to bind cooperatively to DNA. We propose that binding of MyoD complexes to high affinity MyoD binding sites induces conformational changes that facilitate cooperative binding to multiple sites and promote transcriptional activation.

  19. Rate of hydrolysis in ATP synthase is fine-tuned by α-subunit motif controlling active site conformation.

    PubMed

    Beke-Somfai, Tamás; Lincoln, Per; Nordén, Bengt

    2013-02-01

    Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F(o)F(1) ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F(1) performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate. PMID:23345443

  20. Hybrid [FeFe]-hydrogenases with modified active sites show remarkable residual enzymatic activity.

    PubMed

    Siebel, Judith F; Adamska-Venkatesh, Agnieszka; Weber, Katharina; Rumpel, Sigrun; Reijerse, Edward; Lubitz, Wolfgang

    2015-02-24

    [FeFe]-hydrogenases are to date the only enzymes for which it has been demonstrated that the native inorganic binuclear cofactor of the active site Fe2(adt)(CO)3(CN)2 (adt = azadithiolate = [S-CH2-NH-CH2-S](2-)) can be synthesized on the laboratory bench and subsequently inserted into the unmaturated enzyme to yield fully functional holo-enzyme (Berggren, G. et al. (2013) Nature 499, 66-70; Esselborn, J. et al. (2013) Nat. Chem. Biol. 9, 607-610). In the current study, we exploit this procedure to introduce non-native cofactors into the enzyme. Mimics of the binuclear subcluster with a modified bridging dithiolate ligand (thiodithiolate, N-methylazadithiolate, dimethyl-azadithiolate) and three variants containing only one CN(-) ligand were inserted into the active site of the enzyme. We investigated the activity of these variants for hydrogen oxidation as well as proton reduction and their structural accommodation within the active site was analyzed using Fourier transform infrared spectroscopy. Interestingly, the monocyanide variant with the azadithiolate bridge showed ∼50% of the native enzyme activity. This would suggest that the CN(-) ligands are not essential for catalytic activity, but rather serve to anchor the binuclear subsite inside the protein pocket through hydrogen bonding. The inserted artificial cofactors with a propanedithiolate and an N-methylazadithiolate bridge as well as their monocyanide variants also showed residual activity. However, these activities were less than 1% of the native enzyme. Our findings indicate that even small changes in the dithiolate bridge of the binuclear subsite lead to a rather strong decrease of the catalytic activity. We conclude that both the Brønsted base function and the conformational flexibility of the native azadithiolate amine moiety are essential for the high catalytic activity of the native enzyme. PMID:25633077

  1. Site-specific PEGylation of lidamycin and its antitumor activity.

    PubMed

    Li, Liang; Shang, Boyang; Hu, Lei; Shao, Rongguang; Zhen, Yongsu

    2015-05-01

    In this study, N-terminal site-specific mono-PEGylation of the recombinant lidamycin apoprotein (rLDP) of lidamycin (LDM) was prepared using a polyethyleneglycol (PEG) derivative (M w 20 kDa) through a reactive terminal aldehyde group under weak acidic conditions (pH 5.5). The biochemical properties of mPEG-rLDP-AE, an enediyne-integrated conjugate, were analyzed by SDS-PAGE, RP-HPLC, SEC-HPLC and MALDI-TOF. Meanwhile, in vitro and in vivo antitumor activity of mPEG-rLDP-AE was evaluated by MTT assays and in xenograft model. The results indicated that mPEG-rLDP-AE showed significant antitumor activity both in vitro and in vivo. After PEGylation, mPEG-rLDP still retained the binding capability to the enediyne AE and presented the physicochemical characteristics similar to that of native LDP. It is of interest that the PEGylation did not diminish the antitumor efficacy of LDM, implying the possibility that this derivative may function as a payload to deliver novel tumor-targeted drugs. PMID:26579455

  2. Structural analysis of the active site architecture of the VapC toxin from Shigella flexneri.

    PubMed

    Xu, Kehan; Dedic, Emil; Brodersen, Ditlev E

    2016-07-01

    The VapC toxin from the Shigella flexneri 2a virulence plasmid pMYSH6000 belongs to the PIN domain protein family, which is characterized by a conserved fold with low amino acid sequence conservation. The toxin is a bona fide Mg(2+) -dependent ribonuclease and has been shown to target initiator tRNA(fMet) in vivo. Here, we present crystal structures of active site catalytic triad mutants D7A, D7N, and D98N of the VapC toxin in absence of antitoxin. In all structures, as well as in solution, VapC forms a dimer. In the D98N structure, a Hepes molecule occupies both active sites of the dimer and comparison with the structure of RNase H bound to a DNA/RNA hybrid suggests that the Hepes molecule mimics the position of an RNA nucleotide in the VapC active site. Proteins 2016; 84:892-899. © 2016 Wiley Periodicals, Inc. PMID:26833558

  3. Differential Assembly of Catalytic Interactions within the Conserved Active Sites of Two Ribozymes

    PubMed Central

    Herschlag, Daniel

    2016-01-01

    Molecular recognition is central to biology and a critical aspect of RNA function. Yet structured RNAs typically lack the preorganization needed for strong binding and precise positioning. A striking example is the group I ribozyme from Tetrahymena, which binds its guanosine substrate (G) orders of magnitude slower than diffusion. Binding of G is also thermodynamically coupled to binding of the oligonucleotide substrate (S) and further work has shown that the transition from E•G to E•S•G accompanies a conformational change that allows G to make the active site interactions required for catalysis. The group I ribozyme from Azoarcus has a similarly slow association rate but lacks the coupled binding observed for the Tetrahymena ribozyme. Here we test, using G analogs and metal ion rescue experiments, whether this absence of coupling arises from a higher degree of preorganization within the Azoarcus active site. Our results suggest that the Azoarcus ribozyme forms cognate catalytic metal ion interactions with G in the E•G complex, interactions that are absent in the Tetrahymena E•G complex. Thus, RNAs that share highly similar active site architectures and catalyze the same reactions can differ in the assembly of transition state interactions. More generally, an ability to readily access distinct local conformational states may have facilitated the evolutionary exploration needed to attain RNA machines that carry out complex, multi-step processes. PMID:27501145

  4. Crystal Structures of Pseudomonas aeruginosa GIM-1: Active-Site Plasticity in Metallo-β-Lactamases

    PubMed Central

    Borra, Pardha Saradhi; Samuelsen, Ørjan; Spencer, James; Walsh, Timothy R.; Lorentzen, Marit Sjo

    2013-01-01

    Metallo-β-lactamases (MBLs) have rapidly disseminated worldwide among clinically important Gram-negative bacteria and have challenged the therapeutic use of β-lactam antibiotics, particularly carbapenems. The blaGIM-1 gene, encoding one such enzyme, was first discovered in a Pseudomonas aeruginosa isolate from 2002 and has more recently been reported in Enterobacteriaceae. Here, we present crystal structures of GIM-1 in the apo-zinc (metal-free), mono-zinc (where Cys221 was found to be oxidized), and di-zinc forms, providing nine independently refined views of the enzyme. GIM-1 is distinguished from related MBLs in possessing a narrower active-site groove defined by aromatic side chains (Trp228 and Tyr233) at positions normally occupied by hydrophilic residues in other MBLs. Our structures reveal considerable flexibility in two loops (loop 1, residues 60 to 66; loop 2, residues 223 to 242) adjacent to the active site, with open and closed conformations defined by alternative hydrogen-bonding patterns involving Trp228. We suggest that this capacity for rearrangement permits GIM-1 to hydrolyze a wide range of β-lactams in spite of possessing a more constrained active site. Our results highlight the structural diversity within the MBL enzyme family. PMID:23208706

  5. Improved oxygen reduction activity on Pt3Ni(111) via increased surface site availability.

    PubMed

    Stamenkovic, Vojislav R; Fowler, Ben; Mun, Bongjin Simon; Wang, Guofeng; Ross, Philip N; Lucas, Christopher A; Marković, Nenad M

    2007-01-26

    The slow rate of the oxygen reduction reaction (ORR) in the polymer electrolyte membrane fuel cell (PEMFC) is the main limitation for automotive applications. We demonstrated that the Pt3Ni(111) surface is 10-fold more active for the ORR than the corresponding Pt(111) surface and 90-fold more active than the current state-of-the-art Pt/C catalysts for PEMFC. The Pt3Ni(111) surface has an unusual electronic structure (d-band center position) and arrangement of surface atoms in the near-surface region. Under operating conditions relevant to fuel cells, its near-surface layer exhibits a highly structured compositional oscillation in the outermost and third layers, which are Pt-rich, and in the second atomic layer, which is Ni-rich. The weak interaction between the Pt surface atoms and nonreactive oxygenated species increases the number of active sites for O2 adsorption. PMID:17218494

  6. Evidence of positive selection at codon sites localized in extracellular domains of mammalian CC motif chemokine receptor proteins

    PubMed Central

    2010-01-01

    Background CC chemokine receptor proteins (CCR1 through CCR10) are seven-transmembrane G-protein coupled receptors whose signaling pathways are known for their important roles coordinating immune system responses through targeted trafficking of white blood cells. In addition, some of these receptors have been identified as fusion proteins for viral pathogens: for example, HIV-1 strains utilize CCR5, CCR2 and CCR3 proteins to obtain cellular entry in humans. The extracellular domains of these receptor proteins are involved in ligand-binding specificity as well as pathogen recognition interactions. In mammals, the majority of chemokine receptor genes are clustered together; in humans, seven of the ten genes are clustered in the 3p21-24 chromosome region. Gene conversion events, or exchange of DNA sequence between genes, have been reported in chemokine receptor paralogs in various mammalian lineages, especially between the cytogenetically closely located pairs CCR2/5 and CCR1/3. Datasets of mammalian orthologs for each gene were analyzed separately to minimize the potential confounding impact of analyzing highly similar sequences resulting from gene conversion events. Molecular evolution approaches and the software package Phylogenetic Analyses by Maximum Likelihood (PAML) were utilized to investigate the signature of selection that has acted on the mammalian CC chemokine receptor (CCR) gene family. The results of neutral vs. adaptive evolution (positive selection) hypothesis testing using Site Models are reported. In general, positive selection is defined by a ratio of nonsynonymous/synonymous nucleotide changes (dN/dS, or ω) >1. Results Of the ten mammalian CC motif chemokine receptor sequence datasets analyzed, only CCR2 and CCR3 contain amino acid codon sites that exhibit evidence of positive selection using site based hypothesis testing in PAML. Nineteen of the twenty codon sites putatively indentified as likely to be under positive selection code for amino acid

  7. Influence of cysteine 164 on active site structure in rat cysteine dioxygenase.

    PubMed

    Fellner, Matthias; Siakkou, Eleni; Faponle, Abayomi S; Tchesnokov, Egor P; de Visser, Sam P; Wilbanks, Sigurd M; Jameson, Guy N L

    2016-07-01

    Cysteine dioxygenase is a non-heme mononuclear iron enzyme with unique structural features, namely an intramolecular thioether cross-link between cysteine 93 and tyrosine 157, and a disulfide bond between substrate L-cysteine and cysteine 164 in the entrance channel to the active site. We investigated how these posttranslational modifications affect catalysis through a kinetic, crystallographic and computational study. The enzyme kinetics of a C164S variant are identical to WT, indicating that disulfide formation at C164 does not significantly impair access to the active site at physiological pH. However, at high pH, the cysteine-tyrosine cross-link formation is enhanced in C164S. This supports the view that disulfide formation at position 164 can limit access to the active site. The C164S variant yielded crystal structures of unusual clarity in both resting state and with cysteine bound. Both show that the iron in the cysteine-bound complex is a mixture of penta- and hexa-coordinate with a water molecule taking up the final site (60 % occupancy), which is where dioxygen is believed to coordinate during turnover. The serine also displays stronger hydrogen bond interactions to a water bound to the amine of the substrate cysteine. However, the interactions between cysteine and iron appear unchanged. DFT calculations support this and show that WT and C164S have similar binding energies for the water molecule in the final site. This variant therefore provides evidence that WT also exists in an equilibrium between penta- and hexa-coordinate forms and the presence of the sixth ligand does not strongly affect dioxygen binding. PMID:27193596

  8. fRMSDchiral: a novel algorithm to represent differences between positions of stereoisomers in complex with dissymmetric binding site.

    PubMed

    Urniaz, Rafal D; Rutkowska, Ewelina E; Plazinska, Anita; Jozwiak, Krzysztof

    2014-04-01

    The ability of molecules to distinguish between optical isomers is crucial for living systems. The change of position of one enantiomer in respect to the position of the second enantiomer within an asymmetric binding site may be analyzed on different levels. Root Mean Square Deviation (RMSD) may be used for such analyses with low precision. Additional fragment level variants of RMSD allow for more precise definition of differences in location of the main molecular features responsible for recognition of stereoisomers by a selector. Three fRMSDchiral parameters appear to be very useful to precisely quantify the change in orientations of stereoisomers. Proposed calculation emerges as interesting assistance in interpretation of consequences of formation differential interaction(s) responsible for a chiral recognition process. PMID:24631819

  9. Molecular mimicry of substrate oxygen atoms by water molecules in the beta-amylase active site.

    PubMed

    Pujadas, G; Palau, J

    2001-08-01

    Soybean beta-amylase (EC 3.2.1.2) has been crystallized both free and complexed with a variety of ligands. Four water molecules in the free-enzyme catalytic cleft form a multihydrogen-bond network with eight strategic residues involved in enzyme-ligand hydrogen bonds. We show here that the positions of these four water molecules are coincident with the positions of four potential oxygen atoms of the ligands within the complex. Some of these waters are displaced from the active site when the ligands bind to the enzyme. How many are displaced depends on the shape of the ligand. This means that when one of the four positions is not occupied by a ligand oxygen atom, the corresponding water remains. We studied the functional/structural role of these four waters and conclude that their presence means that the conformation of the eight side chains is fixed in all situations (free or complexed enzyme) and preserved from unwanted or forbidden conformational changes that could hamper the catalytic mechanism. The water structure at the active pocket of beta-amylase is therefore essential for providing the ligand recognition process with plasticity. It does not affect the protein active-site geometry and preserves the overall hydrogen-bonding network, irrespective of which ligand is bound to the enzyme. We also investigated whether other enzymes showed a similar role for water. Finally, we discuss the potential use of these results for predicting whether water molecules can mimic ligand atoms in the active center. PMID:11468361

  10. Tedizolid: a novel oxazolidinone with potent activity against multidrug-resistant gram-positive pathogens.

    PubMed

    Zhanel, George G; Love, Riley; Adam, Heather; Golden, Alyssa; Zelenitsky, Sheryl; Schweizer, Frank; Gorityala, Bala; Lagacé-Wiens, Philippe R S; Rubinstein, Ethan; Walkty, Andrew; Gin, Alfred S; Gilmour, Matthew; Hoban, Daryl J; Lynch, Joseph P; Karlowsky, James A

    2015-02-01

    Tedizolid phosphate is a novel oxazolidinone prodrug (converted to the active form tedizolid by phosphatases in vivo) that has been developed and recently approved (June 2014) by the United States FDA for the treatment of acute bacterial skin and skin structure infections (ABSSSIs) caused by susceptible Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Tedizolid is an oxazolidinone, but differs from other oxazolidinones by possessing a modified side chain at the C-5 position of the oxazolidinone nucleus which confers activity against certain linezolid-resistant pathogens and has an optimized C- and D-ring system that improves potency through additional binding site interactions. The mechanism of action of tedizolid is similar to other oxazolidinones and occurs through inhibition of bacterial protein synthesis by binding to 23S ribosomal RNA (rRNA) of the 50S subunit of the ribosome. As with other oxazolidinones, the spontaneous frequency of resistance development to tedizolid is low. Tedizolid is four- to eightfold more potent in vivo than linezolid against all species of staphylococci, enterococci, and streptococci, including drug-resistant phenotypes such as MRSA and vancomycin-resistant enterococci (VRE) and linezolid-resistant phenotypes. Importantly, tedizolid demonstrates activity against linezolid-resistant bacterial strains harboring the horizontally transmissible cfr gene, in the absence of certain ribosomal mutations conferring reduced oxazolidinone susceptibility. With its half-life of approximately 12 h, tedizolid is dosed once daily. It demonstrates linear pharmacokinetics, has a high oral bioavailability of approximately 90 %, and is primarily excreted by the liver as an inactive, non-circulating sulphate conjugate. Tedizolid does not require dosage adjustment in patients with any degree of renal dysfunction or hepatic dysfunction. Studies in animals have demonstrated that the pharmacodynamic parameter most closely

  11. Influence of a mutation in the putative nucleotide binding site of the nitrogen regulatory protein NTRC on its positive control function.

    PubMed Central

    Austin, S; Kundrot, C; Dixon, R

    1991-01-01

    A mutation, serine 170 to alanine, in the proposed ATP binding site of the activator protein NTRC prevents transcriptional activation at sigma 54-dependent promoters both in vivo and in vitro. The rate of phosphorylation of the mutant protein by NTRB and the stability of mutant NTRC-phosphate were similar to those of wild-type NTRC. The phosphorylated mutant protein shows only a slight decrease in affinity (around 2-fold) for tandem NTRC binding sites in the Klebsiella pneumoniae nifL promoter suggesting that the mutation primarily influences the positive control function of NTRC. Moreover the mutant protein is trans dominant to the wild-type protein with respect to transcriptional activation at both the glnAp2 and nifL promoters. In vitro footprinting experiments reveal that the mutant protein is unable to catalyse isomerisation of closed promoter complexes between sigma 54-RNA polymerase and the nifL promoter to open promoter complexes. However, the mutant protein retains the ability to increase the occupancy of the -24, -12 region by sigma 54-RNA polymerase, forming closed complexes at the nifL promoter, which are not detectable in the absence of NTRC. These data support a model in which the activator influences the formation of closed complexes at the nifL promoter in addition to its role in catalysing open complex formation. Images PMID:2041769

  12. Conformational dynamics of the active site loop of S-adenosylmethionine synthetase illuminated by site-directed spin labeling.

    PubMed

    Taylor, John C; Markham, George D

    2003-07-15

    S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase, methionine adenosyltransferase, a.k.a. MAT) is one of numerous enzymes that have a flexible polypeptide loop that moves to gate access to the active site in a motion that is closely coupled to catalysis. Crystallographic studies of this tetrameric enzyme have shown that the loop is closed in the absence of bound substrates. However, the loop must open to allow substrate binding and a variety of data indicate that the loop is closed during the catalytic steps. Previous kinetic studies indicate that during turnover loop motion occurs on a time scale of 10(-2)s, ca. 10-fold faster than chemical transformations and turnover. Site-directed spin labeling has been used to introduce nitroxide groups at two positions in the loop to illuminate how the motion of the loop is affected by substrate binding. The two loop mutants constructed, G105C and D107C, retain wild type levels of MAT activity; attachment of a methanethiosulfonate spin label to convert the cysteine to the "R1" residue reduced the k(cat) only for the labeled D107R1 form (7-fold). The K(m) value for methionine increased 2- to 4-fold for the cysteine mutants and 2- to 7-fold for the labeled proteins, whereas the K(m) for ATP was changed by at most 2-fold. EPR spectra for both labeled proteins are nearly identical and show the presence of two major spin label environments with rotational diffusion rates differing by approximately 10-fold; the slower rate is ca. 4-fold faster than the estimated protein rotational rate. The spectra are not altered by addition of substrates or products. At both positions the less mobile conformation constitutes ca. 65% of the total species, indicating an equilibrium that only slightly favors one form, that in which the label is more immobilized. The equilibrium constant that relates the two forms is comparable to the equilibrium constant of 1.5 for a conformational change that was previously deduced from the

  13. Tertiary Contacts Distant from the Active Site Prime a Ribozyme for Catalysis

    PubMed Central

    Martick, Monika; Scott, William G.

    2015-01-01

    SUMMARY Minimal hammerhead ribozymes have been characterized extensively by static and time-resolved crystallography as well as numerous biochemical analyses, leading to mutually contradictory mechanistic explanations for catalysis. We present the 2.2 Å resolution crystal structure of a full-length Schistosoma mansoni hammerhead ribozyme that permits us to explain the structural basis for its 1000-fold catalytic enhancement. The full-length hammerhead structure reveals how tertiary interactions occurring remotely from the active site prime this ribozyme for catalysis. G-12 and G-8 are positioned consistent with their previously suggested roles in acid-base catalysis, the nucleophile is aligned with a scissile phosphate positioned proximal to the A-9 phosphate, and previously unexplained roles of other conserved nucleotides become apparent within the context of a distinctly new fold that nonetheless accommodates the previous structural studies. These interactions permit us to explain the previously irreconcilable sets of experimental results in a unified, consistent, and unambiguous manner. PMID:16859740

  14. Interaction of mining activities and aquatic environment: A review from Greek mine sites.

    NASA Astrophysics Data System (ADS)

    Vasileiou, Eleni; Kallioras, Andreas

    2016-04-01

    In Greece a significant amount of mineral and ore deposits have been recorded accompanied by large industrial interest and a long mining history. Today many active and/or abandoned mine sites are scattered within the country; while mining activities take place in different sites for exploiting various deposits (clay, limestone, slate, gypsum, kaolin, mixed sulphide ores (lead, zinc, olivine, pozzolan, quartz lignite, nickel, magnesite, aluminum, bauxite, gold, marbles etc). The most prominent recent ones are: (i) the lignite exploitation that is extended in the area of Ptolemais (Western Macedonia) and Megalopolis (Central Peloponnese); and (ii) the major bauxite deposits located in central Greece within the Parnassos-Ghiona geotectonic zone and on Euboea Island. In the latter area, significant ores of magnesite were exploited and mixed sulphide ores. Centuries of intensive mining exploitation and metallurgical treatment of lead-silver deposits in Greece, have also resulted in significant abandoned sites, such as the one in Lavrion. Mining activities in Lavrio, were initiated in ancient times and continued until the 1980s, resulting in the production of significant waste stockpiles deposited in the area, crucial for the local water resources. Ιn many mining sites, environmental pressures are also recorded after the mine closure to the aquatic environment, as the surface waters flow through waste dump areas and contaminated soils. This paper aims to the geospatial visualization of the mining activities in Greece, in connection to their negative (surface- and/or ground-water pollution; overpumping due to extensive dewatering practices) or positive (enhanced groundwater recharge; pit lakes, improvement of water budget in the catchment scale) impacts on local water resources.

  15. Allosteric site-mediated active site inhibition of PBP2a using Quercetin 3-O-rutinoside and its combination.

    PubMed

    Rani, Nidhi; Vijayakumar, Saravanan; P T V, Lakshmi; Arunachalam, Annamalai

    2016-08-01

    Recent crystallographic study revealed the involvement of allosteric site in active site inhibition of penicillin binding protein (PBP2a), where one molecule of Ceftaroline (Cef) binds to the allosteric site of PBP2a and paved way for the other molecule (Cef) to bind at the active site. Though Cef has the potency to inhibit the PBP2a, its adverse side effects are of major concern. Previous studies have reported the antibacterial property of Quercetin derivatives, a group of natural compounds. Hence, the present study aims to evaluate the effect of Quercetin 3-o-rutinoside (Rut) in allosteric site-mediated active site inhibition of PBP2a. The molecular docking studies between allosteric site and ligands (Rut, Que, and Cef) revealed a better binding efficiency (G-score) of Rut (-7.790318) and Cef (-6.194946) with respect to Que (-5.079284). Molecular dynamic (MD) simulation studies showed significant changes at the active site in the presence of ligands (Rut and Cef) at allosteric site. Four different combinations of Rut and Cef were docked and their G-scores ranged between -6.320 and -8.623. MD studies revealed the stability of the key residue (Ser403) with Rut being at both sites, compared to other complexes. Morphological analysis through electron microscopy confirmed that combination of Rut and Cefixime was able to disturb the bacterial cell membrane in a similar fashion to that of Rut and Cefixime alone. The results of this study indicate that the affinity of Rut at both sites were equally good, with further validations Rut could be considered as an alternative for inhibiting MRSA growth. PMID:26360629

  16. Chemotactic Activity of Cyclophilin A in the Skin Mucus of Yellow Catfish (Pelteobagrus fulvidraco) and Its Active Site for Chemotaxis.

    PubMed

    Dawar, Farman Ullah; Tu, Jiagang; Xiong, Yang; Lan, Jiangfeng; Dong, Xing Xing; Liu, Xiaoling; Khattak, Muhammad Nasir Khan; Mei, Jie; Lin, Li

    2016-01-01

    Fish skin mucus is a dynamic barrier for invading pathogens with a variety of anti-microbial enzymes, including cyclophilin A (CypA), a multi-functional protein with peptidyl-prolyl cis/trans isomerase (PPIase) activity. Beside various other immunological functions, CypA induces leucocytes migration in vitro in teleost. In the current study, we have discovered several novel immune-relevant proteins in yellow catfish skin mucus by mass spectrometry (MS). The CypA present among them was further detected by Western blot. Moreover, the CypA present in the skin mucus displayed strong chemotactic activity for yellow catfish leucocytes. Interestingly, asparagine (like arginine in mammals) at position 69 was the critical site in yellow catfish CypA involved in leucocyte attraction. These novel efforts do not only highlight the enzymatic texture of skin mucus, but signify CypA to be targeted for anti-inflammatory therapeutics. PMID:27589721

  17. Active site hydrophobicity is critical to the bioluminescence activity of Vibrio harveyi luciferase.

    PubMed

    Li, Chi-Hui; Tu, Shiao-Chun

    2005-10-01

    Vibrio harveyi luciferase is an alphabeta heterodimer containing a single active site, proposed earlier to be at a cleft in the alpha subunit. In this work, six conserved phenylalanine residues at this proposed active site were subjected to site-directed mutations to investigate their possible functional roles and to delineate the makeup of luciferase active site. After initial screening of Phe --> Ala mutants, alphaF46, alphaF49, alphaF114, and alphaF117 were chosen for additional mutations to Asp, Ser, and Tyr. Comparisons of the general kinetic properties of wild-type and mutated luciferases indicated that the hydrophobic nature of alphaF46, alphaF49, alphaF114, and alphaF117 was important to luciferase V(max) and V(max)/K(m), which were reduced by 3-5 orders of magnitude for the Phe --> Asp mutants. Both alphaF46 and alphaF117 also appeared to be involved in the binding of reduced flavin substrate. Additional studies on the stability and yield of the 4a-hydroperoxyflavin intermediate II and measurements of decanal substrate oxidation by alphaF46D, alphaF49D, alphaF114D, and alphaF117D revealed that their marked reductions in the overall quantum yield (phi( degrees )) were a consequence of diminished yields of luciferase intermediates and, with the exception of alphaF114D, emission quantum yield of the excited emitter due to the replacement of the hydrophobic Phe by the anionic Asp. The locations of these four critical Phe residues in relation to other essential and/or hydrophobic residues are depicted in a refined map of the active site. Functional implications of these residues are discussed. PMID:16185065

  18. Proteolytic regulation of epithelial sodium channels by urokinase plasminogen activator: cutting edge and cleavage sites.

    PubMed

    Ji, Hong-Long; Zhao, Runzhen; Komissarov, Andrey A; Chang, Yongchang; Liu, Yongfeng; Matthay, Michael A

    2015-02-27

    Plasminogen activator inhibitor 1 (PAI-1) level is extremely elevated in the edematous fluid of acutely injured lungs and pleurae. Elevated PAI-1 specifically inactivates pulmonary urokinase-type (uPA) and tissue-type plasminogen activators (tPA). We hypothesized that plasminogen activation and fibrinolysis may alter epithelial sodium channel (ENaC) activity, a key player in clearing edematous fluid. Two-chain urokinase (tcuPA) has been found to strongly stimulate heterologous human αβγ ENaC activity in a dose- and time-dependent manner. This activity of tcuPA was completely ablated by PAI-1. Furthermore, a mutation (S195A) of the active site of the enzyme also prevented ENaC activation. By comparison, three truncation mutants of the amino-terminal fragment of tcuPA still activated ENaC. uPA enzymatic activity was positively correlated with ENaC current amplitude prior to reaching the maximal level. In sharp contrast to uPA, neither single-chain tPA nor derivatives, including two-chain tPA and tenecteplase, affected ENaC activity. Furthermore, γ but not α subunit of ENaC was proteolytically cleaved at ((177)GR↓KR(180)) by tcuPA. In summary, the underlying mechanisms of urokinase-mediated activation of ENaC include release of self-inhibition, proteolysis of γ ENaC, incremental increase in opening rate, and activation of closed (electrically "silent") channels. This study for the first time demonstrates multifaceted mechanisms for uPA-mediated up-regulation of ENaC, which form the cellular and molecular rationale for the beneficial effects of urokinase in mitigating mortal pulmonary edema and pleural effusions. PMID:25555911

  19. Does homeobox-related "positional" genomic information contribute to implantation of metastatic cancer cells at non-random sites?

    PubMed

    Anderson, K M; Darweesh, M; Jajah, A; Tsui, P; Guinan, P; Rubenstein, M

    2007-01-01

    Reasons for the lodgment of metastases from several types of solid cancer at apparently non-random sites have not been established. Recently, a group of genes expressed in human fibroblasts obtained from different anatomic locations was implicated in "positional" genomic information. Essentially, a Cartesian coordinate system identifying fibroblasts originally resident at anterior or more posterior, proximal or distal and dermal or non-dermal (heart, lung, etc.) locations was proposed. The determinants used for these identifications included HOX genes, central to embryonic segmental development, some of which are expressed in differentiated, post-embryonic cells. To the extent that HOX or other homeobox genes are expressed in ectodermal, mesodermal or endodermally-derived, malignantly transformed cells, they might contribute "positional" information to nidation of specific malignant clones at non-random sites. As understood in the past, interdiction of HOX or homeobox-related gene expression might reduce the probability of cancer cell implantation or alter their destinations in complex ways. Ideally, by interfering with HOX or other homeobox gene-related expression of antigenic determinants potentially contributing to their "homing" and nidation, reduced implantation of circulating cancer cells could render them more susceptible to systemic chemotherapy or immunotherapy, as demonstrated in mice. Furthermore, HOX or other homeobox genes or their products could provide novel intra- or extracellular targets for therapy. PMID:17695497

  20. High-magnification vascular imaging to reject false-positive sites in situ during Hexvix® fluorescence cystoscopy

    NASA Astrophysics Data System (ADS)

    Lovisa, Blaise; Jichlinski, Patrice; Weber, Bernd-Claus; Aymon, Daniela; van den Bergh, Hubert; Wagnières, Georges

    2010-09-01

    Fluorescence imaging for detection of non-muscle-invasive bladder cancer is based on the selective production and accumulation of fluorescing porphyrins-mainly, protoporphyrin IX-in cancerous tissues after the instillation of Hexvix®. Although the sensitivity of this procedure is very good, its specificity is somewhat limited due to fluorescence false-positive sites. Consequently, magnification cystoscopy has been investigated in order to discriminate false from true fluorescence positive findings. Both white-light and fluorescence modes are possible with the magnification cystoscope, allowing observation of the bladder wall with magnification ranging between 30× for standard observation and 650×. The optical zooming setup allows adjusting the magnification continuously in situ. In the high-magnification (HM) regime, the smallest diameter of the field of view is 600 microns and the resolution is 2.5 microns when in contact with the bladder wall. With this cystoscope, we characterized the superficial vascularization of the fluorescing sites in order to discriminate cancerous from noncancerous tissues. This procedure allowed us to establish a classification based on observed vascular patterns. Seventy-two patients subject to Hexvix® fluorescence cystoscopy were included in the study. Comparison of HM cystoscopy classification with histopathology results confirmed 32/33 (97%) cancerous biopsies and rejected 17/20 (85%) noncancerous lesions.

  1. A proposed definition of the 'activity' of surface sites on lactose carriers for dry powder inhalation.

    PubMed

    Grasmeijer, Floris; Frijlink, Henderik W; de Boer, Anne H

    2014-06-01

    A new definition of the activity of surface sites on lactose carriers for dry powder inhalation is proposed which relates to drug detachment during dispersion. The new definition is expected to improve the understanding of 'carrier surface site activity', which stimulates the unambiguous communication about this subject and may aid in the rational design and interpretation of future formulation studies. In contrast to the currently prevailing view on carrier surface site activity, it follows from the newly proposed definition that carrier surface site activity depends on more variables than just the physicochemical properties of the carrier surface. Because the term 'active sites' is ambiguous, it is recommended to use the term 'highly active sites' instead to denote carrier surface sites with a relatively high activity. PMID:24613490

  2. Excerpts from "Position Statement on Physical Fitness and Activity in the Context of Leisure Education."

    ERIC Educational Resources Information Center

    Ruskin, Hilleil

    2002-01-01

    Presents a position statement designed to inform governments, non-governmental organizations, and education institutions about the significance and benefits of physical activity for all and establish a clear relationship between physical activity and leisure education. The statement includes specific recommendations for leisure education and…

  3. Positive Behavior Interventions and Support in a Physical Activity Summer Camp

    ERIC Educational Resources Information Center

    Hinton, Vanessa; Buchanan, Alice M.

    2015-01-01

    This purpose of this study was to investigate the implementation of positive behavior interventions and support (PBIS) in a summer camp. The camp provided physical activity opportunities to underserved children attending a summer program at a local, rural public school. Certified physical education teachers led activity stations. Participants in…

  4. Physical Activity and Psychological Benefits. International Society of Sport Psychology Position Statement.

    ERIC Educational Resources Information Center

    Physician and Sportsmedicine, 1992

    1992-01-01

    International Society of Sport Psychology clarifies the psychological benefits of physical activity, noting the positive relationship between physical activity level and mental health. Exercise can reduce anxiety, decrease depression levels, reduce neuroticism and anxiety, reduce stress, and have beneficial emotional effects for both sexes across…

  5. Alignment of Hands-On STEM Engagement Activities with Positive STEM Dispositions in Secondary School Students

    ERIC Educational Resources Information Center

    Christensen, Rhonda; Knezek, Gerald; Tyler-Wood, Tandra

    2015-01-01

    This study examines positive dispositions reported by middle school and high school students participating in programs that feature STEM-related activities. Middle school students participating in school-to-home hands-on energy monitoring activities are compared to middle school and high school students in a different project taking part in…

  6. Improving Functional Annotation in the DRE-TIM Metallolyase Superfamily through Identification of Active Site Fingerprints.

    PubMed

    Kumar, Garima; Johnson, Jordyn L; Frantom, Patrick A

    2016-03-29

    Within the DRE-TIM metallolyase superfamily, members of the Claisen-like condensation (CC-like) subgroup catalyze C-C bond-forming reactions between various α-ketoacids and acetyl-coenzyme A. These reactions are important in the metabolic pathways of many bacterial pathogens and serve as engineering scaffolds for the production of long-chain alcohol biofuels. To improve functional annotation and identify sequences that might use novel substrates in the CC-like subgroup, a combination of structural modeling and multiple-sequence alignments identified active site residues on the third, fourth, and fifth β-strands of the TIM-barrel catalytic domain that are differentially conserved within the substrate-diverse enzyme families. Using α-isopropylmalate synthase and citramalate synthase from Methanococcus jannaschii (MjIPMS and MjCMS), site-directed mutagenesis was used to test the role of each identified position in substrate selectivity. Kinetic data suggest that residues at the β3-5 and β4-7 positions play a significant role in the selection of α-ketoisovalerate over pyruvate in MjIPMS. However, complementary substitutions in MjCMS fail to alter substrate specificity, suggesting residues in these positions do not contribute to substrate selectivity in this enzyme. Analysis of the kinetic data with respect to a protein similarity network for the CC-like subgroup suggests that evolutionarily distinct forms of IPMS utilize residues at the β3-5 and β4-7 positions to affect substrate selectivity while the different versions of CMS use unique architectures. Importantly, mapping the identities of residues at the β3-5 and β4-7 positions onto the protein similarity network allows for rapid annotation of probable IPMS enzymes as well as several outlier sequences that may represent novel functions in the subgroup. PMID:26935545

  7. Negative and positive site-site interactions, and their modulation by pH, insulin analogs, and monoclonal antibodies, are preserved in the purified insulin receptor.

    PubMed Central

    Wang, C C; Goldfine, I D; Fujita-Yamaguchi, Y; Gattner, H G; Brandenburg, D; De Meyts, P

    1988-01-01

    The kinetic properties of the insulin receptor were studied in solution after its purification to homogeneity. Dissociation of 125I-labeled insulin at a 1:50 dilution was not first order; unlabeled insulin at physiological concentrations accelerated the dissociation rate with a maximal effect at approximately 17 nM. At higher concentrations, the unlabeled insulin slowed the dissociation rate. Maximal acceleration was seen at pH 8.0. The ability to accelerate the dissociation rate was diminished with [LeuB24]insulin and suppressed with desoctapeptide, [LeuB25], [LeuB24,B25], desalanine-desasparagine, and desheptapeptide insulins, all of which slowed the dissociation at high concentrations. Monoclonal antibodies to the insulin receptor alpha subunit (MA-5, MA-10, MA-20, and MA-51) all competed for insulin binding to the purified receptor. MA-10 and MA-51 accelerated the dissociation of 125I-labeled insulin, while MA-5 and MA-20 slowed the off rate. Thus, all the aspects of both negatively and positively cooperative site-site interactions previously described in whole cells are present in solubilized purified receptors, demonstrating that these interactions represent intrinsic properties of the receptor molecule, most likely as a result of ligand-induced conformational changes. PMID:3054887

  8. RBF-TSS: identification of transcription start site in human using radial basis functions network and oligonucleotide positional frequencies.

    PubMed

    Mahdi, Rami N; Rouchka, Eric C

    2009-01-01

    Accurate identification of promoter regions and transcription start sites (TSS) in genomic DNA allows for a more complete understanding of the structure of genes and gene regulation within a given genome. Many recently published methods have achieved high identification accuracy of TSS. However, models providing more accurate modeling of promoters and TSS are needed. A novel identification method for identifying transcription start sites that improves the accuracy of TSS recognition for recently published methods is proposed. This method incorporates a metric feature based on oligonucleotide positional frequencies, taking into account the nature of promoters. A radial basis function neural network for identifying transcription start sites (RBF-TSS) is proposed and employed as a classification algorithm. Using non-overlapping chunks (windows) of size 50 and 500 on the human genome, the proposed method achieves an area under the Receiver Operator Characteristic curve (auROC) of 94.75% and 95.08% respectively, providing increased performance over existing TSS prediction methods. PMID:19287502

  9. Strong influence of off-site symmetry positions of hydrogen atoms in ScH3 hcp phases

    NASA Astrophysics Data System (ADS)

    Pakornchote, T.; Bovornratanaraks, T.; Vannarat, S.; Pinsook, U.

    2016-01-01

    We investigate the wave-like arrangements of H atoms around metal plane (Hm) in the ScH3 hcp phase by using the ab-initio method. We found that only P63 / mmc, P 3 bar c 1, P63cm and P63 phases are energetically favorable. The wave-like arrangement allows the off-site symmetry positions of the H atoms, and leads to substantial changes in the pair distribution between Sc and H atoms which are associating with the changes in the electronic structure in such a way that the total energy is lowering. The symmetry breaking from P63mmc is also responsible for the band gap opening. In the P63 structure, the calculated band gap is 0.823 eV and 1.223 eV using GGA and sX-LDA functionals, respectively. This band gap can be compared with 1.7 eV derived from the optical measurement and 1.55 eV from the HSE06 calculation. Thus, the broken symmetry structures can be viewed as Peierls distortion of the P63 / mmc structure. Furthermore, we found that only the P63 structure is dynamically stable, unlike YH3 where the P63cm structure is also stable. The stability of P63 comes from sufficiently strong interactions between two neighboring H atoms at their off-site symmetry positions, i.e. near the metal plane and near the tetragonal site. The P63 phonon density of states is in good agreement with the data from the neutron experiment.

  10. Functional assignment to positively selected sites in the core type III effector RipG7 from Ralstonia solanacearum.

    PubMed

    Wang, Keke; Remigi, Philippe; Anisimova, Maria; Lonjon, Fabien; Kars, Ilona; Kajava, Andrey; Li, Chien-Hui; Cheng, Chiu-Ping; Vailleau, Fabienne; Genin, Stéphane; Peeters, Nemo

    2016-05-01

    The soil-borne pathogen Ralstonia solanacearum causes bacterial wilt in a broad range of plants. The main virulence determinants of R. solanacearum are the type III secretion system (T3SS) and its associated type III effectors (T3Es), translocated into the host cells. Of the conserved T3Es among R. solanacearum strains, the Fbox protein RipG7 is required for R. solanacearum pathogenesis on Medicago truncatula. In this work, we describe the natural ripG7 variability existing in the R. solanacearum species complex. We show that eight representative ripG7 orthologues have different contributions to pathogenicity on M. truncatula: only ripG7 from Asian or African strains can complement the absence of ripG7 in GMI1000 (Asian reference strain). Nonetheless, RipG7 proteins from American and Indonesian strains can still interact with M. truncatula SKP1-like/MSKa protein, essential for the function of RipG7 in virulence. This indicates that the absence of complementation is most likely a result of the variability in the leucine-rich repeat (LRR) domain of RipG7. We identified 11 sites under positive selection in the LRR domains of RipG7. By studying the functional impact of these 11 sites, we show the contribution of five positively selected sites for the function of RipG7CMR15 in M. truncatula colonization. This work reveals the genetic and functional variation of the essential core T3E RipG7 from R. solanacearum. This analysis is the first of its kind on an essential disease-controlling T3E, and sheds light on the co-evolutionary arms race between the bacterium and its hosts. PMID:26300048

  11. Disturbance opens recruitment sites for bacterial colonization in activated sludge.

    PubMed

    Vuono, David C; Munakata-Marr, Junko; Spear, John R; Drewes, Jörg E

    2016-01-01

    Little is known about the role of immigration in shaping bacterial communities or the factors that may dictate success or failure of colonization by bacteria from regional species pools. To address these knowledge gaps, the influence of bacterial colonization into an ecosystem (activated sludge bioreactor) was measured through a disturbance gradient (successive decreases in the parameter solids retention time) relative to stable operational conditions. Through a DNA sequencing approach, we show that the most abundant bacteria within the immigrant community have a greater probability of colonizing the receiving ecosystem, but mostly as low abundance community members. Only during the disturbance do some of these bacterial populations significantly increase in abundance beyond background levels and in few cases become dominant community members post-disturbance. Two mechanisms facilitate the enhanced enrichment of immigrant populations during disturbance: (i) the availability of resources left unconsumed by established species and (ii) the increased availability of niche space for colonizers to establish and displace resident populations. Thus, as a disturbance decreases local diversity, recruitment sites become available to promote colonization. This work advances our understanding of microbial resource management and diversity maintenance in complex ecosystems. PMID:25727891

  12. Construction of DNA recognition sites active in Haemophilus transformation.

    PubMed Central

    Danner, D B; Smith, H O; Narang, S A

    1982-01-01

    Competent Haemophilus cells recognize and preferentially take up Haemophilus DNA during genetic transformation. This preferential uptake is correlated with the presence on incoming DNA of an 11-base-pair (bp) sequence, 5'-A-A-G-T-G-C-G-G-T-C-A-3'. To prove that this sequence is the recognition site that identifies Haemophilus DNA to the competent cell, we have now constructed a series of plasmids, each of which contains the 11-bp sequence. Using two different assay systems we have tested the ability of fragments from these plasmids to compete with cloned Haemophilus DNA fragments that naturally contain the 11-bp sequence. We find that the addition of the 11-bp sequence to a DNA fragment is necessary and sufficient for preferential uptake of that fragment. However, plasmid DNAs containing this sequence may vary as much as 48-fold in uptake activity, and this variation correlates with the A+T-richness of the DNA flanking the 11-mer. Images PMID:6285382

  13. Characterization of active site residues of nitroalkane oxidase.

    PubMed

    Valley, Michael P; Fenny, Nana S; Ali, Shah R; Fitzpatrick, Paul F

    2010-06-01

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by approximately 5-fold and decreases in the rate constant for product release of approximately 2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure. PMID:20056514

  14. Detection limit for activation measurements in ultralow background sites

    NASA Astrophysics Data System (ADS)

    Trache, Livius; Chesneanu, D.; Margineanu, R.; Pantelica, A.; Ghita, D. G.; Burducea, I.; Straticiuc, M.; Tang, X. D.

    2014-09-01

    We used 12C +13C fusion at the beam energies E = 6, 7 and 8 MeV to determine the sensitivity and the limits of activation method measurements in ultralow background sites. A 13C beam of 0.5 μA from the 3 MV Tandem accelerator of the Horia Hulubei National Institute of Physics and Nuclear Engineering - IFIN HH impinged on thick graphite targets. After about 24 hrs of irradiation targets were measured in two different laboratories: one with a heavy shielded Ge detector in the institute (at the surface) and one located underground in the microBequerel laboratory, in the salt mine of Slanic-Prahova, Romania. The 1369- and 2754 keV peaks from 24Na deactivation were clearly observed in the γ-ray spectra obtained for acquisitions lasting a few hours, or a few days. Determination of the detection limit in evaluating the cross sections for the target irradiated at Ec . m = 3 MeV indicates the fact that it is possible to measure gamma spectrum in underground laboratory down to Ec . m = 2 . 6 MeV. Cleaning the spectra with beta-gamma coincidences and increasing beam intensity 20 times will take as further down. The measurements are motivated by the study of the 12 C +12 C reaction at astrophysical energies.

  15. Enzyme-like specificity in zeolites: a unique site position in mordenite for selective carbonylation of methanol and dimethyl ether with CO.

    PubMed

    Boronat, Mercedes; Martínez-Sánchez, Cristina; Law, David; Corma, Avelino

    2008-12-01

    The mechanism of methanol carbonylation at different positions of zeolite MOR is investigated by quantum-chemical methods in order to discover which are the active sites that can selectively catalyze the desired reaction. It is shown that when methanol carbonylation competes with hydrocarbon formation, the first reaction occurs preferentially within 8MR channels. However, the unique selectivity for the carbonylation of methanol and dimethyl ether in mordenite is not only due to the size of the 8MR channel: neither process occurs equally at the two T3-O31 and T3-O33 positions. We show that only the T3-O33 positions are selective and that this selectivity is due to the unusual orientation of the methoxy group in relation to the 8MR channel (parallel to the cylinder axis). Only in this situation does the transition state for the attack of CO fit perfectly in the 8MR channel, while the reaction with methanol or DME is sterically impeded. This result explains why T3-O31, while also located in the 8MR channel of mordenite, is not as selective as the T3-O33 position and why ferrierite, although it contains 8MR channels, is less selective than mordenite. The competing effect of water is explained at the molecular level, and the molecular microkinetic reaction model has been established. PMID:18986144

  16. Get Moving: A Web Site That Increases Physical Activity of Sedentary Employees

    PubMed Central

    Irvine, A. Blair; Philips, Laura; Seeley, John; Wyant, Sara; Duncan, Susan; Moore, Roger W.

    2011-01-01

    Purpose Develop and test a Web site to encourage physical activity (PA) by sedentary workers. Design Randomized control design with 30-day follow-up. Setting Large manufacturing plant. Subjects Included 221 workers; average body mass index was 29.5. Intervention Get Moving was a repeat-visit Web site providing information and support to develop a personalized PA plan. Measures Self-reported: PA, depression, anxiety, stage of change, attitudes, knowledge, self-efficacy, intention, perceived barriers to PA, and motivation. Analysis Multivariate analysis of covariance and univariate analysis of covariance models were used to compare the two study conditions on posttest outcomes, controlling for baseline levels. Results Compared with the control group, the treatment group showed significant improvement. The multivariate test was significant (p < .001), with a large effect size (η2 = .42). The treatment group differed significantly from the control participants on 11 outcomes (p < .005), with large effect sizes for PA status, min/d, and knowledge, attitudes, and behavioral intention. Medium effect sizes were measured for perceived barriers, depressive symptoms, motivation, and self-efficacy. Multiple visits resulted in significantly improved PA, motivation, self-efficacy, and intention, compared with one-time visits. Conclusions The Get Moving Web site had positive effects and was well received. Interventions Web site have potential to increase the PA of sedentary individuals in worksites and elsewhere, but more research is needed into mediators of Web-based interventions. PMID:21192750

  17. Orthogonal electrode catheter array for mapping of endocardial focal site of ventricular activation

    SciTech Connect

    Desai, J.M.; Nyo, H.; Vera, Z.; Seibert, J.A.; Vogelsang, P.J. )

    1991-04-01

    Precise location of the endocardial site of origin of ventricular tachycardia may facilitate surgical and catheter ablation of this arrhythmia. The endocardial catheter mapping technique can locate the site of ventricular tachycardia within 4-8 cm2 of the earliest site recorded by the catheter. This report describes an orthogonal electrode catheter array (OECA) for mapping and radiofrequency ablation (RFA) of endocardial focal site of origin of a plunge electrode paced model of ventricular activation in dogs. The OECA is an 8 F five pole catheter with four peripheral electrodes and one central electrode (total surface area 0.8 cm{sup 2}). In eight mongrel dogs, mapping was performed by arbitrarily dividing the left ventricle (LV) into four segments. Each segment was mapped with OECA to find the earliest segment. Bipolar and unipolar electrograms were obtained. The plunge electrode (not visible on fluoroscopy) site was identified by the earliest wave front arrival times of -30 msec or earlier at two or more electrodes (unipolar electrograms) with reference to the earliest recorded surface ECG (I, AVF, and V1). Validation of the proximity of the five electrodes of the OECA to the plunge electrode was performed by digital radiography and RFA. Pathological examination was performed to document the proximity of the OECA to the plunge electrode and also for the width, depth, and microscopic changes of the ablation. To find the segment with the earliest LV activation a total of 10 {plus minus} 3 (mean {plus minus} SD) positions were mapped. Mean arrival times at the two earlier electrodes were -39 {plus minus} 4 msec and -35 {plus minus} 3 msec. Digital radiography showed the plunge electrode to be within the area covered by all five electrodes in all eight dogs. The plunge electrode was within 1 cm2 area of the region of RFA in all eight dogs.

  18. Sanctuary site leptomeningeal metastases in HER-2 positive breast cancer: A review in the era of trastuzumab.

    PubMed

    Kordbacheh, T; Law, W Y; Smith, I E

    2016-04-01

    The development of trastuzumab and other targeted systemic therapies has transformed the management of HER-2 positive breast cancers. However, as patients live longer and systemic therapies may not cross the blood brain barrier a rising number of patients are developing leptomeningeal metastases and brain metastases as a sanctuary site of disease. Intrathecal trastuzumab has been reported to treat these. We describe a breast cancer patient with HER-2 positive leptomeningeal disease in the spinal cord successfully treated with intrathecal trastuzumab and methotrexate, alongside systemic anti-HER-2 therapy and radiotherapy. We also review the literature to date on the efficacy and safety of intrathecal trastuzumab, and recent evidence suggesting that intrathecal trastuzumab passes via the blood brain barrier into the serum to achieve intravenous concentrations similar to that seen with systemic therapy alone. Overall, intrathecal trastuzumab appears to be a safe and often effective treatment for leptomeningeal metastases in HER-2 positive breast cancer. Ongoing phase I and II studies are required to determine optimum dosing schedules, validate CSF and CSF-to-serum pharmacokinetics, determine efficacy, and to assess the added benefits or disadvantages of prior radiotherapy and concomitant systemic therapy. PMID:27017242

  19. Adaptability and Prediction of Anticipatory Muscular Activity Parameters to Different Movements in the Sitting Position.

    PubMed

    Chikh, Soufien; Watelain, Eric; Faupin, Arnaud; Pinti, Antonio; Jarraya, Mohamed; Garnier, Cyril

    2016-08-01

    Voluntary movement often causes postural perturbation that requires an anticipatory postural adjustment to minimize perturbation and increase the efficiency and coordination during execution. This systematic review focuses specifically on the relationship between the parameters of anticipatory muscular activities and movement finality in sitting position among adults, to study the adaptability and predictability of anticipatory muscular activities parameters to different movements and conditions in sitting position in adults. A systematic literature search was performed using PubMed, Science Direct, Web of Science, Springer-Link, Engineering Village, and EbscoHost. Inclusion and exclusion criteria were applied to retain the most rigorous and specific studies, yielding 76 articles, Seventeen articles were excluded at first reading, and after the application of inclusion and exclusion criteria, 23 were retained. In a sitting position, central nervous system activity precedes movement by diverse anticipatory muscular activities and shows the ability to adapt anticipatory muscular activity parameters to the movement direction, postural stability, or charge weight. In addition, these parameters could be adapted to the speed of execution, as found for the standing position. Parameters of anticipatory muscular activities (duration, order, and amplitude of muscle contractions constituting the anticipatory muscular activity) could be used as a predictive indicator of forthcoming movement. In addition, this systematic review may improve methodology in empirical studies and assistive technology for people with disabilities. PMID:27440765

  20. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  1. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  2. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  3. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  4. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  5. Alignment of Hands-on STEM Engagement Activities with Positive STEM Dispositions in Secondary School Students

    NASA Astrophysics Data System (ADS)

    Christensen, Rhonda; Knezek, Gerald; Tyler-Wood, Tandra

    2015-12-01

    This study examines positive dispositions reported by middle school and high school students participating in programs that feature STEM-related activities. Middle school students participating in school-to-home hands-on energy monitoring activities are compared to middle school and high school students in a different project taking part in activities such as an after-school robotics program. Both groups are compared and contrasted with a third group of high school students admitted at the eleventh grade to an academy of mathematics and science. All students were assessed using the same science, technology, engineering and mathematics (STEM) dispositions instrument. Findings indicate that the after-school group whose participants self-selected STEM engagement activities, and the self-selected academy of mathematics and science group, each had highly positive STEM dispositions comparable to those of STEM professionals, while a subset of the middle school whole-classroom energy monitoring group that reported high interest in STEM as a career, also possessed highly positive STEM dispositions comparable to the STEM Professionals group. The authors conclude that several different kinds of hands-on STEM engagement activities are likely to foster or maintain positive STEM dispositions at the middle school and high school levels, and that these highly positive levels of dispositions can be viewed as a target toward which projects seeking to interest mainstream secondary students in STEM majors in college and STEM careers, can hope to aspire. Gender findings regarding STEM dispositions are also reported for these groups.

  6. Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function

    PubMed Central

    Hobbs, G Aaron; Gunawardena, Harsha P; Baker, Rachael; Campbell, Sharon L

    2013-01-01

    KRas has recently been shown to be activated by monoubiquitination (mUb). Similar to oncogenic mutations, mUb of Ras at position 147 activates Ras by causing a defect in GTPase activating protein (GAP) function. To characterize the mechanism by which mUb impairs GAP-mediated downregulation of Ras, we made various modifications at position 147 of Ras and examined the impact on Ras sensitivity to GAP function. Whereas small modifications (iodoacetamide and glutathione) at position 147 of Ras do not affect GAP-mediated hydrolysis, ligation of Ras to UbG76C (native linker), UbX77C (one residue longer), and PDZ2 (with a native ubiquitin linker) was defective in GAP-mediated GTP hydrolysis. However, restoration of GAP activity was observed for Ras modified with the PDZ2 domain containing a shorter and stiffer linker region than ubiquitin. Therefore, the properties of the linker region dictate whether modification affects GAP-mediated hydrolysis, and our data indicate that the GAP defect requires a minimum linker length of 7 to 8 residues. PMID:24030601

  7. Lateral flow urine lipoarabinomannan assay for detecting active tuberculosis in Hiv-positive adults

    PubMed Central

    Shah, Maunank; Hanrahan, Colleen; Wang, Zhuo Yu; Dendukuri, Nandini; Lawn, Stephen D; Denkinger, Claudia M; Steingart, Karen R

    2016-01-01

    Background Rapid detection of tuberculosis (TB) among people living with human immunodeficiency virus (HIV) is a global health priority. HIV-associated TB may have different clinical presentations and is challenging to diagnose. Conventional sputum tests have reduced sensitivity in HIV-positive individuals, who have higher rates of extrapulmonary TB compared with HIV-negative individuals. The lateral flow urine lipoarabinomannan assay (LF-LAM) is a new, commercially available point-of-care test that detects lipoarabinomannan (LAM), a lipopolysaccharide present in mycobacterial cell walls, in people with active TB disease. Objectives To assess the accuracy of LF-LAM for the diagnosis of active TB disease in HIV-positive adults who have signs and symptoms suggestive of TB (TB diagnosis).To assess the accuracy of LF-LAM as a screening test for active TB disease in HIV-positive adults irrespective of signs and symptoms suggestive of TB (TB screening). Search methods We searched the following databases without language restriction on 5 February 2015: the Cochrane Infectious Diseases Group Specialized Register; MEDLINE (PubMed,1966); EMBASE (OVID, from 1980); Science Citation Index Expanded (SCI-EXPANDED, from 1900), Conference Proceedings Citation Index-Science (CPCI-S, from 1900), and BIOSIS Previews (from 1926) (all three using the Web of Science platform; MEDION; LILACS (BIREME, from 1982); SCOPUS (from 1995); the metaRegister of Controlled Trials (mRCT); the search portal of the World Health Organization International Clinical Trials Registry Platform (WHO ICTRP); and ProQuest Dissertations & Theses A&l (from 1861). Selection criteria Eligible study types included randomized controlled trials, cross-sectional studies, and cohort studies that determined LF-LAM accuracy for TB against a microbiological reference standard (culture or nucleic acid amplification test from any body site). A higher quality reference standard was one in which two or more specimen types were

  8. Active-site mutagenesis of tetanus neurotoxin implicates TYR-375 and GLU-271 in metalloproteolytic activity.

    PubMed

    Rossetto, O; Caccin, P; Rigoni, M; Tonello, F; Bortoletto, N; Stevens, R C; Montecucco, C

    2001-08-01

    Tetanus neurotoxin (TeNT) blocks neurotransmitter release by cleaving VAMP/synaptobrevin, a membrane associated protein involved in synaptic vesicle fusion. Such activity is exerted by the N-terminal 50kDa domain of TeNT which is a zinc-dependent endopeptidase (TeNT-L-chain). Based on the three-dimensional structure of botulinum neurotoxin serotype A (BoNT/A) and serotype B (BoNT/B), two proteins closely related to TeNT, and on X-ray scattering studies of TeNT, we have designed mutations at two active site residues to probe their involvement in activity. The active site of metalloproteases is composed of a primary sphere of residues co-ordinating the zinc atom, and a secondary sphere of residues that determines proteolytic specificity and activity. Glu-261 and Glu-267 directly co-ordinates the zinc atom in BoNT/A and BoNT/B respectively and the corresponding residue of TeNT was replaced by Asp or by the non conservative residue Ala. Tyr-365 is 4.3A away from zinc in BoNT/A, and the corresponding residue of TeNT was replaced by Phe or by Ala. The purified mutants had CD, fluorescence and UV spectra closely similar to those of the wild-type molecule. The proteolytic activity of TeNT-Asp-271 (E271D) is similar to that of the native molecule, whereas that of TeNT-Phe-375 (Y375F) is lower than the control. Interestingly, the two Ala mutants are completely devoid of enzymatic activity. These results demonstrate that both Glu-271 and Tyr-375 are essential for the proteolytic activity of TeNT. PMID:11306125

  9. GAS HYDRATES AT TWO SITES OF AN ACTIVE CONTINENTAL MARGIN.

    USGS Publications Warehouse

    Kvenvolden, K.A.

    1985-01-01

    Sediment containing gas hydrates from two distant Deep Sea Drilling Project sites (565 and 568), located about 670 km apart on the landward flank of the Middle America Trench, was studied to determine the geochemical conditions that characterize the occurrence of gas hydrates. Site 565 was located in the Pacific Ocean offshore the Nicoya Peninsula of Costa Rica in 3,111 m of water. The depth of the hole at this site was 328 m, and gas hydrates were recovered from 285 and 319 m. Site 568 was located about 670 km to the northwest offshore Guatemala in 2,031 m of water. At this site the hole penetrated to 418 m, and gas hydrates were encountered at 404 m.

  10. Dynamically Achieved Active Site Precision in Enzyme Catalysis

    PubMed Central

    2015-01-01

    Conspectus The grand challenge in enzymology is to define and understand all of the parameters that contribute to enzymes’ enormous rate accelerations. The property of hydrogen tunneling in enzyme reactions has moved the focus of research away from an exclusive focus on transition state stabilization toward the importance of the motions of the heavy atoms of the protein, a role for reduced barrier width in catalysis, and the sampling of a protein conformational landscape to achieve a family of protein substates that optimize enzyme–substrate interactions and beyond. This Account focuses on a thermophilic alcohol dehydrogenase for which the chemical step of hydride transfer is rate determining across a wide range of experimental conditions. The properties of the chemical coordinate have been probed using kinetic isotope effects, indicating a transition in behavior below 30 °C that distinguishes nonoptimal from optimal C–H activation. Further, the introduction of single site mutants has the impact of either enhancing or eliminating the temperature dependent transition in catalysis. Biophysical probes, which include time dependent hydrogen/deuterium exchange and fluorescent lifetimes and Stokes shifts, have also been pursued. These studies allow the correlation of spatially resolved transitions in protein motions with catalysis. It is now possible to define a long-range network of protein motions in ht-ADH that extends from a dimer interface to the substrate binding domain across to the cofactor binding domain, over a distance of ca. 30 Å. The ongoing challenge to obtaining spatial and temporal resolution of catalysis-linked protein motions is discussed. PMID:25539048

  11. Cost and threshold analysis of positive charge, a multi-site linkage to HIV care program in the United States.

    PubMed

    Kim, Jeeyon Janet; Maulsby, Cathy; Zulliger, Rose; Jain, Kriti; Charles, Vignetta; Riordan, Maura; Davey-Rothwell, Melissa; Holtgrave, David R

    2015-10-01

    Positive Charge (PC) is a linkage to HIV care initiative implemented by AIDS United with sites in New York, Chicago, Louisiana, North Carolina, and the San Francisco/Bay Area. This study employed standard methods of cost and threshold analyses, as recommended by the US Panel on Cost-effectiveness in Health and Medicine, to calculate cost-saving and cost effective thresholds of the initiative. The overall societal cost of the linkage to care programs ranged from $48,490 to $370,525. The study found that PC's five unique evidence-based linkage to care programs have relatively low costs per client served and highly achievable cost-saving and cost-effectiveness thresholds. The findings from this study suggest that HIV linkage to care programs have the potential to be a highly productive use of public health resources. PMID:26139421

  12. Activity of N-coordinated multi-metal-atom active site structures for Pt-free oxygen reduction reaction catalysis: Role of *OH ligands

    NASA Astrophysics Data System (ADS)

    Holby, Edward F.; Taylor, Christopher D.

    2015-03-01

    We report calculated oxygen reduction reaction energy pathways on multi-metal-atom structures that have previously been shown to be thermodynamically favorable. We predict that such sites have the ability to spontaneously cleave the O2 bond and then will proceed to over-bind reaction intermediates. In particular, the *OH bound state has lower energy than the final 2 H2O state at positive potentials. Contrary to traditional surface catalysts, this *OH binding does not poison the multi-metal-atom site but acts as a modifying ligand that will spontaneously form in aqueous environments leading to new active sites that have higher catalytic activities. These *OH bound structures have the highest calculated activity to date.

  13. Activity of N-coordinated multi-metal-atom active site structures for Pt-free oxygen reduction reaction catalysis: Role of *OH ligands

    PubMed Central

    Holby, Edward F.; Taylor, Christopher D.

    2015-01-01

    We report calculated oxygen reduction reaction energy pathways on multi-metal-atom structures that have previously been shown to be thermodynamically favorable. We predict that such sites have the ability to spontaneously cleave the O2 bond and then will proceed to over-bind reaction intermediates. In particular, the *OH bound state has lower energy than the final 2 H2O state at positive potentials. Contrary to traditional surface catalysts, this *OH binding does not poison the multi-metal-atom site but acts as a modifying ligand that will spontaneously form in aqueous environments leading to new active sites that have higher catalytic activities. These *OH bound structures have the highest calculated activity to date. PMID:25788358

  14. Activity of N-coordinated multi-metal-atom active site structures for Pt-free oxygen reduction reaction catalysis: role of *OH ligands.

    PubMed

    Holby, Edward F; Taylor, Christopher D

    2015-01-01

    We report calculated oxygen reduction reaction energy pathways on multi-metal-atom structures that have previously been shown to be thermodynamically favorable. We predict that such sites have the ability to spontaneously cleave the O2 bond and then will proceed to over-bind reaction intermediates. In particular, the *OH bound state has lower energy than the final 2 H2O state at positive potentials. Contrary to traditional surface catalysts, this *OH binding does not poison the multi-metal-atom site but acts as a modifying ligand that will spontaneously form in aqueous environments leading to new active sites that have higher catalytic activities. These *OH bound structures have the highest calculated activity to date. PMID:25788358

  15. Activity of N-coordinated multi-metal-atom active site structures for Pt-free oxygen reduction reaction catalysis: Role of *OH ligands

    DOE PAGESBeta

    Holby, Edward F.; Taylor, Christopher D.

    2015-03-19

    We report calculated oxygen reduction reaction energy pathways on multi-metal-atom structures that have previously been shown to be thermodynamically favorable. We predict that such sites have the ability to spontaneously cleave the O₂ bond and then will proceed to over-bind reaction intermediates. In particular, the *OH bound state has lower energy than the final 2 H₂O state at positive potentials. Contrary to traditional surface catalysts, this *OH binding does not poison the multi-metal-atom site but acts as a modifying ligand that will spontaneously form in aqueous environments leading to new active sites that have higher catalytic activities. These *OH boundmore » structures have the highest calculated activity to date.« less

  16. Activity of N-coordinated multi-metal-atom active site structures for Pt-free oxygen reduction reaction catalysis: Role of *OH ligands

    SciTech Connect

    Holby, Edward F.; Taylor, Christopher D.

    2015-03-19

    We report calculated oxygen reduction reaction energy pathways on multi-metal-atom structures that have previously been shown to be thermodynamically favorable. We predict that such sites have the ability to spontaneously cleave the O₂ bond and then will proceed to over-bind reaction intermediates. In particular, the *OH bound state has lower energy than the final 2 H₂O state at positive potentials. Contrary to traditional surface catalysts, this *OH binding does not poison the multi-metal-atom site but acts as a modifying ligand that will spontaneously form in aqueous environments leading to new active sites that have higher catalytic activities. These *OH bound structures have the highest calculated activity to date.

  17. Robotics and Automation Activities at the Savannah River Site: A Site Report for SUBWOG 39F

    SciTech Connect

    Teese, G.D.

    1995-09-28

    The Savannah River Site has successfully used robots, teleoperators, and remote video to reduce exposure to ionizing radiation, improve worker safety, and improve the quality of operations. Previous reports have described the use of mobile teleoperators in coping with a high level liquid waste spill, the removal of highly contaminated equipment, and the inspection of nuclear reactor vessels. This report will cover recent applications at the Savannah River, as well as systems which SRS has delivered to other DOE site customers.

  18. Inhibitory activity of the equine infectious anemia virus major 5' splice site in the absence of Rev.

    PubMed Central

    Tan, W; Schalling, M; Zhao, C; Luukkonen, M; Nilsson, M; Fenyö, E M; Pavlakis, G N; Schwartz, S

    1996-01-01

    The major 5' splice site of equine infectious anemia virus (EIAV) conforms to the consensus 5' splice site in eight consecutive positions and is located immediately upstream of the gag AUG. Our results show that the presence of this 5' splice site on the EIAV gag mRNA decreases Gag production 30- to 60-fold. This is caused by inefficient nuclear mRNA export and inefficient mRNA utilization. Inhibition could be overcome by providing human immunodeficiency virus type 1 Rev/Rev-responsive element, human T-cell leukemia virus type 1 Rex/Rex-responsive element, or simian retrovirus type 1 constitutive transport element. In addition, inhibition could be abolished by introducing single point mutations in the 5' splice site or by moving the 5' splice site away from its natural position immediately upstream of the gag AUG. This demonstrates that both maintenance of a perfect consensus 5' splice site and its proper location on the mRNA are important for inhibitory activity of the EIAV major 5' splice site. PMID:8648699

  19. p21-activated kinase 4 regulates mitotic spindle positioning and orientation.

    PubMed

    Bompard, Guillaume; Morin, Nathalie

    2012-01-01

    During mitosis, microtubules (MTs) are massively rearranged into three sets of highly dynamic MTs that are nucleated from the centrosomes to form the mitotic spindle. Tight regulation of spindle positioning in the dividing cell and chromosome alignment at the center of the metaphase spindle are required to ensure perfect chromosome segregation and to position the cytokinetic furrow that will specify the two daughter cells. Spindle positioning requires regulation of MT dynamics, involving depolymerase activities together with cortical and kinetochore-mediated pushing and pulling forces acting on astral MTs and kinetochore fibres. These forces rely on MT motor activities. Cortical pulling forces exerted on astral MTs depend upon dynein/dynactin complexes and are essential in both symmetric and asymmetric cell division. A well-established spindle positioning pathway regulating the cortical targeting of dynein/dynactin involves the conserved LGN (Leu-Gly-Asn repeat-enriched-protein) and NuMA (microtubule binding nuclear mitotic apparatus protein) complex. Spindle orientation is also regulated by integrin-mediated cell adhesion and actin retraction fibres that respond to mechanical stress and are influenced by the microenvironment of the dividing cell. Altering the capture of astral MTs or modulating pulling forces affects spindle position, which can impair cell division, differentiation and embryogenesis. In this general scheme, the activity of mitotic kinases such as Auroras and Plk1 (Polo-like kinase 1) is crucial. Recently, the p21-activated kinases (PAKs) emerged as novel important players in mitotic progression. In our recent article, we demonstrated that PAK4 regulates spindle positioning in symmetric cell division. In this commentary, and in light of recent published studies, we discuss how PAK4 could participate in the regulation of mechanisms involved in spindle positioning and orientation. PMID:22960742

  20. Improving upon Nature: Active site remodeling produces highly efficient aldolase activity towards hydrophobic electrophilic substrates

    PubMed Central

    Cheriyan, Manoj; Toone, Eric J.; Fierke, Carol A.

    2012-01-01

    Substrate specificity of enzymes is frequently narrow and constrained by multiple interactions, limiting the use of natural enzymes in biocatalytic applications. Aldolases have important synthetic applications, but the usefulness of these enzymes is hampered by their narrow reactivity profile with unnatural substrates. To explore the determinants of substrate selectivity and alter the specificity of E. coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, we employed structure-based mutagenesis coupled with library screening of mutant enzymes localized to the bacterial periplasm. We identified two active site mutations (T161S/S184L) that work additively to enhance the substrate specificity of this aldolase to include catalysis of retro-aldol cleavage of (4S)-2-keto-4-hydroxy-4-(2′-pyridyl)butyrate (S-KHPB). These mutations improve the value of kcat/KMS-KHPB by >450-fold, resulting in a catalytic efficiency that is comparable to that of the wild-type enzyme with the natural substrate while retaining high stereoselectivity. Moreover, the value of kcatS-KHPB for this mutant enzyme, a parameter critical for biocatalytic applications, is 3-fold higher than the maximum value achieved by the natural aldolase with any substrate. This mutant also possesses high catalytic efficiency for the retro-aldol cleavage of the natural substrate, KDPG, and a >50-fold improved activity for cleavage of 2-keto-4-hydroxy-octonoate (KHO), a non-functionalized hydrophobic analog. These data suggest a substrate binding mode that illuminates the origin of facial selectivity in aldol addition reactions catalyzed by KDPG and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases. Furthermore, targeting mutations to the active site provides marked improvement in substrate selectivity, demonstrating that structure-guided active site mutagenesis combined with selection techniques can efficiently identify proteins with characteristics that compare favorably to naturally occurring enzymes. PMID

  1. Atomically-thin two-dimensional sheets for understanding active sites in catalysis.

    PubMed

    Sun, Yongfu; Gao, Shan; Lei, Fengcai; Xie, Yi

    2015-02-01

    Catalysis can speed up chemical reactions and it usually occurs on the low coordinated steps, edges, terraces, kinks and corner atoms that are often called "active sites". However, the atomic level interplay between active sites and catalytic activity is still an open question, owing to the large difference between idealized models and real catalysts. This stimulates us to pursue a suitable material model for studying the active sites-catalytic activity relationship, in which the atomically-thin two-dimensional sheets could serve as an ideal model, owing to their relatively simple type of active site and the ultrahigh fraction of active sites that are comparable to the overall atoms. In this tutorial review, we focus on the recent progress in disclosing the factors that affect the activity of reactive sites, including characterization of atomic coordination number, structural defects and disorder in ultrathin two-dimensional sheets by X-ray absorption fine structure spectroscopy, positron annihilation spectroscopy, electron spin resonance and high resolution transmission electron microscopy. Also, we overview their applications in CO catalytic oxidation, photocatalytic water splitting, electrocatalytic oxygen and hydrogen evolution reactions, and hence highlight the atomic level interplay among coordination number, structural defects/disorder, active sites and catalytic activity in the two-dimensional sheets with atomic thickness. Finally, we also present the major challenges and opportunities regarding the role of active sites in catalysis. We believe that this review provides critical insights for understanding the catalysis and hence helps to develop new catalysts with high catalytic activity. PMID:25382246

  2. The active sites of supported silver particle catalysts in formaldehyde oxidation.

    PubMed

    Chen, Yaxin; Huang, Zhiwei; Zhou, Meijuan; Hu, Pingping; Du, Chengtian; Kong, Lingdong; Chen, Jianmin; Tang, Xingfu

    2016-08-01

    Surface silver atoms with upshifted d-orbitals are identified as the catalytically active sites in formaldehyde oxidation by correlating their activity with the number of surface silver atoms, and the degree of the d-orbital upshift governs the catalytic performance of the active sites. PMID:27406403

  3. HIV Serosorting, Status Disclosure, and Strategic Positioning Among Highly Sexually Active Gay and Bisexual Men.

    PubMed

    Grov, Christian; Rendina, H Jonathon; Moody, Raymond L; Ventuneac, Ana; Parsons, Jeffrey T

    2015-10-01

    Researchers have identified harm reduction strategies that gay, bisexual, and other men who have sex with men (GBMSM) use to reduce HIV transmission--including serosorting, status disclosure, and strategic positioning. We report on patterns of these behaviors among 376 highly sexually active (i.e., 9+partners, <90 days) GBMSM: mean age of 37, 49.5% men of color, 87.8% gay identified, 57.5% college educated. We found evidence that many men engaged in serosorting, status disclosure, and strategic positioning; however, rates varied based on the participant's HIV status. HIV-positive and HIV-negative men both engaged in sex with men of similar status more often than they engaged in sex with men known to be a different HIV status (i.e., serosorting). However, HIV-negative men disclosed their HIV-status with about half of their partners, whereas HIV-positive participants disclosed with only about one-third. With regard to strategic positioning, HIV-positive participants were the receptive partner about half the time with their HIV-negative partners and with their HIV-positive partners. In contrast, strategic positioning was very common among HIV-negative participants-they rarely bottomed with HIV-positive partners, bottomed about one-third of the time with status-unknown partners, and 42% of the time (on average) with HIV-negative partners. Highly sexually active GBMSM are a critical population in which to both investigate HIV prevention strategies as well as develop effective intervention programs. Providers and clinicians might be well served to include a wide range of behavioral harm reduction strategies in addition to condom use and biomedical approaches to reduce onward HIV transmission. PMID:26348322

  4. SUMO-activating SAE1 transcription is positively regulated by Myc

    PubMed Central

    Amente, Stefano; Lavadera, Miriam Lubrano; Palo, Giacomo Di; Majello, Barbara

    2012-01-01

    Myc protein plays a fundamental role in regulation of cell cycle, proliferation, differentiation and apoptosis by modulating the expression of a large number of targets. Here we report the transactivation ability of the human Myc protein to activate the SUMO-activating enzyme SAE1 transcription. We found that Myc activates SAE1 transcription via direct binding to canonical E-Boxes sequences located close to the SAE1 transcription start site. A recent report has highlighted the crucial role of the SAE gene expression in Myc mediated oncogenesis. Our study adds new insight in this context since we show here that Myc directly activates SAE1 transcription, suggesting that Myc oncogenic activity which depends on SAE1 is ensured by Myc itself through direct binding and transcriptional activation of SAE1 expression. PMID:22679563

  5. Structural Basis for Substrate and Oxygen Activation in Homoprotocatechuate 2,3-Dioxygenase: Roles of Conserved Active Site Histidine-200

    PubMed Central

    Kovaleva, Elena G.; Rogers, Melanie S.; Lipscomb, John D.

    2015-01-01

    Kinetic and spectroscopic studies have shown that the conserved active site residue His200 of the extradiol ring-cleaving homoprotocatechuate 2,3-dioxygenase (FeHPCD) from Brevibacterium fuscum is critical for efficient catalysis. The roles played by this residue are probed here by analysis of the steady state kinetics, pH dependence, and X-ray crystal structures of the FeHPCD position 200 variants His200Asn, His200Gln, and His200Glu alone and in complex with three catecholic substrates (homoprotocatechuate, 4-sulfonylcatechol, and 4-nitrocatechol) possessing substituents with different inductive capacity. Structures solved at 1.35 –1.75 Å resolution show that there is essentially no change in overall active site architecture or substrate binding mode for these variants when compared to the structures of the wild type enzyme and its analogous complexes. This shows that the maximal 50-fold decrease in kcat for ring cleavage, the dramatic changes in pH dependence, and the switch from ring cleavage to ring oxidation of 4-nitrocatechol by the FeHPCD variants can be attributed specifically to the properties of the altered second sphere residue and the substrate. The results suggest that proton transfer is necessary for catalysis, and that it occurs most efficiently when the substrate provides the proton and His200 serves as a catalyst. However, in the absence of an available substrate proton, a defined proton-transfer pathway in the protein can be utilized. Changes in steric bulk and charge of the residue at position 200 appear capable of altering the rate-limiting step in catalysis, and perhaps, the nature of the reactive species. PMID:26267790

  6. Structural Basis for Substrate and Oxygen Activation in Homoprotocatechuate 2,3-Dioxygenase: Roles of Conserved Active Site Histidine 200.

    PubMed

    Kovaleva, Elena G; Rogers, Melanie S; Lipscomb, John D

    2015-09-01

    Kinetic and spectroscopic studies have shown that the conserved active site residue His200 of the extradiol ring-cleaving homoprotocatechuate 2,3-dioxygenase (FeHPCD) from Brevibacterium fuscum is critical for efficient catalysis. The roles played by this residue are probed here by analysis of the steady-state kinetics, pH dependence, and X-ray crystal structures of the FeHPCD position 200 variants His200Asn, His200Gln, and His200Glu alone and in complex with three catecholic substrates (homoprotocatechuate, 4-sulfonylcatechol, and 4-nitrocatechol) possessing substituents with different inductive capacity. Structures determined at 1.35-1.75 Å resolution show that there is essentially no change in overall active site architecture or substrate binding mode for these variants when compared to the structures of the wild-type enzyme and its analogous complexes. This shows that the maximal 50-fold decrease in kcat for ring cleavage, the dramatic changes in pH dependence, and the switch from ring cleavage to ring oxidation of 4-nitrocatechol by the FeHPCD variants can be attributed specifically to the properties of the altered second-sphere residue and the substrate. The results suggest that proton transfer is necessary for catalysis, and that it occurs most efficiently when the substrate provides the proton and His200 serves as a catalyst. However, in the absence of an available substrate proton, a defined proton-transfer pathway in the protein can be utilized. Changes in the steric bulk and charge of the residue at position 200 appear to be capable of altering the rate-limiting step in catalysis and, perhaps, the nature of the reactive species. PMID:26267790

  7. Identification of catalytically important residues in the active site of Escherichia coli transaldolase.

    PubMed

    Schörken, U; Thorell, S; Schürmann, M; Jia, J; Sprenger, G A; Schneider, G

    2001-04-01

    The roles of invariant residues at the active site of transaldolase B from Escherichia coli have been probed by site-directed mutagenesis. The mutant enzymes D17A, N35A, E96A, T156A, and S176A were purified from a talB-deficient host and analyzed with respect to their 3D structure and kinetic behavior. X-ray analysis showed that side chain replacement did not induce unanticipated structural changes in the mutant enzymes. Three mutations, N35A, E96A, and T156A resulted mainly in an effect on apparent kcat, with little changes in apparent Km values for the substrates. Residues N35 and T156 are involved in the positioning of a catalytic water molecule at the active site and the side chain of E96 participates in concert with this water molecule in proton transfer during catalysis. Substitution of Ser176 by alanine resulted in a mutant enzyme with 2.5% residual activity. The apparent Km value for the donor substrate, fructose 6-phosphate, was increased nearly fivefold while the apparent Km value for the acceptor substrate, erythrose 4-phosphate remained unchanged, consistent with a function for S176 in the binding of the C1 hydroxyl group of the donor substrate. The mutant D17A showed a 300-fold decrease in kcat, and a fivefold increase in the apparent Km value for the acceptor substrate erythrose 4-phosphate, suggesting a role of this residue in carbon-carbon bond cleavage and stabilization of the carbanion/enamine intermediate. PMID:11298760

  8. Mutation of Gly721 Alters DNA Topoisomerase I Active Site Architecture and Sensitivity to Camptothecin*

    PubMed Central

    van der Merwe, Marie; Bjornsti, Mary-Ann

    2015-01-01

    DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA via a concerted mechanism of DNA strand cleavage and religation. Top1p is the cellular target of the anticancer drug camptothecin (CPT), which reversibly stabilizes a covalent enzyme-DNA intermediate. Top1p clamps around duplex DNA, wherein the core and C-terminal domains are connected by extended α-helices (linker domain), which position the active site Tyr of the C-terminal domain within the catalytic pocket. The physical connection of the linker with the Top1p clamp as well as linker flexibility affect enzyme sensitivity to CPT. Crystallographic data reveal that a conserved Gly residue (located at the juncture between the linker and C-terminal domains) is at one end of a short α-helix, which extends to the active site Tyr covalently linked to the DNA. In the presence of drug, the linker is rigid and this α-helix extends to include Gly and the preceding Leu. We report that mutation of this conserved Gly in yeast Top1p alters enzyme sensitivity to CPT. Mutating Gly to Asp, Glu, Asn, Gln, Leu, or Ala enhanced enzyme CPT sensitivity, with the acidic residues inducing the greatest increase in drug sensitivity in vivo and in vitro. By contrast, Val or Phe substituents rendered the enzyme CPT-resistant. Mutation-induced alterations in enzyme architecture preceding the active site Tyr suggest these structural transitions modulate enzyme sensitivity to CPT, while enhancing the rate of DNA cleavage. We postulate that this conserved Gly residue provides a flexible hinge within the Top1p catalytic pocket to facilitate linker dynamics and the structural alterations that accompany drug binding of the covalent enzyme-DNA intermediate. PMID:18056711

  9. Identification of promiscuous ene-reductase activity by mining structural databases using active site constellations

    PubMed Central

    Steinkellner, Georg; Gruber, Christian C.; Pavkov-Keller, Tea; Binter, Alexandra; Steiner, Kerstin; Winkler, Christoph; Łyskowski, Andrzej; Schwamberger, Orsolya; Oberer, Monika; Schwab, Helmut; Faber, Kurt; Macheroux, Peter; Gruber, Karl

    2014-01-01

    The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites (‘catalophores’). As a proof-of-concept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C–C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts. PMID:24954722

  10. A Systematic Review of the Relationship between Socio-Economic Position and Physical Activity

    ERIC Educational Resources Information Center

    Gidlow, Christopher; Johnston, Lynne Halley; Crone, Diane; Ellis, Naomi; James, David

    2006-01-01

    Objective: The aim of the present review was to examine epidemiological evidence to determine if there is strong evidence of a positive gradient of increasing physical activity across the socio-economic strata, and how relationships are affected by socio-economic measurement. Design: Systematic review. Method: A search of major databases was…

  11. Positive Youth Development through an Outdoor Physical Activity Programme: Evidence from a Four-Year Evaluation

    ERIC Educational Resources Information Center

    Armour, Kathleen; Sandford, Rachel

    2013-01-01

    In 2006, Sandford, Armour and Warmington undertook a comprehensive review of the literature on the role of physical activity/sport and physical education in promoting positive development for disaffected youth. This paper revisits the findings of the literature review in light of data from a four-year evaluation of one corporate-sponsored physical…

  12. Enhanced Right Amygdala Activity in Adolescents during Encoding of Positively-Valenced Pictures

    PubMed Central

    Vasa, Roma A.; Pine, Daniel S.; Thorn, Julia M.; Nelson, Tess E.; Spinelli, Simona; Nelson, Eric; Maheu, Francoise S.; Ernst, Monique; Bruck, Maggie; Mostofsky, Stewart H.

    2010-01-01

    While studies among adults implicate the amygdala and interconnecting brain regions in encoding emotional stimuli, few studies have examined whether developmental changes occur within this emotional-memory network during adolescence. The present study examined whether adolescents and adults differentially engaged the amygdala and hippocampus during successful encoding of emotional pictures, with either positive or negative valence. Eighteen adults and twelve adolescents underwent event-related fMRI while encoding emotional pictures. Approximately 30 minutes later, outside the scanner, subjects were asked to recall the pictures seen during the scan. Age group differences in brain activity in the amygdala and hippocampus during encoding of the pictures that were later successfully and unsuccessfully recalled were separately compared for the positive and negative pictures. Adolescents, relative to adults, demonstrated enhanced activity in the right amygdala during encoding of positive pictures that were later recalled compared to not recalled. There were no age group differences in amygdala or hippocampal activity during successful encoding of negative pictures. The findings of preferential activity within the adolescent right amygdala during successful encoding of positive pictures may have implications for the increased reward and novelty seeking behavior, as well as elevated rates of psychopathology, observed during this distinct developmental period. PMID:21127721

  13. Positive Activities: Qualitative Research with Parents. Solutions Research. Research Report. DCSF-RR142

    ERIC Educational Resources Information Center

    Department for Children, Schools and Families, 2009

    2009-01-01

    This research was commissioned by COI and DCSF to understand in depth, the barriers, motivators and messages for parents to encourage participation in positive activities for young people. Within this the research was designed to understand the level of influence of parents in whether a young person participates/what a young person might…

  14. Physical Activity and Positive Youth Development: Impact of a School-Based Program

    ERIC Educational Resources Information Center

    Madsen, Kristine A.; Hicks, Katherine; Thompson, Hannah

    2011-01-01

    Background: Protective factors associated with positive youth development predict health and education outcomes. This study explored trends in these protective factors and in physical activity among low-income students, and determined the impact of a school-based youth development program on these trends. Methods: This study used a…

  15. Students' Use of Extra-Curricular Activities for Positional Advantage in Competitive Job Markets

    ERIC Educational Resources Information Center

    Roulin, Nicolas; Bangerter, Adrian

    2013-01-01

    With the rise of mass higher education, competition between graduates in the labour market is increasing. Students are aware that their degree will not guarantee them a job and realise they should add value and distinction to their credentials to achieve a positional advantage. Participation in extra-curricular activities (ECAs) is one such…

  16. Research on acupuncture points and cortical functional activation position in cats by infrared imaging detection

    NASA Astrophysics Data System (ADS)

    Chen, Shuwang; Sha, Zhanyou; Wang, Shuhai; Wen, Huanming

    2007-12-01

    The research of the brain cognition is mainly to find out the activation position in brain according to the stimulation at present in the world. The research regards the animals as the experimental objects and explores the stimulation response on the cerebral cortex of acupuncture. It provides a new method, which can detect the activation position on the creatural cerebral cortex directly by middle-far infrared imaging. According to the theory of local temperature situation, the difference of cortical temperature maybe associate with the excitement of cortical nerve cells, the metabolism of local tissue and the local hemal circulation. Direct naked detection of temperature variety on cerebral cortex is applied by middle and far infrared imaging technology. So the activation position is ascertained. The effect of stimulation response is superior to other indirect methods. After removing the skulls on the head, full of cerebral cortex of a cat are exposed. By observing the infrared images and measuring the temperatures of the visual cerebral cortex during the process of acupuncturing, the points are used to judge the activation position. The variety in the cortical functional sections is corresponding to the result of the acupuncture points in terms of infrared images and temperatures. According to experimental results, we know that the variety of a cortical functional section is corresponding to a special acupuncture point exactly.

  17. Assessing the Positive Influence of Music Activities in Community Development Programs

    ERIC Educational Resources Information Center

    Dillon, Steve

    2006-01-01

    This article describes a framework for assessing the positive influence of music activities in community development programs. It examines hybrid music, health and rich media approaches to creative case study with the purpose of developing more compelling evidence based advocacy that examines the claims of a causal link. This preliminary study…

  18. Influence of proline position upon the ion channel activity of alamethicin.

    PubMed Central

    Kaduk, C; Duclohier, H; Dathe, M; Wenschuh, H; Beyermann, M; Molle, G; Bienert, M

    1997-01-01

    Alamethicin, a 20-residue peptaibol, induces voltage-dependent ion channels in lipid bilayers according to the barrel-stave model. To study relationships between the proline-14-induced kink region and the channel-forming behavior of the peptide, a set of alamethicin analogs with proline incorporated at positions 11, 12, 13, 14, 15, 16, and 17, respectively, as well as an analog with alanine instead of proline at position 14 were synthesized. Macroscopic conductance experiments show that the voltage dependence of the peptides is conserved although slightly influenced, but the apparent mean number of monomers forming the channels is significantly reduced when proline is not located at position 14. This is confirmed in single-channel experiments. The analogs with proline next to position 14 (i.e., 13, 15, 16) show stable conductance levels, but of reduced number, which follows the order Alam-P14 > Alam-P15 > Alam-P16 > Alam-P13. This reduction in the number of levels is connected with changes in the lifetime of the channels. Analogs with proline at position 11, 12, or 17 produce erratic, extremely short-lived current events that could not be resolved. The changes in functional properties are related to structural properties as probed by circular dichroism. The results indicate that proline at position 14 results in optimal channel activity, whereas channels formed by the analogs bearing proline at different positions are considerably less stable. PMID:9129817

  19. Activity of the Aurora Kinase inhibitor VX-680 against Bcr/Abl positive acute lymphoblastic leukemias

    PubMed Central

    Fei, Fei; Stoddart, Sonia; Groffen, John; Heisterkamp, Nora

    2010-01-01

    The emergence of resistance to tyrosine kinase inhibitors due to point mutations in Bcr/Abl is a challenging problem for Philadelphia-chromosome positive (Ph-positive) acute lymphoblastic leukemia (ALL) patients, especially for those with the T315I mutation, against which neither nilotinib or dasatinib shows significant activity. VX-680 is a pan-Aurora kinase inhibitor active against all Bcr/Abl proteins but has not been extensively examined in preclinical models of Ph-positive ALL. Here, we have tested VX-680 for treatment of Bcr/Abl positive ALL when leukemic cells are protected by the presence of stroma. Under these conditions, VX-680 showed significant effects on primary human Ph-positive ALL cells both with and without the T315I mutation, including ablation of tyrosine phosphorylation downstream of Bcr/Abl, decreased viability and induction of apoptosis. However, drug treatment of human Ph-positive ALL cells for 3 days followed by drug removal allowed the outgrowth of abnormal cells 21 days later, and upon culture of mouse Bcr/Abl ALL cells on stroma with lower concentrations of VX-680, drug-resistant cells emerged. Combined treatment of human ALL cells lacking the T315I mutation with both VX-680 and dasatinib caused significantly more cytotoxicity than each drug alone. We suggest that use of VX-680 together with a second effective drug as first-line treatment for Ph-positive ALL is likely to be safer and more useful than second-line treatment with VX-680 as monotherapy for drug-resistant T315I Ph-positive ALL. PMID:20388735

  20. Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure

    PubMed Central

    O'Farrell, Paul A.; Joshua-Tor, Leemor

    2006-01-01

    Bleomycin hydrolase (BH) is a hexameric papain family cysteine protease which is involved in preparing peptides for antigen presentation and has been implicated in tumour cell resistance to bleomycin chemotherapy. Structures of active-site mutants of yeast BH yielded unexpected results. Replacement of the active-site asparagine with alanine, valine or leucine results in the destabilization of the histidine side chain, demonstrating unambiguously the role of the asparagine residue in correctly positioning the histidine for catalysis. Replacement of the histidine with alanine or leucine destabilizes the asparagine position, indicating a delicate arrangement of the active-site residues. In all of the mutants, the C-terminus of the protein, which lies in the active site, protrudes further into the active site. All mutants were compromised in their catalytic activity. The structures also revealed the importance of a tightly bound water molecule which stabilizes a loop near the active site and which is conserved throughout the papain family. It is displaced in a number of the mutants, causing destabilization of this loop and a nearby loop, resulting in a large movement of the active-site cysteine. The results imply that this water molecule plays a key structural role in this family of enzymes. PMID:17007609

  1. Experimental study on active vibration control using genetic algorithm-based system identification and optimized positive position feedback

    NASA Astrophysics Data System (ADS)

    Orszulik, Ryan R.; Shan, Jinjun

    2012-12-01

    A genetic algorithm is implemented to identify the transfer function of an experimental system consisting of a flexible manipulator with a collocated piezoelectric sensor/actuator pair. A multi-mode positive position feedback controller is then designed based upon the identified transfer function. To this end, the same iteratively implemented genetic algorithm is used to optimize all controller parameters by minimization of the closed loop H∞-norm. The designed controller is then applied for vibration suppression on the experimental system.

  2. Multiple, Ligand-Dependent Routes from the Active Site of Cytochrome P450 2C9

    SciTech Connect

    Cojocaru, Vlad; Winn, Peter J.; Wade, Rebecca C.

    2012-02-13

    The active site of liver-specific, drug-metabolizing cytochrome P450 (CYP) monooxygenases is deeply buried in the protein and is connected to the protein surface through multiple tunnels, many of which were found open in different CYP crystal structures. It has been shown that different tunnels could serve as ligand passage routes in different CYPs. However, it is not understood whether one CYP uses multiple routes for substrate access and product release and whether these routes depend on ligand properties. From 300 ns of molecular dynamics simulations of CYP2C9, the second most abundant CYP in the human liver we found four main ligand exit routes, the occurrence of each depending on the ligand type and the conformation of the F-G loop, which is likely to be affected by the CYP-membrane interaction. A non-helical F-G loop favored exit towards the putative membrane-embedded region. Important protein features that direct ligand exit include aromatic residues that divide the active site and whose motions control access to two pathways. The ligands interacted with positively charged residues on the protein surface through hydrogen bonds that appear to select for acidic substrates. The observation of multiple, ligand-dependent routes in a CYP aids understanding of how CYP mutations affect drug metabolism and provides new possibilities for CYP inhibition.

  3. Active sites of two orthologous cytochromes P450 2E1: Differences revealed by spectroscopic methods

    SciTech Connect

    Anzenbacherova, Eva; Hudecek, Jiri; Murgida, Daniel; Hildebrandt, Peter; Marchal, Stephane; Lange, Reinhard; Anzenbacher, Pavel . E-mail: anzen@tunw.upol.cz

    2005-12-09

    Cytochromes P450 2E1 of human and minipig origin were examined by absorption spectroscopy under high hydrostatic pressure and by resonance Raman spectroscopy. Human enzyme tends to denature to the P420 form more easily than the minipig form; moreover, the apparent compressibility of the heme active site (as judged from a redshift of the absorption maximum with pressure) is greater than that of the minipig counterpart. Relative compactness of the minipig enzyme is also seen in the Raman spectra, where the presence of planar heme conformation was inferred from band positions characteristic of the low-spin heme with high degree of symmetry. In this respect, the CYP2E1 seems to be another example of P450 conformational heterogeneity as shown, e.g., by Davydov et al. for CYP3A4 [Biochem. Biophys. Res. Commun. 312 (2003) 121-130]. The results indicate that the flexibility of the CYP active site is likely one of its basic structural characteristics.

  4. Development of Novel Sugar Isomerases by Optimization of Active Sites in Phosphosugar Isomerases for Monosaccharides

    PubMed Central

    Yeom, Soo-Jin; Kim, Yeong-Su

    2013-01-01

    Phosphosugar isomerases can catalyze the isomerization of not only phosphosugar but also of monosaccharides, suggesting that the phosphosugar isomerases can be used as sugar isomerases that do not exist in nature. Determination of active-site residues of phosphosugar isomerases, including ribose-5-phosphate isomerase from Clostridium difficile (CDRPI), mannose-6-phosphate isomerase from Bacillus subtilis (BSMPI), and glucose-6-phosphate isomerase from Pyrococcus furiosus (PFGPI), was accomplished by docking of monosaccharides onto the structure models of the isomerases. The determinant residues, including Arg133 of CDRPI, Arg192 of BSMPI, and Thr85 of PFGPI, were subjected to alanine substitutions and found to act as phosphate-binding sites. R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI displayed the highest catalytic efficiencies for monosaccharides at each position. These residues exhibited 1.8-, 3.5-, and 4.9-fold higher catalytic efficiencies, respectively, for the monosaccharides than the wild-type enzyme. However, the activities of these 3 variant enzymes for phosphosugars as the original substrates disappeared. Thus, R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI are no longer phosphosugar isomerases; instead, they are changed to a d-ribose isomerase, an l-ribose isomerase, and an l-talose isomerase, respectively. In this study, we used substrate-tailored optimization to develop novel sugar isomerases which are not found in nature based on phosphosugar isomerases. PMID:23204422

  5. The effect of methyl-donated hydrogen bonding on active site conformations of hyaluronate lyase

    NASA Astrophysics Data System (ADS)

    Migues, Angela N.; Vergenz, Robert A.; Moore, Kevin B.

    2010-03-01

    Geometric evidence shows a val-A252 methyl-donated (MD) hydrogen bond (HB) in hyaluronate lyase (Streptococcus pneumoniae) interacts with nearby NH--O and OH--O HBs, distorting active-site helical structure. Results for model fragment A248-254 are based on experimental heavy atom positions with ab initio hydrogen atoms. The MDHB, with (H-O distance, donor-H-O angle) = (2.3å; 174^o), exhibits more favorable geometry than thr-A253 OH--O HB (1.8å; 170^o) to the same ala-249 C=O. Consequently, thr-253 N-H--O interaction is forced closer to lys-250 C=O than ala-249 C=O(2.6 versus 2.7å). A novel method has been developed to quantify the effects of atomic diplacements on motions of neighboring helices. A coordinate system was established to track the movement of specific residues and to ascertain the effect of such motions on active site conformations.

  6. Protein Kinase A and Mitogen-Activated Protein Kinase Pathways Antagonistically Regulate Fission Yeast fbp1 Transcription by Employing Different Modes of Action at Two Upstream Activation Sites

    PubMed Central

    Neely, Lori A.; Hoffman, Charles S.

    2000-01-01

    A significant challenge to our understanding of eukaryotic transcriptional regulation is to determine how multiple signal transduction pathways converge on a single promoter to regulate transcription in divergent fashions. To study this, we have investigated the transcriptional regulation of the Schizosaccharomyces pombe fbp1 gene that is repressed by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway and is activated by a stress-activated mitogen-activated protein kinase (MAPK) pathway. In this study, we identified and characterized two cis-acting elements in the fbp1 promoter required for activation of fbp1 transcription. Upstream activation site 1 (UAS1), located approximately 900 bp from the transcriptional start site, resembles a cAMP response element (CRE) that is the binding site for the atf1-pcr1 heterodimeric transcriptional activator. Binding of this activator to UAS1 is positively regulated by the MAPK pathway and negatively regulated by PKA. UAS2, located approximately 250 bp from the transcriptional start site, resembles a Saccharomyces cerevisiae stress response element. UAS2 is bound by transcriptional activators and repressors regulated by both the PKA and MAPK pathways, although atf1 itself is not present in these complexes. Transcriptional regulation of fbp1 promoter constructs containing only UAS1 or UAS2 confirms that the PKA and MAPK regulation is targeted to both sites. We conclude that the PKA and MAPK signal transduction pathways regulate fbp1 transcription at UAS1 and UAS2, but that the antagonistic interactions between these pathways involve different mechanisms at each site. PMID:10938120

  7. The Role of an Active Site Mg2+ in HDV Ribozyme Self-Cleavage: Insights from QM/MM Calculations

    PubMed Central

    Mlýnský, Vojtěch; Šponer, Jiří

    2014-01-01

    The hepatitis delta virus (HDV) ribozyme is a catalytic RNA motif embedded in the human pathogenic HDV RNA. It catalyzes self-cleavage of its sugar-phosphate backbone with direct participation of the active site cytosine C75. Biochemical and structural data support a general acid role of C75. Here, we used hybrid quantum mechanical/molecular mechanical (QM/MM) calculations to probe the reaction mechanism and changes in Gibbs energy along the ribozyme's reaction pathway with an N3-protonated C75H+ in the active site, which acts as the general acid, and a partially hydrated Mg2+ ion with one deprotonated, inner-shell coordinated water molecule that acts as the general base. We followed eight reaction paths with distinct position and coordination of the catalytically important active site Mg2+ ion. For six of them, we observed feasible activation barriers ranging from 14.2 to 21.9 kcal/mol, indicating that the specific position of the Mg2+ ion in the active site is predicted to strongly affect the kinetics of self-cleavage. The deprotonation of the U-1(2′-OH) nucleophile and the nucleophilic attack of the resulting U-1(2′-O−) on the scissile phosphodiester are found to be separate steps, as deprotonation precedes the nucleophilic attack. This sequential mechanism of the HDV ribozyme differs from the concerted nucleophilic activation and attack suggested for the hairpin ribozyme. We estimated the pKa of the U-1(2′-OH) group to range from 8.8 to 11.2, suggesting that the pKa is lowered by several units from that of a free ribose, comparable to and most likely smaller than the pKa of the solvated active site Mg2+ ion. Our results thus support the notion that the structure of the HDV ribozyme, and particularly the positioning of the active site Mg2+ ion, facilitates deprotonation and activation of the 2′-OH nucleophile. PMID:25412464

  8. Structural mechanism of RuBisCO activation by carbamylation of the active site lysine

    PubMed Central

    Stec, Boguslaw

    2012-01-01

    Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a crucial enzyme in carbon fixation and the most abundant protein on earth. It has been studied extensively by biochemical and structural methods; however, the most essential activation step has not yet been described. Here, we describe the mechanistic details of Lys carbamylation that leads to RuBisCO activation by atmospheric CO2. We report two crystal structures of nitrosylated RuBisCO from the red algae Galdieria sulphuraria with O2 and CO2 bound at the active site. G. sulphuraria RuBisCO is inhibited by cysteine nitrosylation that results in trapping of these gaseous ligands. The structure with CO2 defines an elusive, preactivation complex that contains a metal cation Mg2+ surrounded by three H2O/OH molecules. Both structures suggest the mechanism for discriminating gaseous ligands by their quadrupole electric moments. We describe conformational changes that allow for intermittent binding of the metal ion required for activation. On the basis of these structures we propose the individual steps of the activation mechanism. Knowledge of all these elements is indispensable for engineering RuBisCO into a more efficient enzyme for crop enhancement or as a remedy to global warming. PMID:23112176

  9. Is carbon sequestration on post mining sites driven by earthworm activity?

    NASA Astrophysics Data System (ADS)

    Frouz, J.; Pizl, V.

    2009-04-01

    Carbon storage was measured in seven types of forest (alder, oak, lime, willow-birch, pine, spruce and larch) about 30 years old developed in on e large post mining site as split plot design. The carbon storage in soil wary substantial and represent 10-100% of carbon storage in aboveground wood biomass. Carbon storage in soil do not show any correlation with litter input but correlate significantly and positively with earthworm abundance, and micromorphological traces of earthworm activity. Field and laboratory microcosm experiment showed that earthworm mediated soil mixing support carbon storage in soil. Detailed study of soil aggregates created by worms and other forces indicated that worm aggregates contain much larger content of POC. This indicate that soil bioturbation by earthworms may significantly increase carbon storage in soil.

  10. Active vibration suppression through positive acceleration feedback on a building-like structure: An experimental study

    NASA Astrophysics Data System (ADS)

    Enríquez-Zárate, J.; Silva-Navarro, G.; Abundis-Fong, H. F.

    2016-05-01

    This work deals with the structural and dynamic analysis of a building-like structure consisting of a three-story building with one active vibration absorber. The base of the structure is perturbed using an electromagnetic shaker, which provides forces with a wide range of excitation frequencies, including some resonance frequencies of the structure. One beam-column of the structure is coupled with a PZT stack actuator to reduce the vibrations. The overall mechanical structure is modeled using Euler-Lagrange methodology and validated using experimental modal analysis and Fine Element Method (FEM) techniques. The active control laws are synthesized to actively attenuate the vibration system response via the PZT stack actuator, caused by excitation forces acting on the base of the structure. The control scheme is obtained using Positive Acceleration Feedback (PAF) and Multiple Positive Acceleration Feedback (MPAF) to improve the closed-loop system response. Some experimental results are included to illustrate the overall system performance.

  11. Positive And Negative Feedback Loops Coupled By Common Transcription Activator And Repressor

    NASA Astrophysics Data System (ADS)

    Sielewiesiuk, Jan; Łopaciuk, Agata

    2015-03-01

    Dynamical systems consisting of two interlocked loops with negative and positive feedback have been studied using the linear analysis of stability and numerical solutions. Conditions for saddle-node bifurcation were formulated in a general form. Conditions for Hopf bifurcations were found in a few symmetrical cases. Auto-oscillations, when they exist, are generated by the negative feedback repressive loop. This loop determines the frequency and amplitude of oscillations. The positive feedback loop of activation slightly modifies the oscillations. Oscillations are possible when the difference between Hilll's coefficients of the repression and activation is sufficiently high. The highly cooperative activation loop with a fast turnover slows down or even makes the oscillations impossible. The system under consideration can constitute a component of epigenetic or enzymatic regulation network.

  12. 78 FR 33908 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-05

    ... identified Wind Energy Area (WEA) on the OCS offshore Rhode Island (RI) and Massachusetts (MA). The revised... from leasing, site characterization, and site assessment in and around the Call Area (76 FR 51391). The... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on...

  13. 77 FR 39508 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-03

    ... specific project proposals on those leases) in an identified Wind Energy Area (WEA) on the OCS offshore..., site characterization, and site assessment in and around the Call Area (76 FR 51391). The Call Area is... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on...

  14. Active Layer and Moisture Measurements for Intensive Site 0 and 1, Barrow, Alaska

    DOE Data Explorer

    John Peterson

    2015-04-17

    These are measurements of Active Layer Thickness collected along several lines beginning in September, 2011 to the present. The data were collected at several time periods along the Site0 L2 Line, the Site1 AB Line, and an ERT Monitoring Line near Area A in Site1.

  15. Nuclear Site Security in the Event of Terrorist Activity

    SciTech Connect

    Thomson, M.L.; Sims, J.

    2008-07-01

    This paper, presented as a poster, identifies why ballistic protection should now be considered at nuclear sites to counter terrorist threats. A proven and flexible form of multi purpose protection is described in detail with identification of trial results that show its suitability for this role. (authors)

  16. Preliminary siting activities for new waste handling facilities at the Idaho National Engineering Laboratory

    SciTech Connect

    Taylor, D.D.; Hoskinson, R.L.; Kingsford, C.O.; Ball, L.W.

    1994-09-01

    The Idaho Waste Processing Facility, the Mixed and Low-Level Waste Treatment Facility, and the Mixed and Low-Level Waste Disposal Facility are new waste treatment, storage, and disposal facilities that have been proposed at the Idaho National Engineering Laboratory (INEL). A prime consideration in planning for such facilities is the selection of a site. Since spring of 1992, waste management personnel at the INEL have been involved in activities directed to this end. These activities have resulted in the (a) identification of generic siting criteria, considered applicable to either treatment or disposal facilities for the purpose of preliminary site evaluations and comparisons, (b) selection of six candidate locations for siting,and (c) site-specific characterization of candidate sites relative to selected siting criteria. This report describes the information gathered in the above three categories for the six candidate sites. However, a single, preferred site has not yet been identified. Such a determination requires an overall, composite ranking of the candidate sites, which accounts for the fact that the sites under consideration have different advantages and disadvantages, that no single site is superior to all the others in all the siting criteria, and that the criteria should be assigned different weighing factors depending on whether a site is to host a treatment or a disposal facility. Stakeholder input should now be solicited to help guide the final selection. This input will include (a) siting issues not already identified in the siting, work to date, and (b) relative importances of the individual siting criteria. Final site selection will not be completed until stakeholder input (from the State of Idaho, regulatory agencies, the public, etc.) in the above areas has been obtained and a strategy has been developed to make a composite ranking of all candidate sites that accounts for all the siting criteria.

  17. Cumulative activation during positive and negative events and state anxiety predicts subsequent inertia of amygdala reactivity

    PubMed Central

    Miendlarzewska, Ewa A.; Eryilmaz, Hamdi; Vuilleumier, Patrik

    2015-01-01

    Inertia, together with intensity and valence, is an important component of emotion. We tested whether positive and negative events generate lingering changes in subsequent brain responses to unrelated threat stimuli and investigated the impact of individual anxiety. We acquired fMRI data while participants watched positive or negative movie-clips and subsequently performed an unrelated task with fearful and neutral faces. We quantified changes in amygdala reactivity to fearful faces as a function of the valence of preceding movies and cumulative neural activity evoked during them. We demonstrate that amygdala responses to emotional movies spill over to subsequent processing of threat information in a valence-specific manner: negative movies enhance later amygdala activation whereas positive movies attenuate it. Critically, the magnitude of such changes is predicted by a measure of cumulative amygdala responses to the preceding positive or negative movies. These effects appear independent of overt attention, are regionally limited to amygdala, with no changes in functional connectivity. Finally, individuals with higher state anxiety displayed stronger modulation of amygdala reactivity by positive movies. These results suggest that intensity and valence of emotional events as well as anxiety levels promote local changes in amygdala sensitivity to threat, highlighting the importance of past experience in shaping future affective reactivity. PMID:24603023

  18. The divide within: Older active ICT users position themselves against different 'Others'.

    PubMed

    Kania-Lundholm, Magdalena; Torres, Sandra

    2015-12-01

    Although research into older people's internet usage patterns is rapidly growing, their understandings of digital technologies, particularly in relation to how these are informed by their understandings of aging and old age, remain unexplored. This is the case because research on older active ICT users tends to regard old age as an empirically interesting part of the life-course as opposed to a theoretically profuse source of information about why and how older people engage with digital technologies. This article explores - through focus group interviews with 30 older adults (aged 66-89) - the ways in which the social position of old age is used by older active ICT users in order to make sense of how and why they engage with these technologies. In this article, positioning theory is used to shed light on how the older people interviewed positioned themselves as 'active older users' in the interviews. The analysis brings to the fore the divide that older people themselves create as they discursively position themselves against different types of ICT users and non-users (young and old) when describing how and why they engage with digital technologies. PMID:26568212

  19. Effect of sugar positions in ginsenosides and their inhibitory potency on Na+/K+-ATPase activity

    PubMed Central

    Chen, Ronald JY; Chung, Tse-yu; Li, Feng-yin; Lin, Nan-hei; Tzen, Jason TC

    2009-01-01

    Aim: To determine whether ginsenosides with various sugar attachments may act as active components responsible for the cardiac therapeutic effects of ginseng and sanqi (the roots of Panax ginseng and Panax notoginseng) via the same molecular mechanism triggered by cardiac glycosides, such as ouabain and digoxin. Methods: The structural similarity between ginsenosides and ouabain was analyzed. The inhibitory potency of ginsenosides and ouabain on Na+/K+-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of ginsenosides to Na+/K+-ATPase. Results: Ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure, equivalent to the sugar position in cardiac glycosides, and possessed inhibitory potency on Na+/K+-ATPase activity. However, their inhibitory potency was significantly reduced or completely abolished when a monosaccharide was linked to the C-6 or C-20 position of the steroid-like structure; replacement of the monosaccharide with a disaccharide molecule at either of these positions caused the disappearance of the inhibitory potency. Molecular modeling and docking confirmed that the difference in Na+/K+-ATPase inhibitory potency among ginsenosides was due to the steric hindrance of sugar attachment at the C-6 and C-20 positions of the steroid-like structure. Conclusion: The cardiac therapeutic effects of ginseng and sanqi should be at least partly attributed to the effective inhibition of Na+/K+-ATPase by their metabolized ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure. PMID:19060914

  20. Blogs and Social Network Sites as Activity Systems: Exploring Adult Informal Learning Process through Activity Theory Framework

    ERIC Educational Resources Information Center

    Heo, Gyeong Mi; Lee, Romee

    2013-01-01

    This paper uses an Activity Theory framework to explore adult user activities and informal learning processes as reflected in their blogs and social network sites (SNS). Using the assumption that a web-based space is an activity system in which learning occurs, typical features of the components were investigated and each activity system then…

  1. Kinetic and structural evaluation of selected active site mutants of the Aspergillus fumigatus KDNase (sialidase).

    PubMed

    Yeung, Juliana H F; Telford, Judith C; Shidmoossavee, Fahimeh S; Bennet, Andrew J; Taylor, Garry L; Moore, Margo M

    2013-12-23

    Aspergillus fumigatus is an airborne fungal pathogen. We previously cloned and characterized an exo-sialidase from A. fumigatus and showed that it preferred 2-keto-3-deoxynononic acid (KDN) as a substrate to N-acetylneuraminic acid (Neu5Ac). The purpose of this study was to investigate the structure-function relationships of critical catalytic site residues. Site-directed mutagenesis was used to create three mutant recombinant enzymes: the catalytic nucleophile (Y358H), the general acid/base catalyst (D84A), and an enlargement of the binding pocket to attempt to accommodate the N-acetyl group of Neu5Ac (R171L). Crystal structures for all enzymes were determined. The D84A mutation had an effect in decreasing the activity of AfKDNase that was stronger than that of the same mutation in the structurally similar sialidase from the bacterium Micromonospora viridifaciens. These data suggest that the catalytic acid is more important in the reaction of AfKDNase and that catalysis is less dependent on nucleophilic or electrostatic stabilization of the developing positive charge at the transition state for hydrolysis. Removal of the catalytic nucleophile (Y358H) significantly lowered the activity of the enzyme, but this mutant remained a retaining glycosidase as demonstrated by nuclear magnetic resonance spectroscopic analysis. This is a novel finding that has not been shown with other sialidases. Kinetic activity measured at pH 5.2 revealed that R171L had higher activity on a Neu5Ac-based substrate than wild-type KDNase; hence, leucine in place of arginine in the binding pocket improved catalysis toward Neu5Ac substrates. Hence, whether a sialidase is primarily a KDNase or a neuraminidase is due in part to the presence of an amino acid that creates a steric clash with the N-acetyl group. PMID:24295366

  2. Defining the Structural Parameters that Confer Anticonvulsant Activity by the Site-by-Site Modification of (R)-N′-Benzyl 2- Amino-3-methylbutanamide

    PubMed Central

    King, Amber; De Ryck, Marc; Kaminski, Rafal; Valade, Anne; Stables, James P.; Kohn, Harold

    2011-01-01

    Primary Amino Acid Derivatives (PAADs) (N′-benzyl 2-substituted 2-amino acetamides) are structurally related to Functionalized Amino Acids (FAAs) (N′-benzyl 2- substituted 2-acetamido acetamides) but differ by the absence of the terminal N-acetyl group. Both classes exhibit potent anticonvulsant activities in the maximal electroshock seizure animal model and the reported structure-activity relationships (SARs) of PAADs and FAAs differ in significant ways. Recently, we documented that PAAD efficacy was associated with a hydrocarbon moiety at the C(2)-carbon, while in the FAAs, a substituted heteroatom one atom removed from the C(2)-center was optimal. Previously in this issue, we showed that PAAD activity was dependent upon the electronic properties of the 4′-N′-benzylamide substituent, while FAA activity was insensitive to electronic changes at this site. In this study, we prepared analogs of (R)-N′-benzyl 2-amino-3-methylbutanamide to identify the structural components for maximal anticonvulsant activity. We demonstrated that the SAR of PAADs and FAAs diverged at the terminal amide site and that PAADs had considerably more structural latitude in the types of units that could be incorporated at this position, suggesting that these compounds function according to different mechanism(s). PMID:21861466

  3. Preferential Acquisition and Activation of Plasminogen Glycoform II by PAM Positive Group A Streptococcal Isolates.

    PubMed

    De Oliveira, David M P; Law, Ruby H P; Ly, Diane; Cook, Simon M; Quek, Adam J; McArthur, Jason D; Whisstock, James C; Sanderson-Smith, Martina L

    2015-06-30

    Plasminogen (Plg) circulates in the host as two predominant glycoforms. Glycoform I Plg (GI-Plg) contains glycosylation sites at Asn289 and Thr346, whereas glycoform II Plg (GII-Plg) is exclusively glycosylated at Thr346. Surface plasmon resonance experiments demonstrated that Plg binding group A streptococcal M protein (PAM) exhibits comparative equal affinity for GI- and GII-Plg in the "closed" conformation (for GII-Plg, KD = 27.4 nM; for GI-Plg, KD = 37.0 nM). When Plg was in the "open" conformation, PAM exhibited an 11-fold increase in affinity for GII-Plg (KD = 2.8 nM) compared with that for GI-Plg (KD = 33.2 nM). The interaction of PAM with Plg is believed to be mediated by lysine binding sites within kringle (KR) 2 of Plg. PAM-GI-Plg interactions were fully inhibited with 100 mM lysine analogue ε-aminocaproic acid (εACA), whereas PAM-GII-Plg interactions were shown to be weakened but not inhibited in the presence of 400 mM εACA. In contrast, binding to the KR1-3 domains of GII-Plg (angiostatin) by PAM was completely inhibited in the presence 5 mM εACA. Along with PAM, emm pattern D GAS isolates express a phenotypically distinct SK variant (type 2b SK) that requires Plg ligands such as PAM to activate Plg. Type 2b SK was able to generate an active site and activate GII-Plg at a rate significantly higher than that of GI-Plg when bound to PAM. Taken together, these data suggest that GAS selectively recruits and activates GII-Plg. Furthermore, we propose that the interaction between PAM and Plg may be partially mediated by a secondary binding site outside of KR2, affected by glycosylation at Asn289. PMID:26029848

  4. Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae

    PubMed Central

    Daniel, Bastian; Wallner, Silvia; Steiner, Barbara; Oberdorfer, Gustav; Kumar, Prashant; van der Graaff, Eric; Roitsch, Thomas; Sensen, Christoph W.; Gruber, Karl; Macheroux, Peter

    2016-01-01

    Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family. PMID:27276217

  5. CRP-dependent positive autoregulation and proteolytic degradation regulate competence activator Sxy of Escherichia coli.

    PubMed

    Jaskólska, Milena; Gerdes, Kenn

    2015-03-01

    Natural competence, the ability of bacteria to take up exogenous DNA and incorporate it into their chromosomes, is in most bacteria a transient phenomenon under complex genetic and environmental control. In the Gram-negative bacteria Haemophilus influenzae and Vibrio cholerae, the master regulator Sxy/TfoX controls competence development. Although not known to be naturally competent, Escherichia coli possesses a Sxy homologue and a competence regulon containing the genes required for DNA uptake. Here, we show that in contrast to other characterised Gamma-proteobacteria, E. coli Sxy is positively autoregulated at the level of transcription by a mechanism that requires cAMP receptor protein (CRP), cyclic AMP (cAMP) and a CRP-S site in the sxy promoter. Similarly, we found no evidence that Sxy expression in E. coli was regulated at the translational level. However, our analysis revealed that Sxy is an unstable protein and that its cellular level is negatively regulated at the post-translational level via degradation by Lon protease. Interestingly, in the Gram-positive model organism Bacillus subtilis, the competence master regulator ComK is also positively autoregulated at the level of transcription and negatively regulated by proteolysis. Together, these findings reveal striking similarities between the competence regulons of a Gram-positive and a Gram-negative bacterium. PMID:25491382

  6. Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process

    SciTech Connect

    Miao, Yinglong; Baudry, Jerome Y

    2011-01-01

    Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site.

  7. Loop substitution as a tool to identify active sites of interleukin-1 beta.

    PubMed

    Palla, E; Bensi, G; Solito, E; Buonamassa, D T; Fassina, G; Raugei, G; Spano, F; Galeotti, C; Mora, M; Domenighini, M

    1993-06-25

    By computer analysis of the amino acid sequence of human interleukin-1 beta (IL-1 beta) and of the human type I IL-1 receptor (IL-1RI), we have identified two hydropathically complementary peptides (Fassina, G., Roller, P. P., Olson, A. D., Thorgeirsson, S. S., and Omichinski, J. G. (1989) J. Biol. Chem. 264, 11252-11257) capable of binding to each other. The sequence of the IL-1 beta peptide corresponds to that of residues 88-99 (loop 7 of the crystal structure of mature IL-1 beta) of mature IL-1 beta, one of the exposed and highly charged regions of the molecule. The substitution of this loop with an amino acid sequence of the same length but different hydropathic profile generates a mutant with drastically reduced binding activity to IL-1RI. In contrast, the binding affinity to the type II IL-1R (IL-1RII) is the same as that of wild type IL-1 beta. The results show that 1) loop 7 is part of the binding site of IL-1 beta to IL-1RI, but not to IL-1RII. 2) The structure of the mutant protein is not grossly altered except locally at the position of the substituted loop. 3) The substitution of amino acids by site-directed mutagenesis of the loop 7 region generates mutants with binding affinity constants slightly lower than that of wild type IL-1 beta and not comparable to that of the loop substitution analogue. 4. All mutants analyzed, including the loop substitutions, are biologically active, confirming the structural integrity of the proteins. We propose a binding site in which the cooperation of several low energy bonds extended over a wide area results in a high affinity complex between IL-1 and the type I receptor. PMID:7685764

  8. Structural Diversity Within the Mononuclear and Binuclear Active Sites of N-Acetyl-D-Glucosamine-6-Phosphate Deacetylase

    SciTech Connect

    Hall,R.; Brown, S.; Fedorov, A.; Fedorov, E.; Xu, C.; Babbitt, P.; Almo, S.; Raushel, F.

    2007-01-01

    NagA catalyzes the hydrolysis of N-acetyl-D-glucosamine-6-phosphate to D-glucosamine-6-phosphate and acetate. X-ray crystal structures of NagA from Escherichia coli were determined to establish the number and ligation scheme for the binding of zinc to the active site and to elucidate the molecular interactions between the protein and substrate. The three-dimensional structures of the apo-NagA, Zn-NagA, and the D273N mutant enzyme in the presence of a tight-binding N-methylhydroxyphosphinyl-D-glucosamine-6-phosphate inhibitor were determined. The structure of the Zn-NagA confirms that this enzyme binds a single divalent cation at the beta-position in the active site via ligation to Glu-131, His-195, and His-216. A water molecule completes the ligation shell, which is also in position to be hydrogen bonded to Asp-273. In the structure of NagA bound to the tight binding inhibitor that mimics the tetrahedral intermediate, the methyl phosphonate moiety has displaced the hydrolytic water molecule and is directly coordinated to the zinc within the active site. The side chain of Asp-273 is positioned to activate the hydrolytic water molecule via general base catalysis and to deliver this proton to the amino group upon cleavage of the amide bond of the substrate. His-143 is positioned to help polarize the carbonyl group of the substrate in conjunction with Lewis acid catalysis by the bound zinc. The inhibitor is bound in the {alpha}-configuration at the anomeric carbon through a hydrogen bonding interaction of the hydroxyl group at C-1 with the side chain of His-251. The phosphate group of the inhibitor attached to the hydroxyl at C-6 is ion paired with Arg-227 from the adjacent subunit. NagA from Thermotoga maritima was shown to require a single divalent cation for full catalytic activity.

  9. Structural diversity within the mononuclear and binuclear active sites of N-acetyl-D-glucosamine-6-phosphate deacetylase.

    PubMed

    Hall, Richard S; Brown, Shoshana; Fedorov, Alexander A; Fedorov, Elena V; Xu, Chengfu; Babbitt, Patricia C; Almo, Steven C; Raushel, Frank M

    2007-07-10

    NagA catalyzes the hydrolysis of N-acetyl-d-glucosamine-6-phosphate to d-glucosamine-6-phosphate and acetate. X-ray crystal structures of NagA from Escherichia coli were determined to establish the number and ligation scheme for the binding of zinc to the active site and to elucidate the molecular interactions between the protein and substrate. The three-dimensional structures of the apo-NagA, Zn-NagA, and the D273N mutant enzyme in the presence of a tight-binding N-methylhydroxyphosphinyl-d-glucosamine-6-phosphate inhibitor were determined. The structure of the Zn-NagA confirms that this enzyme binds a single divalent cation at the beta-position in the active site via ligation to Glu-131, His-195, and His-216. A water molecule completes the ligation shell, which is also in position to be hydrogen bonded to Asp-273. In the structure of NagA bound to the tight binding inhibitor that mimics the tetrahedral intermediate, the methyl phosphonate moiety has displaced the hydrolytic water molecule and is directly coordinated to the zinc within the active site. The side chain of Asp-273 is positioned to activate the hydrolytic water molecule via general base catalysis and to deliver this proton to the amino group upon cleavage of the amide bond of the substrate. His-143 is positioned to help polarize the carbonyl group of the substrate in conjunction with Lewis acid catalysis by the bound zinc. The inhibitor is bound in the alpha-configuration at the anomeric carbon through a hydrogen bonding interaction of the hydroxyl group at C-1 with the side chain of His-251. The phosphate group of the inhibitor attached to the hydroxyl at C-6 is ion paired with Arg-227 from the adjacent subunit. NagA from Thermotoga maritima was shown to require a single divalent cation for full catalytic activity. PMID:17567048

  10. Active site densities, oxygen activation and adsorbed reactive oxygen in alcohol activation on npAu catalysts.

    PubMed

    Wang, Lu-Cun; Friend, C M; Fushimi, Rebecca; Madix, Robert J

    2016-07-01

    The activation of molecular O2 as well as the reactivity of adsorbed oxygen species is of central importance in aerobic selective oxidation chemistry on Au-based catalysts. Herein, we address the issue of O2 activation on unsupported nanoporous gold (npAu) catalysts by applying a transient pressure technique, a temporal analysis of products (TAP) reactor, to measure the saturation coverage of atomic oxygen, its collisional dissociation probability, the activation barrier for O2 dissociation, and the facility with which adsorbed O species activate methanol, the initial step in the catalytic cycle of esterification. The results from these experiments indicate that molecular O2 dissociation is associated with surface silver, that the density of reactive sites is quite low, that adsorbed oxygen atoms do not spill over from the sites of activation onto the surrounding surface, and that methanol reacts quite facilely with the adsorbed oxygen atoms. In addition, the O species from O2 dissociation exhibits reactivity for the selective oxidation of methanol but not for CO. The TAP experiments also revealed that the surface of the npAu catalyst is saturated with adsorbed O under steady state reaction conditions, at least for the pulse reaction. PMID:27376884

  11. Active Site Structure and Peroxidase Activity of Oxidatively Modified Cytochrome c Species in Complexes with Cardiolipin.

    PubMed

    Capdevila, Daiana A; Oviedo Rouco, Santiago; Tomasina, Florencia; Tortora, Verónica; Demicheli, Verónica; Radi, Rafael; Murgida, Daniel H

    2015-12-29

    We report a resonance Raman and UV-vis characterization of the active site structure of oxidatively modified forms of cytochrome c (Cyt-c) free in solution and in complexes with cardiolipin (CL). The studied post-translational modifications of Cyt-c include methionine sulfoxidation and tyrosine nitration, which lead to altered heme axial ligation and increased peroxidase activity with respect to those of the wild-type protein. In spite of the structural and activity differences between the protein variants free in solution, binding to CL liposomes induces in all cases the formation of a spectroscopically identical bis-His axial coordination conformer that more efficiently promotes lipid peroxidation. The spectroscopic results indicate that the bis-His form is in equilibrium with small amounts of high-spin species, thus suggesting a labile distal His ligand as the basis for the CL-induced increase in enzymatic activity observed for all protein variants. For Cyt-c nitrated at Tyr74 and sulfoxidized at Met80, the measured apparent binding affinities for CL are ∼4 times larger than for wild-type Cyt-c. On the basis of these results, we propose that these post-translational modifications may amplify the pro-apoptotic signal of Cyt-c under oxidative stress conditions at CL concentrations lower than for the unmodified protein. PMID:26620444

  12. [Comparative urinary bactericidal activity of oral antibiotics against gram-positive pathogens].

    PubMed

    Bedenić, Branka; Budimir, Ana; Gverić, Ana; Plecko, Vanda; Vranes, Jasmina; Bubonja-Sonje, Marina; Kalenić, Smilja

    2012-01-01

    In routine bacteriological laboratories the antibacterial activity of antibiotics is determined by in vitro testing, usually by disk-diffusion test. However, in vitro testing does not always reflect antibacterial efficiency of antibiotics in vivo. In this investigation, the urine samples obtained in a single oral dose pharmacokinetic study were examined for their bactericidal activity against a range of relevant Gram-positive urinary tract pathogens. Urinary bactericidal activity of linezolid had been previously compared with ciprofloxacin but not with other oral antibiotics such as beta-lactams. Linezolid showed satisfactory urinary bactericidal titres throughout the whole testing period against all Gram-positive cocci. Fluoroquinolones displayed high and persisting levels of urinary bactericidal activity against staphylococci, but their activity against enterococci was weaker. According to the results of ex-vivo testing amoxycillin could be recommended only for infections caused by E. faecalis. Amoxycillin combined with clavulanic acid can be considered as a therapeutic option for infections caused by S. saprophyticus and E. faecalis. Older cephalosporins had high titres only against S. saprophyticus. Their drawback is a short elimination half-time in urine resulting in rapid decrease of urinary bactericidal titers during dosing interval. Furthermore, they do not show activity against enterococci due to their intrinsic resistance to cephalosporins. PMID:22930932

  13. Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin

    PubMed Central

    Gasymov, Oktay K.; Abduragimov, Adil R.; Glasgow, Ben J.

    2008-01-01

    The absorption spectra of N-acetyl-L-tryptophanamide in various solvents were resolved into the sums of the 1La and 1Lb components. The relative intensities of the 0-0 transitions of the 1Lb bands correlate linearly with the solvent polarity values (ETN). A novel strategy, which utilizes a set of the experimental 1Lb bands, was employed to resolve the near-UV CD spectra of tryptophanyl residues. Resolved spectral parameters from the single-tryptophan mutants of tear lipocalin (TL), F99W and Y87W, corroborate the fluorescence as well as structural data of TL. Analysis of the 1Lb bands of the Trp CD spectra in proteins is a valuable tool to obtain the local features. The “DMSO-like” 1Lb band of Trp CD spectra may be used as a “fingerprint” to identify the tryptophanyl side chains in situations where the benzene rings of Trp have van der Waals interactions with the side chains of its nearest neighbor. In addition, the signs and intensities of the components hold information about the side-chain conformations and dynamics in proteins. Combined with Trp mutagenesis, this method we call site-directed circular dichroism is broadly applicable to various proteins to obtain the position-specific data. PMID:18047823

  14. Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin.

    PubMed

    Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J

    2008-03-15

    The absorption spectra of N-acetyl-L-tryptophanamide in various solvents were resolved into the sums of the (1)L(a) and (1)L(b) components. The relative intensities of the 0-0 transitions of the (1)L(b) bands correlate linearly with the solvent polarity values (E(T)(N)). A novel strategy that uses a set of the experimental (1)L(b) bands was employed to resolve the near-UV circular dichroism (CD) spectra of tryptophanyl residues. Resolved spectral parameters from the single-tryptophan mutants of tear lipocalin (TL), F99W and Y87W, corroborate the fluorescence and structural data of TL. Analysis of the (1)L(b) bands of the Trp CD spectra in proteins is a valuable tool to obtain the local features. The dimethyl sulfoxide (DMSO)-like (1)L(b) band of Trp CD spectra may be used as a "fingerprint" to identify the tryptophanyl side chains in situations where the benzene rings of Trp have van der Waals interactions with the side chains of its nearest neighbor. In addition, the signs and intensities of the components hold information about the side chain conformations and dynamics in proteins. Combined with Trp mutagenesis, this method, which we call site-directed circular dichroism, is broadly applicable to various proteins to obtain the position-specific data. PMID:18047823

  15. Fine particulate matter source apportionment for the chemical speciation trends network site at Birmingham, Alabama, using positive matrix factorization

    SciTech Connect

    Baumann, K.; Jayanty, R.K.; Flanagan, J.B.

    2008-01-15

    The Positive Matrix Factorization (PMF) receptor model version 1.1 was used with data from the fine particulate matter (PM2.5) Chemical Speciation Trends Network (STN) to estimate source contributions to ambient PM2.5 in a highly industrialized urban setting in the southeastern United States. Model results consistently resolved 10 factors that are interpreted as two secondary, five industrial, one motor vehicle, one road dust, and one biomass burning sources. It was found that most PMF factors did not cleanly represent single source types and instead are 'contaminated' by other sources. Secondary particulate matter formed by atmospheric processes, such as sulfate and secondary OC, contribute the majority of ambient PM2.5 and exhibit strong seasonality 37 {+-} 10% winter vs. 55 {+-} 16% summer average. Motor vehicle emissions constitute the biggest primary PM2.5 mass contribution. In summary, this study demonstrates the utility of the EC tracer method to effectively blank-correct the OC concentrations in the STN dataset. In addition, examination of the effect of input uncertainty estimates on model results indicates that the estimated uncertainties currently being provided with the STN data may be somewhat lower than the levels needed for optimum modeling results. An appendix , available to members on the website www.awma lists stationary sources of PM2.5 within 10 km of the NHBM site and PM2.5 emissions greater than 1 ton per year. 71 refs., 7 figs., 9 tabs.

  16. Gram-Positive Nickel Resistant Bacteria Isolated from Riparian Sediments Contaminated with Ni and U on the Savannah River Site

    NASA Astrophysics Data System (ADS)

    Sowder, A. G.; Khijniak, T. V.; van Nostrand, J.; Bertsch, P. M.; Morris, P. J.

    2002-12-01

    The natural attenuation of pollutants in riparian and wetland systems is driven in large part by the services provided by the diverse microbial communities that thrive in these nutritionally and chemically complex environments. For co-contaminated systems, the presence of heavy metals at excessive levels may alter the structure and function of microbial communities that are essential for the immobilization of inorganics and degradation of organic contaminants. We examined riparian sediments heavily contaminated with U and Ni (1000's of mg/kg) from a small stream on the U.S. Department of Energy's Savannah River Site that received metallurgical process effluents wastewater over a thirty-year period associated with the production of nuclear materials. Four gram positive bacteria were isolated that displayed marked resistance (5000 mg/kg) to Ni relative to organisms from uncontaminated control locations: Arthrobacter oxydans, Streptomyces galbus, Streptomyces aureofaciens, and Kitasatospora cystarginea. The metal resistance of S. aureofaciens and K. cystarginea was further characterized in growth experiments for resistance to other metals. Ongoing geochemical characterization of U and Ni in terms of solid phase partitioning and aqueous phase speciation and solubility indicate that Ni is more chemically labile and, by extension, bioavailable than U in these aged-contaminated sediments. Accordingly, the isolation of Ni resistant organisms is consistent with greater selective pressure from Ni as a result of its greater bioavailability. These results are placed in context of environmental management and remediation of co-contaminated, biogeochemically complex environments.

  17. Exploring pharmacological activities and signaling of morphinans substituted in position 6 as potent agonists interacting with the μ opioid receptor

    PubMed Central

    2014-01-01

    Background Opioid analgesics are the most effective drugs for the treatment of moderate to severe pain. However, they also produce several adverse effects that can complicate pain management. The μ opioid (MOP) receptor, a G protein-coupled receptor, is recognized as the opioid receptor type which primarily mediates the pharmacological actions of clinically used opioid agonists. The morphinan class of analgesics including morphine and oxycodone are of main importance as therapeutically valuable drugs. Though the natural alkaloid morphine contains a C-6-hydroxyl group and the semisynthetic derivative oxycodone has a 6-carbonyl function, chemical approaches have uncovered that functionalizing position 6 gives rise to a range of diverse activities. Hence, position 6 of N-methylmorphinans is one of the most manipulated sites, and is established to play a key role in ligand binding at the MOP receptor, efficacy, signaling, and analgesic potency. We have earlier reported on a chemically innovative modification in oxycodone resulting in novel morphinans with 6-acrylonitrile incorporated substructures. Results This study describes in vitro and in vivo pharmacological activities and signaling of new morphinans substituted in position 6 with acrylonitrile and amido functions as potent agonists and antinociceptive agents interacting with MOP receptors. We show that the presence of a 6-cyano group in N-methylmorphinans has a strong influence on the binding to the opioid receptors and post-receptor signaling. One 6-cyano-N-methylmorphinan of the series was identified as the highest affinity and most selective MOP agonist, and very potent in stimulating G protein coupling and intracellular calcium release through the MOP receptor. In vivo, this MOP agonist showed to be greatly effective against thermal and chemical nociception in mice with marked increased antinociceptive potency than the lead molecule oxycodone. Conclusion Development of such novel chemotypes by targeting

  18. Identification of Ice Nucleation Active Sites on Feldspar Dust Particles

    PubMed Central

    2015-01-01

    Mineral dusts originating from Earth’s crust are known to be important atmospheric ice nuclei. In agreement with earlier studies, feldspar was found as the most active of the tested natural mineral dusts. Here we investigated in closer detail the reasons for its activity and the difference in the activity of the different feldspars. Conclusions are drawn from scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, and oil-immersion freezing experiments. K-feldspar showed by far the highest ice nucleation activity. Finally, we give a potential explanation of this effect, finding alkali-metal ions having different hydration shells and thus an influence on the ice nucleation activity of feldspar surfaces. PMID:25584435

  19. Passive damping to enhance active positioning of a prototype lithography platen

    SciTech Connect

    Segalman, D.J.; Kipp, R.L.; Gregory, D.L.

    1994-12-31

    A viscoelastic tuned-mass damper was used to suppress specific structural modes of a prototype lithography platen. The platen is magnetically levitated and it is repositioned and held in position by a closed-loop feedback control system. Important capabilities of the platen control system are precise positioning and rapid repositioning, which tend to require high frequency bandwidth. The high bandwidth excites structural vibration modes which are disruptive to the control system. The present work was to develop and demonstrate a means to suppress these modes using passive vibration damping techniques. The motivation is to increase the robustness of the platen positioning and control system by reducing unwanted modal accelerations excited by high control system bandwidth. Activities performed and discussed in this paper include the analytical design of viscoelastic tuned-mass dampers and the demonstration/testing of their effectiveness on the platen while levitated and controlled.

  20. Individual Public Transportation Accessibility is Positively Associated with Self-Reported Active Commuting

    PubMed Central

    Djurhuus, Sune; Hansen, Henning Sten; Aadahl, Mette; Glümer, Charlotte

    2014-01-01

    Background: Active commuters have lower risk of chronic disease. Understanding which of the, to some extent, modifiable characteristics of public transportation that facilitate its use is thus important in a public health perspective. The aim of the study was to examine the association between individual public transportation accessibility and self-reported active commuting, and whether the associations varied with commute distance, age, and gender. Methods: Twenty-eight thousand nine hundred twenty-eight commuters in The Capital Region of Denmark reported self-reported time spent either walking or cycling to work or study each day and the distance to work or study. Data were obtained from the Danish National Health Survey collected in February to April 2010. Individual accessibility by public transportation was calculated using a multi-modal network in a GIS. Multilevel logistic regression was used to analyze the association between accessibility, expressed as access area, and being an active commuter. Results: Public transport accessibility area based on all stops within walking and cycling distance was positively associated with being an active commuter. Distance to work, age, and gender modified the associations. Residing within 10 km commute distance and in areas of high accessibility was associated with being an active commuter and meeting the recommendations of physical activity. For the respondents above 29 years, individual public transportation accessibility was positively associated with being an active commuter. Women having high accessibility had significantly higher odds of being an active commuter compared to having a low accessibility. For men, the associations were insignificant. Conclusion: This study extends the knowledge about the driving forces of using public transportation for commuting by examining the individual public transportation accessibility. Findings suggest that transportation accessibility supports active commuting and planning

  1. Early Site Permit Demonstration Program: Recommendations for communication activities and public participation in the Early Site Permit Demonstration Program

    SciTech Connect

    Not Available

    1993-01-27

    On October 24, 1992, President Bush signed into law the National Energy Policy Act of 1992. The bill is a sweeping, comprehensive overhaul of the Nation`s energy laws, the first in more than a decade. Among other provisions, the National Energy Policy Act reforms the licensing process for new nuclear power plants by adopting a new approach developed by the US Nuclear Regulatory Commission (NRC) in 1989, and upheld in court in 1992. The NRC 10 CFR Part 52 rule is a three-step process that guarantees public participation at each step. The steps are: early site permit approval; standard design certifications; and, combined construction/operating licenses for nuclear power reactors. Licensing reform increases an organization`s ability to respond to future baseload electricity generation needs with less financial risk for ratepayers and the organization. Costly delays can be avoided because design, safety and siting issues will be resolved before a company starts to build a plant. Specifically, early site permit approval allows for site suitability and acceptability issues to be addressed prior to an organization`s commitment to build a plant. Responsibility for site-specific activities, including communications and public participation, rests with those organizations selected to try out early site approval. This plan has been prepared to assist those companies (referred to as sponsoring organizations) in planning their communications and public involvement programs. It provides research findings, information and recommendations to be used by organizations as a resource and starting point in developing their own plans.

  2. Ultrafast ligand binding dynamics in the active site of native bacterial nitric oxide reductase.

    PubMed

    Kapetanaki, Sofia M; Field, Sarah J; Hughes, Ross J L; Watmough, Nicholas J; Liebl, Ursula; Vos, Marten H

    2008-01-01

    The active site of nitric oxide reductase from Paracoccus denitrificans contains heme and non-heme iron and is evolutionarily related to heme-copper oxidases. The CO and NO dynamics in the active site were investigated using ultrafast transient absorption spectroscopy. We find that, upon photodissociation from the active site heme, 20% of the CO rebinds in 170 ps, suggesting that not all the CO transiently binds to the non-heme iron. The remaining 80% does not rebind within 4 ns and likely migrates out of the active site without transient binding to the non-heme iron. Rebinding of NO to ferrous heme takes place in approximately 13 ps. Our results reveal that heme-ligand recombination in this enzyme is considerably faster than in heme-copper oxidases and are consistent with a more confined configuration of the active site. PMID:18420024

  3. Positively Valenced Stimuli Facilitate Creative Novel Metaphoric Processes by Enhancing Medial Prefrontal Cortical Activation

    PubMed Central

    Subramaniam, Karuna; Beeman, Mark; Faust, Miriam; Mashal, Nira

    2013-01-01

    A metaphor is a figure of speech in which a subject is symbolic of another unrelated object. In the present study, we examined neural patterns associated with both novel unfamiliar and conventional familiar metaphoric processing, and how these patterns are modulated by affective valence. Prior to fMRI scanning, participants received a list of word pairs (novel unfamiliar metaphors as well as conventional familiar metaphors) and were asked to denote the valence (positive, negative, or neutral) of each word pair. During scanning, participants had to decide whether the word pairs formed meaningful or meaningless expressions. Results indicate that participants were faster and more accurate at deciding that positively valenced metaphors were meaningful compared to neutral metaphors. These behavioral findings were accompanied by increased activation in the medial prefrontal cortex (mPFC), posterior cingulate cortex (PCC), and the right inferior parietal lobe (RIPL). Specifically, positively valenced novel unfamiliar metaphors elicited activation in these brain regions in addition to the left superior temporal gyrus when compared to neutral novel metaphors. We also found that the mPFC and PCC mediated the processing of positively valenced metaphors when compared to negatively valenced metaphors. Positively valenced conventional metaphors, however, elicited different neural signatures when contrasted with either neutral or negatively valenced conventional metaphors. Together, our results indicate that positively valenced stimuli facilitate creative metaphoric processes (specifically novel metaphoric processes) by mediating attention and cognitive control processes required for the access, integration, and selection of semantic associations via modulation of the mPFC. The present study is important for the development of neural accounts of emotion-cognition interactions required for creativity, language, and successful social functioning in general. PMID:23637686

  4. Binding stability of peptides derived from 1ALA residue and 7GLY residues to sites near active center of fluctuating papain

    NASA Astrophysics Data System (ADS)

    Nishiyama, Katsuhiko

    2012-05-01

    We investigated the binding stability of peptides derived from 1ALA residue and 7GLY residues to sites near active center of fluctuating papain via molecular dynamics and docking simulations. Replacing GLY residue in 8GLY with ALA residue had a positive effect on binding stability to the sites in some cases although the replacing had a negative effect on it in other cases. Furthermore the replacing had a negative effect on the chance of binding to the sites. Residue in peptide should be replaced on the basis of systematic exploration of its position.

  5. Identification of novel aminopiperidine derivatives for antibacterial activity against Gram-positive bacteria.

    PubMed

    Lee, Hee-Yeol; An, Kyung-Mi; Jung, Juyoung; Koo, Je-Min; Kim, Jeong-Geun; Yoon, Jong-Min; Lee, Myong-Jae; Jang, HyeonSoo; Lee, Hong-Sub; Park, Soobong; Kang, Jae-Hoon

    2016-07-01

    We have previously reported amidopiperidine derivatives as a novel peptide deformylase (PDF) inhibitor and evaluated its antibacterial activity against Gram-positive bacteria, but poor pharmacokinetic profiles have resulted in low efficacy in in vivo mouse models. In order to overcome these weaknesses, we newly synthesized aminopiperidine derivatives with remarkable antimicrobial properties and oral bioavailability, and also identified their in vivo efficacy against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE) and penicillin-resistant Streptococcus pneumoniae (PRSP). PMID:27173797

  6. Dynamic detection of window starting positions and its implementation within an activity recognition framework.

    PubMed

    Ni, Qin; Patterson, Timothy; Cleland, Ian; Nugent, Chris

    2016-08-01

    Activity recognition is an intrinsic component of many pervasive computing and ambient intelligent solutions. This has been facilitated by an explosion of technological developments in the area of wireless sensor network, wearable and mobile computing. Yet, delivering robust activity recognition, which could be deployed at scale in a real world environment, still remains an active research challenge. Much of the existing literature to date has focused on applying machine learning techniques to pre-segmented data collected in controlled laboratory environments. Whilst this approach can provide valuable ground truth information from which to build recognition models, these techniques often do not function well when implemented in near real time applications. This paper presents the application of a multivariate online change detection algorithm to dynamically detect the starting position of windows for the purposes of activity recognition. PMID:27392647

  7. Graduated exposure and positive reinforcement to overcome setting and activity avoidance in an adolescent with autism.

    PubMed

    Schmidt, Jonathan D; Luiselli, James K; Rue, Hanna; Whalley, Katherine

    2013-01-01

    Some students who have developmental disabilities avoid settings and activities that can improve their learning and quality of life. This two-phase study concerned an adolescent boy with autism who avoided the gross-motor exercise room, gymnasium, and music room at his school; he demonstrated distress, agitation, and problem behaviors when prompted to enter these areas. Using graduated exposure combined with positive reinforcement, he learned to enter these settings without resisting and eventually to participate in activities within the settings. This article discusses this intervention approach for reducing and eliminating avoidant behavior. PMID:22987915

  8. Structure-Activity Analysis of Gram-positive Bacterium-producing Lasso Peptides with Anti-mycobacterial Activity

    PubMed Central

    Inokoshi, Junji; Koyama, Nobuhiro; Miyake, Midori; Shimizu, Yuji; Tomoda, Hiroshi

    2016-01-01

    Lariatin A, an 18-residue lasso peptide encoded by the five-gene cluster larABCDE, displays potent and selective anti-mycobacterial activity. The structural feature is an N-terminal macrolactam ring, through which the C-terminal passed to form the rigid lariat-protoknot structure. In the present study, we established a convergent expression system by the strategy in which larA mutant gene-carrying plasmids were transformed into larA-deficient Rhodococcus jostii, and generated 36 lariatin variants of the precursor protein LarA to investigate the biosynthesis and the structure-activity relationships. The mutational analysis revealed that four amino acid residues (Gly1, Arg7, Glu8, and Trp9) in lariatin A are essential for the maturation and production in the biosynthetic machinery. Furthermore, the study on structure-activity relationships demonstrated that Tyr6, Gly11, and Asn14 are responsible for the anti-mycobacterial activity, and the residues at positions 15, 16 and 18 in lariatin A are critical for enhancing the activity. This study will not only provide a useful platform for genetically engineering Gram-positive bacterium-producing lasso peptides, but also an important foundation to rationally design more promising drug candidates for combatting tuberculosis. PMID:27457620

  9. Structure-Activity Analysis of Gram-positive Bacterium-producing Lasso Peptides with Anti-mycobacterial Activity

    NASA Astrophysics Data System (ADS)

    Inokoshi, Junji; Koyama, Nobuhiro; Miyake, Midori; Shimizu, Yuji; Tomoda, Hiroshi

    2016-07-01

    Lariatin A, an 18-residue lasso peptide encoded by the five-gene cluster larABCDE, displays potent and selective anti-mycobacterial activity. The structural feature is an N-terminal macrolactam ring, through which the C-terminal passed to form the rigid lariat-protoknot structure. In the present study, we established a convergent expression system by the strategy in which larA mutant gene-carrying plasmids were transformed into larA-deficient Rhodococcus jostii, and generated 36 lariatin variants of the precursor protein LarA to investigate the biosynthesis and the structure-activity relationships. The mutational analysis revealed that four amino acid residues (Gly1, Arg7, Glu8, and Trp9) in lariatin A are essential for the maturation and production in the biosynthetic machinery. Furthermore, the study on structure-activity relationships demonstrated that Tyr6, Gly11, and Asn14 are responsible for the anti-mycobacterial activity, and the residues at positions 15, 16 and 18 in lariatin A are critical for enhancing the activity. This study will not only provide a useful platform for genetically engineering Gram-positive bacterium-producing lasso peptides, but also an important foundation to rationally design more promising drug candidates for combatting tuberculosis.

  10. TNF activation of NF-κB is essential for development of single-positive thymocytes.

    PubMed

    Webb, Louise V; Ley, Steven C; Seddon, Benedict

    2016-07-25

    NF-κB activation has been implicated at multiple stages of thymic development of T cells, during which it is thought to mediate developmental signals originating from the T cell receptor (TCR). However, the Card11-Bcl10-Malt1 (CBM) complex that is essential for TCR activation of NF-κB in peripheral T cells is not required for thymocyte development. It has remained unclear whether the TCR activates NF-κB independent of the CBM complex in thymocyte development or whether another NF-κB activating receptor is involved. In the present study, we generated mice in which T cells lacked expression of both catalytic subunits of the inhibitor of κB kinase (IKK) complex, IKK1 and IKK2, to investigate this question. Although early stages of T cell development were unperturbed, maturation of CD4 and CD8 single-positive (SP) thymocytes was blocked in mice lacking IKK1/2 in the T cell lineage. We found that IKK1/2-deficient thymocytes were specifically sensitized to TNF-induced cell death in vitro. Furthermore, the block in thymocyte development in IKK1/2-deficient mice could be rescued by blocking TNF with anti-TNF mAb or by ablation of TNFRI expression. These experiments reveal an essential role for TNF activation of NF-κB to promote the survival and development of single positive T cells in the thymus. PMID:27432943

  11. Conversed mutagenesis of an inactive peptide to ASIC3 inhibitor for active sites determination.

    PubMed

    Osmakov, Dmitry I; Koshelev, Sergey G; Andreev, Yaroslav A; Dyachenko, Igor A; Bondarenko, Dmitry A; Murashev, Arkadii N; Grishin, Eugene V; Kozlov, Sergey A

    2016-06-15

    Peptide Ugr9-1 from the venom of sea anemone Urticina grebelnyi selectively inhibits the ASIC3 channel and significantly reverses inflammatory and acid-induced pain in vivo. A close homolog peptide Ugr 9-2 does not have these features. To find the pharmacophore residues and explore structure-activity relationships of Ugr 9-1, we performed site-directed mutagenesis of Ugr 9-2 and replaced several positions by the corresponding residues from Ugr 9-1. Mutant peptides Ugr 9-2 T9F and Ugr 9-2 Y12H were able to inhibit currents of the ASIC3 channels 2.2 times and 1.3 times weaker than Ugr 9-1, respectively. Detailed analysis of the spatial models of Ugr 9-1, Ugr 9-2 and both mutant peptides revealed the presence of the basic-aromatic clusters on opposite sides of the molecule, each of which is responsible for the activity. Additionally, Ugr9-1 mutant with truncated N- and C-termini retained similar with the Ugr9-1 action in vitro and was equally potent in vivo model of thermal hypersensitivity. All together, these results are important for studying the structure-activity relationships of ligand-receptor interaction and for the future development of peptide drugs from animal toxins. PMID:26686983

  12. The effects of rock type and landscape position on solution chemistry of soils in the Biosphere 2 Desert Site of the Santa Catalina Mountains Critical Zone Observatory

    NASA Astrophysics Data System (ADS)

    Penprase, S. B.; Abramson, N.; LaSharr, K.; Chorover, J.

    2014-12-01

    The interaction of near surface soil water with surrounding rock and soil matter plays a crucial role in determining the chemical composition of biogeological systems. This interaction drives subsurface processes such as erosion, mineralization, and depletion. However, how and why soil pore water chemistry fluctuates based on localized conditions such as rock type and landscape position is not fully understood. This study examines the role these two factors play in altering soil water chemistry by analyzing samples collected from schist and granite field sites within the Biosphere 2 Desert Site of the Santa Catalina Mountains Critical Zone Observatory. We hypothesized that soil water from the schist site would have higher solute concentrations than the granite site because schist is a more weatherable rock and, thus, is more susceptible to chemical erosion. We also hypothesized that soil water from convergent positions would have higher solute concentrations than those from divergent positions due to a longer upgradient flow path. Each field site was situated within a Zero Order Basin (ZOB) with seven Zero Tension Lysimeters (ZTL). At the schist site, there were 3 convergent (SC 1-3) and 4 divergent (SD 1-4) ZTL positions. For the granite site, there were 4 convergent (GC 1-2, 4-5) and 3 divergent (GD 1-3) ZTLs. Samples were collected following rainstorms from July 2011-July 2013. Each solution sample was analyzed for major and trace cations, anions, pH, EC, and organic and inorganic carbon. Comparisons between SC and GC and all schist and all granite are consistent with the hypotheses for multiple elements. Results also indicate higher solute levels for SC relative to SD. Thus, our analyses suggest that rock type and landscape position influence the chemical composition of soil water at these two sites.

  13. Kinetics of Hydrogen Atom Abstraction from Substrate by an Active Site Thiyl Radical in Ribonucleotide Reductase

    PubMed Central

    2015-01-01

    Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. Active E. coli class Ia RNR is an α2β2 complex that undergoes reversible, long-range proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (β-Y122 → [β-W48] → β-Y356 → α-Y731 → α-Y730 → α-C439) that spans ∼35 Å. To unmask PCET kinetics from rate-limiting conformational changes, we prepared a photochemical RNR containing a [ReI] photooxidant site-specifically incorporated at position 355 ([Re]-β2), adjacent to PCET pathway residue Y356 in β. [Re]-β2 was further modified by replacing Y356 with 2,3,5-trifluorotyrosine to enable photochemical generation and spectroscopic observation of chemically competent tyrosyl radical(s). Using transient absorption spectroscopy, we compare the kinetics of Y· decay in the presence of substrate and wt-α2, Y731F-α2 ,or C439S-α2, as well as with 3′-[2H]-substrate and wt-α2. We find that only in the presence of wt-α2 and the unlabeled substrate do we observe an enhanced rate of radical decay indicative of forward radical propagation. This observation reveals that cleavage of the 3′-C–H bond of substrate by the transiently formed C439· thiyl radical is rate-limiting in forward PCET through α and has allowed calculation of a lower bound for the rate constant associated with this step of (1.4 ± 0.4) × 104 s–1. Prompting radical propagation with light has enabled observation of PCET events heretofore inaccessible, revealing active site chemistry at the heart of RNR catalysis. PMID:25353063

  14. Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts.

    PubMed

    Sahraie, Nastaran Ranjbar; Kramm, Ulrike I; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

    2015-01-01

    Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and (57)Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity. PMID:26486465

  15. Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts

    NASA Astrophysics Data System (ADS)

    Sahraie, Nastaran Ranjbar; Kramm, Ulrike I.; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

    2015-10-01

    Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and 57Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity.

  16. Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts

    PubMed Central

    Sahraie, Nastaran Ranjbar; Kramm, Ulrike I.; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

    2015-01-01

    Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and 57Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity. PMID:26486465

  17. Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP.

    PubMed

    Miner, Kyle D; Kurtz, Donald M

    2016-02-16

    HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'→5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites. PMID:26786892

  18. Assessment of activation products in the Savannah River Site environment

    SciTech Connect

    Carlton, W.H.; Denham, M.

    1996-07-01

    This document assesses the impact of radioactive activation products released from SRS facilities since the first reactor became operational late in 1953. The isotopes reported here are those whose release resulted in the highest dose to people living near SRS: {sup 32}P, {sup 51}Cr, {sup 60}C, and {sup 65}Zn. Release pathways, emission control features, and annual releases to the aqueous and atmospheric environments are discussed. No single incident has resulted in a major acute release of activation products to the environment. The releases were the result of normal operations of the reactors and separations facilities. Releases declined over the years as better controls were established and production was reduced. The overall radiological impact of SRS activation product atmospheric releases from 1954 through 1994 on the offsite maximally exposed individual can be characterized by a total dose of 0.76 mrem. During the same period, such an individual received a total dose of 14,400 mrem from non-SRS sources of ionizing radiation present in the environment. SRS activation product aqueous releases between 1954 and 1994 resulted in a total dose of 54 mrem to the offsite maximally exposed individual. The impact of SRS activation product releases on offsite populations also has been evaluated.

  19. The DNA-binding domain of the transcriptional activator protein NifA resides in its carboxy terminus, recognises the upstream activator sequences of nif promoters and can be separated from the positive control function of NifA.

    PubMed Central

    Morett, E; Cannon, W; Buck, M

    1988-01-01

    The positive control protein NifA activates transcription of nitrogen fixation promoters in Klebsiella pneumoniae. NifA is believed to bind to specific sites, the upstream activator sequences (UAS's), of the nif promoters which it activates. We have now shown by mutation of the carboxy terminus of NifA that this is the DNA-binding domain and that the DNA-binding and positive activator functions of NifA can be separated. Mutational analysis of the nifH UAS and in vivo methylation protection analysis of the interaction of NifA with the nifH promoter demonstrates that the UAS is recognised by the carboxy terminus of NifA. The UAS's of K. pneumoniae nif promoters are also required for activation by the Rhizobium meliloti NifA indicating that this activator also possesses DNA-binding activity. Images PMID:3062575

  20. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand.

    PubMed

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins' active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes. PMID:25449264

  1. Active site proton delivery and the lyase activity of human CYP17A1

    SciTech Connect

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.; Denisov, Ilia G.; Sligar, Stephen G.

    2014-01-03

    equivalents and protons are funneled into non-productive pathways. This is similar to previous work with other P450 catalyzed hydroxylation. However, catalysis of carbon–carbon bond scission by the T306A mutant was largely unimpeded by disruption of the CYP17A1 acid-alcohol pair. The unique response of CYP17A1 lyase activity to mutation of Thr306 is consistent with a reactive intermediate formed independently of proton delivery in the active site, and supports involvement of a nucleophilic peroxo-anion rather than the traditional Compound I in catalysis.

  2. Characterization of an Active Thermal Erosion Site, Caribou Creek, Alaska

    NASA Astrophysics Data System (ADS)

    Busey, R.; Bolton, W. R.; Cherry, J. E.; Hinzman, L. D.

    2013-12-01

    The goal of this project is to estimate volume loss of soil over time from this site, provide parameterizations on erodibility of ice rich permafrost and serve as a baseline for future landscape evolution simulations. Located in the zone of discontinuous permafrost, the interior region of Alaska (USA) is home to a large quantity of warm, unstable permafrost that is both high in ice content and has soil temperatures near the freezing point. Much of this permafrost maintains a frozen state despite the general warming air temperature trend in the region due to the presence of a thick insulating organic mat and a dense root network in the upper sub-surface of the soil column. At a rapidly evolving thermo-erosion site, located within the Caribou-Poker Creeks Research Watershed (part of the Bonanza Creek LTER) near Chatanika, Alaska (N65.140, W147.570), the protective organic layer and associated plants were disturbed by an adjacent traditional use trail and the shifting of a groundwater spring. These triggers have led to rapid geomorphological change on the landscape as the soil thaws and sediment is transported into the creek at the valley bottom. Since 2006 (approximately the time of initiation), the thermal erosion has grown to 170 meters length, 3 meters max depth, and 15 meters maximum width. This research combines several data sets: DGPS survey, imagery from an extremely low altitude pole-based remote sensing (3 to 5 meters above ground level), and imagery from an Unmanned Aerial System (UAS) at about 60m altitude.

  3. Activity in ventromedial prefrontal cortex during self-related processing: positive subjective value or personal significance?

    PubMed

    Kim, Kyungmi; Johnson, Marcia K

    2015-04-01

    Well-being and subjective experience of a coherent world depend on our sense of 'self' and relations between the self and the environment (e.g. people, objects and ideas). The ventromedial prefrontal cortex (vMPFC) is involved in self-related processing, and disrupted vMPFC activity is associated with disruptions of emotional/social functioning (e.g. depression and autism). Clarifying precise function(s) of vMPFC in self-related processing is an area of active investigation. In this study, we sought to more specifically characterize the function of vMPFC in self-related processing, focusing on two alternative accounts: (i) assignment of positive subjective value to self-related information and (ii) assignment of personal significance to self-related information. During functional magnetic resonance imaging (fMRI), participants imagined owning objects associated with either their perceived ingroup or outgroup. We found that for ingroup-associated objects, vMPFC showed greater activity for objects with increased than decreased post-ownership preference. In contrast, for outgroup-associated objects, vMPFC showed greater activity for objects with decreased than increased post-ownership preference. Our findings support the idea that the function of vMPFC in self-related processing may not be to represent/evaluate the 'positivity' or absolute preference of self-related information but to assign personal significance to it based on its meaning/function for the self. PMID:24837477

  4. Triterpene sapogenin-polyarginine conjugates exhibit promising antibacterial activity against Gram-positive strains.

    PubMed

    Na, Heiya; Li, Xiangpeng; Zou, Cunbin; Wang, Chenhong; Wang, Chao; Liu, Keliang

    2016-07-01

    Triterpene sapogenins are a group of biologically active compounds with antibacterial activity. However, the limited solubility and poor bioavailability of triterpene sapogenins restrict their therapeutic application. Polyarginine peptides are small cationic peptides with high affinities for multiple negatively charged cell membranes and possess moderate antibacterial activities. In this study, we designed and synthesized a series of sapogenin-polyarginine conjugates in which the triterpene sapogenin moiety was covalently appended to the positively charged polyarginine via click chemistry. A clear synergistic effect was found, and the conjugates exhibited potent and selective antibacterial activity against Gram-positive strains. Among them, BAc-R3 was the most promising compound, which was also proven to be nontoxic toward mammalian cells as well as stable in plasma. The mechanism of BAc-R3 primarily involves an interaction with the bacterial membrane, similar to that of antimicrobial peptides (AMPs). This scaffold design opens an avenue for the further development of novel antibiotics comprised of the combination of a peptide and a natural product. PMID:27209170

  5. Marine Biology Field Trip Sites. Ocean Related Curriculum Activities.

    ERIC Educational Resources Information Center

    Pauls, John

    The ocean affects all of our lives. Therefore, awareness of and information about the interconnections between humans and oceans are prerequisites to making sound decisions for the future. Project ORCA (Ocean Related Curriculum Activities) has developed interdisciplinary curriculum materials designed to meet the needs of students and teachers…

  6. Litter effects on seedling establishment interact with seed position and earthworm activity.

    PubMed

    Donath, T W; Eckstein, R L

    2012-01-01

    Seedling establishment is influenced by litter cover and by seed predators, but little is known about interactions between these two factors. We tested their effects on emergence of five typical grassland species in a microcosm experiment. We manipulated the amounts of grass litter, seed sowing position and earthworm activity to determine whether: (i) the protective effect of litter against seed predation depends on cover amount and seed sowing position, i.e., on top or beneath litter; (ii) seed transport by earthworms changes the effect of seed sowing position on seedling emergence; and (iii) seeds transported into deeper soil layers by earthworms are still germinable. Litter cover and presence of earthworms lowered seedling emergence. The impact of seed position increased with seed size. Emergence of large-seeded species was reduced when sown on the surface. Additionally, we found an important seed position × earthworm interaction related to seed size. Emergence of large-seeded species sown on top of the litter was up to three times higher when earthworms were present than without earthworms. Earthworms also significantly altered the depth distribution of seeds in the soil and across treatments: on average 6% of seeds germinated after burial. In contrast to the seed position effect, we found no size effect on mobility and germinability of seeds after burial in the soil. Nevertheless, the fate of different-sized seeds may differ. While burial will remove large seeds from the regeneration pool, it may enhance seed bank build up in small-seeded species. Consequently, changes in the amount of litter cover and the invertebrate community play a significant role in plant community composition. PMID:21972886

  7. Respiratory Motion of The Heart and Positional Reproducibility Under Active Breathing Control

    SciTech Connect

    Jagsi, Reshma; Moran, Jean M.; Kessler, Marc L.; Marsh, Robin B. C; Balter, James M.; Pierce, Lori J. . E-mail: ljpierce@umich.edu

    2007-05-01

    Purpose: To reduce cardiotoxicity from breast radiotherapy (RT), innovative techniques are under investigation. Information about cardiac motion with respiration and positional reproducibility under active breathing control (ABC) is necessary to evaluate these techniques. Methods and Materials: Patients requiring loco-regional RT for breast cancer were scanned by computed tomography using an ABC device at various breath-hold states, before and during treatment. Ten patients were studied. For each patient, 12 datasets were analyzed. Mutual information-based regional rigid alignment was used to determine the magnitude and reproducibility of cardiac motion as a function of breathing state. For each scan session, motion was quantified by evaluating the displacement of a point along the left anterior descending artery (LAD) with respect to its position at end expiration. Long-term positional reproducibility was also assessed. Results: Displacement of the LAD was greatest in the inferior direction, moderate in the anterior direction, and lowest in the left-right direction. At shallow breathing states, the average displacement of LAD position was up to 6 mm in the inferior direction. The maximum displacement in any patient was 2.8 cm in the inferior direction, between expiration and deep-inspiration breath hold. At end expiration, the long-term reproducibility (SD) of the LAD position was 3 mm in the A-P, 6 mm in the S-I, and 4 mm in the L-R directions. At deep-inspiration breath hold, long-term reproducibility was 3 mm in the A-P, 7 mm in the S-I, and 3 mm in the L-R directions. Conclusions: These data demonstrate the extent of LAD displacement that occurs with shallow breathing and with deep-inspiration breath hold. This information may guide optimization studies considering the effects of respiratory motion and reproducibility of cardiac position on cardiac dose, both with and without ABC.

  8. Hydride binding to the active site of [FeFe]-hydrogenase.

    PubMed

    Chernev, Petko; Lambertz, Camilla; Brünje, Annika; Leidel, Nils; Sigfridsson, Kajsa G V; Kositzki, Ramona; Hsieh, Chung-Hung; Yao, Shenglai; Schiwon, Rafael; Driess, Matthias; Limberg, Christian; Happe, Thomas; Haumann, Michael

    2014-11-17

    [FeFe]-hydrogenase from green algae (HydA1) is the most efficient hydrogen (H2) producing enzyme in nature and of prime interest for (bio)technology. Its active site is a unique six-iron center (H-cluster) composed of a cubane cluster, [4Fe4S]H, cysteine-linked to a diiron unit, [2Fe]H, which carries unusual carbon monoxide (CO) and cyanide ligands and a bridging azadithiolate group. We have probed the molecular and electronic configurations of the H-cluster in functional oxidized, reduced, and super-reduced or CO-inhibited HydA1 protein, in particular searching for intermediates with iron-hydride bonds. Site-selective X-ray absorption and emission spectroscopy were used to distinguish between low- and high-spin iron sites in the two subcomplexes of the H-cluster. The experimental methods and spectral simulations were calibrated using synthetic model complexes with ligand variations and bound hydride species. Distinct X-ray spectroscopic signatures of electronic excitation or decay transitions in [4Fe4S]H and [2Fe]H were obtained, which were quantitatively reproduced by density functional theory calculations, thereby leading to specific H-cluster model structures. We show that iron-hydride bonds are absent in the reduced state, whereas only in the super-reduced state, ligand rotation facilitates hydride binding presumably to the Fe-Fe bridging position at [2Fe]H. These results are in agreement with a catalytic cycle involving three main intermediates and at least two protonation and electron transfer steps prior to the H2 formation chemistry in [FeFe]-hydrogenases. PMID:25369169

  9. Dual functional activity of semaphorin 3B is required for positioning the anterior commissure.

    PubMed

    Falk, Julien; Julien, Falk; Bechara, Ahmad; Fiore, Roberto; Nawabi, Homaira; Zhou, Heather; Hoyo-Becerra, Carolina; Bozon, Muriel; Rougon, Geneviève; Grumet, Martin; Püschel, Andreas W; Sanes, Joshua R; Castellani, Valérie

    2005-10-01

    Chemorepulsion by semaphorins plays a critical role during the development of neuronal projections. Although semaphorin-induced chemoattraction has been reported in vitro, the contribution of this activity to axon pathfinding is still unclear. Using genetic and culture models, we provide evidence that both attraction and repulsion by Sema3B, a secreted semaphorin, are critical for the positioning of a major brain commissural projection, the anterior commissure (AC). NrCAM, an immunoglobulin superfamily adhesion molecule of the L1 subfamily, associates with neuropilin-2 and is a component of a receptor complex for Sema3B and Sema3F. Finally, we show that activation of the FAK/Src signaling cascade distinguishes Sema3B-mediated attractive from repulsive axonal responses of neurons forming the AC, revealing a mechanism underlying the dual activity of this guidance cue. PMID:16202709

  10. Reduction of urease activity by interaction with the flap covering the active site.

    PubMed

    Macomber, Lee; Minkara, Mona S; Hausinger, Robert P; Merz, Kenneth M

    2015-02-23

    With the increasing appreciation for the human microbiome coupled with the global rise of antibiotic resistant organisms, it is imperative that new methods be developed to specifically target pathogens. To that end, a novel computational approach was devised to identify compounds that reduce the activity of urease, a medically important enzyme of Helicobacter pylori, Proteus mirabilis, and many other microorganisms. Urease contains a flexible loop that covers its active site; Glide was used to identify small molecules predicted to lock this loop in an open conformation. These compounds were screened against the model urease from Klebsiella aerogenes, and the natural products epigallocatechin and quercetin were shown to inhibit at low and high micromolar concentrations, respectively. These molecules exhibit a strong time-dependent inactivation of urease that was not due to their oxygen sensitivity. Rather, these compounds appear to inactivate urease by reacting with a specific Cys residue located on the flexible loop. Substitution of this cysteine by alanine in the C319A variant increased the urease resistance to both epigallocatechin and quercetin, as predicted by the computational studies. Protein dynamics are integral to the function of many enzymes; thus, identification of compounds that lock an enzyme into a single conformation presents a useful approach to define potential inhibitors. PMID:25594724

  11. Reduction of Urease Activity by Interaction with the Flap Covering the Active Site

    PubMed Central

    Macomber, Lee; Minkara, Mona S.; Hausinger, Robert P.; Merz, Kenneth M.

    2015-01-01

    With the increasing appreciation for the human microbiome coupled with the global rise of antibiotic resistant organisms, it is imperative that new methods be developed to specifically target pathogens. To that end, a novel computational approach was devised to identify compounds that reduce the activity of urease, a medically important enzyme of Helicobacter pylori, Proteus mirabilis, and many other microorganisms. Urease contains a flexible loop that covers its active site; Glide was used to identify small molecules predicted to lock this loop in an open conformation. These compounds were screened against the model urease from Klebsiella aerogenes and the natural products epigallocatechin and quercetin were shown to inhibit at low and high micromolar concentrations, respectively. These molecules exhibit a strong time-dependent inactivation of urease that was not due to their oxygen sensitivity. Rather, these compounds appear to inactivate urease by reacting with a specific Cys residue located on the flexible loop. Substitution of this cysteine by alanine in the C319A variant increased the urease resistance to both epigallocatechin and quercetin, as predicted by the computational studies. Protein dynamics are integral to the function of many enzymes; thus, identification of compounds that lock an enzyme into a single conformation presents a useful approach to define potential inhibitors. PMID:25594724

  12. Encroachment of Human Activity on Sea Turtle Nesting Sites

    NASA Astrophysics Data System (ADS)

    Ziskin, D.; Aubrecht, C.; Elvidge, C.; Tuttle, B.; Baugh, K.; Ghosh, T.

    2008-12-01

    The encroachment of anthropogenic lighting on sea turtle nesting sites poses a serious threat to the survival of these animals [Nicholas, 2001]. This danger is quantified by combining two established data sets. The first is the Nighttime Lights data produced by the NOAA National Geophysical Data Center [Elvidge et al., 1997]. The second is the Marine Turtle Database produced by the World Conservation Monitoring Centre (WCMC). The technique used to quantify the threat of encroachment is an adaptation of the method described in Aubrecht et al. [2008], which analyzes the stress on coral reef systems by proximity to nighttime lights near the shore. Nighttime lights near beaches have both a direct impact on turtle reproductive success since they disorient hatchlings when they mistake land-based lights for the sky-lit surf [Lorne and Salmon, 2007] and the lights are also a proxy for other anthropogenic threats. The identification of turtle nesting sites with high rates of encroachment will hopefully steer conservation efforts to mitigate their effects [Witherington, 1999]. Aubrecht, C, CD Elvidge, T Longcore, C Rich, J Safran, A Strong, M Eakin, KE Baugh, BT Tuttle, AT Howard, EH Erwin, 2008, A global inventory of coral reef stressors based on satellite observed nighttime lights, Geocarto International, London, England: Taylor and Francis. In press. Elvidge, CD, KE Baugh, EA Kihn, HW Kroehl, ER Davis, 1997, Mapping City Lights with Nighttime Data from the DMSP Operational Linescan System, Photogrammatic Engineering and Remote Sensing, 63:6, pp. 727-734. Lorne, JK, M Salmon, 2007, Effects of exposure to artificial lighting on orientation of hatchling sea turtles on the beach and in the ocean, Endangered Species Research, Vol. 3: 23-30. Nicholas, M, 2001, Light Pollution and Marine Turtle Hatchlings: The Straw that Breaks the Camel's Back?, George Wright Forum, 18:4, p77-82. Witherington, BE, 1999, Reducing Threats To Nesting Habitat, Research and Management Techniques for

  13. Small Molecule Active Site Directed Tools for Studying Human Caspases.

    PubMed

    Poreba, Marcin; Szalek, Aleksandra; Kasperkiewicz, Paulina; Rut, Wioletta; Salvesen, Guy S; Drag, Marcin

    2015-11-25

    Caspases are proteases of clan CD and were described for the first time more than two decades ago. They play critical roles in the control of regulated cell death pathways including apoptosis and inflammation. Due to their involvement in the development of various diseases like cancer, neurodegenerative diseases, or autoimmune disorders, caspases have been intensively investigated as potential drug targets, both in academic and industrial laboratories. This review presents a thorough, deep, and systematic assessment of all technologies developed over the years for the investigation of caspase activity and specificity using substrates and inhibitors, as well as activity based probes, which in recent years have attracted considerable interest due to their usefulness in the investigation of biological functions of this family of enzymes. PMID:26551511

  14. Activation of brown adipose tissue mitochondrial GDP binding sites

    SciTech Connect

    Swick, A.G.

    1987-01-01

    The primary function of brown adipose tissue (BAT) is heat production. This ability is attributed to the existence of a unique inner mitochondrial membrane protein termed the uncoupling protein or thermogenin. This protein is permeable to H+ and thus allows respiration (and therefore thermogenesis) to proceed at a rapid rate, independent of ADP phosphorylation. Proton conductance can be inhibited by the binding of purine nucleotides to the uncoupling protein. The binding of (/sup 3/H)-GDP to BAT mitochondria is frequently used as a measure of BAT thermogenic activity. Rats fed a diet that was low but adequate in protein exhibited a decrease in feed efficiency. In addition, BAT thermogenesis was activated as indicated by an elevation in the level of GDP binding to BAT mitochondria. This phenomena occurred in older rats and persisted over time.

  15. Activation of the Klebsiella pneumoniae nifU promoter: identification of multiple and overlapping upstream NifA binding sites.

    PubMed Central

    Cannon, W V; Kreutzer, R; Kent, H M; Morett, E; Buck, M

    1990-01-01

    The Klebsiella pneumoniae nifU promoter is positively controlled by the NifA protein and requires a form of RNA polymerase holoenzyme containing the rpoN encoded sigma factor, sigma 54. Occupancy of the K. pneumoniae nifU promoter by NifA was examined using in vivo dimethyl sulphate footprinting. Three binding sites for NifA (Upstream Activator Sequences, UASs 1, 2 and 3) located at -125, -116 and -72 were identified which conform to the UAS consensus sequence TGT-N10-ACA. An additional NifA binding site was identified at position -90. The UASs located at -125 (UAS1) and -116 (UAS2) overlap and do not appear to bind NifA as independent sites. They may represent a NifA binding site interacting with two NifA dimers. UAS3 is located at -72, and abuts a binding site for integration host factor (IHF) and is not normally highly occupied by NifA. In the absence of IHF UAS3 showed increased occupancy by NifA. Mutational and footprinting analysis of the three UASs indicates (1) IHF and NifA can compete for binding and that this competition influences the level of expression from the nifU promoter (2) that UAS2 is a principle sequence of the UAS 1,2 region required for activation and (3) that none of the NifA binding sites interacts with NifA independently. In vivo KMnO4 footprinting demonstrated that NifA catalyses open complex formation at the nifU promoter. IHF was required for maximal expression from the nifU and nifH promoters in Escherichia coli, and for the establishment of a Nif+ phenotype in E. coli from the nif plasmid pRD1. Images PMID:2186362

  16. Physical Activity in the Prevention of Benign Paroxysmal Positional Vertigo: Probable Association

    PubMed Central

    Bazoni, Jéssica Aparecida; Mendes, William Siqueira; Meneses-Barriviera, Caroline Luiz; Melo, Juliana Jandre; Costa, Viviane de Souza Pinho; Teixeira, Denilson de Castro; Marchiori, Luciana Lozza de Moraes

    2014-01-01

    Introduction Physical inactivity is an important risk factor for many age-related diseases and symptoms such as dizziness and vertigo. Objective The aim of the study was to investigate the possible association between benign paroxysmal positional vertigo (BPPV) and regular physical activity in elderly subjects. Methods This cross-sectional study included 491 elderly individuals who lived independently. Physical exercise was assessed through a questionnaire and BPPV by history and the Dix-Hallpike maneuver. Results The present study indicates no significant association between BPPV with lack of physical activity in men and in the total population. We have confirmed associations between BPPV with lack of physical activity in women (p = 0.01). Women with a sedentary lifestyle who do not practice physical activity are 2.62 more likely to have BPPV than those with regular physical activity. Conclusion These results highlight the importance of identifying risk factors for BPPV that can be modified through specific interventions. Regular physical activity is a lifestyle with potential to decrease the risk of vertigo in women. PMID:25992128

  17. Localization of Physical Activity in Primary School Children Using Accelerometry and Global Positioning System

    PubMed Central

    Bürgi, Rahel; Tomatis, Laura; Murer, Kurt; de Bruin, Eling D.

    2015-01-01

    Background Ecological approaches have highlighted the importance of the built environment as a factor affecting physical activity. However, knowledge on children’s activity patterns is still incomplete. Particularly, data on the spatial context of physical activity is limited, which limits the potential to design location-based interventions effectively. Using global positioning system (GPS) and accelerometry, this study aimed to identify locations where children engage in moderate-vigorous physical activity (MVPA). Methods Participants included 119 children (11–14 years, 57% girls) from public schools in Winterthur, Switzerland. During a regular school week between February and April 2013, children wore an accelerometer and GPS sensor for seven consecutive days. Time-matched accelerometer and GPS data was mapped with a geographic information system and each data point was assigned to one of seven defined activity settings. Both the absolute amount of MVPA and proportion of time in MVPA were calculated for every setting. Multilevel analyses accounting for the hierarchical structure of the data were conducted to investigate any gender differences. Results Children achieved most MVPA on streets (34.5%) and on school grounds (33.4%). The proportion children spent in MVPA was highest in recreational facilities (19.4%), at other schools (19.2%) and on streets (18.6%). Boys accumulated significantly more MVPA overall and on other school grounds (p < 0.05) and showed a significantly higher proportion of time in MVPA at own school and outside of Winterthur (p < 0.05). Conclusions The results indicate the importance of streets and school grounds as activity-promoting environments. The high use of streets may be an indicator for active transportation, which appears to contribute to an active lifestyle in both genders. In contrast, the school setting is more likely to encourage physical activity in boys. Recreational facilities seem to be conducive for MVPA among both

  18. Crystal structure of the ectoine hydroxylase, a snapshot of the active site.

    PubMed

    Höppner, Astrid; Widderich, Nils; Lenders, Michael; Bremer, Erhard; Smits, Sander H J

    2014-10-24

    Ectoine and its derivative 5-hydroxyectoine are compatible solutes that are widely synthesized by bacteria to cope physiologically with osmotic stress. They also serve as chemical chaperones and maintain the functionality of macromolecules. 5-Hydroxyectoine is produced from ectoine through a stereo-specific hydroxylation, an enzymatic reaction catalyzed by the ectoine hydroxylase (EctD). The EctD protein is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily and is evolutionarily well conserved. We studied the ectoine hydroxylase from the cold-adapted marine ultra-microbacterium Sphingopyxis alaskensis (Sa) and found that the purified SaEctD protein is a homodimer in solution. We determined the SaEctD crystal structure in its apo-form, complexed with the iron catalyst, and in a form that contained iron, the co-substrate 2-oxoglutarate, and the reaction product of EctD, 5-hydroxyectoine. The iron and 2-oxoglutarate ligands are bound within the EctD active site in a fashion similar to that found in other members of the dioxygenase superfamily. 5-Hydroxyectoine, however, is coordinated by EctD in manner different from that found in high affinity solute receptor proteins operating in conjunction with microbial import systems for ectoines. Our crystallographic analysis provides a detailed view into the active site of the ectoine hydroxylase and exposes an intricate network of interactions between the enzyme and its ligands that collectively ensure the hydroxylation of the ectoine substrate in a position- and stereo-specific manner. PMID:25172507

  19. Crystal Structure of the Ectoine Hydroxylase, a Snapshot of the Active Site*

    PubMed Central

    Höppner, Astrid; Widderich, Nils; Lenders, Michael; Bremer, Erhard; Smits, Sander H. J.

    2014-01-01

    Ectoine and its derivative 5-hydroxyectoine are compatible solutes that are widely synthesized by bacteria to cope physiologically with osmotic stress. They also serve as chemical chaperones and maintain the functionality of macromolecules. 5-Hydroxyectoine is produced from ectoine through a stereo-specific hydroxylation, an enzymatic reaction catalyzed by the ectoine hydroxylase (EctD). The EctD protein is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily and is evolutionarily well conserved. We studied the ectoine hydroxylase from the cold-adapted marine ultra-microbacterium Sphingopyxis alaskensis (Sa) and found that the purified SaEctD protein is a homodimer in solution. We determined the SaEctD crystal structure in its apo-form, complexed with the iron catalyst, and in a form that contained iron, the co-substrate 2-oxoglutarate, and the reaction product of EctD, 5-hydroxyectoine. The iron and 2-oxoglutarate ligands are bound within the EctD active site in a fashion similar to that found in other members of the dioxygenase superfamily. 5-Hydroxyectoine, however, is coordinated by EctD in manner different from that found in high affinity solute receptor proteins operating in conjunction with microbial import systems for ectoines. Our crystallographic analysis provides a detailed view into the active site of the ectoine hydroxylase and exposes an intricate network of interactions between the enzyme and its ligands that collectively ensure the hydroxylation of the ectoine substrate in a position- and stereo-specific manner. PMID:25172507

  20. The Positive Transcription Elongation Factor b Is an Essential Cofactor for the Activation of Transcription by Myocyte Enhancer Factor 2

    PubMed Central

    Nojima, Masanori; Huang, Yehong; Tyagi, Mudit; Kao, Hung-Ying; Fujinaga, Koh

    2014-01-01

    The positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 and cyclin T1, stimulates the elongation of transcription by hyperphosphorylating the C-terminal region of RNA polymerase II. Aberrant activation of P-TEFb results in manifestations of cardiac hypertrophy in mice, suggesting that P-TEFb is an essential factor for cardiac myocyte function and development. Here, we present evidence that P-TEFb selectively activates transcription mediated by the myocyte enhancer factor 2 (MEF2) family of transcription factors, key regulatory factors for myocyte development. Knockdown of endogenous cyclin T1 in murine C2C12 cells abolishes MEF2-dependent reporter gene expression as well as transcription of endogenous MEF2 target genes, whereas overexpression of P-TEFb enhances MEF2-dependent transcription. P-TEFb interacts with MEF2 both in vitro and in vivo. Activation of MEF2-dependent transcription induced by serum starvation is mediated by a rapid dissociation of P-TEFb from its inhibitory subunit, HEXIM1, and a subsequent recruitment of P-TEFb to MEF2 binding sites in the promoter region of MEF2 target genes. These results indicate that recruitment of P-TEFb is a critical step for stimulation of MEF2-dependent transcription, therefore providing a fundamentally important regulatory mechanism underlying the transcriptional program in muscle cells. PMID:18662700

  1. Force-Based Puncture Detection and Active Position Holding for Assisted Retinal Vein Cannulation*

    PubMed Central

    Gonenc, Berk; Tran, Nhat; Riviere, Cameron N.; Gehlbach, Peter; Taylor, Russell H.; Iordachita, Iulian

    2016-01-01

    Retinal vein cannulation is a demanding procedure proposed to treat retinal vein occlusion by direct therapeutic agent delivery methods. Challenges in identifying the moment of venous puncture, achieving cannulation and maintaining cannulation during drug delivery currently limit the feasibility of the procedure. In this study, we respond to these problems with an assistive system combining a handheld micromanipulator, Micron, with a force-sensing microneedle. The integrated system senses the instant of vein puncture based on measured forces and the position of the needle tip. The system actively holds the cannulation device securely in the vein following cannulation and during drug delivery. Preliminary testing of the system in a dry phantom, stretched vinyl membranes, demonstrates a significant improvement in the total time the needle could be maintained stably inside of the vein. This was especially evident in smaller veins and is attributed to decreased movement of the positioned cannula following venous cannulation. PMID:27127804

  2. A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes.

    PubMed

    Gryaznova, Yuliya; Koca Caydasi, Ayse; Malengo, Gabriele; Sourjik, Victor; Pereira, Gislene

    2016-01-01

    The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control. PMID:27159239

  3. A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes

    PubMed Central

    Gryaznova, Yuliya; Caydasi, Ayse Koca; Malengo, Gabriele; Sourjik, Victor; Pereira, Gislene

    2016-01-01

    The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control. DOI: http://dx.doi.org/10.7554/eLife.14029.001 PMID:27159239

  4. Active and latent tuberculosis among HIV-positive injecting drug users in Indonesia

    PubMed Central

    Meijerink, Hinta; Wisaksana, Rudi; Lestari, Mery; Meilana, Intan; Chaidir, Lydia; van der Ven, Andre JAM; Alisjahbana, Bachti; van Crevel, Reinout

    2015-01-01

    Introduction Injecting drug use (IDU) is associated with tuberculosis but few data are available from low-income settings. We examined IDU in relation to active and latent tuberculosis (LTBI) among HIV-positive individuals in Indonesia, which has a high burden of tuberculosis and a rapidly growing HIV epidemic strongly driven by IDU. Methods Active tuberculosis was measured prospectively among 1900 consecutive antiretroviral treatment (ART)-naïve adult patients entering care in a clinic in West Java. Prevalence of LTBI was determined cross-sectionally in a subset of 518 ART-experienced patients using an interferon-gamma release assay. Results Patients with a history of IDU (53.1%) more often reported a history of tuberculosis treatment (34.8% vs. 21.9%, p<0.001), more often received tuberculosis treatment during follow-up (adjusted HR=1.71; 95% CI: 1.25–2.35) and more often had bacteriologically confirmed tuberculosis (OR=1.67; 95% CI: 0.94–2.96). LTBI was equally prevalent among people with and without a history of IDU (29.1 vs. 30.4%, NS). The risk estimates did not change after adjustment for CD4 cell count or ART. Conclusions HIV-positive individuals with a history of IDU in Indonesia have more active tuberculosis, with similar rates of LTBI. Within the HIV clinic, LTBI screening and isoniazid preventive therapy may be prioritized to patients with a history of IDU. PMID:25690530

  5. A structural model for across membrane coupling between the Qo and Qi active sites of cytochrome bc1.

    PubMed

    Cooley, Jason W

    2010-12-01

    The two spatially distant quinone-binding sites of the ubihydroquinone: cytochrome c oxidoreductase (cyt bc(1)) complex have been shown to influence one another in some fashion. This transmembrane communication alters cofactor and redox partner binding interactions and could potentially influence the timing or 'concerted' steps involved in the steady-state turnover of the homodimeric enzymes. Yet, despite several lines of evidence corroborating the coupling of the quinone binding active sites to one another, little to no testable hypothesis has been offered to explain how such a "signal" might be transmitted across the presumably rigid hydrophobic domain of the enzyme. Recently, it has been shown that this interquinone binding sites communication influences the steady-state position of the mobile [2Fe-2S] cluster containing iron sulfur protein (Sarewicz M., Dutka M., Froncisz W., Osyczka A. (2009) Biochemistry 48, 5708-5720) as mediated by at least one transmembrane helix of the b-type cyt containing subunit (Cooley, J. W., Lee, D. W., and Daldal, F. (2009) Biochemistry 48, 1988-1999). Here we provide an overview of the evidence supporting the structural coupling of these sites and provide a theoretical framework for how the redox state of a quinone at one cofactor binding site might influence the cofactor-, inhibitor-, and/or protein-protein interactions at the structurally distant opposing Q binding site. PMID:20513347

  6. School Pharmacist/School Environmental Hygienic Activities at School Site.

    PubMed

    Muramatsu, Akiyoshi

    2016-01-01

    The "School Health and Safety Act" was enforced in April 2009 in Japan, and "school environmental health standards" were established by the Minister of Education, Culture, Sports, Science and Technology. In Article 24 of the Enforcement Regulations, the duties of the school pharmacist have been clarified; school pharmacists have charged with promoting health activities in schools and carrying out complete and regular checks based on the "school environmental health standards" in order to protect the health of students and staff. In supported of this, the school pharmacist group of Japan Pharmaceutical Association has created and distributed digital video discs (DVDs) on "check methods of school environmental health standards" as support material. We use the DVD to ensure the basic issues that school pharmacists deal with, such as objectives, criteria, and methods for each item to be checked, advice, and post-measures. We conduct various workshops and classes, and set up Q&A committees so that inquiries from members are answered with the help of such activities. In addition, school pharmacists try to improve the knowledge of the school staff on environmental hygiene during their in-service training. They also conduct "drug abuse prevention classes" at school and seek to improve knowledge and recognition of drugs, including "dangerous drugs". PMID:27252053

  7. Anomalous scattering analysis of Agrobacterium radiobacter phosphotriesterase: the prominent role of iron in the heterobinuclear active site

    PubMed Central

    Jackson, Colin J.; Carr, Paul D.; Kim, Hye-Kyung; Liu, Jian-Wei; Herrald, Paul; Mitić, Nataša; Schenk, Gerhard; Smith, Clyde A.; Ollis, David L.

    2006-01-01

    Bacterial phosphotriesterases are binuclear metalloproteins for which the catalytic mechanism has been studied with a variety of techniques, principally using active sites reconstituted in vitro from apoenzymes. Here, atomic absorption spectroscopy and anomalous X-ray scattering have been used to determine the identity of the metals incorporated into the active site in vivo. We have recombinantly expressed the phosphotriesterase from Agrobacterium radiobacter (OpdA) in Escherichia coli grown in medium supplemented with 1 mM CoCl2 and in unsupplemented medium. Anomalous scattering data, collected from a single crystal at the Fe–K, Co–K and Zn–K edges, indicate that iron and cobalt are the primary constituents of the two metal-binding sites in the catalytic centre (α and β) in the protein expressed in E. coli grown in supplemented medium. Comparison with OpdA expressed in unsupplemented medium demonstrates that the cobalt present in the supplemented medium replaced zinc at the β-position of the active site, which results in an increase in the catalytic efficiency of the enzyme. These results suggest an essential role for iron in the catalytic mechanism of bacterial phosphotriesterases, and that these phosphotriesterases are natively heterobinuclear iron–zinc enzymes. PMID:16686603

  8. An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors*

    PubMed Central

    Wang, Jingyi; Kuryatov, Alexander; Sriram, Aarati; Jin, Zhuang; Kamenecka, Theodore M.; Kenny, Paul J.; Lindstrom, Jon

    2015-01-01

    Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets. PMID:25869137

  9. The active site loop of S-adenosylmethionine synthetase modulates catalytic efficiency.

    PubMed

    Taylor, John C; Takusagawa, Fusao; Markham, George D

    2002-07-30

    glycine mutants causes the activation enthalpy of the reaction to approximately double in each case, while the activation entropy changes from negative in the wild-type enzyme to positive in the mutants. The favorable activation entropy in the mutant-catalyzed reactions may reflect release of water during catalysis, while the negative activation entropy in the reaction catalyzed by the wild-type enzyme apparently reflects reorganization of the loop. The observations point to how nature can fine-tune the activity of an enzyme by modifying substrate and product access to the active site rather than by altering the enzyme x substrate contacts or the catalytic machinery itself. PMID:12135357

  10. Isolated metal active site concentration and stability control catalytic CO2 reduction selectivity.

    PubMed

    Matsubu, John C; Yang, Vanessa N; Christopher, Phillip

    2015-03-01

    CO2 reduction by H2 on heterogeneous catalysts is an important class of reactions that has been studied for decades. However, atomic scale details of structure-function relationships are still poorly understood. Particularly, it has been suggested that metal particle size plays a unique role in controlling the stability of CO2 hydrogenation catalysts and the distribution of active sites, which dictates reactivity and selectivity. These studies often have not considered the possible role of isolated metal active sites in the observed dependences. Here, we utilize probe molecule diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) with known site-specific extinction coefficients to quantify the fraction of Rh sites residing as atomically dispersed isolated sites (Rhiso), as well as Rh sites on the surface of Rh nanoparticles (RhNP) for a series of TiO2 supported Rh catalysts. Strong correlations were observed between the catalytic reverse water gas shift turn over frequency (TOF) and the fraction of Rhiso sites and between catalytic methanation TOF and the fraction of RhNP sites. Furthermore, it was observed that reaction condition-induced disintegration of Rh nanoparticles, forming Rhiso active sites, controls the changing reactivity with time on stream. This work demonstrates that isolated atoms and nanoparticles of the same metal on the same support can exhibit uniquely different catalytic selectivity in competing parallel reaction pathways and that disintegration of nanoparticles under reaction conditions can play a significant role in controlling stability. PMID:25671686

  11. The influence of substituents in 3-position on the activity of chroman-type potassium channel activators.

    PubMed

    Bergmann, R; Gericke, R

    1994-03-01

    Swern oxidation of chromanol 1 led to ketone 3 with concomitant chlorination of the adjacent 4-position. Using Leuckart conditions, chromanone 2 was converted to enamine 5.--4-Bromochromene-3-carbaldehyde 8, which was obtained by Vilsmeier-Arnold reaction from 7, turned out to be a suitable intermediate for the insertion of the pyridone residue. 3-Chloro derivatives 16 and 19 resulted on heating the mesylate or tosylate with LiCl in DMF. Bromination of chromene 20 led to 21.--All compounds were tested for oral antihypertensive activity in spontaneously hypertensive rats with a dose of 1 mg/kg. PMID:8179475

  12. Classical-quantum arbitrarily varying wiretap channel: Ahlswede dichotomy, positivity, resources, super-activation

    NASA Astrophysics Data System (ADS)

    Boche, Holger; Cai, Minglai; Deppe, Christian; Nötzel, Janis

    2016-08-01

    We establish the Ahlswede dichotomy for arbitrarily varying classical-quantum wiretap channels, i.e., either the deterministic secrecy capacity of the channel is zero, or it equals its randomness-assisted secrecy capacity. We analyze the secrecy capacity of these channels when the sender and the receiver use various resources. It turns out that randomness, common randomness, and correlation as resources are very helpful for achieving a positive secrecy capacity. We prove the phenomenon "super-activation" for arbitrarily varying classical-quantum wiretap channels, i.e., two channels, both with zero deterministic secrecy capacity, if used together allow perfect secure transmission.

  13. The balance of flexibility and rigidity in the active site residues of hen egg white lysozyme

    NASA Astrophysics Data System (ADS)

    Qi, Jian-Xun; Jiang, Fan

    2011-05-01

    The crystallographic temperature factors (B factor) of individual atoms contain important information about the thermal motion of the atoms in a macromolecule. Previously the theory of flexibility of active site has been established based on the observation that the enzyme activity is sensitive to low concentration denaturing agents. It has been found that the loss of enzyme activity occurs well before the disruption of the three-dimensional structural scaffold of the enzyme. To test the theory of conformational flexibility of enzyme active site, crystal structures were perturbed by soaking in low concentration guanidine hydrochloride solutions. It was found that many lysozyme crystals tested could still diffract until the concentration of guanidine hydrochloride reached 3 M. It was also found that the B factors averaged over individually collected data sets were more accurate. Thus it suggested that accurate measurement of crystal temperature factors could be achieved for medium-high or even medium resolution crystals by averaging over multiple data sets. Furthermore, we found that the correctly predicted active sites included not only the more flexible residues, but also some more rigid residues. Both the flexible and the rigid residues in the active site played an important role in forming the active site residue network, covering the majority of the substrate binding residues. Therefore, this experimental prediction method may be useful for characterizing the binding site and the function of a protein, such as drug targeting.

  14. S-Nitrosylation Positively Regulates Ascorbate Peroxidase Activity during Plant Stress Responses1

    PubMed Central

    Yang, Huanjie; Mu, Jinye; Chen, Lichao; Feng, Jian; Hu, Jiliang; Li, Lei; Zhou, Jian-Min; Zuo, Jianru

    2015-01-01

    Nitric oxide (NO) and reactive oxygen species (ROS) are two classes of key signaling molecules involved in various developmental processes and stress responses in plants. The burst of NO and ROS triggered by various stimuli activates downstream signaling pathways to cope with abiotic and biotic stresses. Emerging evidence suggests that the interplay of NO and ROS plays a critical role in regulating stress responses. However, the underpinning molecular mechanism remains poorly understood. Here, we show that NO positively regulates the activity of the Arabidopsis (Arabidopsis thaliana) cytosolic ascorbate peroxidase1 (APX1). We found that S-nitrosylation of APX1 at cysteine (Cys)-32 enhances its enzymatic activity of scavenging hydrogen peroxide, leading to the increased resistance to oxidative stress, whereas a substitution mutation at Cys-32 causes the reduction of ascorbate peroxidase activity and abolishes its responsiveness to the NO-enhanced enzymatic activity. Moreover, S-nitrosylation of APX1 at Cys-32 also plays an important role in regulating immune responses. These findings illustrate a unique mechanism by which NO regulates hydrogen peroxide homeostasis in plants, thereby establishing a molecular link between NO and ROS signaling pathways. PMID:25667317

  15. Gramicidin A Mutants with Antibiotic Activity against Both Gram-Positive and Gram-Negative Bacteria.

    PubMed

    Zerfas, Breanna L; Joo, Yechaan; Gao, Jianmin

    2016-03-17

    Antimicrobial peptides (AMPs) have shown potential as alternatives to traditional antibiotics for fighting infections caused by antibiotic-resistant bacteria. One promising example of this is gramicidin A (gA). In its wild-type sequence, gA is active by permeating the plasma membrane of Gram-positive bacteria. However, gA is toxic to human red blood cells at similar concentrations to those required for it to exert its antimicrobial effects. Installing cationic side chains into gA has been shown to lower its hemolytic activity while maintaining the antimicrobial potency. In this study, we present the synthesis and the antibiotic activity of a new series of gA mutants that display cationic side chains. Specifically, by synthesizing alkylated lysine derivatives through reductive amination, we were able to create a broad selection of structures with varied activities towards Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). Importantly, some of the new mutants were observed to have an unprecedented activity towards important Gram-negative pathogens, including Escherichia coli, Klebsiella pneumoniae and Psuedomonas aeruginosa. PMID:26918268

  16. The calculation of surface orbital energies for specific types of active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.; Cole, F.

    1992-11-01

    An angular overlap calculation has been used to determine the s, p, and d orbital energy levels of the different types of surface sites present on dispersed metal catalysts. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  17. The calculation of surface orbital energies for specific types of active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.; Cole, F.

    1992-01-01

    An angular overlap calculation has been used to determine the s, p, and d orbital energy levels of the different types of surface sites present on dispersed metal catalysts. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  18. Extending the Diffuse Layer Model of Surface Acidity Behavior: III. Estimating Bound Site Activity Coefficients

    EPA Science Inventory

    Although detailed thermodynamic analyses of the 2-pK diffuse layer surface complexation model generally specify bound site activity coefficients for the purpose of accounting for those non-ideal excess free energies contributing to bound site electrochemical potentials, in applic...

  19. A switch region determines the cell type-specific positive or negative action of YY1 on the activity of the human papillomavirus type 18 promoter.

    PubMed Central

    Bauknecht, T; Jundt, F; Herr, I; Oehler, T; Delius, H; Shi, Y; Angel, P; Zur Hausen, H

    1995-01-01

    YY1 is a zinc finger transcription factor which acts as either a repressor or an activator dependent on the promoter context. YY1 is a potent activator of the genuine human papillomavirus type 18 (HPV-18) upstream regulatory region (URR) in HeLa cells, which are known for high-level expression of the HPV-18 early genes. The activating activity of YY1 is dependent on the presence of a newly identified switch region located upstream of the YY1 binding site. Deletion of this region causes YY1 to act as a repressor of HPV-18 promoter activity. In vivo footprinting of the HPV-18 URR and an in vitro electrophoretic mobility shift assay identified proteins binding to the switch region. Site-directed mutagenesis of the switch region and YY1 binding sites suggests that these two regions work in concert to yield high-level HPV-18 URR activity in HeLa cells but not in HepG2 cells, where HPV-18 is almost inactive. These data identified a novel mode of cell type-specific regulation of HPV-18 promoter activity by positive or negative action of YY1, determined by the switch region binding factor(s). PMID:7983700

  20. 1993 annual report of hazardous waste activities for the Oak Ridge K-25 site

    SciTech Connect

    Not Available

    1994-02-01

    This report is a detailed listing of all of the Hazardous Waste activities occurring at Martin Marietta`s K-25 site. Contained herein are hazardous waste notification forms, waste stream reports, generator fee forms and various TSDR reports.

  1. Chemical modification studies on arginine kinase: essential cysteine and arginine residues at the active site.

    PubMed

    Zhu, Wen-Jing; Li, Miao; Wang, Xiao-Yun

    2007-12-01

    Chemical modification was used to elucidate the essential amino acids in the catalytic activity of arginine kinase (AK) from Migratoria manilensis. Among six cysteine (Cys) residues only one Cys residue was determined to be essential in the active site by Tsou's method. Furthermore, the AK modified by DTNB can be fully reactivated by dithiothreitol (DTT) in a monophasic kinetic course. At the same time, this reactivation can be slowed down in the presence of ATP, suggesting that the essential Cys is located near the ATP binding site. The ionizing groups at the AK active site were studied and the standard dissociation enthalpy (DeltaH degrees ) was 12.38kcal/mol, showing that the dissociation group may be the guanidino of arginine (Arg). Using the specific chemical modifier phenylglyoxal (PG) demonstrated that only one Arg, located near the ATP binding site, is essential for the activity of AK. PMID:17765964

  2. 78 FR 8190 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-05

    ...BOEM is reopening the comment period announced in the Notice of Intent to Prepare an Environmental Assessment (EA) for Commercial Wind Leasing and Site Assessment Activities on the OCS Offshore North...

  3. Effects of Cycling Conditions of Active Material From Discharged Ni Positive Plates Studied by Inelastic Neutron Scattering Spectroscopy

    NASA Technical Reports Server (NTRS)

    Eckert, Juergen; Varma, Ravi; Diebolt, Lisa; Reid, Margaret

    1998-01-01

    The objectives of this presentation are: identify atomic-level signatures of electrochemical activity of the active material on the Ni positive plates of Ni-H2 batteries, relate finding to cycling conditions and histories, and develop INS spectroscopy as a non-destructive testing technique for the evaluation of Ni-positive plates of Ni-H2 batteries.

  4. Structural Dissection of the Active Site of Thermotoga maritima β-Galactosidase Identifies Key Residues for Transglycosylating Activity.

    PubMed

    Talens-Perales, David; Polaina, Julio; Marín-Navarro, Julia

    2016-04-13

    Glycoside hydrolases, specifically β-galactosidases, can be used to synthesize galacto-oligosaccharides (GOS) due to the transglycosylating (secondary) activity of these enzymes. Site-directed mutagenesis of a thermoresistant β-galactosidase from Thermotoga maritima has been carried out to study the structural basis of transgalactosylation and to obtain enzymatic variants with better performance for GOS biosynthesis. Rational design of mutations was based on homologous sequence analysis and structural modeling. Analysis of mutant enzymes indicated that residue W959, or an alternative aromatic residue at this position, is critical for the synthesis of β-3'-galactosyl-lactose, the major GOS obtained with the wild-type enzyme. Mutants W959A and W959C, but not W959F, showed an 80% reduced synthesis of this GOS. Other substitutions, N574S, N574A, and F571L, increased the synthesis of β-3'-galactosyl-lactose about 40%. Double mutants F571L/N574S and F571L/N574A showed an increase of about 2-fold. PMID:26998654

  5. Exogenous Magnesium Chloride Reduces the Activated Partial Thromboplastin Times of Lupus Anticoagulant-Positive Patients

    PubMed Central

    Tokutake, Takayoshi; Baba, Hisami; Shimada, Yuji; Takeda, Wataru; Sato, Keijiro; Hiroshima, Yuki; Kirihara, Takehiko; Shimizu, Ikuo; Nakazawa, Hideyuki; Kobayashi, Hikaru; Ishida, Fumihiro

    2016-01-01

    The activated partial thromboplastin time (APTT) assay is a basic hemostatic assay based on the time it takes for clots to form in plasma samples after the addition of calcium chloride. It is used to screen for various coagulation disorders. Several previous reports have suggested that magnesium (Mg) might contribute to coagulation reactions by binding to specific coagulation proteins. We investigated the effects of Mg on the APTT. In healthy controls, the APTT was significantly prolonged in proportion to the increase in the concentration of magnesium chloride in the range from 2.1 to 16.7 mmol/L. Among eight samples from patients with various disorders that exhibited prolonged APTT, two samples demonstrated shorter APTT when Mg was added, both of which were from patients that were positive for lupus anticoagulant. When we examined 206 clinical APTT samples, we found that Mg shortened the APTT of two samples. These two samples were also from lupus anticoagulant-positive patients (p-value: <0.003). Our findings regarding the unique effects of exogenous Mg on the APTT of lupus anticoagulant-positive patients might shed light on the role of Mg in APTT assays and lead to the development of a novel screening method for lupus anticoagulant. PMID:27355205

  6. Calculation of Vibrational Shifts of Nitrile Probes in the Active Site of Ketosteroid Isomerase upon Ligand Binding

    PubMed Central

    Layfield, Joshua P.

    2012-01-01

    The vibrational Stark effect provides insight into the roles of hydrogen bonding, electrostatics, and conformational motions in enzyme catalysis. In a recent application of this approach to the enzyme ketosteroid isomerase (KSI), thiocyanate probes were introduced in site-specific positions throughout the active site. This paper implements a quantum mechanical/molecular mechanical (QM/MM) approach for calculating the vibrational shifts of nitrile (CN) probes in proteins. This methodology is shown to reproduce the experimentally measured vibrational shifts upon binding of the intermediate analog equilinen to KSI for two different nitrile probe positions. Analysis of the molecular dynamics simulations provides atomistic insight into the roles that key residues play in determining the electrostatic environment and hydrogen-bonding interactions experienced by the nitrile probe. For the M116C-CN probe, equilinen binding reorients an active site water molecule that is directly hydrogen bonded to the nitrile probe, resulting in a more linear CNH angle and increasing the CN frequency upon binding. For the F86C-CN probe, equilinen binding orients the Asp103 residue, decreasing the hydrogen-bonding distance between the Asp103 backbone and the nitrile probe and slightly increasing the CN frequency. This QM/MM methodology is applicable to a wide range of biological systems and has the potential to assist in the elucidation of the fundamental principles underlying enzyme catalysis. PMID:23210919

  7. Reproducibility of liver position using active breathing coordinator for liver cancer radiotherapy

    SciTech Connect

    Eccles, Cynthia; Brock, Kristy K.; Bissonnette, Jean-Pierre; Hawkins, Maria; Dawson, Laura A. . E-mail: laura.dawson@rmp.uhn.on.ca

    2006-03-01

    Purpose: To measure the intrabreath-hold liver motion and the intrafraction and interfraction reproducibility of liver position relative to vertebral bodies using an active breathing coordinator (ABC) in patients with unresectable liver cancer treated with hypofractionated stereotactic body radiation therapy (SBRT). Methods: Tolerability of ABC and organ motion during ABC was assessed using kV fluoroscopy in 34 patients. For patients treated with ABC, repeat breath-hold CT scans in the ABC breath-hold position were acquired at simulation to estimate the volumetric intrafraction reproducibility of the liver relative to the vertebral bodies. In addition, preceding each radiation therapy fraction, with the liver immobilized using ABC, repeat anteroposterior (AP) megavoltage verification images were obtained. Off-line alignments were completed to determine intrafraction reproducibility (from repeat images obtained before one treatment) and interfraction reproducibility (from comparisons of the final image for each fraction with the AP) of diaphragm position relative to vertebral bodies. For each image set, the vertebral bodies were aligned, and the resultant craniocaudal (CC) offset in diaphragm position was measured. Liver position during ABC was also evaluated from kV fluoroscopy acquired at the time of simulation, kV fluoroscopy at the time of treatment, and from MV beam's-eye view movie loops acquired during treatment. Results: Twenty-one of 34 patients were screened to be suitable for ABC. The average free breathing range of these patients was 13 mm (range, 5-1 mm). Fluoroscopy revealed that the average maximal diaphragm motion during ABC breath-hold was 1.4 mm (range, 0-3.4 mm). The MV treatment movie loops confirmed diaphragm stability during treatment. For a measure of intrafraction reproducibility, an analysis of 36 repeat ABC computed tomography (CT) scans in 14 patients was conducted. The average mean difference in the liver surface position was -0.9 mm, -0

  8. Anisotropic Covalency Contributions to Superexchange Pathways in Type One Copper Active Sites

    PubMed Central

    2015-01-01

    Type one (T1) Cu sites deliver electrons to catalytic Cu active sites: the mononuclear type two (T2) Cu site in nitrite reductases (NiRs) and the trinuclear Cu cluster in the multicopper oxidases (MCOs). The T1 Cu and the remote catalytic sites are connected via a Cys-His intramolecular electron-transfer (ET) bridge, which contains two potential ET pathways: P1 through the protein backbone and P2 through the H-bond between the Cys and the His. The high covalency of the T1 Cu–S(Cys) bond is shown here to activate the T1 Cu site for hole superexchange via occupied valence orbitals of the bridge. This covalency-activated electronic coupling (HDA) facilitates long-range ET through both pathways. These pathways can be selectively activated depending on the geometric and electronic structure of the T1 Cu site and thus the anisotropic covalency of the T1 Cu–S(Cys) bond. In NiRs, blue (π-type) T1 sites utilize P1 and green (σ-type) T1 sites utilize P2, with P2 being more efficient. Comparing the MCOs to NiRs, the second-sphere environment changes the conformation of the Cys-His pathway, which selectively activates HDA for superexchange by blue π sites for efficient turnover in catalysis. These studies show that a given protein bridge, here Cys-His, provides different superexchange pathways and electronic couplings depending on the anisotropic covalencies of the donor and acceptor metal sites. PMID:25310460

  9. Site-directed mutagenesis of Lysine{sup 382}, the activator-binding site, of ADP-Glucose pyrophosphorylase from Anabaena PCC 6120

    SciTech Connect

    Sheng, Jun; Charng, Yee-yung; Preiss, J.

    1996-03-05

    Previous studies have shown that a highly conserved lysyl residue (Lys{sup 419}) near the C-terminus of Anabaena ADP-glucose pyrophosphorylase is involved in the binding of 3-P-glycerate, the allosteric activator. Phosphopyridoxylation of the K419R mutant enzyme modified another conserved lysyl residue (Lys{sup 382}), suggesting that this residue might be also located within the activator-binding site. Site-directed mutagenesis of Lys{sup 382} of the Anabaena enzyme was performed to determine the role of this residue. Replacing Lys{sup 382} with either arginine, alanine, or glutamine produced mutant enzymes with apparent affinities for 3-P-glycerate 10-160-fold lower than that of the wild-type enzyme. The glutamic acid mutant enzyme was inhibited by 3-P-glycerate. These mutations had lesser impact on the kinetic constants for the substrates and inhibitor, P{sub i}, and on the thermal stability. These results indicate that both the charge and size of the residue at position 382 influence the binding of 3-P-glycerate. Site-directed mutagenesis was also performed to obtain a K382R-K419R double mutant. The apparent affinity for 3-P-glycerate of this double-mutant enzyme was 104-fold lower than that of the wild-type enzyme, and the specificity for activator of this mutant enzyme was altered. The K382R-K419R enzyme could not be phosphopyridoxylated, suggesting that other lysine residues are not involved in the binding of 3-P-glycerate. 32 refs., 2 figs., 3 tabs.

  10. Picornaviruses and nuclear functions: targeting a cellular compartment distinct from the replication site of a positive-strand RNA virus

    PubMed Central

    Flather, Dylan; Semler, Bert L.

    2015-01-01

    The compartmentalization of DNA replication and gene transcription in the nucleus and protein production in the cytoplasm is a defining feature of eukaryotic cells. The nucleus functions to maintain the integrity of the nuclear genome of the cell and to control gene expression based on intracellular and environmental signals received through the cytoplasm. The spatial separation of the major processes that lead to the expression of protein-coding genes establishes the necessity of a transport network to allow biomolecules to translocate between these two regions of the cell. The nucleocytoplasmic transport network is therefore essential for regulating normal cellular functioning. The Picornaviridae virus family is one of many viral families that disrupt the nucleocytoplasmic trafficking of cells to promote viral replication. Picornaviruses contain positive-sense, single-stranded RNA genomes and replicate in the cytoplasm of infected cells. As a result of the limited coding capacity of these viruses, cellular proteins are required by these intracellular parasites for both translation and genomic RNA replication. Being of messenger RNA polarity, a picornavirus genome can immediately be translated upon entering the cell cytoplasm. However, the replication of viral RNA requires the activity of RNA-binding proteins, many of which function in host gene expression, and are consequently localized to the nucleus. As a result, picornaviruses disrupt nucleocytoplasmic trafficking to exploit protein functions normally localized to a different cellular compartment from which they translate their genome to facilitate efficient replication. Furthermore, picornavirus proteins are also known to enter the nucleus of infected cells to limit host-cell transcription and down-regulate innate antiviral responses. The interactions of picornavirus proteins and host-cell nuclei are extensive, required for a productive infection, and are the focus of this review. PMID:26150805

  11. Preferable sites and orientations of transgene inserted in the adenovirus vector genome: The E3 site may be unfavorable for transgene position

    PubMed Central

    Suzuki, M; Kondo, S; Pei, Z; Maekawa, A; Saito, I; Kanegae, Y

    2015-01-01

    The adenovirus vector (AdV) can carry two transgenes in its genome, the therapeutic gene and a reporter gene, for example. The E3 insertion site has often been used for the expression of the second transgene. A transgene can be inserted at six different sites/orientations: E1, E3 and E4 sites, and right and left orientations. However, the best combination of the insertion sites and orientations as for the titers and the expression levels has not sufficiently been studied. We attempted to construct 18 AdVs producing GFP or LacZ gene driven by the EF1α promoter and Cre gene driven by the α-fetoprotein promoter. The AdV containing GFP gene at E3 in the rightward orientation (GFP-E3R) was not available. The LacZ-E3R AdV showed 20-fold lower titer and 50-fold lower level of fiber mRNA than the control E1L AdV. Notably, we found four aberrantly spliced mRNAs in the LacZ-E3L/R AdVs, probably explaining their very low titers. Although the transgene expression levels in the E4R AdVs were about threefold lower than those in the E1L AdVs, their titers are comparable with that of E1L AdVs. We concluded that E1L and E4R sites/orientations are preferable for expressing the main target gene and a second gene, respectively. PMID:25588742

  12. The Three Mycobacterium tuberculosis Antigen 85 Isoforms Have Unique Substrates and Activities Determined by Non-active Site Regions*

    PubMed Central

    Backus, Keriann M.; Dolan, Michael A.; Barry, Conor S.; Joe, Maju; McPhie, Peter; Boshoff, Helena I. M.; Lowary, Todd L.; Davis, Benjamin G.; Barry, Clifton E.

    2014-01-01

    The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro216–Phe228 loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors. PMID:25028517

  13. 'Unconventional' coordination chemistry by metal chelating fragments in a metalloprotein active site.

    PubMed

    Martin, David P; Blachly, Patrick G; Marts, Amy R; Woodruff, Tessa M; de Oliveira, César A F; McCammon, J Andrew; Tierney, David L; Cohen, Seth M

    2014-04-01

    The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn(2+) ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a 'flattened' mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal-ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal-ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins. PMID:24635441

  14. Two Active Forms of UDP-N-Acetylglucosamine Enolpyruvyl Transferase in Gram-Positive Bacteria

    PubMed Central

    Du, Wensheng; Brown, James R.; Sylvester, Daniel R.; Huang, Jianzhong; Chalker, Alison F.; So, Chi Y.; Holmes, David J.; Payne, David J.; Wallis, Nicola G.

    2000-01-01

    Gene sequences encoding the enzymes UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from many bacterial sources were analyzed. It was shown that whereas gram-negative bacteria have only one murA gene, gram-positive bacteria have two distinct genes encoding these enzymes which have possibly arisen from gene duplication. The two murA genes of the gram-positive organism Streptococcus pneumoniae were studied further. Each of the murA genes was individually inactivated by allelic replacement. In each case, the organism was viable despite losing one of its murA genes. However, when attempts were made to construct a double-deletion strain, no mutants were obtained. This indicates that both genes encode active enzymes that can substitute for each other, but that the presence of a MurA function is essential to the organism. The two genes were further cloned and overexpressed, and the enzymes they encode were purified. Both enzymes catalyzed the transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine, confirming they are both active UDP-N-acetylglucosamine enolpyruvyl transferases. The catalytic parameters of the two enzymes were similar, and they were both inhibited by the antibiotic fosfomycin. PMID:10894720

  15. Activated WNT signaling in postnatal SOX2-positive dental stem cells can drive odontoma formation

    PubMed Central

    Xavier, Guilherme M.; Patist, Amanda L.; Healy, Chris; Pagrut, Ankita; Carreno, Gabriela; Sharpe, Paul T.; Pedro Martinez-Barbera, Juan; Thavaraj, Selvam; Cobourne, Martyn T.; Andoniadou, Cynthia L.

    2015-01-01

    In common with most mammals, humans form only two dentitions during their lifetime. Occasionally, supernumerary teeth develop in addition to the normal complement. Odontoma represent a small group of malformations containing calcified dental tissues of both epithelial and mesenchymal origin, with varying levels of organization, including tooth-like structures. The specific cell type responsible for the induction of odontoma, which retains the capacity to re-initiate de novo tooth development in postnatal tissues, is not known. Here we demonstrate that aberrant activation of WNT signaling by expression of a non-degradable form of β-catenin specifically in SOX2-positive postnatal dental epithelial stem cells is sufficient to generate odontoma containing multiple tooth-like structures complete with all dental tissue layers. Genetic lineage-tracing confirms that odontoma form in a similar manner to normal teeth, derived from both the mutation-sustaining epithelial stem cells and adjacent mesenchymal tissues. Activation of the WNT pathway in embryonic SOX2-positive progenitors results in ectopic expression of secreted signals that promote odontogenesis throughout the oral cavity. Significantly, the inductive potential of epithelial dental stem cells is retained in postnatal tissues, and up-regulation of WNT signaling specifically in these cells is sufficient to promote generation and growth of ectopic malformations faithfully resembling human odontoma. PMID:26411543

  16. Activated WNT signaling in postnatal SOX2-positive dental stem cells can drive odontoma formation.

    PubMed

    Xavier, Guilherme M; Patist, Amanda L; Healy, Chris; Pagrut, Ankita; Carreno, Gabriela; Sharpe, Paul T; Martinez-Barbera, Juan Pedro; Thavaraj, Selvam; Cobourne, Martyn T; Andoniadou, Cynthia L

    2015-01-01

    In common with most mammals, humans form only two dentitions during their lifetime. Occasionally, supernumerary teeth develop in addition to the normal complement. Odontoma represent a small group of malformations containing calcified dental tissues of both epithelial and mesenchymal origin, with varying levels of organization, including tooth-like structures. The specific cell type responsible for the induction of odontoma, which retains the capacity to re-initiate de novo tooth development in postnatal tissues, is not known. Here we demonstrate that aberrant activation of WNT signaling by expression of a non-degradable form of β-catenin specifically in SOX2-positive postnatal dental epithelial stem cells is sufficient to generate odontoma containing multiple tooth-like structures complete with all dental tissue layers. Genetic lineage-tracing confirms that odontoma form in a similar manner to normal teeth, derived from both the mutation-sustaining epithelial stem cells and adjacent mesenchymal tissues. Activation of the WNT pathway in embryonic SOX2-positive progenitors results in ectopic expression of secreted signals that promote odontogenesis throughout the oral cavity. Significantly, the inductive potential of epithelial dental stem cells is retained in postnatal tissues, and up-regulation of WNT signaling specifically in these cells is sufficient to promote generation and growth of ectopic malformations faithfully resembling human odontoma. PMID:26411543

  17. Knowing good from bad: differential activation of human cortical areas by positive and negative outcomes.

    PubMed

    Nieuwenhuis, Sander; Slagter, Heleen A; von Geusau, Niels J Alting; Heslenfeld, Dirk J; Holroyd, Clay B

    2005-06-01

    Previous research has identified a component of the event-related brain potential (ERP), the feedback-related negativity, that is elicited by feedback stimuli associated with unfavourable outcomes. In the present research we used event-related functional magnetic resonance imaging (fMRI) and electroencephalographic (EEG) recordings to test the common hypothesis that this component is generated in the caudal anterior cingulate cortex. The EEG results indicated that our paradigm, a time estimation task with trial-to-trial performance feedback, elicited a large feedback-related negativity (FRN). Nevertheless, the fMRI results did not reveal any area in the caudal anterior cingulate cortex that was differentially activated by positive and negative performance feedback, casting doubt on the notion that the FRN is generated in this brain region. In contrast, we found a number of brain areas outside the posterior medial frontal cortex that were activated more strongly by positive feedback than by negative feedback. These included areas in the rostral anterior cingulate cortex, posterior cingulate cortex, right superior frontal gyrus, and striatum. An anatomically constrained source model assuming equivalent dipole generators in the rostral anterior cingulate, posterior cingulate, and right superior frontal gyrus produced a simulated scalp distribution that corresponded closely to the observed scalp distribution of the FRN. These results support a new hypothesis regarding the neural generators of the FRN, and have important implications for the use of this component as an electrophysiological index of performance monitoring and reward processing. PMID:15978024

  18. Deep-Sea Magnetics on Active and Fossil Hydrothermal Sites: a Tool to Detect and Characterize Submarine Ore Deposits

    NASA Astrophysics Data System (ADS)

    Dyment, J.; Szitkar, F.; Fouquet, Y.; Choi, Y.

    2011-12-01

    Since the first discoveries of hydrothermal sites at mid-ocean ridges in the 70s, international efforts in the deep seafloor exploration have unravelled a wide variety of hydrothermal sites in terms of geological settings, physical parameters, and biological communities as well. Such efforts, coordinated in the InterRidge program since 1992, are becoming even more important when the increasing need in metals for developing economies makes the exploitation of metal sulfides accumulated at deep-sea hydrothermal sites a realistic target. The usual method to find hydrothermal sites is to detect the associated chemical plumes enriched in manganese, methane, hydrogen, helium 3, in the water column. How efficient it has been proven, such a method is limited to the search for active hydrothermal vents. Active vents, however, are not the best places for mining the seafloor, because (1) they host massive sulfides deposits in the making and may not represent the largest accumulation; (2) they are still very hot and would rapidly damage the mining tools; and, last but not the least, (3) they host fragile and precious ecosystem that could be dramatically affected by mining operations. Methods to find fossil hydrothermal sites (i.e. colder and devoid of specific ecosystems) include systematic rock sampling - a very tedious endeavour - and high resolution, near seafloor geophysical surveys. Existing magnetic surveys on basalt-hosted, peridotite-hosted and sediment-hosted sites revealed different types of signatures, which reflect the magnetizations of the host rock and the ore deposit, among others. Basalt-hosted sites exhibit negative magnetic anomalies, i.e. a deficit of magnetization, due to thermal demagnetization and hydrothermal alteration of the highly magnetic basalt, whereas both peridotite-hosted and sediment-hosted sites show positive anomalies, i.e. an excess of magnetization, clearly associated with the ore deposit. Results from recent cruises Serpentine (R

  19. Active sites for NO reduction over Fe-ZSM-5 catalysts.

    PubMed

    Schwidder, M; Santhosh Kumar, M; Brückner, A; Grünert, W

    2005-02-14

    A study of Fe-ZSM-5 catalysts with variable amounts of isolated, oligomeric and heavily aggregated Fe3+ oxo sites (as evidenced by UV-Vis and EPR spectroscopic data) and their catalytic properties in the selective catalytic reduction of NO by isobutane or by NH3 is presented, which allows development of a unified concept of the active Fe sites in these reactions, according to which isolated Fe sites catalyse both SCR reactions while oligomeric sites, though also involved in the selective reduction path, limit the catalyst performance by causing the total oxidation of the reductant. PMID:15685345

  20. Kinetics of positive ions and electrically neutral active particles in afterglow in neon at low pressure

    NASA Astrophysics Data System (ADS)

    Pejović, Milić M.; Nešić, Nikola T.; Pejović, Momčilo M.

    2014-04-01

    Kinetics of positive ions and electrically neutral active particles formed during breakdown and successive discharge in neon-filled tube at 6.6 millibars pressure had been analyzed. This analysis was performed on the basis of mean value of electrical breakdown time delay t¯d dependence on afterglow period τ (memory curve). It was shown that positive ions are present in the 1μs < τ < 30 ms interval, which is manifested through t ¯d slow increase with the increase of τ. A rapid t¯d increase in the 30 ms < τ < 3 s interval is a consequence of significant decrease of positive ions concentration and dominant role in breakdown initiation have ground state nitrogen atoms, which further release secondary electrons from the cathode by catalytic recombination process. These atoms are formed during discharge by dissociation of ground state nitrogen molecules that are present as impurities in neon. For τ > 3 s, breakdown is initiated by cosmic rays and natural radioactivity. The increase of discharge current leads to decrease of t¯d due to the increase of positive ions concentration in inter electrode gap. The increase of applied voltage also decreases t¯d for τ > 30 ms due to the increase of the probability for initial electron to initiate breakdown. The presence of UV radiation leads to the decrease of t¯d due to the increased electron yield caused by photoelectrons. The influence of photoelectrons on breakdown initiation can be noticed for τ > 0.1 ms, while they dominantly determine t¯d for τ > 30 ms.

  1. Kinetics of positive ions and electrically neutral active particles in afterglow in neon at low pressure

    SciTech Connect

    Pejović, Milić M. Nešić, Nikola T.; Pejović, Momčilo M.

    2014-04-15

    Kinetics of positive ions and electrically neutral active particles formed during breakdown and successive discharge in neon-filled tube at 6.6 millibars pressure had been analyzed. This analysis was performed on the basis of mean value of electrical breakdown time delay t{sup ¯}{sub d} dependence on afterglow period τ (memory curve). It was shown that positive ions are present in the 1μs < τ < 30 ms interval, which is manifested through t{sup ¯}{sub d} slow increase with the increase of τ. A rapid t{sup ¯}{sub d} increase in the 30 ms < τ < 3 s interval is a consequence of significant decrease of positive ions concentration and dominant role in breakdown initiation have ground state nitrogen atoms, which further release secondary electrons from the cathode by catalytic recombination process. These atoms are formed during discharge by dissociation of ground state nitrogen molecules that are present as impurities in neon. For τ > 3 s, breakdown is initiated by cosmic rays and natural radioactivity. The increase of discharge current leads to decrease of t{sup ¯}{sub d} due to the increase of positive ions concentration in inter electrode gap. The increase of applied voltage also decreases t{sup ¯}{sub d} for τ > 30 ms due to the increase of the probability for initial electron to initiate breakdown. The presence of UV radiation leads to the decrease of t{sup ¯}{sub d} due to the increased electron yield caused by photoelectrons. The influence of photoelectrons on breakdown initiation can be noticed for τ > 0.1 ms, while they dominantly determine t{sup ¯}{sub d} for τ > 30 ms.

  2. Magnitudes of muscle activation of spine stabilizers, gluteals, and hamstrings during supine bridge to neutral position.

    PubMed

    Youdas, James W; Hartman, James P; Murphy, Brooke A; Rundle, Ashley M; Ugorowski, Jenna M; Hollman, John H

    2015-01-01

    The aim of this study was to compare the magnitude of selective core muscle activation during supine bridging to neutral exercises (three on a stable and three on an unstable surface). Surface EMG analysis was performed on the lumbar multifidus, gluteus medius, gluteus maximus, and hamstrings from 13 male and 13 female subjects. Lumbar multifidus recruitment was not influenced by exercise or condition and ranged between 29.2 and 35.9% of maximum voluntary isometric contraction (MVIC). Peak gluteus medius activation (42.0% MVIC) occurred in unstable single-leg bridge. Maximum recruitment of gluteus maximus (32.6% MVIC) appeared during stable single-leg bridge. Peak hamstring activation (59.6% MVIC) occurred during stable double-leg hamstring curl. Regardless of condition, hamstrings demonstrated high (51.9-59.6% MVIC) muscle recruitment during double-leg hamstring curls compared with the single-leg bridge or double-leg bridge. Various supine bridging to neutral exercises activated the hamstrings at levels conducive to strengthening, whereas recruitment of lumbar multifidus, gluteus medius, and gluteus maximus promoted endurance training. Clinically, we were unable to conclude the unstable support surface was preferable to the stable surface for boosting muscle recruitment of spine stabilizers, gluteals, and hamstring muscles during supine bridge to neutral position. PMID:25671354

  3. Ionospheric plasma deterioration in the area of enhanced seismic activity as compared to antipodal sites far from seismicity

    NASA Astrophysics Data System (ADS)

    Gulyaeva, Tamara; Arikan, Feza; Poustovalova, Ljubov; Stanislawska, Iwona

    2016-07-01

    The early magnetogram records from two nearly antipodal sites at Greenwich and Melbourne corresponding to the activity level at the invariant magnetic latitude of 50 deg give a long series of geomagnetic aa indices since 1868. The aa index derived from magnetic perturbation values at only two observatories (as distinct from the planetary ap index) experiences larger extreme values if either input site is well situated to the overhead ionospheric and/or field aligned current systems producing the magnetic storm effects. Analysis of the earthquakes catalogues since 1914 has shown the area of the peak global earthquake occurrence in the Pacific Ocean southwards from the magnetic equator, and, in particular, at Australia. In the present study the ionospheric critical frequency, foF2, is analyzed from the ionosonde measurements at the nearby observatories, Canberra and Slough (Chilton), and Moscow (control site) since 1944 to 2015. The daily-hourly-annual percentage occurrence of positive ionospheric W index (pW+) and negative index (pW-) is determined. It is found that the ionospheric plasma depletion pW- of the instant foF2 as compared to the monthly median is well correlated to the aa index at all three sites but the positive storm signatures show drastic difference at Canberra (no correlation of pW+ with aa index) as compared to two other sites where the high correlation is found of the ionospheric plasma density enhancement with the geomagnetic activity. A possible suppression of the enhanced ionospheric variability over the region of intense seismicity is discussed in the paper. This study is supported by TUBITAK EEEAG 115E915.

  4. Mapping the agonist binding site of the nicotinic acetylcholine receptor. Orientation requirements for activation by covalent agonist.

    PubMed

    Sullivan, D A; Cohen, J B

    2000-04-28

    To characterize the structural requirements for ligand orientation compatible with activation of the Torpedo nicotinic acetylcholine receptor (nAChR), we used Cys mutagenesis in conjunction with sulfhydryl-reactive reagents to tether primary or quaternary amines at defined positions within the agonist binding site of nAChRs containing mutant alpha- or gamma-subunits expressed in Xenopus oocytes. 4-(N-Maleimido)benzyltrimethylammonium and 2-aminoethylmethanethiosulfonate acted as irreversible antagonists when tethered at alphaY93C, alphaY198C, or gammaE57C, as well as at alphaN94C (2-aminoethylmethanethiosulfonate only). [2-(Trimethylammonium)-ethyl]-methanethiosulfonate (MTSET), which attaches thiocholine to binding site Cys, also acted as an irreversible antagonist when tethered at alphaY93C, alphaN94C, or gammaE57C. However, MTSET modification of alphaY198C resulted in prolonged activation of the nAChR not reversible by washing but inhibitable by subsequent exposure to non-competitive antagonists. Modification of alphaY198C (or any of the other positions tested) by [(trimethylammonium)methyl]methanethiosulfonate resulted only in irreversible inhibition, while modification of alphaY198C by [3-(trimethylammonium)propyl]methanethiosulfonate resulted in irreversible activation of nAChR, but at lower efficacy than by MTSET. Thus changing the length of the tethering arm by less than 1 A in either direction markedly effects the ability of the covalent trimethylammonium to activate the nAChR, and agonist activation depends on a very selective orientation of the quaternary ammonium within the agonist binding site. PMID:10777557

  5. Randomized Controlled Trial of Positive Affect Induction to Promote Physical Activity After Percutaneous Coronary Intervention

    PubMed Central

    Peterson, Janey C.; Charlson, Mary E.; Hoffman, Zachary; Wells, Martin T.; Wong, Shing-Chiu; Hollenberg, James P.; Jobe, Jared B.; Boschert, Kathryn A.; Isen, Alice M.; Allegrante, John P.

    2013-01-01

    Background Within 1 year after percutaneous coronary intervention, more than 20% of patients experience new adverse events. Physical activity confers a 25% reduction in mortality; however, physical activity is widely underused. Thus, there is a need for more powerful behavioral interventions to promote physical activity. Our objective was to motivate patients to achieve an increase in expenditure of 336 kcal/wk or more at 12 months as assessed by the Paffenbarger Physical Activity and Exercise Index. Methods Two hundred forty-two patients were recruited immediately after percutaneous coronary intervention between October 2004 and October 2006. Patients were randomized to 1 of 2 groups. The patient education (PE) control group (n=118) (1) received an educational workbook, (2) received a pedometer, and (3) set a behavioral contract for a physical activity goal. The positive affect/self-affirmation (PA) intervention group (n=124) received the 3 PE control components plus (1) a PA workbook chapter, (2) bimonthly induction of PA by telephone, and (3) small mailed gifts. All patients were contacted with standardized bimonthly telephone follow-up for 12 months. Results Attrition was 4.5%, and 2.1% of patients died. Significantly more patients in the PA intervention group increased expenditure by 336 kcal/wk or more at 12 months, our main outcome, compared with the PE control group (54.9% vs 37.4%, P=.007). The PA intervention patients were 1.7 times more likely to reach the goal of a 336-kcal/wk or more increase by 12 months, controlling for demographic and psychosocial measures. In multivariate analysis, the PA intervention patients had nearly double the improvement in kilocalories per week at 12 months compared with the PE control patients (602 vs 328, P=.03). Conclusion Patients who receive PA intervention after percutaneous coronary intervention are able to achieve a sustained and clinically significant increase in physical activity by 12 months. Trial Registration

  6. Pharmacological activity of a Bv8 analogue modified in position 24

    PubMed Central

    Lattanzi, R; Sacerdote, P; Franchi, S; Canestrelli, M; Miele, R; Barra, D; Visentin, S; DeNuccio, C; Porreca, F; De Felice, M; Guida, F; Luongo, L; de Novellis, V; Maione, S; Negri, L

    2012-01-01

    BACKGROUND AND PURPOSE The amphibian peptide Bv8 induces potent nociceptive sensitization in rodents. Its mammalian homologue, prokineticin 2 (PROK2), is strongly up-regulated in inflamed tissues and is a major determinant in triggering inflammatory pain. Bv8 and PROK2 activate two closely related GPCRs, PK1 and PK2, in a relatively non-selective fashion. To characterize better the roles of the two receptors in hyperalgesia and to obtain ligands whose binding affinity and efficacy differed for the two receptors, we modified the Bv8 molecule in regions essential for receptor recognition and activation. EXPERIMENTAL APPROACH We modified the Bv8 molecule by substituting Trp in position 24 with Ala (A-24) and compared it with Bv8 for binding and activating PK1 and PK2 receptors in cell preparations and in affecting nociceptive thresholds in rodents. KEY RESULTS A-24 preferentially bound to PK2 receptors and activated them with a lower potency (5-fold) than Bv8. When systemically injected, A-24 induced Bv8-like hyperalgesia in rats and in mice, at doses 100 times higher than Bv8. Locally and systemically injected at inactive doses, A-24 antagonized Bv8-induced hyperalgesia. In rat and mouse models of inflammatory and post-surgical pain, A-24 showed potent and long-lasting anti-hyperalgesic activity. Unlike Bv8, A-24 increased β-endorphin levels in mouse brain. CONCLUSIONS AND IMPLICATIONS A-24 induced its anti-hyperalgesic effect in rodents by directly blocking nociceptor PK1 receptors and by activating the central opioid system and the descending pain control pathway through brain PK2 receptors. PMID:22122547

  7. Reconstructing liver shape and position from MR image slices using an active shape model

    NASA Astrophysics Data System (ADS)

    Fenchel, Matthias; Thesen, Stefan; Schilling, Andreas

    2008-03-01

    We present an algorithm for fully automatic reconstruction of 3D position, orientation and shape of the human liver from a sparsely covering set of n 2D MR slice images. Reconstructing the shape of an organ from slice images can be used for scan planning, for surgical planning or other purposes where 3D anatomical knowledge has to be inferred from sparse slices. The algorithm is based on adapting an active shape model of the liver surface to a given set of slice images. The active shape model is created from a training set of liver segmentations from a group of volunteers. The training set is set up with semi-manual segmentations of T1-weighted volumetric MR images. Searching for the optimal shape model that best fits to the image data is done by maximizing a similarity measure based on local appearance at the surface. Two different algorithms for the active shape model search are proposed and compared: both algorithms seek to maximize the a-posteriori probability of the grey level appearance around the surface while constraining the surface to the space of valid shapes. The first algorithm works by using grey value profile statistics in normal direction. The second algorithm uses average and variance images to calculate the local surface appearance on the fly. Both algorithms are validated by fitting the active shape model to abdominal 2D slice images and comparing the shapes, which have been reconstructed, to the manual segmentations and to the results of active shape model searches from 3D image data. The results turn out to be promising and competitive to active shape model segmentations from 3D data.

  8. β-Amyrin Biosynthesis: Promiscuity for Steric Bulk at Position 23 in the Oxidosqualene Substrate and the Significance of Hydrophobic Interaction between the Methyl Group at Position 30 and the Binding Site.

    PubMed

    Kaneko, Ikki; Hoshino, Tsutomu

    2016-08-01

    To examine how the sterics at the 23 position of (3S)-2,3-oxidosqualene 1 influence the polycyclization cascade in β-amyrin biosynthesis, substrate analogues substituted with an ethyl group (10, 11), a hydrogen atom (12, 13), or a propyl residue (14) at the 23 position were incubated with β-amyrin synthase. The bulkier ethyl group was accepted as a substrate, leading to formation of the β-amyrin skeleton (42, 43) without truncation of the multiple cyclization reactions. Analogue 13, possessing a hydrogen atom and an ethyl group at the 23E and 23Z positions, respectively, was also converted into the β-amyrin skeleton 45. However, the analogue lacking an ethyl group at the 23Z position (12) underwent almost no conversion, strongly indicating that an alkyl group must exist at the Z position. The cyclization of the analogue with a propyl substituent at the Z position (14) was poor. Analogue 15 possessing CH2OH at the 23E position afforded a new compound 47 in a high yield as a result of trapping of the final oleanyl cation. Conversely, 16 with 23Z-CH2OH afforded novel compounds 48-50 in low yields, which resulted from the intermediary dammarenyl and baccharenyl cations. Therefore, the hydrophobic interaction between the 23Z-alkyl group and its binding site (possibly via CH/π interaction) is critical for adopting the correct chair-chair-chair-boat-boat conformation and for the full cyclization cascade. PMID:27419810

  9. Site-directed mutagenesis of dicarboxylic acids near the active site of Bacillus cereus 5/B/6 beta-lactamase II.

    PubMed Central

    Lim, H M; Iyer, R K; Pène, J J

    1991-01-01

    An amino acid residue functioning as a general base has been proposed to assist in the hydrolysis of beta-lactam antibiotics by the zinc-containing Bacillus cereus beta-lactamase II [Bicknell & Waley (1985) Biochemistry 24, 6876-6887]. Oligonucleotide-directed mutagenesis of cloned Bacillus cereus 5/B/6 beta-lactamase II was used in an 'in vivo' study to investigate the role of carboxy-group-containing amino acids near the active site of the enzyme. Substitution of asparagine for the wild-type aspartic acid residue at position 81 resulted in fully functional enzyme. An aspartic acid residue at position 90 is essential for beta-lactamase II to confer any detectable ampicillin and cephalosporin C resistance to Escherichia coli. Conversion of Asp90 into Asn90 or Glu90 lead to the synthesis of inactive enzyme, suggesting that the spatial position of the beta-carboxy group of Asp90 is critical for enzyme function. Images Fig. 2. Fig. 3. PMID:1904717

  10. Oxygenation, EMG and position sense during computer mouse work. Impact of active versus passive pauses.

    PubMed

    Crenshaw, A G; Djupsjöbacka, M; Svedmark, A

    2006-05-01

    We investigated the effects of active versus passive pauses implemented during computer mouse work on muscle oxygenation and EMG of the forearm extensor carpi radialis muscle, and on wrist position sense. Fifteen healthy female subjects (age: 19-24 years) performed a 60-min mouse-operated computer task, divided into three 20 min periods, on two occasions separated by 3-6 days. On one occasion a passive pause (subjects resting) was implemented at the end of each 20-min period, and on another occasion an active pause (subjects performed a number of high intensity extensions of the forearm) was implemented. Also at the end of each 20-min period, test contractions were conducted and subjective ratings of fatigue and stress were obtained. Another parameter of interest was total haemoglobin calculated as the summation of oxy-and deoxy-haemoglobin, since it reflects blood volume changes. The most interesting findings were an overall increasing trend in total haemoglobin throughout the mouse work (P<0.001), and that this trend was greater for the active pause as compared to the passive pause (P<0.01). These data were accompanied by an overall increase in oxygen saturation (P<0.001), with a tendency, albeit not significant, toward a higher increase for the active pause (P=0.13). EMG amplitude and median frequency tended to decrease (P=0.08 and 0.05, respectively) during the mouse work but was not different between pause types. Borg ratings of forearm fatigue showed an overall increase during the activity (P<0.001), but the perceptions of stress did not change. Position sense did not change due to the mouse work for either pause type. While increasing trends were found for both pause types, the present study lends support to the hypothesis of an enhancement in oxygenation and blood volume for computer mouse work implemented with active pauses. However, a presumption of an association between this enhancement and attenuated fatigue during the mouse work was not supported

  11. The complexities of measuring access to parks and physical activity sites in New York City: a quantitative and qualitative approach

    PubMed Central

    Maroko, Andrew R; Maantay, Juliana A; Sohler, Nancy L; Grady, Kristen L; Arno, Peter S

    2009-01-01

    Background Proximity to parks and physical activity sites has been linked to an increase in active behaviors, and positive impacts on health outcomes such as lower rates of cardiovascular disease, diabetes, and obesity. Since populations with a low socio-economic status as well as racial and ethnic minorities tend to experience worse health outcomes in the USA, access to parks and physical activity sites may be an environmental justice issue. Geographic Information systems were used to conduct quantitative and qualitative analyses of park accessibility in New York City, which included kernel density estimation, ordinary least squares (global) regression, geographically weighted (local) regression, and longitudinal case studies, consisting of field work and archival research. Accessibility was measured by both density of park acreage and density of physical activity sites. Independent variables included percent non-Hispanic black, percent Hispanic, percent below poverty, percent of adults without high school diploma, percent with limited English-speaking ability, and population density. Results The ordinary least squares linear regression found weak relationships in both the park acreage density and the physical activity site density models (Ra2 = .11 and .23, respectively; AIC = 7162 and 3529, respectively). Geographically weighted regression, however, suggested spatial non-stationarity in both models, indicating disparities in accessibility that vary over space with respect to magnitude and directionality of the relationships (AIC = 2014 and -1241, respectively). The qualitative analysis supported the findings of the local regression, confirming that although there is a geographically inequitable distribution of park space and physical activity sites, it is not globally predicted by race, ethnicity, or socio-economic status. Conclusion The combination of quantitative and qualitative analyses demonstrated the complexity of the issues around racial and ethnic

  12. Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site

    PubMed Central

    Grossman, Moran; Born, Benjamin; Heyden, Matthias; Tworowski, Dmitry; Fields, Gregg B; Sagi, Irit; Havenith, Martina

    2012-01-01

    Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site. PMID:21926991

  13. Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site

    SciTech Connect

    Grossman, Moran; Born, Benjamin; Heyden, Matthias; Tworowski, Dmitry; Fields, Gregg B.; Sagi, Irit; Havenith, Martina

    2011-09-18

    Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site.

  14. The Influence of Artificially Introduced N-Glycosylation Sites on the In Vitro Activity of Xenopus laevis Erythropoietin

    PubMed Central

    Nagasawa, Kazumichi; Meguro, Mizue; Sato, Kei; Tanizaki, Yuta; Nogawa-Kosaka, Nami; Kato, Takashi

    2015-01-01

    Erythropoietin (EPO), the primary regulator of erythropoiesis, is a heavily glycosylated protein found in humans and several other mammals. Intriguingly, we have previously found that EPO in Xenopus laevis (xlEPO) has no N-glycosylation sites, and cross-reacts with the human EPO (huEPO) receptor despite low homology with huEPO. In this study, we introduced N-glycosylation sites into wild-type xlEPO at the positions homologous to those in huEPO, and tested whether the glycosylated mutein retained its biological activity. Seven xlEPO muteins, containing 1–3 additional N-linked carbohydrates at positions 24, 38, and/or 83, were expressed in COS-1 cells. The muteins exhibited lower secretion efficiency, higher hydrophilicity, and stronger acidic properties than the wild type. All muteins stimulated the proliferation of both cell lines, xlEPO receptor-expressing xlEPOR-FDC/P2 cells and huEPO receptor-expressing UT-7/EPO cells, in a dose-dependent manner. Thus, the muteins retained their in vitro biological activities. The maximum effect on xlEPOR-FDC/P2 proliferation was decreased by the addition of N-linked carbohydrates, but that on UT-7/EPO proliferation was not changed, indicating that the muteins act as partial agonists to the xlEPO receptor, and near-full agonists to the huEPO receptor. Hence, the EPO-EPOR binding site in X. laevis locates the distal region of artificially introduced three N-glycosylation sites, demonstrating that the vital conformation to exert biological activity is conserved between humans and X. laevis, despite the low similarity in primary structures of EPO and EPOR. PMID:25898205

  15. HIV integration site distributions in resting and activated CD4+ T cells infected in culture

    PubMed Central

    Brady, Troy; Agosto, Luis M.; Malani, Nirav; Berry, Charles C.; O'Doherty, Una; Bushman, Frederic

    2010-01-01

    Objective The goal of this study was to investigate whether the location of HIV integration differs in resting versus activated T cells, a feature that could contribute to the formation of latent viral reservoirs via effects on integration targeting. Design Primary resting or activated CD4+ T cells were infected with purified X4-tropic HIV in the presence and absence of nucleoside triphosphates and genomic locations of integrated provirus determined. Methods We sequenced and analyzed a total of 2661 HIV integration sites using linker-mediated PCR and 454 sequencing. Integration site data sets were then compared to each other and to computationally generated random distributions. Results HIV integration was favored in active transcription units in both cell types, but integration sites from activated cells were found more often in genomic regions that were dense in genes, dense in CpG islands, and enriched in G/C bases. Integration sites from activated cells were also more strongly correlated with histone methylation patterns associated with active genes. Conclusion These data indicate that integration site distributions show modest but significant differences between resting and activated CD4+ T cells, and that integration in resting cells occurs more often in regions that may be suboptimal for proviral gene expression. PMID:19550285

  16. Source identification of water-soluble organic aerosols at a roadway site using a positive matrix factorization analysis.

    PubMed

    Park, Seungshik; Cho, Sung Yong; Bae, Min-Suk

    2015-11-15

    Daily PM2.5 measurements were carried out at a local roadway every sixth day from May 2011 to August 2013 to obtain seasonal quantitative information on the primary and secondary sources of two water-soluble organic carbon (WSOC) fractions. Filter samples were analyzed for OC, elemental carbon (EC), WSOC, hydrophilic and hydrophobic WSOC fractions (WSOC(HPI) and WSOC(HPO)), and ionic species. An XAD solid phase extraction method and a total organic carbon analyzer were used to isolate the two WSOC fractions and determine their amounts, respectively. The WSOC/OC and WSOC(HPI)/WSOC ratios were 0.62±0.13 and 0.47±0.14, respectively. Similar seasonal profiles in EC, OC, and WSOC concentrations were observed, with higher concentrations occurring in the cold season and lower concentrations in the warm season. However, opposite results were obtained in WSOC/OC and WSOC(HPI)/WSOC ratios, with the higher in the warm season and the lower in the cold season. Correlation analyses indicated that two WSOC fractions in winter were likely attributed to secondary formation processes, biomass burning (BB), and traffic emissions, while WSOC(HPI) observed in other seasons were associated with secondary formation processes similar to those of oxalate and secondary inorganic species. A positive matrix factorization (PMF) model was employed to investigate the sources of two WSOC fractions. PMF indicated that concentrations of WSOC fractions were affected by five sources: secondary NO3(-) related, secondary SO4(2-) and oxalate related, traffic emissions, BB emissions, and sea-salt. Throughout the study period, secondary organic aerosols were estimated to be the most dominant contributor of WSOC fractions, with higher contributions occurring in the warm seasons. The contribution of secondary aerosol formation processes (NO3(-) related+SO4(2-) and oxalate related) to WSOC(HPI) and WSOC(HPO) was on an average 56.2% (45.0-73.8%) and 47.7% (39.6-52.1%), respectively. The seasonal average

  17. Subtle Changes in Motif Positioning Cause Tissue-Specific Effects on Robustness of an Enhancer's Activity

    PubMed Central

    Erceg, Jelena; Saunders, Timothy E.; Girardot, Charles; Devos, Damien P.; Hufnagel, Lars; Furlong, Eileen E. M.

    2014-01-01

    Deciphering the specific contribution of individual motifs within cis-regulatory modules (CRMs) is crucial to understanding how gene expression is regulated and how this process is affected by sequence variation. But despite vast improvements in the ability to identify where transcription factors (TFs) bind throughout the genome, we are limited in our ability to relate information on motif occupancy to function from sequence alone. Here, we engineered 63 synthetic CRMs to systematically assess the relationship between variation in the content and spacing of motifs within CRMs to CRM activity during development using Drosophila transgenic embryos. In over half the cases, very simple elements containing only one or two types of TF binding motifs were capable of driving specific spatio-temporal patterns during development. Different motif organizations provide different degrees of robustness to enhancer activity, ranging from binary on-off responses to more subtle effects including embryo-to-embryo and within-embryo variation. By quantifying the effects of subtle changes in motif organization, we were able to model biophysical rules that explain CRM behavior and may contribute to the spatial positioning of CRM activity in vivo. For the same enhancer, the effects of small differences in motif positions varied in developmentally related tissues, suggesting that gene expression may be more susceptible to sequence variation in one tissue compared to another. This result has important implications for human eQTL studies in which many associated mutations are found in cis-regulatory regions, though the mechanism for how they affect tissue-specific gene expression is often not understood. PMID:24391522

  18. CCL21/IL21-armed oncolytic adenovirus enhances antitumor activity against TERT-positive tumor cells.

    PubMed

    Li, Yang; Li, Yi-Fei; Si, Chong-Zhan; Zhu, Yu-Hui; Jin, Yan; Zhu, Tong-Tong; Liu, Ming-Yuan; Liu, Guang-Yao

    2016-07-15

    Multigene-armed oncolytic adenoviruses are capable of efficiently generating a productive antitumor immune response. The chemokine (C-C motif) ligand 21 (CCL21) binds to CCR7 on naïve T cells and dendritic cells (DCs) to promote their chemoattraction to the tumor and resultant antitumor activity. Interleukin 21 (IL21) promotes survival of naïve T cells while maintaining their CCR7 surface expression, which increases their capacity to transmigrate in response to CCL21 chemoattraction. IL21 is also involved in NK cell differentiation and B cell activation and proliferation. The generation of effective antitumor immune responses is a complex process dependent upon coordinated interactions of various subsets of effector cells. Using the AdEasy system, we aimed to construct an oncolytic adenovirus co-expressing CCL21 and IL21 that could selectively replicate in TERTp-positive tumor cells (Ad-CCL21-IL21 virus). The E1A promoter of these oncolytic adenoviruses was replaced by telomerase reverse transcriptase promoter (TERTp). Ad-CCL21-IL21 was constructed from three plasmids, pGTE-IL21, pShuttle-CMV-CCL21 and AdEasy-1 and was homologously recombined and propagated in the Escherichia coli strain BJ5183 and the packaging cell line HEK-293, respectively. Our results showed that our targeted and armed oncolytic adenoviruses Ad-CCL21-IL21 can induce apoptosis in TERTp-positive tumor cells to give rise to viral propagation, in a dose-dependent manner. Importantly, we confirm that these modified oncolytic adenoviruses do not replicate efficiently in normal cells even under high viral loads. Additionally, we investigate the role of Ad-CCL21-IL21 in inducing antitumor activity and tumor specific cytotoxicity of CTLs in vitro. This study suggests that Ad-CCL21-IL21 is a promising targeted tumor-specific oncolytic adenovirus. PMID:27157859

  19. Determination of the protease cleavage site repertoire—The RNase H but not the RT domain is essential for foamy viral protease activity

    SciTech Connect

    Spannaus, Ralf; Bodem, Jochen

    2014-04-15

    In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2' and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies. The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation. - Highlights: • Unprocessed Gag is required for optimal infectivity of foamy viruses. • Positions P2 and P2' are invariant in the foamy viral cleavage sites. • The RNaseH domain is essential for protease activity. • The RNaseH domains of other retroviruses support foamy viral protease activity.

  20. Deep Sequencing of Random Mutant Libraries Reveals the Active Site of the Narrow Specificity CphA Metallo-β-Lactamase is Fragile to Mutations.

    PubMed

    Sun, Zhizeng; Mehta, Shrenik C; Adamski, Carolyn J; Gibbs, Richard A; Palzkill, Timothy

    2016-01-01

    CphA is a Zn(2+)-dependent metallo-β-lactamase that efficiently hydrolyzes only carbapenem antibiotics. To understand the sequence requirements for CphA function, single codon random mutant libraries were constructed for residues in and near the active site and mutants were selected for E. coli growth on increasing concentrations of imipenem, a carbapenem antibiotic. At high concentrations of imipenem that select for phenotypically wild-type mutants, the active-site residues exhibit stringent sequence requirements in that nearly all residues in positions that contact zinc, the substrate, or the catalytic water do not tolerate amino acid substitutions. In addition, at high imipenem concentrations a number of residues that do not directly contact zinc or substrate are also essential and do not tolerate substitutions. Biochemical analysis confirmed that amino acid substitutions at essential positions decreased the stability or catalytic activity of the CphA enzyme. Therefore, the CphA active - site is fragile to substitutions, suggesting active-site residues are optimized for imipenem hydrolysis. These results also suggest that resistance to inhibitors targeted to the CphA active site would be slow to develop because of the strong sequence constraints on function. PMID:27616327

  1. Assessment of the site of ventricular activation by Fourier analysis of gated blood-pool studies

    SciTech Connect

    Links, J.M.; Raichlen, J.S.; Wagner, H.N. Jr.; Reid, P.R.

    1985-01-01

    The authors studied the use of first-harmonic Fourier analysis of gated blood-pool images to assess the site of ventricular activation in a group of 12 patients undergoing electrophysiologic pacing studies. They acquired gated blood-pool studies during pacing at up to four sites at each of two different rates. A total of 50 studies were made. At a pacing rate of 100 beats/min, when the pacing electrode was the right-ventricular outflow tract, 7/8; at the anterolateral left-ventricular wall, 4/4. When the Fourier activation site was at the right-ventricular apex, 9/9 times the pacing electrode was there; at the right-ventricular outflow tract, 7/10; in the left ventricle, 4/4. Fourier analysis of gated blood-pool studies can help identify the site of ventricular activation but is not sufficiently accurate to fully replace endocardial mapping.

  2. Tandem mass spectrometry-based detection of c4'-oxidized abasic sites at specific positions in DNA fragments.

    PubMed

    Chowdhury, Goutam; Guengerich, F Peter

    2009-07-01

    Oxidative damage to DNA has been linked to aging, cancer, and other biological processes. Reactive oxygen species and various antitumor agents including bleomycin and ionizing radiation have been shown to cause oxidative DNA sugar damage. Detection of DNA lesions is important for understanding the toxicological or therapeutic consequences associated with such agents. C4'-oxidized abasic sites (C4-AP) are produced by the antitumor drug bleomycin and ionizing radiation. The currently available methods for the detection of C4-AP cannot provide both structural and sequence information. We have developed an LC-ESI-MS-based approach for specific detection and mapping of C4-AP from a mixture of lesions. We show using Fe-bleomycin-damaged DNA that C4-AP can be detected at cytosine and thymine sites by direct MS analysis. Our results reveal that collision-induced dissociation of C4-AP-containing oligonucleotides results in preferential fragmentation at C4-AP sites with the formation of the unique a* ions (18 amu more than the a-B ions) that allow mapping of the C4-AP sites. Various chemical modification strategies (e.g., reduction with NaBH4 and NaBD4 and derivatization with methoxyamine and hydrazine, followed by LC-MS analysis) were also used for unambiguous detection of C4-AP sites. Finally, we show that the methods described here can detect the presence of C4-AP at specific sites in a complex sample such as hydroxyl radical-damaged DNA. The LC-MS approach was also used for the simultaneous detection of the other C4'-oxidation end product, 3'-phosphoglycolate, at a specific site in hydroxyl radical-damaged DNA. Thus, LC-MS provides a rapid and direct approach for the detection and mapping of oxidative DNA lesions. PMID:19496605

  3. Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions

    SciTech Connect

    Crichlow, G.; Lubetsky, J; Leng, L; Bucala, R; Lolis, E

    2009-01-01

    Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic data indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.

  4. Active Site Inhibitors Protect Protein Kinase C from Dephosphorylation and Stabilize Its Mature Form*

    PubMed Central

    Gould, Christine M.; Antal, Corina E.; Reyes, Gloria; Kunkel, Maya T.; Adams, Ryan A.; Ziyar, Ahdad; Riveros, Tania; Newton, Alexandra C.

    2011-01-01

    Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C. PMID:21715334

  5. Trait positive and negative emotionality differentially associate with diurnal cortisol activity.

    PubMed

    Miller, Karissa G; Wright, Aidan G C; Peterson, Laurel M; Kamarck, Thomas W; Anderson, Barbara A; Kirschbaum, Clemens; Marsland, Anna L; Muldoon, Matthew F; Manuck, Stephen B

    2016-06-01

    Inter-individual variability in metrics of hypothalamic-pituitary-adrenocortical (HPA) activity, such as the slope of the diurnal decline in cortisol, cortisol awakening response (CAR), and total cortisol output, have been found to associate inversely with trait ratings of extraversion and positive affect (E/PA) and positively with neuroticism and negative affect (N/NA) in some, but not all, investigations. These inconsistencies may partly reflect varied intensity of cortisol sampling among studies and reliance on self-rated traits, which are subject to reporting biases and limitations of introspection. Here, we further examined dispositional correlates of HPA activity in 490 healthy, employed midlife volunteers (M age=43 years; 54% Female; 86% white). Trait ratings were requested from participants and 2 participant-elected informants using the Positive and Negative Affect Schedule (PANAS) and Extraversion and Neuroticism dimensions of NEO personality inventories. CAR was assessed as percent increase in cortisol levels from awakening to 30min after awakening; and the diurnal slope and total output of cortisol [Area Under the Curve (AUC)] were determined from cortisol measurements taken at awakening, +4 and +9h later, and bedtime, across 3 workdays. Structural equation modeling was used to estimate multi-informant E/PA and N/NA factors. We used 3days of measurement as indicators to model each of the three latent cortisol factors (slope, CAR, and AUC). With the two latent emotionality and three latent cortisol indices included there was good fit to the data (χ(2)(200)=278.38, p=0.0002; RMSEA=0.028, 90% CI=0.02-0.04; CFI/TLI=0.97/0.96; SRMR=0.04). After controlling for covariates (age, sex, race), results showed higher latent E/PA associated with a steeper diurnal slope (Standardized β=-0.19, p=0.02) and smaller CAR (Standardized β=-0.26, p=0.004), whereas N/NA did not associate with any cortisol metric (Standardized β's=-0.12 to 0.13, p's=0.10 to 0.53). These

  6. Site-Specific Expression of Gelatinolytic Activity during Morphogenesis of the Secondary Palate in the Mouse Embryo

    PubMed Central

    Gkantidis, Nikolaos; Blumer, Susan; Katsaros, Christos; Graf, Daniel; Chiquet, Matthias

    2012-01-01

    Morphogenesis of the secondary palate in mammalian embryos involves two major events: first, reorientation of the two vertically oriented palatal shelves into a horizontal position above the tongue, and second, fusion of the two shelves at the midline. Genetic evidence in humans and mice indicates the involvement of matrix metalloproteinases (MMPs). As MMP expression patterns might differ from sites of activity, we used a recently developed highly sensitive in situ zymography technique to map gelatinolytic MMP activity in the developing mouse palate. At embryonic day 14.5 (E14.5), we detected strong gelatinolytic activity around the lateral epithelial folds of the nasopharyngeal cavity, which is generated as a consequence of palatal shelf elevation. Activity was concentrated in the basement membrane of the epithelial fold but extended into the adjacent mesenchyme, and increased in intensity with lateral outgrowth of the cavity at E15.5. Gelatinolytic activity at this site was not the consequence of epithelial fold formation, as it was also observed in Bmp7-deficient embryos where shelf elevation is delayed. In this case, gelatinolytic activity appeared in vertical shelves at the exact position where the epithelial fold will form during elevation. Mmp2 and Mmp14 (MT1-MMP), but not Mmp9 and Mmp13, mRNAs were expressed in the mesenchyme around the epithelial folds of the elevated palatal shelves; this was confirmed by immunostaining for MMP-2 and MT1-MMP. Weak gelatinolytic activity was also found at the midline of E14.5 palatal shelves, which increased during fusion at E15.5. Whereas MMPs have been implicated in palatal fusion before, this is the first report showing that gelatinases might contribute to tissue remodeling during early stages of palatal shelf elevation and formation of the nasopharynx. PMID:23091646

  7. Site-specific expression of gelatinolytic activity during morphogenesis of the secondary palate in the mouse embryo.

    PubMed

    Gkantidis, Nikolaos; Blumer, Susan; Katsaros, Christos; Graf, Daniel; Chiquet, Matthias

    2012-01-01

    Morphogenesis of the secondary palate in mammalian embryos involves two major events: first, reorientation of the two vertically oriented palatal shelves into a horizontal position above the tongue, and second, fusion of the two shelves at the midline. Genetic evidence in humans and mice indicates the involvement of matrix metalloproteinases (MMPs). As MMP expression patterns might differ from sites of activity, we used a recently developed highly sensitive in situ zymography technique to map gelatinolytic MMP activity in the developing mouse palate. At embryonic day 14.5 (E14.5), we detected strong gelatinolytic activity around the lateral epithelial folds of the nasopharyngeal cavity, which is generated as a consequence of palatal shelf elevation. Activity was concentrated in the basement membrane of the epithelial fold but extended into the adjacent mesenchyme, and increased in intensity with lateral outgrowth of the cavity at E15.5. Gelatinolytic activity at this site was not the consequence of epithelial fold formation, as it was also observed in Bmp7-deficient embryos where shelf elevation is delayed. In this case, gelatinolytic activity appeared in vertical shelves at the exact position where the epithelial fold will form during elevation. Mmp2 and Mmp14 (MT1-MMP), but not Mmp9 and Mmp13, mRNAs were expressed in the mesenchyme around the epithelial folds of the elevated palatal shelves; this was confirmed by immunostaining for MMP-2 and MT1-MMP. Weak gelatinolytic activity was also found at the midline of E14.5 palatal shelves, which increased during fusion at E15.5. Whereas MMPs have been implicated in palatal fusion before, this is the first report showing that gelatinases might contribute to tissue remodeling during early stages of palatal shelf elevation and formation of the nasopharynx. PMID:23091646

  8. Intervention to increase physical activity in irritable bowel syndrome shows long-term positive effects

    PubMed Central

    Johannesson, Elisabet; Ringström, Gisela; Abrahamsson, Hasse; Sadik, Riadh

    2015-01-01

    AIM: To assess the long-term effects of physical activity on irritable bowel syndrome (IBS) symptoms and on quality of life, fatigue, depression and anxiety. METHODS: Seventy-six patients from a previous randomized controlled interventional study on increased physical activity in IBS were asked to participate in this long-term follow-up study. The included patients attended one visit in which they filled out questionnaires and they underwent a submaximal cycle ergometer test. The primary end point was the change in the IBS Severity Scoring System (IBS-SSS) at baseline, i.e., before the intervention and at follow-up. The secondary endpoints were changes in quality of life, fatigue, depression and anxiety. RESULTS: A total of 39 [32 women, median age 45 (28-61) years] patients were included in this follow-up. Median follow-up time was 5.2 (range: 3.8-6.2) years. The IBS symptoms were improved compared with baseline [IBS-SSS: 276 (169-360) vs 218 (82-328), P = 0.001]. This was also true for the majority of the dimensions of psychological symptoms such as disease specific quality of life, fatigue, depression and anxiety. The reported time of physical activity during the week before the visit had increased from 3.2 (0.0-10.0) h at baseline to 5.2 (0.0-15.0) h at follow-up, P = 0.019. The most common activities reported were walking, aerobics and cycling. There was no significant difference in the oxygen uptake 31.8 (19.7-45.8) mL per min per kg at baseline vs 34.6 (19.0-54.6) mL/min per kg at follow-up. CONCLUSION: An intervention to increase physical activity has positive long-term effects on IBS symptoms and psychological symptoms. PMID:25593485

  9. Clusterless Decoding of Position From Multiunit Activity Using A Marked Point Process Filter

    PubMed Central

    Deng, Xinyi; Liu, Daniel F.; Kay, Kenneth; Frank, Loren M.; Eden, Uri T.

    2016-01-01

    Point process filters have been applied successfully to decode neural signals and track neural dynamics. Traditionally, these methods assume that multiunit spiking activity has already been correctly spike-sorted. As a result, these methods are not appropriate for situations where sorting cannot be performed with high precision such as real-time decoding for brain-computer interfaces. As the unsupervised spike-sorting problem remains unsolved, we took an alternative approach that takes advantage of recent insights about clusterless decoding. Here we present a new point process decoding algorithm that does not require multiunit signals to be sorted into individual units. We use the theory of marked point processes to construct a function that characterizes the relationship between a covariate of interest (in this case, the location of a rat on a track) and features of the spike waveforms. In our example, we use tetrode recordings, and the marks represent a four-dimensional vector of the maximum amplitudes of the spike waveform on each of the four electrodes. In general, the marks may represent any features of the spike waveform. We then use Bayes’ rule to estimate spatial location from hippocampal neural activity. We validate our approach with a simulation study and with experimental data recorded in the hippocampus of a rat moving through a linear environment. Our decoding algorithm accurately reconstructs the rat’s position from unsorted multiunit spiking activity. We then compare the quality of our decoding algorithm to that of a traditional spike-sorting and decoding algorithm. Our analyses show that the proposed decoding algorithm performs equivalently or better than algorithms based on sorted single-unit activity. These results provide a path toward accurate real-time decoding of spiking patterns that could be used to carry out content-specific manipulations of population activity in hippocampus or elsewhere in the brain. PMID:25973549

  10. Structure of the E. Coli Bifunctional GlmU Acetyltransferase Active Site with Substrates and Products

    SciTech Connect

    Olsen,L.; Vetting, M.; Roderick, S.

    2007-01-01

    The biosynthesis of UDP-GlcNAc in bacteria is carried out by GlmU, an essential bifunctional uridyltransferase that catalyzes the CoA-dependent acetylation of GlcN-1-PO{sub 4} to form GlcNAc-1-PO{sub 4} and its subsequent condensation with UTP to form pyrophosphate and UDP-GlcNAc. As a metabolite, UDP-GlcNAc is situated at a branch point leading to the biosynthesis of lipopolysaccharide and peptidoglycan. Consequently, GlmU is regarded as an important target for potential antibacterial agents. The crystal structure of the Escherichia coli GlmU acetyltransferase active site has been determined in complexes with acetyl-CoA, CoA/GlcN-1-PO{sub 4}, and desulpho-CoA/GlcNAc-1-PO{sub 4}. These structures reveal the enzyme groups responsible for binding the substrates. A superposition of these complex structures suggests that the 2-amino group of GlcN-1-PO{sub 4} is positioned in proximity to the acetyl-CoA to facilitate direct attack on its thioester by a ternary complex mechanism.

  11. Identification of essential active-site residues in the cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Crantz) by site-directed mutagenesis.

    PubMed Central

    Keresztessy, Z; Brown, K; Dunn, M A; Hughes, M A

    2001-01-01

    The coding sequence of the mature cyanogenic beta-glucosidase (beta-glucoside glucohydrolase, EC 3.2.1.21; linamarase) was cloned into the vector pYX243 modified to contain the SUC2 yeast secretion signal sequence and expressed in Saccharomyces cerevisiae. The recombinant enzyme is active, glycosylated and showed similar stability to the plant protein. Michaelis constants for hydrolysis of the natural substrate, linamarin (K(m)=1.06 mM) and the synthetic p-nitrophenyl beta-D-glucopyranoside (PNP-Glc; K(m)=0.36 mM), as well as apparent pK(a) values of the free enzyme and the enzyme-substrate complexes (pK(E)(1)=4.4-4.8, pK(E)(2)=6.7-7.2, pK(ES)(1)=3.9-4.4, pK(ES)(2)=8.3) were very similar to those of the plant enzyme. Site-directed mutagenesis was carried out to study the function of active-site residues based on a homology model generated for the enzyme using the MODELLER program. Changing Glu-413 to Gly destroyed enzyme activity, consistent with it being the catalytic nucleophile. The Gln-339Glu mutation also abolished activity, confirming a function in positioning the catalytic diad. The Ala-201Val mutation shifted the pK(a) of the acid/base catalyst Glu-198 from 7.22 to 7.44, reflecting a change in its hydrophobic environment. A Phe-269Asn change increased K(m) for linamarin hydrolysis 16-fold (16.1 mM) and that for PNP-Glc only 2.5-fold (0.84 mM), demonstrating that Phe-269 contributes to the cyanogenic specificity of the cassava beta-glucosidase. PMID:11139381

  12. Rethinking procrastination: positive effects of "active" procrastination behavior on attitudes and performance.

    PubMed

    Chu, Angela Hsin Chun; Choi, Jin Nam

    2005-06-01

    Researchers and practitioners have long regarded procrastination as a self-handicapping and dysfunctional behavior. In the present study, the authors proposed that not all procrastination behaviors either are harmful or lead to negative consequences. Specifically, the authors differentiated two types of procrastinators: passive procrastinators versus active procrastinators. Passive procrastinators are procrastinators in the traditional sense. They are paralyzed by their indecision to act and fail to complete tasks on time. In contrast, active procrastinators are a "positive" type of procrastinator. They prefer to work under pressure, and they make deliberate decisions to procrastinate. The present results showed that although active procrastinators procrastinate to the same degree as passive procrastinators, they are more similar to nonprocrastinators than to passive procrastinators in terms of purposive use of time, control of time, self-efficacy belief, coping styles, and outcomes including academic performance. The present findings offer a more sophisticated understanding of procrastination behavior and indicate a need to reevaluate its implications for outcomes of individuals. PMID:15959999

  13. Active-site motions and polarity enhance catalytic turnover of hydrated subtilisin dissolved in organic solvents.

    PubMed

    Hudson, Elton P; Eppler, Ross K; Beaudoin, Julianne M; Dordick, Jonathan S; Reimer, Jeffrey A; Clark, Douglas S

    2009-04-01

    The enzyme subtilisin Carlsberg was surfactant-solubilized into two organic solvents, isooctane and tetrahydrofuran, and hydrated through stepwise changes in the thermodynamic water activity, a(w). The apparent turnover number k(cat)(app) in these systems ranged from 0.2 to 80 s(-1) and increased 11-fold in isooctane and up to 50-fold in tetrahydrofuran with increasing a(w). (19)F NMR relaxation experiments employing an active-site inhibitor were used to assess the dependence of active-site motions on a(w). The rates of NMR-derived fast (k > 10(7) s(-1)) and slow (k < 10(4) s(-1)) active-site motions increased in both solvents upon hydration, but only the slow motions correlated with k(cat). The (19)F chemical shift was a sensitive probe of the local electronic environment and provided an empirical measure of the active-site dielectric constant epsilon(as), which increased with hydration to epsilon(as) approximately 13 in each solvent. In both solvents, the transition state free energy data and epsilon(as) followed Kirkwood's model for the continuum solvation of a dipole, indicating that water also enhanced catalysis by altering the active-site's electronic environment and increasing its polarity to better stabilize the transition state. These results reveal that favorable dynamic and electrostatic effects both contribute to accelerated catalysis by solubilized subtilisin Carlsberg upon hydration in organic solvents. PMID:19317505

  14. Enhanced Enzyme Kinetic Stability by Increasing Rigidity within the Active Site*

    PubMed Central

    Xie, Yuan; An, Jiao; Yang, Guangyu; Wu, Geng; Zhang, Yong; Cui, Li; Feng, Yan

    2014-01-01

    Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser105 residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T5015, the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability. PMID:24448805

  15. Effects of resource activities upon repository siting and waste containment with reference to bedded salt

    SciTech Connect

    Ashby, J.; Rowe, J.

    1980-02-01

    The primary consideration for the suitability of a nuclear waste repository site is the overall ability of the repository to safely contain radioactive waste. This report is a discussion of the past, present, and future effects of resource activities on waste containment. Past and present resource activities which provide release pathways (i.e., leaky boreholes, adjacent mines) will receive initial evaluation during the early stages of any repository site study. However, other resource activities which may have subtle effects on containment (e.g., long-term pumping causing increased groundwater gradients, invasion of saline water causing lower retardation) and all potential future resource activities must also be considered during the site evaluation process. Resource activities will affect both the siting and the designing of repositories. Ideally, sites should be located in areas of low resource activity and low potential for future activity, and repository design should seek to eliminate or minimize the adverse effects of any resource activity. Buffer zones should be created to provide areas in which resource activities that might adversely affect containment can be restricted or curtailed. This could mean removing large areas of land from resource development. The impact of these frozen assets should be assessed in terms of their economic value and of their effect upon resource reserves. This step could require a major effort in data acquisition and analysis followed by extensive numerical modeling of regional fluid flow and mass transport. Numerical models should be used to assess the effects of resource activity upon containment and should include the cumulative effects of different resource activities. Analysis by other methods is probably not possible except for relatively simple cases.

  16. A caspase active site probe reveals high fractional inhibition needed to block DNA fragmentation.

    PubMed

    Méthot, Nathalie; Vaillancourt, John P; Huang, JingQi; Colucci, John; Han, Yongxin; Ménard, Stéphane; Zamboni, Robert; Toulmond, Sylvie; Nicholson, Donald W; Roy, Sophie

    2004-07-01

    Apoptotic markers consist of either caspase substrate cleavage products or phenotypic changes that manifest themselves as a consequence of caspase-mediated substrate cleavage. We have shown recently that pharmacological inhibitors of caspase activity prevent the appearance of two such apoptotic manifestations, alphaII-spectrin cleavage and DNA fragmentation, but that blockade of the latter required a significantly higher concentration of inhibitor. We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [(125)I]M808, which acts as a caspase active site probe. [(125)I]M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells. Moreover, [(125)I]M808 detected active caspases in septic mice when injected intravenously. Using this caspase probe, an active site occupancy assay was developed and used to measure the fractional inhibition required to block apoptosis-induced DNA fragmentation. In thymocytes, occupancy of up to 40% of caspase active sites had no effect on DNA fragmentation, whereas inhibition of half of the DNA cleaving activity required between 65 and 75% of active site occupancy. These results suggest that a high and persistent fractional inhibition will be required for successful caspase inhibition-based therapies. PMID:15067000

  17. Long-term active layer and ground surface temperature trends: results of 12 years of observations at Alaskan CALM sites

    NASA Astrophysics Data System (ADS)

    Shiklomanov, N. I.; Nelson, F. E.; Streletskyi, D. A.; Klene, A. E.; Schimek, M.; Little, J.

    2006-12-01

    The uppermost layer of seasonal thawing above permafrost (the active layer) is an important regulator of energy and mass fluxes between the surface and the atmosphere in the polar regions. The Circumpolar Active Layer Monitoring (CALM) program is a network of sites at which data about active-layer thickness (ALT) and dynamics are collected. CALM was established in the 1990s to observe and detect the long-term response of the active layer and near-surface permafrost to changes in climate. Active layer monitoring is an important component of efforts to assess the effects of global change in permafrost environments. CALM strategies are evolving; this presentation showcases some additions to CALM observation procedures designed to monitor processes and detect changes not anticipated in the original CALM protocol of the early 1990s. In this study we used data from 12 (1995-2006) years of extensive, spatially oriented field observations at CALM sites in northern Alaska to examine landscape-specific spatial and temporal trends in active-layer thickness and air and ground surface temperature. Despite an observed increase in air temperature, active-layer thickness exhibited a decreasing trend over the study period. This result indicates that soil consolidation accompanying penetration of thaw into an ice-rich stratum at the base of the active layer has resulted in subsidence of the surface with little or no apparent thickening of the active layer, as traditionally defined. Differential Global Positioning Systems (DGPS) technology was used to detect frost heave and thaw settlement within representative landscapes. Preliminary results indicate that heave and settlement follow patterns of spatial variation similar to those of active-layer thickness. To evaluate the effect of vegetation on ground surface temperature, several heat-transfer coefficients were estimated, including land cover specific thermal diffusivity and empirical n-factors.

  18. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes.

    PubMed

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  19. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    PubMed Central

    Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  20. Computational approaches to the determination of active site structures and reaction mechanisms in heterogeneous catalysts.

    PubMed

    Catlow, C R A; French, S A; Sokol, A A; Thomas, J M

    2005-04-15

    We apply quantum chemical methods to the study of active site structures and reaction mechanisms in mesoporous silica and metal oxide catalysts. Our approach is based on the use of both molecular cluster and embedded cluster (QM/MM) techniques, where the active site and molecular complex are described using density functional theory (DFT) and the embedding matrix simulated by shell model potentials. We consider three case studies: alkene epoxidation over the microporous TS-1 catalyst; methanol synthesis on ZnO and Cu/ZnO and C-H bond activation over Li-doped MgO. PMID:15901543

  1. Binding of the wheat basic leucine zipper protein EmBP-1 to nucleosomal binding sites is modulated by nucleosome positioning.

    PubMed Central

    Niu, X; Adams, C C; Workman, J L; Guiltinan, M J

    1996-01-01

    To investigate interactions of the basic leucine zipper transcription factor EmBP-1 with its recognition sites in nucleosomal DNA, we reconstituted an abscisic acid response element and a high-affinity binding site for EmBP-1 into human and wheat nucleosome cores in vitro. DNA binding studies demonstrated that nucleosomal elements can be bound by EmBP-1 at reduced affinities relative to naked DNA. EmBP-1 affinity was lowest when the recognition sites were positioned near the center of the nucleosome. Binding was achieved with a truncated DNA binding domain; however, binding of full-length EmBP-1 caused additional strong DNase I hypersensitivity flanking the binding sites. Similar results were observed with nucleosomes reconstituted with either human or wheat histones, demonstrating a conserved mechanism of transcription factor-nucleosome interactions. We conclude that positioning of recognition sequences on a nucleosome may play an important role in regulating interactions of EmBP-1 with its target sites in plant cells. PMID:8837510

  2. Automated docking of {alpha}-(1,4)- and {alpha}-(1,6)-linked glucosyl trisaccharides in the glucoamylase active site

    SciTech Connect

    Countinho, P.M.; Reilly, P.J.; Dowd, M.K.

    1998-06-01

    Low-energy conformers of five {alpha}-(1,4)- and {alpha}-(1,6)-linked glucosyl trisaccharides were flexibly docked into the glucoamylase active site using AutoDock 2.2. To ensure that all significant conformational space was searched, the starting trisaccharide conformers for docking were all possible combinations of the corresponding disaccharide low-energy conformers. All docked trisaccharides occupied subsites {minus}1 and +1 in very similar modes to those of corresponding nonreducing-end disaccharides. For linear substrates, full binding at subsite +2 occurred only when the substrate reducing end was {alpha}-(1,4)-linked, with hydrogen-bonding with the hydroxy-methyl group being the only polar interaction there. Given the absence of other important interactions at this subsite, multiple substrate conformations are allowed. For the one docked branched substrate, steric hindrance in the {alpha}-(1,6)-glycosidic oxygen suggests that the active-site residues have to change position for hydrolysis to occur. Subsite +1 of the glucoamylase active site allows flexibility in binding but, at least in Aspergillus glucoamylases, subsite +2 selectively binds substrates {alpha}-(1,4)-linked between subsites +1 and +2. Enzyme engineering to limit substrate flexibility at subsite +2 could improve glucoamylase industrial properties.

  3. Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules

    PubMed Central

    Davidge, Kelly S; Singh, Sandip; Bowman, Lesley AH; Tinajero-Trejo, Mariana; Carballal, Sebastián; Radi, Rafael; Poole, Robert K; Dikshit, Kanak; Estrin, Dario A; Marti, Marcelo A; Boechi, Leonardo

    2015-01-01

    Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and •NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels that are partially blocked by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify •NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, •NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site. PMID:26478812

  4. Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules.

    PubMed

    Boron, Ignacio; Bustamante, Juan Pablo; Davidge, Kelly S; Singh, Sandip; Bowman, Lesley Ah; Tinajero-Trejo, Mariana; Carballal, Sebastián; Radi, Rafael; Poole, Robert K; Dikshit, Kanak; Estrin, Dario A; Marti, Marcelo A; Boechi, Leonardo

    2015-01-01

    Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and (•)NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels that are partially blocked by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify (•)NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, (•)NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations introduce modifications in both tunnel topologies and affect the incoming ligand capacity to displace retained water molecules at the active site. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site. PMID:26478812

  5. Size controlled protein nanoemulsions for active targeting of folate receptor positive cells.

    PubMed

    Loureiro, Ana; Nogueira, Eugénia; Azoia, Nuno G; Sárria, Marisa P; Abreu, Ana S; Shimanovich, Ulyana; Rollett, Alexandra; Härmark, Johan; Hebert, Hans; Guebitz, Georg; Bernardes, Gonçalo J L; Preto, Ana; Gomes, Andreia C; Cavaco-Paulo, Artur

    2015-11-01

    Bovine serum albumin (BSA) nanoemulsions were produced by high pressure homogenization with a tri-block copolymer (Poloxamer 407), which presents a central hydrophobic chain of polyoxypropylene (PPO) and two identical lateral hydrophilic chains of polyethylene glycol (PEG). We observed a linear correlation between tri-block copolymer concentration and size - the use of 5mg/mL of Poloxamer 407 yields nanoemulsions smaller than 100nm. Molecular dynamics and fluorescent tagging of the tri-block copolymer highlight their mechanistic role on the size of emulsions. This novel method enables the fabrication of highly stable albumin emulsions in the nano-size range, highly desirable for controlled drug delivery. Folic Acid (FA)-tagged protein nanoemulsions were shown to promote specific folate receptor (FR)-mediated targeting in FR positive cells. The novel strategy presented here enables the construction of size controlled, functionalized protein-based nanoemulsions with excellent characteristics for active targeting in cancer therapy. PMID:26241920

  6. Earthquake lights and the stress-activation of positive hole charge carriers in rocks

    USGS Publications Warehouse

    St-Laurent, F.; Derr, J.S.; Freund, F.T.

    2006-01-01

    Earthquake-related luminous phenomena (also known as earthquake lights) may arise from (1) the stress-activation of positive hole (p-hole) charge carriers in igneous rocks and (2) the accumulation of high charge carrier concentrations at asperities in the crust where the stress rates increase very rapidly as an earthquake approaches. It is proposed that, when a critical charge carrier concentration is reached, the p-holes form a degenerated solid state plasma that can break out of the confined rock volume and propagate as a rapidly expanding charge cloud. Upon reaching the surface the charge cloud causes dielectric breakdown at the air-rock interface, i.e. corona discharges, accompanied by the emission of light and high frequency electromagnetic radiation. ?? 2006 Elsevier Ltd. All rights reserved.

  7. Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells

    PubMed Central

    Lin, Congping; Schuster, Martin; Guimaraes, Sofia Cunha; Ashwin, Peter; Schrader, Michael; Metz, Jeremy; Hacker, Christian; Gurr, Sarah Jane; Steinberg, Gero

    2016-01-01

    Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ∼95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes. PMID:27251117

  8. Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells

    NASA Astrophysics Data System (ADS)

    Lin, Congping; Schuster, Martin; Guimaraes, Sofia Cunha; Ashwin, Peter; Schrader, Michael; Metz, Jeremy; Hacker, Christian; Gurr, Sarah Jane; Steinberg, Gero

    2016-06-01

    Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ~95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes.

  9. Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells.

    PubMed

    Lin, Congping; Schuster, Martin; Guimaraes, Sofia Cunha; Ashwin, Peter; Schrader, Michael; Metz, Jeremy; Hacker, Christian; Gurr, Sarah Jane; Steinberg, Gero

    2016-01-01

    Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ∼95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes. PMID:27251117

  10. Interferon-α/β enhances temozolomide activity against MGMT-positive glioma stem-like cells.

    PubMed

    Shen, Dong; Guo, Cheng-Cheng; Wang, Jing; Qiu, Zhi-Kun; Sai, Ke; Yang, Qun-Ying; Chen, Yin-Sheng; Chen, Fu-Rong; Wang, Jie; Panasci, Lawrence; Chen, Zhong-Ping

    2015-11-01

    Glioma is one of the most common primary tumors of the central nervous system in adults. Glioblastoma (GBM) is the most lethal type of glioma, whose 5-year survival is 9.8% at best. Glioma stem-like cells (GSCs) play an important role in recurrence and treatment resistance. MGMT is a DNA repair protein that removes DNA adducts and therefore attenuates treatment efficiency. It has been reported that interferon-α/β (IFN-α/β) downregulates the level of MGMT and sensitizes glioma cells to temozolomide. In the present study, we assessed whether IFN-α/β is able to sensitize GSCs to temozolomide by modulating MGMT expression. Upon the treatment of IFN-α/β, the efficacy of temozolomide against MGMT‑positive GSCs was markedly enhanced by combination treatment with IFN-α/β when compared with the temozolomide single agent group, and MGMT expression was markedly decreased at the same time. Further mechanistic study showed that IFN-α/β suppressed the NF-κB activity, which further mediated the sensitization of MGMT‑positive GSCs to temozolomide. Our data therefore demonstrated that the application of IFN-α/β is a promising agent with which to enhance temozolomide efficiency and reduce drug resistance, and our findings shed light on improving clinical outcomes and prolonging the survival of patients with malignant gliomas. PMID:26329778

  11. Activity of T-DM1 in HER-2 positive central nervous system breast cancer metastases

    PubMed Central

    Torres, Sofia; Maralani, Pejman; Verma, Sunil

    2014-01-01

    A 55-year-old woman with metastatic human epidermal growth factor receptor 2 (HER-2) positive breast cancer (BC) to the lungs and bones was diagnosed with central nervous system (CNS) metastases in November 2011. The MRI showed a right parietal lobe mass with adjacent leptomeningeal disease and several small bilateral cerebellar metastases. She was treated with whole brain irradiation (WBI), followed by capecitabine and lapatinib (December 2011-March 2013) and trastuzumab and lapatinib (May 2013-August 2013). Then, the brain MRI showed progression. I