WorldWideScience

Sample records for active site insights

  1. Mechanochemical coupling in the myosin motor domain. I. Insights from equilibrium active-site simulations.

    Haibo Yu

    2007-02-01

    Full Text Available Although the major structural transitions in molecular motors are often argued to couple to the binding of Adenosine triphosphate (ATP, the recovery stroke in the conventional myosin has been shown to be dependent on the hydrolysis of ATP. To obtain a clearer mechanistic picture for such "mechanochemical coupling" in myosin, equilibrium active-site simulations with explicit solvent have been carried out to probe the behavior of the motor domain as functions of the nucleotide chemical state and conformation of the converter/relay helix. In conjunction with previous studies of ATP hydrolysis with different active-site conformations and normal mode analysis of structural flexibility, the results help establish an energetics-based framework for understanding the mechanochemical coupling. It is proposed that the activation of hydrolysis does not require the rotation of the lever arm per se, but the two processes are tightly coordinated because both strongly couple to the open/close transition of the active site. The underlying picture involves shifts in the dominant population of different structural motifs as a consequence of changes elsewhere in the motor domain. The contribution of this work and the accompanying paper [] is to propose the actual mechanism behind these "population shifts" and residues that play important roles in the process. It is suggested that structural flexibilities at both the small and large scales inherent to the motor domain make it possible to implement tight couplings between different structural motifs while maintaining small free-energy drops for processes that occur in the detached states, which is likely a feature shared among many molecular motors. The significantly different flexibility of the active site in different X-ray structures with variable level arm orientations supports the notation that external force sensed by the lever arm may transmit into the active site and influence the chemical steps (nucleotide

  2. Widely available active sites on Ni2P for electrochemical hydrogen evolution--insights from first principles calculations.

    Hansen, Martin H; Stern, Lucas-Alexandre; Feng, Ligang; Rossmeisl, Jan; Hu, Xile

    2015-04-28

    We present insights into the mechanism and the active site for hydrogen evolution on nickel phosphide (Ni2P). Ni2P was recently discovered to be a very active non-precious hydrogen evolution catalyst. Current literature attributes the activity of Ni2P to a particular site on the (0001) facet. In the present study, using Density Functional Theory (DFT) calculations, we show that several widely available low index crystal facets on Ni2P have better properties for a high catalytic activity. DFT calculations were used to identify moderately bonding nickel bridge sites and nickel hollow sites for hydrogen adsorption and to calculate barriers for the Tafel pathway. The investigated surfaces in this study were the (101̅0), (1̅1̅20), (112̅0), (112̅1) and (0001) facets of the hexagonal Ni2P crystal. In addition to the DFT results, we present experiments on Ni2P nanowires growing along the 〈0001〉 direction, which are shown as efficient hydrogen evolution catalysts. The experimental results add these nanowires to a variety of different morphologies of Ni2P, which are all active for HER. PMID:25812670

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

    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

  4. The Active Site of Oligogalacturonate Lyase Provides Unique Insights into Cytoplasmic Oligogalacturonate β-Elimination*

    Abbott, D. Wade; Gilbert, Harry J.; Boraston, Alisdair B.

    2010-01-01

    Oligogalacturonate lyases (OGLs; now also classified as pectate lyase family 22) are cytoplasmic enzymes found in pectinolytic members of Enterobacteriaceae, such as the enteropathogen Yersinia enterocolitica. OGLs utilize a β-elimination mechanism to preferentially catalyze the conversion of saturated and unsaturated digalacturonate into monogalacturonate and the 4,5-unsaturated monogalacturonate-like molecule, 5-keto-4-deoxyuronate. To provide mechanistic insights into the specificity of th...

  5. Structural Insights into the Protease-like Antigen Plasmodium falciparum SERA5 and Its Noncanonical Active-Site Serine

    Hodder, Anthony N.; Malby, Robyn L.; Clarke, Oliver B.; Fairlie, W. Douglas; Colman, Peter M.; Crabb, Brendan S.; Smith, Brian J.; (WEHIMR); (Melbourne)

    2009-08-28

    The sera genes of the malaria-causing parasite Plasmodium encode a family of unique proteins that are maximally expressed at the time of egress of parasites from infected red blood cells. These multi-domain proteins are unique, containing a central papain-like cysteine-protease fragment enclosed between the disulfide-linked N- and C-terminal domains. However, the central fragment of several members of this family, including serine repeat antigen 5 (SERA5), contains a serine (S596) in place of the active-site cysteine. Here we report the crystal structure of the central protease-like domain of Plasmodium falciparum SERA5, revealing a number of anomalies in addition to the putative nucleophilic serine: (1) the structure of the putative active site is not conducive to binding substrate in the canonical cysteine-protease manner; (2) the side chain of D594 restricts access of substrate to the putative active site; and (3) the S{sub 2} specificity pocket is occupied by the side chain of Y735, reducing this site to a small depression on the protein surface. Attempts to determine the structure in complex with known inhibitors were not successful. Thus, despite having revealed its structure, the function of the catalytic domain of SERA5 remains an enigma.

  6. Widely available active sites on Ni2P for electrochemical hydrogen evolution - insights from first principles calculations

    Hansen, Martin Hangaard; Stern, Lucas-Alexandre; Feng, Ligang;

    2015-01-01

    the present study, using Density Functional Theory (DFT) calculations, we show that several widely available low index crystal facets on Ni2P have better properties for a high catalytic activity. DFT calculations were used to identify moderately bonding nickel bridge sites and nickel hollow sites for...

  7. Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism.

    Zhao, Haiyan; Lin, Zihan; Lynn, Anna Y; Varnado, Brittany; Beutler, John A; Murelli, Ryan P; Le Grice, Stuart F J; Tang, Liang

    2015-12-15

    Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Here we show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg(2+). A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 Å. Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg(2+) from Ca(2+). Using a metal ion chelator β-thujaplicinol as a molecular probe, we observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiled mechanistic details of the two-metal-ion catalysis at atomic resolution. PMID:26450964

  8. Three-Dimensional Model of Lanosterol 14α-Demethylase from Cryptococcus neoformans: Active-Site Characterization and Insights into Azole Binding ▿ †

    Sheng, Chunquan; Miao, Zhenyuan; Ji, Haitao; Yao, Jianzhong; Wang, Wenya; Che, Xiaoying; Dong, Guoqiang; Lü, Jiaguo; Guo, Wei; Zhang, Wannian

    2009-01-01

    Cryptococcus neoformans is one of the most important causes of life-threatening fungal infections in immunocompromised patients. Lanosterol 14α-demethylase (CYP51) is the target of azole antifungal agents. This study describes, for the first time, the 3-dimensional model of CYP51 from Cryptococcus neoformans (CnCYP51). The model was further refined by energy minimization and molecular-dynamics simulations. The active site of CnCYP51 was well characterized by multiple-copy simultaneous-search ...

  9. Theoretic Insight into CO2 Reduction at Active Sites of Molybdenum and Tungsten Enzymes: a {\\pi} Interaction between CO2 and Tungsten Bis-Dithiolene Complexes

    Yan, Yong

    2014-01-01

    Active sites of molybdenum and tungsten enzymes, particularly mononuclear tungsten formate dehydrogenase (FDH) have been theoretically investigated towards their interaction with CO2. Obvious {\\pi} interaction has been found between the 2e reduced metallodithiole moiety and the molecular CO2. This weak {\\pi} bonding is predicated both at gas phase, noted as -6.0 kcal/mol and aqueous solvation level, -3.6 kcal/mol. Such interaction is not only limited to CO2, but also to the CO2 reduced product, i.e. formate, in the form of anion- {\\pi} interaction, noted as -6.8 kcal/mol and -4.1 kcal/mol respectively in gas and aqueous solvation model. The Bailar twisted angles from 60o to 0o, governing structure preference of tungsten dithiolene from octahedron to triangle prism in their restricted structures, has been explored to evaluate such {\\pi} in-terrelations with CO2 and formate. An octahedral structure with 3 kcal/mol energy lower is preferred over the triangle prismatic when such interactions are concerned.

  10. Inhibition of CK2 Activity by TCDD via Binding to ATP-competitive Binding Site of Catalytic Subunit:Insight from Computational Studies

    XU Xian-jin; CANNISTRARO Salvatore; BIZZARRI Anna-rita; ZENG Yi; CHEN Wei-zu; WANG Cun-xin

    2013-01-01

    Alternative mechanisms of toxic effects induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD),instead of the binding to aryl hydrocarbon receptor(AhR),have been taken into consideration.It has been recently shown that TCDD reduces rapidly the activity of CK2(casein kinase Ⅱ) both in vivo and in vitro.It is found that TCDD has high molecular similarities to the known inhibitors of CK2 catalytic subunit(CK2α).This suggests that TCDD could also be an ATP-competitive inhibitor of CK2α.In this work,docking TCDD to CK2 was carried out based on the two structures of CK2α from maize and human,respectively.The binding free energies of the predicted CK2α-TCDD complexes estimated by the molecular mechanics/Poisson-Boltzmann surface area(MM/PBSA) method are from -85.1 kJ/mol to-114.3 kJ/mol for maize and are from-96.1 kJ/mol to-118.2 kJ/mol for human,which are comparable to those estimated for the known inhibitor and also ATP with CK2α.The energetic analysis also reveals that the van der Waals interaction is the dominant contribution to the binding free energy.These results are also useful for designing new drugs for a target of overexpressing CK2 in cancers.

  11. New insight into the dynamic properties and the active site architecture of H-Ras p21 revealed by X-ray crystallography at very high resolution

    Klink Björn U

    2010-10-01

    Full Text Available Abstract Background In kinetic crystallography, the usually static method of X-ray diffraction is expanded to allow time-resolved analysis of conformational rearrangements in protein structures. To achieve this, reactions have to be triggered within the protein crystals of interest, and optical spectroscopy can be used to monitor the reaction state. For this approach, a modified form of H-Ras p21 was designed which allows reaction initiation and fluorescence readout of the initiated GTPase reaction within the crystalline state. Rearrangements within the crystallized protein due to the progressing reaction and associated heterogeneity in the protein conformations have to be considered in the subsequent refinement processes. Results X-ray diffraction experiments on H-Ras p21 in different states along the reaction pathway provide detailed information about the kinetics and mechanism of the GTPase reaction. In addition, a very high data quality of up to 1.0 Å resolution allowed distinguishing two discrete subconformations of H-Ras p21, expanding the knowledge about the intrinsic flexibility of Ras-like proteins, which is important for their function. In a complex of H-Ras•GppNHp (guanosine-5'-(β,γ-imido-triphosphate, a second Mg2+ ion was found to be coordinated to the γ-phosphate group of GppNHp, which positions the hydrolytically active water molecule very close to the attacked γ-phosphorous atom. Conclusion For the structural analysis of very high-resolution data we have used a new 'two-chain-isotropic-refinement' strategy. This refinement provides an alternative and easy to interpret strategy to reflect the conformational variability within crystal structures of biological macromolecules. The presented fluorescent form of H-Ras p21 will be advantageous for fluorescence studies on H-Ras p21 in which the use of fluorescent nucleotides is not feasible.

  12. DOE site performance assessment activities

    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

  13. Neural Activity When People Solve Verbal Problems with Insight

    Jung-Beeman Mark

    2004-01-01

    Full Text Available People sometimes solve problems with a unique process called insight, accompanied by an "Aha!" experience. It has long been unclear whether different cognitive and neural processes lead to insight versus noninsight solutions, or if solutions differ only in subsequent subjective feeling. Recent behavioral studies indicate distinct patterns of performance and suggest differential hemispheric involvement for insight and noninsight solutions. Subjects solved verbal problems, and after each correct solution indicated whether they solved with or without insight. We observed two objective neural correlates of insight. Functional magnetic resonance imaging (Experiment 1 revealed increased activity in the right hemisphere anterior superior temporal gyrus for insight relative to noninsight solutions. The same region was active during initial solving efforts. Scalp electroencephalogram recordings (Experiment 2 revealed a sudden burst of high-frequency (gamma-band neural activity in the same area beginning 0.3 s prior to insight solutions. This right anterior temporal area is associated with making connections across distantly related information during comprehension. Although all problem solving relies on a largely shared cortical network, the sudden flash of insight occurs when solvers engage distinct neural and cognitive processes that allow them to see connections that previously eluded them.

  14. Probing the active sites for CO dissociation on ruthenium nanoparticles

    Strebel, Christian Ejersbo; Murphy, Shane; Nielsen, Rasmus Munksgård;

    2012-01-01

    The active sites for CO dissociation were probed on mass-selected Ru nanoparticles on a HOPG support by temperature programmed desorption spectroscopy using isotopically labelled CO. Combined with transmission electron microscopy we gain insight on how the size and morphology of the nanoparticles...

  15. Active Site Engineering in Electrocatalysis

    Verdaguer Casadevall, Arnau; Stephens, Ifan; Chorkendorff, Ib

    The overall goal of this thesis has been to design better catalysts for electrochemical reactions through a fundamental understanding of the materials at atomic scale. This has been achieved by combining electrochemical measurements with a variety of characterization techniques, often in ultra high...... under reaction conditions, which is ultimately controlled by the crystal structure of the underlying alloy.• Oxygen reduction to hydrogen peroxide has been investigated on single site catalysts, mainly alloys of noble metals with Hg. This resulted in a very special structure with isolated atoms of Pt or......, inexistent in other forms of Cu. The presence of strong CO binding sites correlates well with electrochemical activity, which paves the way for the rational development of even better electrocatalysts....

  16. Forecasting the solar activity cycle: new insights

    Nandy, Dibyendu; Karak, Bidya Binay

    2013-01-01

    Having advanced knowledge of solar activity is important because the Sun's magnetic output governs space weather and impacts technologies reliant on space. However, the irregular nature of the solar cycle makes solar activity predictions a challenging task. This is best achieved through appropriately constrained solar dynamo simulations and as such the first step towards predictions is to understand the underlying physics of the solar dynamo mechanism. In Babcock-Leighton type dynamo models, ...

  17. Forecasting the solar activity cycle: new insights

    Nandy, Dibyendu

    2013-01-01

    Having advanced knowledge of solar activity is important because the Sun's magnetic output governs space weather and impacts technologies reliant on space. However, the irregular nature of the solar cycle makes solar activity predictions a challenging task. This is best achieved through appropriately constrained solar dynamo simulations and as such the first step towards predictions is to understand the underlying physics of the solar dynamo mechanism. In Babcock-Leighton type dynamo models, the poloidal field is generated near the solar surface whereas the toroidal field is generated in the solar interior. Therefore a finite time is necessary for the coupling of the spatially segregated source layers of the dynamo. This time delay introduces a memory in the dynamo mechanism which allows forecasting of future solar activity. Here we discuss how this forecasting ability of the solar cycle is affected by downward turbulent pumping of magnetic flux. With significant turbulent pumping the memory of the dynamo is ...

  18. A Carbon Flux Super Site. New Insights and Innovative Atmosphere-Terrestrial Carbon Exchange Measurements and Modeling

    Leclerc, Monique Y. [The University of Georgia Research Foundation, Athens, GA (United States)

    2014-11-17

    This final report presents the main activities and results of the project “A Carbon Flux Super Site: New Insights and Innovative Atmosphere-Terrestrial Carbon Exchange Measurements and Modeling” from 10/1/2006 to 9/30/2014. It describes the new AmeriFlux tower site (Aiken) at Savanna River Site (SC) and instrumentation, long term eddy-covariance, sodar, microbarograph, soil and other measurements at the site, and intensive field campaigns of tracer experiment at the Carbon Flux Super Site, SC, in 2009 and at ARM-CF site, Lamont, OK, and experiments in Plains, GA. The main results on tracer experiment and modeling, on low-level jet characteristics and their impact on fluxes, on gravity waves and their influence on eddy fluxes, and other results are briefly described in the report.

  19. Active zone stability:insights from fly neuromuscular junction

    Xiaolin Tian; Chunlai Wu

    2015-01-01

    The presynaptic active zone is a dynamic structure that orchestrates regulated release of neurotrans-mitters. Developmental and aging processes, and changes in neuronal network activity can all modulate the number, size and composition of active zone and thereby synaptic efifcacy. However, very little is known about the mechanism that controls the structural stability of active zone. By study-ing a model synapse, theDrosophila neuromuscular junction, our recent work shed light on how two scaffolding proteins at the active zone regulate active zone stability by promoting a localized dephos-phorylation event at the nerve terminal. Here we discuss the major insights from our ifndings and their implications for future research.

  20. Characterization of Reuse Activities at Contaminated Sites

    Angela Vitulli; Charlotte Dougherty; Kimberly Bosworth

    2004-01-01

    Given the increased focus on reuse activity within EPA and state site cleanup programs, policy makers would benefit from looking across programs to better understand the extent and nature of reuse; examine site characteristics that influence reuse; leverage lessons learned; and coordinate reuse activities, data collection, and information management. This research paper begins to examine these issues. It reports the results of a preliminary review and analysis of available EPA and state progr...

  1. Fingerprinting differential active site constraints of ATPases

    Hacker, Stephan M.; Hardt, Norman; Buntru, Alexander; Pagliarini, Dana; Möckel, Martin; Mayer, Thomas U; Scheffner, Martin; Hauck, Christof R.; Marx, Andreas

    2013-01-01

    The free energy provided by adenosine triphosphate (ATP) hydrolysis is central to many cellular processes and, therefore, the number of enzymes utilizing ATP as a substrate is almost innumerable. Modified analogues of ATP are a valuable means to understand the biological function of ATPases. Although these enzymes have evolved towards binding to ATP, large differences in active site architectures were found. In order to systematically access the specific active site constraints of different A...

  2. Dust devil track survey at Elysium Planitia, Mars: Implications for the InSight landing sites

    Reiss, Dennis; Lorenz, Ralph D.

    2016-03-01

    The InSight (Interior Exploration using Seismic Investigations, Geodesy and Heat Transport) robotic lander is scheduled to land in Elysium Planitia on Mars in September 2016. InSight will perform the first comprehensive surface-based geophysical investigation including seismic measurements. Knowledge about encounter rates of dust devils with the InSight lander are important for two main reasons: (1) dust devils will affect the scientific measurements, i.e., wind-induced seismic noise, and (2) the power-supply of the InSight lander and instruments is provided by solar arrays and previous landers and rovers on Mars were affected by a steady decline in electrical power output due to atmospheric dust deposition on the solar panels. Long term science operations were only made possible by dust clearing events of the solar arrays caused by wind gusts and dust devils. In this study we analyzed dust devil tracks (DDTs) at the final InSight landing site region in Elysium Planitia. Formation of DDTs is caused by the removal of a layer of dust by passing dust devils, hence in principle the same process as clearing of dust from solar panels. We mapped the number, size (width and length), and orientation of DDTs in repeat observations using High Resolution Imaging Science Experiment (HiRISE) images covering the exact same surface area acquired within a relatively short time span (solar panel clearing recurrence interval estimate of ∼11 Mars years using the mean annual DDT formation rate, and the mean DDT width and length from all measured DDTs. Due to several uncertainties this solar panel clearing recurrence interval for the InSight landing should be seen as an upper limit estimate.

  3. Methane to acetic acid over Cu-exchanged zeolites: mechanistic insights from a site-specific carbonylation reaction.

    Narsimhan, Karthik; Michaelis, Vladimir K; Mathies, Guinevere; Gunther, William R; Griffin, Robert G; Román-Leshkov, Yuriy

    2015-02-11

    The selective low temperature oxidation of methane is an attractive yet challenging pathway to convert abundant natural gas into value added chemicals. Copper-exchanged ZSM-5 and mordenite (MOR) zeolites have received attention due to their ability to oxidize methane into methanol using molecular oxygen. In this work, the conversion of methane into acetic acid is demonstrated using Cu-MOR by coupling oxidation with carbonylation reactions. The carbonylation reaction, known to occur predominantly in the 8-membered ring (8MR) pockets of MOR, is used as a site-specific probe to gain insight into important mechanistic differences existing between Cu-MOR and Cu-ZSM-5 during methane oxidation. For the tandem reaction sequence, Cu-MOR generated drastically higher amounts of acetic acid when compared to Cu-ZSM-5 (22 vs 4 μmol/g). Preferential titration with sodium showed a direct correlation between the number of acid sites in the 8MR pockets in MOR and acetic acid yield, indicating that methoxy species present in the MOR side pockets undergo carbonylation. Coupled spectroscopic and reactivity measurements were used to identify the genesis of the oxidation sites and to validate the migration of methoxy species from the oxidation site to the carbonylation site. Our results indicate that the Cu(II)-O-Cu(II) sites previously associated with methane oxidation in both Cu-MOR and Cu-ZSM-5 are oxidation active but carbonylation inactive. In turn, combined UV-vis and EPR spectroscopic studies showed that a novel Cu(2+) site is formed at Cu/Al <0.2 in MOR. These sites oxidize methane and promote the migration of the product to a Brønsted acid site in the 8MR to undergo carbonylation. PMID:25562431

  4. Managing Siting Activities for Nuclear Power Plants

    One of the IAEA's statutory objectives is to ''seek to accelerate and enlarge the contribution of atomic energy to peace, health and prosperity throughout the world''. One way this objective is achieved is through the publication of a range of technical series. Two of these are the IAEA Nuclear Energy Series and the IAEA Safety Standards Series. According to Article III.A.6 of the IAEA Statute, the safety standards establish 'standards of safety for protection of health and minimization of danger to life and property.' The safety standards include the Safety Fundamentals, Safety Requirements and Safety Guides. These standards are written primarily in a regulatory style, and are binding on the IAEA for its own programmes. The principal users are the regulatory bodies in Member States and other national authorities. The IAEA Nuclear Energy Series comprises reports designed to encourage and assist R and D on, and application of, nuclear energy for peaceful uses. This includes practical examples to be used by owners and operators of utilities in Member States, implementing organizations, academia, and government officials, among others. This information is presented in guides, reports on technology status and advances, and best practices for peaceful uses of nuclear energy based on inputs from international experts. The IAEA Nuclear Energy Series complements the IAEA Safety Standards Series. The introduction of nuclear power brings new challenges to States - one of them being the selection of appropriates sites. It is a project that needs to begin early, be well managed, and deploy good communications with all stakeholders; including regulators. This is important, not just for those States introducing nuclear power for the first time, but for any State looking to build a new nuclear power plant. The purpose of the siting activities goes beyond choosing a suitable site and acquiring a licence. A large part of the project is about producing and maintaining a validated

  5. Efficient oxygen electrocatalysis on special active sites

    Halck, Niels Bendtsen

    cobalt incorporated in ruthenium dioxide at high overpotentials during the oxygen reduction reaction (ORR). Density functional theory calculations were used to explain this phenomenon. The special active sites concepts are used to propose a general unified approach to increase the efficiency for oxygen...

  6. Efficient oxygen electrocatalysis on special active sites

    Halck, Niels Bendtsen

    Oxygen electrocatalysis will be pivotal in future independent of fossil fuels. Renewable energy production will rely heavily on oxygen electrocatalysis as a method for storing energy from intermittent energy sources such as the wind and sun in the form of chemical bonds and to release the energy ...... electrocatalysis (ORR and OER) using organic functional groups on another class of catalysts. These consist of graphene sheets modified to have a local porphyrine site with different transition metals ions as model systems....... stored in these bonds in an eco-friendly fashion in fuel cells. This thesis explores catalysts for oxygen electrocatalysis and how carefully designed local structures on catalysts surfaces termed special active sites can influence the activity. Density functional theory has been used as a method...... throughout this thesis to understand these local structure effects and their influence on surface reactions. The concept of these special active sites is used to explain how oxygen evolution reaction (OER) catalysts can have activities beyond the limits of what was previously thought possible. The concept is...

  7. Structural insights of the ssDNA binding site in the multifunctional endonuclease AtBFN2 from Arabidopsis thaliana.

    Tsung-Fu Yu

    Full Text Available The multi S1/P1 nuclease AtBFN2 (EC 3.1.30.1 encoded by the Arabidopsis thaliana At1g68290 gene is a glycoprotein that digests RNA, ssDNA, and dsDNA. AtBFN2 depends on three zinc ions for cleaving DNA and RNA at 3'-OH to yield 5'-nucleotides. In addition, AtBFN2's enzymatic activity is strongly glycan dependent. Plant Zn(2+-dependent endonucleases present a unique fold, and belong to the Phospholipase C (PLC/P1 nuclease superfamily. In this work, we present the first complete, ligand-free, AtBFN2 crystal structure, along with sulfate, phosphate and ssDNA co-crystal structures. With these, we were able to provide better insight into the glycan structure and possible enzymatic mechanism. In comparison with other nucleases, the AtBFN2/ligand-free and AtBFN2/PO4 models suggest a similar, previously proposed, catalytic mechanism. Our data also confirm that the phosphate and vanadate can inhibit the enzyme activity by occupying the active site. More importantly, the AtBFN2/A5T structure reveals a novel and conserved secondary binding site, which seems to be important for plant Zn(2+-dependent endonucleases. Based on these findings, we propose a rational ssDNA binding model, in which the ssDNA wraps itself around the protein and the attached surface glycan, in turn, reinforces the binding complex.

  8. BET is active on Sellafield site

    Several companies, all part of BET Plant Services are carrying out work at the British Nuclear Fuels (BNFL) site at Sellafield, Cumbria, on one of the largest construction projects in Europe. The main development scheme is the THORP (Thermal Oxide Reprocessing Plant) buildings. One of the BET companies has the contract to paint the inside of the fuel storage ponds. It will also coat the surfaces of the MASWEP (Medium Active Solid Waste Encapsulation Plant) complex. Other work includes insulation and fire prevention installation. Scaffolding at the EARP (Enhanced Actinide Removal Plant) site is being provided on a common user basis so all the contractors can use the scaffolding and share the cost. Temporary office and living accommodation blocks have been provide by another BET company. (author)

  9. Immunodetection of phosphorylation sites gives new insights into the mechanisms underlying phospholamban phosphorylation in the intact heart.

    Mundiña-Weilenmann, C; Vittone, L; Ortale, M; de Cingolani, G C; Mattiazzi, A

    1996-12-27

    Phosphorylation site-specific antibodies, quantification of 32P incorporation into phospholamban, and simultaneous measurements of mechanical activity were used in Langendorff-perfused rat hearts to provide further insights into the underlying mechanisms of phospholamban phosphorylation. Immunological detection of phospholamban phosphorylation sites showed that the isoproterenol concentration-dependent increase in phospholamban phosphorylation was due to increases in phosphorylation of both Ser16 and Thr17 residues. When isoproterenol concentration was increased at extremely low Ca2+ supply to the myocardium, phosphorylation of Thr17 was virtually absent. Under these conditions, 32P incorporation into phospholamban, due to Ser16, decreased by 50%. Changes in Ca2+ supply to the myocardium either at constant beta-adrenergic stimulation or in the presence of okadaic acid, a phosphatase inhibitor, exclusively modified Thr17 phosphorylation. Changes in phospholamban phosphorylation due to either Ser16 and/or Thr17 were paralleled by changes in myocardial relaxation. The results indicate that cAMP- (Ser16) and Ca2+-calmodulin (Thr17)-dependent pathways of phospholamban phosphorylation can occur independently of each other. However, in the absence of beta-adrenergic stimulation, phosphorylation of Thr17 could only be detected after simultaneous activation of Ca2+-calmodulin-dependent protein kinase and inactivation of phosphatase. It is suggested that under physiological conditions, this requisite is only filled by cAMP-dependent mechanisms. PMID:8969222

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

    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

  11. Addressing Community Concerns about Lead Contamination in Soil: Insights for Site Cleanup

    Health risks associated with contaminated sites are a key driver for cleanup decisions and determinations about alternate land use of areas released to the public, particularly in heavily populated metropolitan areas. To guide risk management and future use decisions at contaminated sites, insights can be gained from community-based research. These evaluations can also help ensure that assessments and decisions developed for urban sites consider input received from community members. In order to evaluate the potential risk due to consumption of plants home-grown in lead-contaminated soil, a pilot study was conducted over a period of two summers in a Chicago, IL neighborhood. This survey included analyses of lead concentrations in a convenience sampling of edible fruits, vegetables, and herbs and also examined how the sample preparation method affected the lead concentrations detected in plant materials. A pattern of lead transfer from soil through the root to the stem and leaves of garden crops was found. This pattern is a concern particularly for plants in which the roots, stems, stalks, or leaves are consumed. Analyses of fruiting vegetables indicated that concentrations were below the limit of detection. Depending on the soil lead level and specific plant, the contamination found in some leafy vegetables and herbs may exceed the body's daily excretion rate and could contribute to the total body burden of lead, especially in children. Finally, washing edible portions did not necessarily eliminate the risk, indicating that the lead was located both on and in the plant tissue. This research was conducted in coordination with health experts from the community, and local citizens were involved in discussions on the research and implications for their health protection measures. In certain residential locations, identifying and understanding the potential source of lead contamination provides information for the community such that simple measures can be applied for

  12. Promoter proximal polyadenylation sites reduce transcription activity

    Andersen, Pia Kjølhede; Lykke-Andersen, Søren; Jensen, Torben Heick

    2012-01-01

    transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ∼500...

  13. Activities on the site during construction phase

    A survey is given of the work done on the site from site-opening till turn over of the plant to the client. After a short introduction to time schedules, manpower on site, site facilities and civil work and constructions, the commissioning and trial operation phase is discussed in detail. This phase begins with finishing the assembly of individual systems and components and ends with the trial operation and the acceptance measurement. During this period the subsystems are started-up in a useful sequence, first from cold, then from hot conditions and are finally operated as a total with nuclear energy. The single steps are: a) commissioning of indivudal systems; b) hot functional test 1 (without fuels) c) baseline inspection at the reactor pressure vessel; d) hot functional test 2 (with fuels); e) preparation for first criticality; f) postcriticality test program; g) trial operation: h) acceptance measurement. (HP)

  14. Crystallographic B factor of critical residues at enzyme active site

    张海龙; 宋时英; 林政炯

    1999-01-01

    Thirty-seven sets of crystallographic enzyme data were selected from Protein Data Bank (PDB, 1995). The average temperature factors (B) of the critical residues at the active site and the whole molecule of those enzymes were calculated respectively. The statistical results showed that the critical residues at the active site of most of the enzymes had lower B factors than did the whole molecules, indicating that in the crystalline state the critical residues at the active site of the natural enzymes possess more stable conformation than do the whole molecules. The flexibility of the active site during the unfolding by denaturing was also discussed.

  15. Probability of solar panel clearing events at the Insight landing sites (Mars) from a dust devil track survey

    Reiss, D.; Lorenz, R. D.

    2015-10-01

    The InSight robotic lander is scheduled to land on Mars in September 2016. InSight was designed to perform the first comprehensive surface-based geophysical investigation of Mars [1]. Passage of vortices may have a number of influences on the geophysical measurements to be made by InSight. Seismic data could be influenced by dust devils and vortices via several mechanisms such as loading of the elastic ground by a surface pressure field which causes a local tilt [e.g. 2]. In addition, the power supply of the InSight instruments is provided by solar arrays. Solar-powered missions on Mars like the Sojourner rover in 1997 were affected by a decline in electrical power output by 0.2-0.3 %per day caused by steadily dust deposition on its horizontal solar panel [3]. The solar-powered Mars Exploration Rovers (MERs) Spirit and Opportunity experienced similar dust deposition rates [4] which led to steady power decrease over time endangering longer rover operation times. The much longer operation times of the rovers were made possible by unanticipated 'dust clearing events' of the solar arrays by wind gust or dust devils [5]. Recent studies imply that dust devils are primarily responsible for those recurrent 'dust clearing events' [6]. In this study we investigate the potential frequency of intense dust devil occurrences at the InSight landing site regions, which are able to remove dust from its solar panels. We analyzed newly formed dust devil tracks within a given time span using multi-temporal HiRISE image data covering the same surface area. Based on these measurements we will give encounter rate predictions of intense (high tangential speed and high pressure drop) dust devils with the InSight lander.

  16. Ecological Insights from Pelagic Habitats Acquired Using Active Acoustic Techniques

    Benoit-Bird, Kelly J.; Lawson, Gareth L.

    2016-01-01

    Marine pelagic ecosystems present fascinating opportunities for ecological investigation but pose important methodological challenges for sampling. Active acoustic techniques involve producing sound and receiving signals from organisms and other water column sources, offering the benefit of high spatial and temporal resolution and, via integration into different platforms, the ability to make measurements spanning a range of spatial and temporal scales. As a consequence, a variety of questions concerning the ecology of pelagic systems lend themselves to active acoustics, ranging from organism-level investigations and physiological responses to the environment to ecosystem-level studies and climate. As technologies and data analysis methods have matured, the use of acoustics in ecological studies has grown rapidly. We explore the continued role of active acoustics in addressing questions concerning life in the ocean, highlight creative applications to key ecological themes ranging from physiology and behavior to biogeography and climate, and discuss emerging avenues where acoustics can help determine how pelagic ecosystems function.

  17. Interferon Gamma Release Assays in active Tuberculosis: new medical insights

    Sandro Pierdomenico

    2011-09-01

    Full Text Available Since first presentation, Interferon γ Release Assays (IGRAs have had basic and wide application to LTBI, in accordance with international consensus and CDC recommendations, leaving their use in active TB to the field of study and research.We reviewed the results of 633 patients investigated from 2004 to 2008 targeting active TB, with the objective to highlight immunological data supporting test performances.We evaluated Quantiferon TB Gold (1st generation IGRA kit in association to Culture (MGIT 960 and Lowenstein Jensen and PCR (Probetec-ET having the positivity of culture plus clinical diagnosis as the standard true value to compare. QTB Gold was studied in 69 TB positive patients (42 pulmonary and 27 extra-pulmonary, with Sensitivity, Specificity, PPV and NPV average to 61.8%, 94.5%, 54.3% and 95.9% respectively, after indeterminate results discharging. Significant statistical differences didn’t emerge between pulmonary and extra-pulmonary infections (CI 95%.The overall indeterminate ratio arose up to 20.3% in patients with active TB vs 2.7% of global population (p<0.001. In 22% of patients with active pulmonary disease, IGRA conversed to positivity after 15 days in replicated tests, in spite of current treatment. 4 patients, with pulmonary TB and Quantiferon persistent negativities, underwent 18 months follow-up as not respondent although SIRE phenotypic susceptibilities and enough DOT compliance. Molecular DST documented hetero resistance for rpoB (MUT 1, MUT 3 plus wild lines and katG (MUT 1 plus wild in association to lack of inhA wild lines (Genotype MTBDR plus, Hain Lifescience. These reports suggest a mutational relationship between Rv3874 – 3875 cassette, encoding ESAT-6 / CFP-10, and rpoB, katG, inhA genes plausibly implying weak or absent selective clonal Th 1 activation to IGRA antigens. Our data seem to point out: 1 positive results are able to match true active TB in less than 50% of patients; 2 negative results could leave

  18. Insights into Animal and Plant Lectins with Antimicrobial Activities

    Renata de Oliveira Dias

    2015-01-01

    Full Text Available Lectins are multivalent proteins with the ability to recognize and bind diverse carbohydrate structures. The glyco -binding and diverse molecular structures observed in these protein classes make them a large and heterogeneous group with a wide range of biological activities in microorganisms, animals and plants. Lectins from plants and animals are commonly used in direct defense against pathogens and in immune regulation. This review focuses on sources of animal and plant lectins, describing their functional classification and tridimensional structures, relating these properties with biotechnological purposes, including antimicrobial activities. In summary, this work focuses on structural-functional elucidation of diverse lectin groups, shedding some light on host-pathogen interactions; it also examines their emergence as biotechnological tools through gene manipulation and development of new drugs.

  19. Recent insight into the biological activities of synthetic xanthone derivatives.

    Shagufta; Ahmad, Irshad

    2016-06-30

    Xanthones are a class of oxygen containing heterocyclic compounds with a broad range of biological activities, and they have prominent significance in the field of medicinal chemistry. Xanthone is an attractive scaffold for the design and development of new drugs due to its promising biological activities, primarily as anticancer, antimalarial, antimicrobial, anti-HIV, anticonvulsant, anticholinesterase, antioxidant, anti-inflammatory, and as inhibitors of several enzymes like α-glycosidase, topoisomerase, protein kinase, aromatase, etc. In this review, we have compiled and discussed recent developments on the pharmacological profile of synthetic xanthone derivatives for different therapeutic targets. The review highlights the therapeutic significance of xanthones and offers support in the development of new xanthone derivatives as therapeutic agents. PMID:27111599

  20. Antibacterial activity of silver nanoparticles: A surface science insight

    Le Ouay, Benjamin; Stellacci, Francesco

    2015-01-01

    Silver nanoparticles constitute a very promising approach for the development of new antimicrobial systems. Nanoparticulate objects can bring significant improvements in the antibacterial activity of this element, through specific effect such as an adsorption at bacterial surfaces. However, the mechanism of action is essentially driven by the oxidative dissolution of the nanoparticles, as indicated by recent direct observations. The rote of Ag+ release in the action mechanism was also indirec...

  1. Insights into Animal and Plant Lectins with Antimicrobial Activities

    Renata de Oliveira Dias; Leandro dos Santos Machado; Ludovico Migliolo; Octavio Luiz Franco

    2015-01-01

    Lectins are multivalent proteins with the ability to recognize and bind diverse carbohydrate structures. The glyco -binding and diverse molecular structures observed in these protein classes make them a large and heterogeneous group with a wide range of biological activities in microorganisms, animals and plants. Lectins from plants and animals are commonly used in direct defense against pathogens and in immune regulation. This review focuses on sources of animal and plant lectins, describing...

  2. Proteomics reliability for micropollutants degradation insight into activated sludge systems.

    Buttiglieri, Gianluigi; Collado, Neus; Casas, Nuria; Comas, Joaquim; Rodriguez-Roda, Ignasi

    2015-01-01

    Little information is available on pharmaceutical trace compounds degradation pathways in wastewater. The potential of the proteomics approach has been evaluated to extract information on activated sludge microbial metabolism in degrading a trace concentration of a pharmaceutical compound (ibuprofen). Ibuprofen is one of the most consumed pharmaceuticals, measured in wastewater at very high concentrations and, despite its high removal rates, found in different environmental compartments. Aerated and completely mixed activated sludge batch tests were spiked with ibuprofen at 10 and 1,000 μg L(-1). Ibuprofen concentrations were determined in the liquid phase: 100% removal was observed and the kinetics were estimated. The solid phase was sampled for proteomics purposes. The first objective was to apply proteomics to evaluate protein profile variations in a complex matrix such as activated sludge. The second objective was to determine, at different ibuprofen concentrations, which proteins followed pre-defined trends. No newly expressed proteins were found. Nonetheless, the obtained results suggest that proteomics itself is a promising methodology to be applied in this field. Statistical and comparative studies analyses provided, in fact, useful information on biological reproducibility and permitted us to detect 62 proteins following coherent and plausible expected trends in terms of presence and intensity change. PMID:26360747

  3. Insight into GATA1 transcriptional activity through interrogation of cis elements disrupted in human erythroid disorders.

    Wakabayashi, Aoi; Ulirsch, Jacob C; Ludwig, Leif S; Fiorini, Claudia; Yasuda, Makiko; Choudhuri, Avik; McDonel, Patrick; Zon, Leonard I; Sankaran, Vijay G

    2016-04-19

    Whole-exome sequencing has been incredibly successful in identifying causal genetic variants and has revealed a number of novel genes associated with blood and other diseases. One limitation of this approach is that it overlooks mutations in noncoding regulatory elements. Furthermore, the mechanisms by which mutations in transcriptionalcis-regulatory elements result in disease remain poorly understood. Here we used CRISPR/Cas9 genome editing to interrogate three such elements harboring mutations in human erythroid disorders, which in all cases are predicted to disrupt a canonical binding motif for the hematopoietic transcription factor GATA1. Deletions of as few as two to four nucleotides resulted in a substantial decrease (>80%) in target gene expression. Isolated deletions of the canonical GATA1 binding motif completely abrogated binding of the cofactor TAL1, which binds to a separate motif. Having verified the functionality of these three GATA1 motifs, we demonstrate strong evolutionary conservation of GATA1 motifs in regulatory elements proximal to other genes implicated in erythroid disorders, and show that targeted disruption of such elements results in altered gene expression. By modeling transcription factor binding patterns, we show that multiple transcription factors are associated with erythroid gene expression, and have created predictive maps modeling putative disruptions of their binding sites at key regulatory elements. Our study provides insight into GATA1 transcriptional activity and may prove a useful resource for investigating the pathogenicity of noncoding variants in human erythroid disorders. PMID:27044088

  4. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    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

  5. Savannah River Site prioritization of transition activities

    Effective management of SRS conversion from primarily a production facility to other missions (or Decontamination and Decommissioning (D ampersand 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

  6. Savannah River Site prioritization of transition activities

    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.

  7. Metaproteomics provides functional insight into activated sludge wastewater treatment.

    Paul Wilmes

    Full Text Available BACKGROUND: Through identification of highly expressed proteins from a mixed culture activated sludge system this study provides functional evidence of microbial transformations important for enhanced biological phosphorus removal (EBPR. METHODOLOGY/PRINCIPAL FINDINGS: A laboratory-scale sequencing batch reactor was successfully operated for different levels of EBPR, removing around 25, 40 and 55 mg/l P. The microbial communities were dominated by the uncultured polyphosphate-accumulating organism "Candidatus Accumulibacter phosphatis". When EBPR failed, the sludge was dominated by tetrad-forming alpha-Proteobacteria. Representative and reproducible 2D gel protein separations were obtained for all sludge samples. 638 protein spots were matched across gels generated from the phosphate removing sludges. 111 of these were excised and 46 proteins were identified using recently available sludge metagenomic sequences. Many of these closely match proteins from "Candidatus Accumulibacter phosphatis" and could be directly linked to the EBPR process. They included enzymes involved in energy generation, polyhydroxyalkanoate synthesis, glycolysis, gluconeogenesis, glycogen synthesis, glyoxylate/TCA cycle, fatty acid beta oxidation, fatty acid synthesis and phosphate transport. Several proteins involved in cellular stress response were detected. CONCLUSIONS/SIGNIFICANCE: Importantly, this study provides direct evidence linking the metabolic activities of "Accumulibacter" to the chemical transformations observed in EBPR. Finally, the results are discussed in relation to current EBPR metabolic models.

  8. Lipid interaction sites on channels, transporters and receptors: Recent insights from molecular dynamics simulations.

    Hedger, George; Sansom, Mark S P

    2016-10-01

    Lipid molecules are able to selectively interact with specific sites on integral membrane proteins, and modulate their structure and function. Identification and characterization of these sites are of importance for our understanding of the molecular basis of membrane protein function and stability, and may facilitate the design of lipid-like drug molecules. Molecular dynamics simulations provide a powerful tool for the identification of these sites, complementing advances in membrane protein structural biology and biophysics. We describe recent notable biomolecular simulation studies which have identified lipid interaction sites on a range of different membrane proteins. The sites identified in these simulation studies agree well with those identified by complementary experimental techniques. This demonstrates the power of the molecular dynamics approach in the prediction and characterization of lipid interaction sites on integral membrane proteins. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg. PMID:26946244

  9. The Surface Groups and Active Site of Fibrous Mineral Materials

    DONG Fa-qin; WAN Pu; FENG Qi-ming; SONG Gong-bao; PENG Tong-jiang; LI Ping; LI Guo-wu

    2004-01-01

    The exposed and transformed groups of fibrous brucite,wollastonite,chrysotile asbestos,sepiolite,palygorskite,clinoptilolite,crocidolite and diatomaceous earth mineral materials are analyzed by IR spectra after acid and alikali etching,strong mechanical and polarity molecular interaction.The results show the active sites concentrate on the ends in stick mineral materials and on the defect or hole edge in pipe mineral materials.The inside active site of mineral materials plays a main role in small molecular substance.The shape of minerals influence their distribution and density of active site.The strong mechanical impulsion and weak chemical force change the active site feature of minerals,the powder process enables minerals exposed more surface group and more combined types.The surface processing with the small polarity molecular or the brand of middle molecular may produce ionation and new coordinate bond,and change the active properties and level of original mineral materials.

  10. Landscapes, depositional environments and human occupation at Middle Paleolithic open-air sites in the southern Levant, with new insights from Nesher Ramla, Israel

    Zaidner, Yossi; Frumkin, Amos; Friesem, David; Tsatskin, Alexander; Shahack-Gross, Ruth

    2016-04-01

    Middle Paleolithic human occupation in the Levant (250-50 ka ago) has been recorded in roofed (cave and rockshelter) and open-air sites. Research at these different types of sites yielded different perspectives on the Middle Paleolithic human behavior and evolution. Until recently, open-air Middle Paleolithic sites in the Levant were found in three major sedimentary environments: fluvial, lake-margin and spring. Here we describe a unique depositional environment and formation processes at the recently discovered open-air site of Nesher Ramla (Israel) and discuss their contribution to understanding site formation processes in open-air sites in the Levant. The site is 8-m-thick Middle Paleolithic sequence (OSL dated to 170-80 ka) that is located in a karst sinkhole formed by gravitational deformation and sagging into underground voids. The sedimentary sequence was shaped by gravitational collapse, cyclic colluviation of soil and gravel into the depression, waterlogging, in situ pedogenesis and human occupation. Original bedding and combustion features are well-preserved in the Lower archaeological sequence, a rare occurrence in comparison to other open-air archaeological sites. This phenomenon coincides with episodes of fast sedimentation/burial, which also allowed better preservation of microscopic remains such as ash. The Upper archaeological sequence does not exhibit bedding or preservation of ash, despite presence of heat-affected lithic artifacts, which makes it similar to other open-air sites in the Levant. We suggest that rate of burial is the major factor that caused the difference between the Upper and Lower sequences. The differences in the burial rate may be connected to environmental and vegetation changes at the end of MIS 6. We also identified an interplay between sediment in-wash and density of human activity remains, i.e. during episodes of low natural sediment input the density of artifacts is higher relative to episodes with high rate of sediment in

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

    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

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

    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

  13. Evaluation of two statistical methods provides insights into the complex patterns of alternative polyadenylation site switching.

    Jie Li

    Full Text Available Switching between different alternative polyadenylation (APA sites plays an important role in the fine tuning of gene expression. New technologies for the execution of 3'-end enriched RNA-seq allow genome-wide detection of the genes that exhibit significant APA site switching between different samples. Here, we show that the independence test gives better results than the linear trend test in detecting APA site-switching events. Further examination suggests that the discrepancy between these two statistical methods arises from complex APA site-switching events that cannot be represented by a simple change of average 3'-UTR length. In theory, the linear trend test is only effective in detecting these simple changes. We classify the switching events into four switching patterns: two simple patterns (3'-UTR shortening and lengthening and two complex patterns. By comparing the results of the two statistical methods, we show that complex patterns account for 1/4 of all observed switching events that happen between normal and cancerous human breast cell lines. Because simple and complex switching patterns may convey different biological meanings, they merit separate study. We therefore propose to combine both the independence test and the linear trend test in practice. First, the independence test should be used to detect APA site switching; second, the linear trend test should be invoked to identify simple switching events; and third, those complex switching events that pass independence testing but fail linear trend testing can be identified.

  14. Counting Active Sites on Titanium Oxide-Silica Catalysts for Hydrogen Peroxide Activation through In Situ Poisoning with Phenylphosphonic Acid

    Eaton, Todd R.; Boston, Andrew M.; Thompson, Anthony B.; Gray, Kimberly A.; Notestein, Justin M. [NWU

    2015-06-04

    Quantifying specific active sites in supported catalysts improves our understanding and assists in rational design. Supported oxides can undergo significant structural changes as surface densities increase from site-isolated cations to monolayers and crystallites, which changes the number of kinetically relevant sites. Herein, TiOx domains are titrated on TiOx–SiO2 selectively with phenylphosphonic acid (PPA). An ex situ method quantifies all fluid-accessible TiOx, whereas an in situ titration during cis-cyclooctene epoxidation provides previously unavailable values for the number of tetrahedral Ti sites on which H2O2 activation occurs. We use this method to determine the active site densities of 22 different catalysts with different synthesis methods, loadings, and characteristic spectra and find a single intrinsic turnover frequency for cis-cyclooctene epoxidation of (40±7) h-1. This simple method gives molecular-level insight into catalyst structure that is otherwise hidden when bulk techniques are used.

  15. Fluorescence energy transfer studies on the active site of papain

    Henes, Jill B.; Briggs, Martha S.; Sligar, Stephen G.; Fruton, Joseph S.

    1980-01-01

    Measurements have been performed of the excited-state lifetimes and fluorescence yields of papain tryptophan units when acyl derivatives of Phe-glycinal are bound at the active site of the enzyme. The enhancement of tryptophan fluorescence in complexes of papain with the acetyl or benzyloxycarbonyl derivatives is not stereospecific with respect to the configuration of the phenylalanyl residue, and the L and D isomers are equally effective as active-site-directed inhibitors of papain action. E...

  16. What causes low magnetization at basalt-hosted hydrothermal sites? Insights from inactive site Krasnov (MAR 16°38′N)

    Szitkar, Florent; Dyment, Jerome; Choi, Yujin; Fouquet, Yves

    2014-01-01

    High-resolution magnetic surveys acquired near the seafloor show that active basalt-hosted hydrothermal sites are associated with zones of lower magnetization. This observation may reflect the thermal demagnetization of a hot hydrothermal zone, the alteration of basalt affected by hydrothermal circulation, and/or the presence of thick, nonmagnetic hydrothermal deposits. In order to discriminate among these inferences, we acquired vector magnetic data 50 m above inactive hydrothermal site Kras...

  17. Decision precision or holistic heuristic?: Insights on on-site selection of student nurses and midwives.

    Macduff, Colin; Stephen, Audrey; Taylor, Ruth

    2016-01-01

    Concerns about quality of care delivery in the UK have led to more scrutiny of criteria and methods for the selection of student nurses. However few substantive research studies of on-site selection processes exist. This study elicited and interpreted perspectives on interviewing processes and related decision making involved in on-site selection of student nurses and midwives. Individual and focus group interviews were undertaken with 36 lecturers, 5 clinical staff and 72 students from seven Scottish universities. Enquiry focused primarily on interviewing of candidates on-site. Qualitative content analysis was used as a primary strategy, followed by in-depth thematic analysis. Students had very mixed experiences of interview processes. Staff typically took into account a range of candidate attributes that they valued in order to achieve holistic assessments. These included: interpersonal skills, team working, confidence, problem-solving, aptitude for caring, motivations, and commitment. Staff had mixed views of the validity and reliability of interview processes. A holistic heuristic for overall decision making predominated over belief in the precision of, and evidence base for, particular attribute measurement processes. While the development of measurement tools for particular attributes continues apace, tension between holism and precision is likely to persist within on-site selection procedures. PMID:26213147

  18. Key messages from active CO2 storage sites

    Wildenborg, T.; Wollenweber, J. [TNO, Princetonlaan 6, 3584 CB Utrecht (Netherlands); Chadwick, A. [BGS, Environmental Science Centre, Keyworth, Nottingham, NG12 5GG (United Kingdom); Deflandre, J.P. [IFP Energies nouvelles, 1-4 avenue de Bois Preau, 92852 Rueil-Malmaison (France); Eiken, O. [Statoil Research Centre, Rotvoll, Arkitekt Ebbells vei 10, 7005 Trondheim (Norway); Mathieson, A. [BP, Alternative Energy, Chertsey Road, Sunbury on Thames (United Kingdom); Metcalfe, R. [QUINTESSA, The Hub, 14 Station Road, Henley-on-Thames, Oxfordshire (United Kingdom); Schmidt Hattenberger, C. [GFZ German Research Centre for Geosciences, Centre for CO2Storage, Potsdam (Germany)

    2013-07-01

    An extensive programme of modelling, monitoring and verification activities was deployed at a set of active storage sites worldwide including Sleipner, In Salah, Ketzin, Weyburn, K12-B and Snoehvit (EU CO2ReMoVe project). All investigated storage sites were well managed and did not have a negative impact on humans or the environment. Time-lapse seismic and pressure monitoring are key in verifying the deep subsurface performance of the storage sites. Evidence gathered during the site characterisation and operational phases is key to handover responsibility of the storage site to governmental authorities after injection has definitely ceased, which is the focus of the follow-up EU project CO2CARE.

  19. Klipperaas study site. Scope of activities and main results

    During the period from 1977 - 1986 SKB (Swedish Nuclear Fuel and Waste Management Co.) performed surface and borehole investigations of 14 study sites for the purpose of assessing their suitability for a repository of spent nuclear fuel. The next phase in the SKB site selection rpogramme will be to perform detailed characterisation, including characterization from shafts and/or tunnels, of two or three sites. The detailed investigations will continue over several years to provide all the data needed for a licensing application to build a repository. Such an application is foreseen to be given to the authorities around the year 2003. It is presently not clear if any of the study sites will be selected as a site for detailed characterization. Other sites with geological and/or socio-economical characteristics judged more favorable may very well be the ones selected. However, as a part of the background documentation needed for the site selection studies to come, summary reports will be prepared for most study sites. These reports will include scope of activities, main results, uncertainties and need of complementary investigations. This report concern the Klipperaas study site. The main topics are the scope of activities, geologic model, geohydrological model, groundwater chemistry, assessment of solute transport, and rock mechanics

  20. Dashboard applications to monitor experiment activities at sites

    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.

  1. Dashboard applications to monitor experiment activities at sites

    Andreeva, J; Boehm, M; Casajus, A; Flix, J; Gaidioz, B; Grigoras, C; Kokoszkiewicz, L; Lanciotti, E; Rocha, R; Saiz, P; Santinelli, R; Sidorova, I; Sciabà, A; Tsaregorodtsev, A

    2010-01-01

    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.

  2. Dashboard applications to monitor experiment activities at sites

    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.

  3. Toward understanding the active SETI debate: Insights from risk communication and perception

    Korbitz, Adam

    2014-12-01

    Insights from the robust field of risk communication and perception have to date been almost totally absent from the policy debate regarding the relative risks and merits of Active SETI or Messaging to Extraterrestrial Intelligence (METI). For many years, the practice (or proposed practice) of Active SETI has generated a vigorous and sometimes heated policy debate within the scientific community. There have also been some negative reactions in the media toward the activities of those engaged in Active SETI. Risk communication is a scientific approach to communication regarding situations involving potentially sensitive or controversial situations in which there may be high public concern and low public trust. The discipline has found wide acceptance and utility in fields such as public health, industrial regulation and environmental protection. Insights from the scientific field of risk communication (such as omission bias, loss aversion, the availability heuristic, probability neglect, and the general human preference for voluntary over involuntary risks) may help those who have participated in either side of the debate over Active SETI to better understand why the debate has taken on this posture. Principles of risk communication and risk perception may also help those engaged in Active SETI to communicate more effectively with other scientists, the public, with the media, and with policy makers regarding their activities and to better understand and respond to concerns expressed regarding the activity.

  4. New insights on the wooden weapons from the Paleolithic site of Schöningen.

    Schoch, Werner H; Bigga, Gerlinde; Böhner, Utz; Richter, Pascale; Terberger, Thomas

    2015-12-01

    The Paleolithic site of Schöningen is famous for the earliest known, completely preserved wooden weapons. Here we present recent results of an ongoing analysis of the nine spears, one lance, a double pointed stick, and a burnt stick dating to the Holsteinian, c. 300 kyr. Macroscopic and microscopic analyses, as well as studies of thin sections, contribute to a better understanding of the manufacture of the wooden weapons. They were deposited in organic sediments at a former lakeshore among numerous bones of butchered horses. In general, the spears are extremely well-preserved and show no or little sign of taphonomic alteration, although some of the weapons are broken and parts were slightly moved, probably by water action. The excellent preservation conditions provide considerable information on the operational sequence of production. The hunters selected thin trunks of spruce or pine and initially stripped off the bark. Traces of cutting, scraping, and smoothing can be observed on the spear surfaces in detail. In the case of spear X, repeated use of the weapon is implied by re-sharpening of the tip. Analyses of wood anatomy provide information on climatic conditions and contribute to the better understanding of the development of the site. PMID:26442632

  5. Gideaa study site. Scope of activities and main results

    During the period from 1977-1986 SKB (Swedish Nuclear Fuel and Waste Management Co) performed surface and borehole investigations of 14 study sites for the purpose of assessing their suitability for a repository of spent nuclear fuel. The next phase in the SKB site selection programme will be to perform detailed characterization, including characterization from shafts and/or tunnels, of two or three sites. The detailed investigations will continue over several years to provide all the data needed for a licensing application to build a repository. Such an application is foreseen to be given to the authorities around the year 2003. It is presently not clear if anyone of the study sites will be selected as a site for detailed characterization. Other site with geological and/or socio-economical characteristics judged more favourable may very well be the ones selected. However, as a part of the background documentation needed for the site selection studies to come, summary reports will be prepared for most study sites. These reports will include scope of activities, main results, uncertainties and need of complementary investigations. This report concerns the Gideaa study site. (au)

  6. Fjaellveden study site. Scope of activities and main results

    During the period from 1977-1986 SKB (Swedish Nuclear Fuel and Waste Management CO) performed surface and borehole investigations of 14 study sites for the purpose of assessing their suitability for a repository of spent nuclear fuel. The next phase in the SKB site selection programme will be to perform detailed characterization, including characterization from shafts and/or tunnels, of two or three sites. The detailed investigations will continue over several years to provide all the data needed for a licensing application to build repository. Such an application is foreseen to be given to the authorities around the year 2003. It is presently not clear if anyone of the study sites will be selected as a site for detailed characterization. Other sites with geological and/or socio-economical characteristics judged more favourable may very well be the ones selected. However, as a part of the background documentation needed for the site selection studies to come, summary reports will be prepared for most study sites. These reports will include scope of activities, main results, uncertainties and need for complementary investigations. This report concerns the Fjaellveden study site. (au)

  7. Kamlunge study site. Scope of activities and main results

    During the period from 1977-1986 SKB (Swedish nuclear Fuel and Waste Management Co.) performed surface and borehole investigations of 14 study sites for the purpose of assessing their suitability for a repository of spent nuclear fuel. The next phase in the SKB site selection programme will be to perform detailed characterization, including characterization from shafts and/or tunnels, of two or three sites. The detailed investigations will continue over several years to provide all the data needed for a licensing application to build a repository. Such an application is foreseen to be given to the authorities around the year 2003. It is presently not clear if anyone of the study sites will be selected as a site for detailed characterization. Other sites with geological and/or socio-economical characteristics judged more favourable may very well be selected. However, as a part of the background documentation needed for the site selection studies to come, summary reports will be prepared for most study sites. These reports will include scope of activities, main results, uncertainties and need of complementary investigations. This report concerns the Kamlunge study site. (79 refs.) (au)

  8. Polymorphisms in miRNA binding site: new insight into small cell lung cancer susceptibility

    Hong-yu LIU; Jun CHEN

    2011-01-01

    Lung cancer is a leading cause in cancer-related deaths with less than 15% five-year survival worldwide.Small cell lung cancer (SCLC),which accounts for about 15%-18% of lung cancer,carries the worst prognosis within the lung cancer patients.SCLC differs from other lung cancers,so called non-small cell lung cancers (NSCLCs),in the specifically clinical and biologic characteristics.It exhibits aggressive behavior,with rapid growth,early spread to distant sites.Although exquisite sensitive to chemotherapy and radiation,SCLC recurs rapidly with only 5% of patients surviving five years and frequent association with distinct paraneoplastic syndromes[1].

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

    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.

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

    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

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

    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.

  12. Insights into site formation at Rose Cottage Cave, South Africa, based on the analysis of sediment peels

    Kloos, Peter; Miller, Christopher E.; Kritikakis, Panagiotis; Wadley, Lyn

    2016-04-01

    few artefacts and implies that there may have been more human activity at the site during this time than has previously been suggested.

  13. Effect of Siloxane Ring Strain and Cation Charge Density on the Formation of Coordinately Unsaturated Metal Sites on Silica: Insights from DFT Studies

    Das, Ujjal; Zhang, Guanghui; Hu, Bo; Hock, Adam S.; Redfern, Paul C.; Miller, Jeffrey T.; Curtiss, Larry A.

    2015-12-01

    Amorphous silica (SiO2) is commonly used as a support in heterogeneous catalysis. However, due to the structural disorder and temperature induced change of surface morphology, the structures of silica supported metal catalysts are difficult to determine. Most studies are primarily focused on understanding the interactions of different types of surface hydroxyl groups with metal ions. In comparison, the effect of siloxane ring size on the structure of silica supported metal catalysts and how it affects catalytic activity is poorly understood. Here, we have used density functional theory calculations to understand the effect of siloxane ring strain on structure and activity of different monomeric Lewis acid metal sites on silica. In particular, we have found that large siloxane rings favor strong dative bonding interaction between metal ion and surface hydroxyls, leading to the formation of high-coordinate metal sites. In comparison, metal-silanol interaction is weak in small siloxane rings, resulting in low-coordinate metal sites. The physical origin of this size dependence is associated with siloxane ring strain, and, a correlation between metal-silanol interaction energy and ring strain energy has been observed. In addition to ring strain, the strength of the metal-silanol interaction also depends on the positive charge density of the cations. In fact, a correlation also exists between metal-silanol interaction energy and charge density of several first-row transition and post-transition metals. The theoretical results are compared with the EXAFS data of monomeric Zn(II) and Ga(III) ions grafted on silica. The molecular level insights of how metal ion coordination on silica depends on siloxane ring strain and cation charge density will be useful in the synthesis of new catalysts.

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

    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…

  15. Active site modeling in copper azurin molecular dynamics simulations

    Rizzuti, B; Swart, M; Sportelli, L; Guzzi, R

    2004-01-01

    Active site modeling in molecular dynamics simulations is investigated for the reduced state of copper azurin. Five simulation runs (5 ns each) were performed at room temperature to study the consequences of a mixed electrostatic/constrained modeling for the coordination between the metal and the po

  16. Active Sites Environmental Monitoring Program: FY 1990 annual report

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

    1991-10-01

    Chapter 3 of US Department of Energy (DOE) Order 5820.2A (DOE 1988) sets forth requirements for environmental monitoring of active low-level waste (LLW) disposal sites. Active sites are defined as those LLW facilities that were in use on or after the date of the order (September 1988). The transuranic (TRU) waste storage areas in Solid Waste Storage Area (SWSA) 5 North are covered by Chap. 2 of the order. In both chapters, monitoring is required to provide for early warning of leaks before those leaks pose a threat to human health or the environment. Chapter 3 also requires that monitoring be conducted to evaluate the short- and long-term performance of LLW disposal facilities. In accordance with this order, the Solid Waste Operations Department at Oak Ridge National Laboratory (ORNL) has established an Active Sites Environmental Monitoring Program (ASEMP) that is implemented by staff of the Environmental Sciences Division (ESD) at ORNL. This report summarizes data from ASEMP monitoring activities for the final 6 months of FY 1990. A brief summary of the monitoring methodology for each site is presented also.

  17. The nature of the active site in heterogeneous metal catalysis

    Nørskov, Jens Kehlet; Bligaard, Thomas; Larsen, Britt Hvolbæk;

    2008-01-01

    This tutorial review, of relevance for the surface science and heterogeneous catalysis communities, provides a molecular-level discussion of the nature of the active sites in metal catalysis. Fundamental concepts such as "Bronsted-Evans-Polanyi relations'' and "volcano curves'' are introduced, and...

  18. Active Sites Environmental Monitoring Program: FY 1990 annual report

    Chapter 3 of US Department of Energy (DOE) Order 5820.2A (DOE 1988) sets forth requirements for environmental monitoring of active low-level waste (LLW) disposal sites. Active sites are defined as those LLW facilities that were in use on or after the date of the order (September 1988). The transuranic (TRU) waste storage areas in Solid Waste Storage Area (SWSA) 5 North are covered by Chap. 2 of the order. In both chapters, monitoring is required to provide for early warning of leaks before those leaks pose a threat to human health or the environment. Chapter 3 also requires that monitoring be conducted to evaluate the short- and long-term performance of LLW disposal facilities. In accordance with this order, the Solid Waste Operations Department at Oak Ridge National Laboratory (ORNL) has established an Active Sites Environmental Monitoring Program (ASEMP) that is implemented by staff of the Environmental Sciences Division (ESD) at ORNL. This report summarizes data from ASEMP monitoring activities for the final 6 months of FY 1990. A brief summary of the monitoring methodology for each site is presented also

  19. Flexibility and Stability Trade-Off in Active Site of Cold-Adapted Pseudomonas mandelii Esterase EstK.

    Truongvan, Ngoc; Jang, Sei-Heon; Lee, ChangWoo

    2016-06-28

    Cold-adapted enzymes exhibit enhanced conformational flexibility, especially in their active sites, as compared with their warmer-temperature counterparts. However, the mechanism by which cold-adapted enzymes maintain their active site stability is largely unknown. In this study, we investigated the role of conserved D308-Y309 residues located in the same loop as the catalytic H307 residue in the cold-adapted esterase EstK from Pseudomonas mandelii. Mutation of D308 and/or Y309 to Ala or deletion resulted in increased conformational flexibility. Particularly, the D308A or Y309A mutant showed enhanced substrate affinity and catalytic rate, as compared with wild-type EstK, via enlargement of the active site. However, all mutant EstK enzymes exhibited reduced thermal stability. The effect of mutation was greater for D308 than Y309. These results indicate that D308 is not preferable for substrate selection and catalytic activity, whereas hydrogen bond formation involving D308 is critical for active site stabilization. Taken together, conformation of the EstK active site is constrained via flexibility-stability trade-off for enzyme catalysis and thermal stability. Our study provides further insights into active site stabilization of cold-adapted enzymes. PMID:27259687

  20. Activity of rhodium-catalyzed hydroformylation: added insight and predictions from theory.

    Sparta, Manuel; Børve, Knut J; Jensen, Vidar R

    2007-07-11

    We have performed a density functional theory investigation of hydroformylation of ethylene for monosubstituted rhodium-carbonyl catalysts, HRh(CO)3L, where the modifying ligand, L, is a phosphite (L = P(OMe)3, P(OPh)3, or P(OCH2CF3)3), a phosphine (L = PMe3, PEt3, PiPr3, or PPh3), or a N-heterocyclic carbene (NHC) based on the tetrahydropyrimidine, imidazol, or tetrazol ring, respectively. The study follows the Heck and Breslow mechanism. Excellent correspondence between our calculations and existing experimental information is found, and the present results constitute the first example of a realistic quantum chemical description of the catalytic cycle of hydroformylation using ligand-modified rhodium carbonyl catalysts. This description explains the mechanistic and kinetic basis of the contemporary understanding of this class of reaction and offers unprecedented insight into the electronic and steric factors governing catalytic activity. The insight has been turned into structure-activity relationships and used as guidelines when also subjecting to calculation phosphite and NHC complexes that have yet to be reported experimentally. The latter calculations illustrate that it is possible to increase the electron-withdrawing capacity of both phosphite and NHC ligands compared to contemporary ligands through directed substitution. Rhodium complexes of such very electron-withdrawing ligands are predicted to be more active than contemporary catalysts for hydroformylation. PMID:17555314

  1. In Situ Probing of the Active Site Geometry of Ultrathin Nanowires for the Oxygen Reduction Reaction.

    Liu, Haiqing; An, Wei; Li, Yuanyuan; Frenkel, Anatoly I; Sasaki, Kotaro; Koenigsmann, Christopher; Su, Dong; Anderson, Rachel M; Crooks, Richard M; Adzic, Radoslav R; Liu, Ping; Wong, Stanislaus S

    2015-10-01

    To create truly effective electrocatalysts for the cathodic reaction governing proton exchange membrane fuel cells (PEMFC), namely the oxygen reduction reaction (ORR), necessitates an accurate and detailed structural understanding of these electrocatalysts, especially at the nanoscale, and to precisely correlate that structure with demonstrable performance enhancement. To address this key issue, we have combined and interwoven theoretical calculations with experimental, spectroscopic observations in order to acquire useful structural insights into the active site geometry with implications for designing optimized nanoscale electrocatalysts with rationally predicted properties. Specifically, we have probed ultrathin (∼2 nm) core-shell Pt∼Pd9Au nanowires, which have been previously shown to be excellent candidates for ORR in terms of both activity and long-term stability, from the complementary perspectives of both DFT calculations and X-ray absorption spectroscopy (XAS). The combination and correlation of data from both experimental and theoretical studies has revealed for the first time that the catalytically active structure of our ternary nanowires can actually be ascribed to a PtAu∼Pd configuration, comprising a PtAu binary shell and a pure inner Pd core. Moreover, we have plausibly attributed the resulting structure to a specific synthesis step, namely the Cu underpotential deposition (UPD) followed by galvanic replacement with Pt. Hence, the fundamental insights gained into the performance of our ultrathin nanowires from our demonstrated approach will likely guide future directed efforts aimed at broadly improving upon the durability and stability of nanoscale electrocatalysts in general. PMID:26402364

  2. Insights into Regulated Ligand Binding Sites from the Structure of ZO-1 Src Homology 3-Guanylate Kinase Module

    Lye, Ming F.; Fanning, Alan S.; Su, Ying; Anderson, James M.; Lavie, Arnon (UNC); (UIC)

    2010-11-09

    Tight junctions are dynamic components of epithelial and endothelial cells that regulate the paracellular transport of ions, solutes, and immune cells. The assembly and permeability of these junctions is dependent on the zonula occludens (ZO) proteins, members of the membrane-associated guanylate kinase homolog (MAGUK) protein family, which are characterized by a core Src homology 3 (SH3)-GUK module that coordinates multiple protein-protein interactions. The structure of the ZO-1 SH3-GUK domain confirms that the interdependent folding of the SH3 and GUK domains is a conserved feature of MAGUKs, but differences in the orientation of the GUK domains in three different MAGUKs reveal interdomain flexibility of the core unit. Using pull-down assays, we show that an effector loop, the U6 region in ZO-1, forms a novel intramolecular interaction with the core module. This interaction is divalent cation-dependent and overlaps with the binding site for the regulatory molecule calmodulin on the GUK domain. These findings provide insight into the previously observed ability of the U6 region to regulate TJ assembly in vivo and the structural basis for the complex protein interactions of the MAGUK family.

  3. Mapping the active site of vaccinia virus RNA triphosphatase

    The RNA triphosphatase component of vaccinia virus mRNA capping enzyme (the product of the viral D1 gene) belongs to a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, Chlorella virus, and baculoviruses. The family is defined by two glutamate-containing motifs (A and C) that form the metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight-stranded β barrel (the so-called ''triphosphate tunnel''). Here we queried whether vaccinia virus capping enzyme is a member of the tunnel subfamily, via mutational mapping of amino acids required for vaccinia triphosphatase activity. We identified four new essential side chains in vaccinia D1 via alanine scanning and illuminated structure-activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight a constellation of six acidic and three basic amino acids that likely compose the vaccinia triphosphatase active site (Glu37, Glu39, Arg77, Lys107, Glu126, Asp159, Lys161, Glu192, and Glu194). These nine essential residues are conserved in all vertebrate and invertebrate poxvirus RNA capping enzymes. We discerned no pattern of clustering of the catalytic residues of the poxvirus triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). We infer that the poxvirus triphosphatases are a distinct lineage within the metal-dependent RNA triphosphatase family. Their unique active site, which is completely different from that of the host cell's capping enzyme, recommends the poxvirus RNA triphosphatase as a molecular target for antipoxviral drug discovery

  4. Genome wide transcription start sites analysis of Xanthomonas campestris pv. campestris B100 with insights into the gum gene cluster directing the biosynthesis of the exopolysaccharide xanthan.

    Alkhateeb, Rabeaa S; Vorhölter, Frank-Jörg; Rückert, Christian; Mentz, Almut; Wibberg, Daniel; Hublik, Gerd; Niehaus, Karsten; Pühler, Alfred

    2016-05-10

    Xanthomonas campestris pv. campestris (Xcc) is the major producer of the exopolysaccharide xanthan, the commercially most important natural polysaccharide of microbial origin. The current work provides deeper insights into the yet uncharacterized transcriptomic features of the xanthan producing strain Xcc-B100. Towards this goal, RNA sequencing of a library based on the selective enrichment of the 5' ends of native transcripts was performed. This approach resulted in the genome wide identification of 3067 transcription start sites (TSSs) that were further classified based on their genomic positions. Among them, 1545 mapped upstream of an actively transcribed CDS and 1363 were classified as novel TSSs representing antisense, internal, and TSSs belonging to previously unidentified genomic features. Analyzing the transcriptional strength of primary and antisense TSSs revealed that in some instances antisense transcription seemed to be initiated at a higher level than its sense counterpart. Mapping the exact positions of TSSs aided in the identification of promoter consensus motifs, ribosomal binding sites, and enhanced the genome annotation of 159 in silico predicted translational start (TLS) sites. The global view on length distribution of the 5' untranslated regions (5'-UTRs) deduced from the data pointed to the occurrence of leaderless transcripts and transcripts with unusually long 5'-UTRs, in addition to identifying seven putative riboswitch elements for Xcc-B100. Concerning the biosynthesis of xanthan, we focused on the transcriptional organization of the gum gene cluster. Under the conditions tested, we present evidence for a complex transcription pattern of the gum genes with multiple TSSs and an obvious considerable role of antisense transcription. The gene gumB, encoding an outer membrane xanthan exporter, is presented here as an example for genes that possessed a strong antisense TSS. PMID:26975844

  5. Decommissioning and decontamination activity, Gnome Site, Eddy County, New Mexico

    The purpose of this assessment is to present a brief description of the proposed activity and its potential impacts on the environment. This assessment will constitute an evaluation as to whether or not a formal Environmental Statement need be prepared. As background to the proposed activity, Project Gnome was an underground nuclear test conducted in December 1961 as part of the PLOWSHARE Program. The project site is located about 25 miles southeast of Carlsbad, New Mexico. By means of an excavated shaft and tunnel, a 3-kiloton nuclear explosive was emplaced and detonated in a salt bed about 1200 feet below the surface. The uncontaminated rock and salt muck from the original excavation and subsequent contaminated muck and minor construction debris from reentry activities into the nuclear cavity is commingled and stored in a pile near the Gnome/Coach Shaft. Other areas on the site are known to have been contaminated. In 1969, a program was conducted to cleanup and dispose of all surface contamination to whatever depth it occurred in excess of 0.1 mR/hr. Contaminated materials and soil were collected and disposed into the Gnome shaft, which was filled and sealed. Since then, NV has proposed to DOE/HQ much lower criteria for residual radioactive contamination for the Gnome Site. These proposed criteria were to collect and dispose of surficial materials which contain more than 2 x 10-5 microcuries per gram of soil for beta/gamma emitters and 3 x 10-2 microcuries per milliliter of tritium in soil moisture. According to the latest reconnaissance in 1972, low concentrations of Cs-137, Sr-90 and tritium were present at various locations on the site in excess of these proposed guidelines. Other operational areas within the site are suspected of containing radioactive contamination in much lesser volume, which are to be determined by careful probing and monitoring, as described in the next section

  6. Methodology for contaminated sites of military activity territories restoration

    Major part of Eastern Europe countries meet environmental problems related to sites of military activity. Major part of these sites is characterised with degradation of natural landscapes and contamination of geological environment with toxic and hazardous waste representing actual and potential danger for population and environment. Actual danger is caused with localisation of toxic waste, hazardous materials and waste which are preventing normal land use. Potential danger is related to successive dispersion of contamination in biosphere as well as origin of new derivatives and products having toxic and hazardous properties. The list of such sites and objects comprises bases of land, air and naval forces. These objects include a network of infrastructures: storages of fuels and lubricants (surface, underground), filling stations, pipe lines, reparation stations, garages, decontamination stations, underground storages of different purposes, depots (for ammunition, chemical products), hospitals, constructions, firing grounds (tank, artillery, aircraft bombing etc.) and waste disposal sites. Special programs aimed at military industries and bases contaminated sites remediation have been carrying out in developed countries (USA, United Kingdom, Germany etc.). This experience was used in the frames of joint programs having been founded in several countries of Central and Eastern Europe (Chesh Republic, Slovakia, Lithuania etc.). (author)

  7. Exploiting Innocuous Activity for Correlating Users Across Sites

    Goga, Oana; Lei, Howard; Parthasarathi, Sree Hari Krishnan; Friedland, Gerald; Sommer, Robin; Teixeira, Renata

    2013-01-01

    International audience We study how potential attackers can identify accounts on different social network sites that all belong to the same user, exploiting only innocuous activity that inherently comes with posted content. We examine three specific features on Yelp, Flickr, and Twitter: the geo-location attached to a user's posts, the timestamp of posts, and the user's writing style as captured by language models. We show that among these three features the location of posts is the most po...

  8. Active sites in char gasification: Final technical report

    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.

  9. Resolving the Structure of Active Sites on Platinum Catalytic Nanoparticles

    Chang, Lan Yun; Barnard, Amanda S.; Gontard, Lionel Cervera; Dunin-Borkowski, Rafal E.

    2010-01-01

    Accurate understanding of the structure of active sites is fundamentally important in predicting catalytic properties of heterogeneous nanocatalysts. We present an accurate determination of both experimental and theoretical atomic structures of surface monatomic steps on industrial platinum...... nanoparticles. This comparison reveals that the edges of nanoparticles can significantly alter the atomic positions of monatomic steps in their proximity, which can lead to substantial deviations in the catalytic properties compared with the extended surfaces....

  10. The purification of affinity-labelled active-site peptides

    The isolation of the labelled peptide from the protein digest, following the affinity labelling of the active sites of enzymes or antibodies, is described. Single-step affinity chromatography utilises the affinity of the native enzymes or antibody for the ligand used to label the same protein. The labelled peptide is the only one in the digest that displays affinity for the immobilised protein and can be released with eluants that dissociate the protein-ligand complex. (Auth.)

  11. Structural Insights into the Activation of Human Relaxin Family Peptide Receptor 1 by Small-Molecule Agonists.

    Hu, Xin; Myhr, Courtney; Huang, Zaohua; Xiao, Jingbo; Barnaeva, Elena; Ho, Brian A; Agoulnik, Irina U; Ferrer, Marc; Marugan, Juan J; Southall, Noel; Agoulnik, Alexander I

    2016-03-29

    The GPCR relaxin family peptide receptor 1 (RXFP1) mediates the action of relaxin peptide hormone, including its tissue remodeling and antifibrotic effects. The peptide has a short half-life in plasma, limiting its therapeutic utility. However, small-molecule agonists of human RXFP1 can overcome this limitation and may provide a useful therapeutic approach, especially for chronic diseases such as heart failure and fibrosis. The first small-molecule agonists of RXFP1 were recently identified from a high-throughput screening, using a homogeneous cell-based cAMP assay. Optimization of the hit compounds resulted in a series of highly potent and RXFP1 selective agonists with low cytotoxicity, and excellent in vitro ADME and pharmacokinetic properties. Here, we undertook extensive site-directed mutagenesis studies in combination with computational modeling analysis to probe the molecular basis of the small-molecule binding to RXFP1. The results showed that the agonists bind to an allosteric site of RXFP1 in a manner that closely interacts with the seventh transmembrane domain (TM7) and the third extracellular loop (ECL3). Several residues were determined to play an important role in the agonist binding and receptor activation, including a hydrophobic region at TM7 consisting of W664, F668, and L670. The G659/T660 motif within ECL3 is crucial to the observed species selectivity of the agonists for RXFP1. The receptor binding and activation effects by the small molecule ML290 were compared with the cognate ligand, relaxin, providing valuable insights on the structural basis and molecular mechanism of receptor activation and selectivity for RXFP1. PMID:26866459

  12. Insight into the mechanism of biological methanol activation based on the crystal structure of the methanol-cobalamin methyltransferase complex.

    Hagemeier, Christoph H; Krer, Markus; Thauer, Rudolf K; Warkentin, Eberhard; Ermler, Ulrich

    2006-12-12

    Some methanogenic and acetogenic microorganisms have the catalytic capability to cleave heterolytically the C O bond of methanol. To obtain insight into the elusive enzymatic mechanism of this challenging chemical reaction we have investigated the methanol-activating MtaBC complex from Methanosarcina barkeri composed of the zinc-containing MtaB and the 5-hydroxybenzimidazolylcobamide-carrying MtaC subunits. Here we report the 2.5-A crystal structure of this complex organized as a (MtaBC)(2) heterotetramer. MtaB folds as a TIM barrel and contains a novel zinc-binding motif. Zinc(II) lies at the bottom of a funnel formed at the C-terminal beta-barrel end and ligates to two cysteinyl sulfurs (Cys-220 and Cys-269) and one carboxylate oxygen (Glu-164). MtaC is structurally related to the cobalamin-binding domain of methionine synthase. Its corrinoid cofactor at the top of the Rossmann domain reaches deeply into the funnel of MtaB, defining a region between zinc(II) and the corrinoid cobalt that must be the binding site for methanol. The active site geometry supports a S(N)2 reaction mechanism, in which the C O bond in methanol is activated by the strong electrophile zinc(II) and cleaved because of an attack of the supernucleophile cob(I)amide. The environment of zinc(II) is characterized by an acidic cluster that increases the charge density on the zinc(II), polarizes methanol, and disfavors deprotonation of the methanol hydroxyl group. Implications of the MtaBC structure for the second step of the reaction, in which the methyl group is transferred to coenzyme M, are discussed. PMID:17142327

  13. O2 activation by binuclear Cu sites: Noncoupled versus exchange coupled reaction mechanisms

    Chen, Peng; Solomon, Edward I.

    2004-09-01

    Binuclear Cu proteins play vital roles in O2 binding and activation in biology and can be classified into coupled and noncoupled binuclear sites based on the magnetic interaction between the two Cu centers. Coupled binuclear Cu proteins include hemocyanin, tyrosinase, and catechol oxidase. These proteins have two Cu centers strongly magnetically coupled through direct bridging ligands that provide a mechanism for the 2-electron reduction of O2 to a µ-2:2 side-on peroxide bridged species. This side-on bridged peroxo-CuII2 species is activated for electrophilic attack on the phenolic ring of substrates. Noncoupled binuclear Cu proteins include peptidylglycine -hydroxylating monooxygenase and dopamine -monooxygenase. These proteins have binuclear Cu active sites that are distant, that exhibit no exchange interaction, and that activate O2 at a single Cu center to generate a reactive CuII/O2 species for H-atom abstraction from the C-H bond of substrates. O2 intermediates in the coupled binuclear Cu enzymes can be trapped and studied spectroscopically. Possible intermediates in noncoupled binuclear Cu proteins can be defined through correlation to mononuclear CuII/O2 model complexes. The different intermediates in these two classes of binuclear Cu proteins exhibit different reactivities that correlate with their different electronic structures and exchange coupling interactions between the binuclear Cu centers. These studies provide insight into the role of exchange coupling between the Cu centers in their reaction mechanisms.

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

    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

  15. Site characterization techniques used in environmental remediation activities

    As a result of decades of nuclear energy research, weapons production, as well as ongoing operations, a significant amount of radioactive contamination has occurred throughout the United States Department of Energy (DOE) complex. DOE facility are in the process of assessing and potentially remediating various sites according to the regulations imposed by a Federal Facility Agreement and Consent order (FFA/CO) between DOE, the state in which the facility is located, and the U.S. Environmental Protection Agency (EPA). In support of these active site remediation efforts, the DOE has devoted considerable resources towards the development of innovative site characterization techniques that support environmental restoration activities. These resources and efforts have focused on various aspects of this complex problem. Research and technology development conducted at the Idaho National Engineering and Environmental Laboratory (INEEL) has resulted in the ability and state-of-the-art equipment required to obtain real-time, densely spaced, in situ characterization data (i.e. detection, speciation, and location) of various radionuclides and contaminants. The Remedial Action Monitoring System (RAMS), developed by the INEEL, consists of enhanced sensor technology, measurement modeling and interpretation techniques, and a suite of deployment platforms which can be interchanged to directly support remedial cleanup and site verification operations. In situ characterization techniques have advanced to the point where they are being actively deployed in support of remedial operations. The INEEL has deployed its system at various DOE and international sites. The deployment of in situ characterization systems during environmental restoration operations has shown that this approach results in several significant benefits versus conventional sampling techniques. A flexible characterization system permits rapid modification to satisfy physical site conditions, available site resources

  16. Seismic activity parameters of the Finnish potential repository sites

    Posiva Oy has started a project for estimating the possible earthquake induced rock movements on the deposition holes containing canisters of spent nuclear fuel. These estimates will be made for the four investigation sites, Romuvaara, Kivetty, Olkiluoto and Haestholmen. This study deals with the current and future seismicity associated with the above mentioned sites. Seismic belts that participate the seismic behaviour of the studied sites have been identified and the magnitude-frequency distributions of these belts have been estimated. The seismic activity parameters of the sites have been deduced from the characteristics of the seismic belts in order to forecast the seismicity during the next 100,000 years. The report discusses the possible earthquakes induced by future glaciation. The seismic interpretation seems to indicate that the previous postglacial faults in Finnish Lapland have been generated in compressional environment. The orientation of the rather uniform compression has been NW-SE, which coincide with the current stress field. It seems that, although the impact of postglacial crustal rebound must have been significant, the impact of plate tectonics has been dominant. A major assumption of this study has been that future seismicity will generally resemble the current seismicity. However, when the postglacial seismicity is concerned, the magnitude-frequency distribution is likely different and the expected maximum magnitude will be higher. Maximum magnitudes of future postglacial earthquakes have been approximated by strain release examinations. Seismicity has been examined within the framework of the lineament maps, in order to associate the future significant earthquakes with active fault zones in the vicinity of the potential repository sites. (orig.)

  17. Seismic activity parameters of the Finnish potential repository sites

    Saari, J. [Fortum Engineering Oy, Vantaa (Finland)

    2000-10-01

    Posiva Oy has started a project for estimating the possible earthquake induced rock movements on the deposition holes containing canisters of spent nuclear fuel. These estimates will be made for the four investigation sites, Romuvaara, Kivetty, Olkiluoto and Haestholmen. This study deals with the current and future seismicity associated with the above mentioned sites. Seismic belts that participate the seismic behaviour of the studied sites have been identified and the magnitude-frequency distributions of these belts have been estimated. The seismic activity parameters of the sites have been deduced from the characteristics of the seismic belts in order to forecast the seismicity during the next 100,000 years. The report discusses the possible earthquakes induced by future glaciation. The seismic interpretation seems to indicate that the previous postglacial faults in Finnish Lapland have been generated in compressional environment. The orientation of the rather uniform compression has been NW-SE, which coincide with the current stress field. It seems that, although the impact of postglacial crustal rebound must have been significant, the impact of plate tectonics has been dominant. A major assumption of this study has been that future seismicity will generally resemble the current seismicity. However, when the postglacial seismicity is concerned, the magnitude-frequency distribution is likely different and the expected maximum magnitude will be higher. Maximum magnitudes of future postglacial earthquakes have been approximated by strain release examinations. Seismicity has been examined within the framework of the lineament maps, in order to associate the future significant earthquakes with active fault zones in the vicinity of the potential repository sites. (orig.)

  18. 77 FR 40638 - Syniverse Technologies, Inc., Including On-Site Leased Workers From Insight Global Stone Staffing...

    2012-07-10

    ... 19, 2012 (Vol. 77, No. 76 FR 23510). At the request of State Workforce Office, the Department... Insight Global Stone Staffing, and Randstad Formerly Known as Sapphire Technologies, Watertown, MA... workers from Insight Global, Stone Staffing, Randstad formerly known as Sapphire Technologies,...

  19. Design Insights for Tuning the Electrocatalytic Activity of Perovskite Oxides for the Oxygen Evolution Reaction

    Malkhandi, S; Trinh, P; Manohar, AK; Manivannan, A; Balasubramanian, M; Prakash, GKS; Narayanan, SR

    2015-04-16

    Rechargeable metal-air batteries and water electrolyzers based on aqueous alkaline electrolytes hold the potential to be sustainable solutions to address the challenge of storing large amounts of electrical energy generated from solar and wind resources. For these batteries and electrolyzers to be economically viable, it is essential to have efficient, durable, and inexpensive electrocatalysts for the oxygen evolution reaction. In this article, we describe new insights for predicting and tuning the activity of inexpensive transition metal oxides for designing efficient and inexpensive electrocatalysts. We have focused on understanding the factors determining the electrocatalytic activity for oxygen evolution in a strong alkaline medium. To this end, we have conducted a systematic investigation of nanophase calcium-doped lanthanum cobalt manganese oxide, an example of a mixed metal oxide that can be tuned for its electrocatalytic activity by varying the transition metal composition. Using X-ray absorption spectroscopy (XANES), X-ray photoelectron spectroscopy (XPS), electrochemical polarization experiments, and analysis of mechanisms, we have identified the key determinants of electrocatalytic activity. We have found that the Tafel slopes are determined by the oxidation states and the bond energy of the surface intermediates of Mn-OH and Co-OH bonds while the catalytic activity increased with the average d-electron occupancy of the sigma* orbital of the M-OH bond. We anticipate that such understanding will be very useful in predicting the behavior of other transition metal oxide catalysts.

  20. Oxygen reduction and evolution at single-metal active sites

    Calle-Vallejo, F.; Martínez, J.I.; García Lastra, Juan Maria;

    2013-01-01

    A worldwide spread of clean technologies such as low-temperature fuel cells and electrolyzers depends strictly on their technical reliability and economic affordability. Currently, both conditions are hardly fulfilled mainly due to the same reason: the oxygen electrode, which has large overpotent......A worldwide spread of clean technologies such as low-temperature fuel cells and electrolyzers depends strictly on their technical reliability and economic affordability. Currently, both conditions are hardly fulfilled mainly due to the same reason: the oxygen electrode, which has large...... overpotentials and is made of precious materials. A possible solution is the use of non-noble electrocatalysts with single-metal active sites. Here, on the basis of DFT calculations of adsorbed intermediates and a thermodynamic analysis, we compare the oxygen reduction (ORR) and evolution (OER) activities of...... functionalized graphitic materials and gas-phase porphyrins with late transition metals. We find that both kinds of materials follow approximately the same activity trends, and active sites with transition metals from groups 7 to 9 may be good ORR and OER electrocatalysts. However, spin analyses show more...

  1. Identification of residues involved in nucleotidyltransferase activity of JHP933 from helicobacter pyloriby site-directed mutagenesis

    Ye Xianren

    2016-01-01

    Full Text Available Helicobacter pylori is a well-known bacterial pathogen involved in the development of peptic ulcer, gastric adenocarcinoma and other forms of gastric cancer. Evidence has suggested that certain strain-specific genes in the plasticity region may play key roles in the pathogenesis of H. pylori-associated gastroduodenal diseases. Therefore there is considerable interest in the strain-specific genes located in the plasticity regions of H. pylori. JHP933 is encoded by the gene in the plasticity region of H. pylori strain J99. Recently, the crystal structure of JHP933 has confirmed it as a nucleotidyltransferase (NTase superfamily protein and a putative active site has been proposed. However, no evidence from direct functional assay has been presented to confirm the active site and little is known about the functional mechanism of JHP933. Here, through superimposition with Cid1/NTP complex structures, we modelled the complex structures of JHP933 with different NTPs. Based on the models and using rational site-directed mutagenesis combined with enzymatic activity assays, we confirm the active site and identify several residues important for the nucleotidyl transferring function of JHP933. Furthermore, mutations of these active site residues result in the abolishment of the nucleotidyltransferase activity of JHP933. This work provides preliminary insight into the molecular mechanism underlying the pathophysiological role in H. pylori infection of JHP933 as a novel NTase superfamily protein.

  2. HDAC Inhibitors without an Active Site Zn2+-Binding Group

    Vickers, Chris J.; Olsen, Christian Adam; Leman, Luke J.;

    2012-01-01

    Natural and synthetic histone deacetylase (HDAC) inhibitors generally derive their strong binding affinity and high potency from a key functional group that binds to the Zn2+ ion within the enzyme active site. However, this feature is also thought to carry the potential liability of undesirable off......-target interactions with other metalloenzymes. As a step toward mitigating this issue, here, we describe the design, synthesis, and structure−activity characterizations of cyclic α3β-tetrapeptide HDAC inhibitors that lack the presumed indispensable Zn2+-binding group. The lead compounds (e.g., 15 and 26) display good...... potency against class 1 HDACs and are active in tissue culture against various human cancer cell lines. Importantly, enzymological analysis of 26 indicates that the cyclic α3β-tetrapeptide is a fast-on/ off competitive inhibitor of HDACs 1−3 with Ki values of 49, 33, and 37 nM, respectively. Our proof...

  3. Crystal structures of yellowtail ascites virus VP4 protease: trapping an internal cleavage site trans acyl-enzyme complex in a native Ser/Lys dyad active site.

    Chung, Ivy Yeuk Wah; Paetzel, Mark

    2013-05-01

    Yellowtail ascites virus (YAV) is an aquabirnavirus that causes ascites in yellowtail, a fish often used in sushi. Segment A of the YAV genome codes for a polyprotein (pVP2-VP4-VP3), where processing by its own VP4 protease yields the capsid protein precursor pVP2, the ribonucleoprotein-forming VP3, and free VP4. VP4 protease utilizes the rarely observed serine-lysine catalytic dyad mechanism. Here we have confirmed the existence of an internal cleavage site, preceding the VP4/VP3 cleavage site. The resulting C-terminally truncated enzyme (ending at Ala(716)) is active, as shown by a trans full-length VP4 cleavage assay and a fluorometric peptide cleavage assay. We present a crystal structure of a native active site YAV VP4 with the internal cleavage site trapped as trans product complexes and trans acyl-enzyme complexes. The acyl-enzyme complexes confirm directly the role of Ser(633) as the nucleophile. A crystal structure of the lysine general base mutant (K674A) reveals the acyl-enzyme and empty binding site states of VP4, which allows for the observation of structural changes upon substrate or product binding. These snapshots of three different stages in the VP4 protease reaction mechanism will aid in the design of anti-birnavirus compounds, provide insight into previous site-directed mutagenesis results, and contribute to understanding of the serine-lysine dyad protease mechanism. In addition, we have discovered that this protease contains a channel that leads from the enzyme surface (adjacent to the substrate binding groove) to the active site and the deacylating water. PMID:23511637

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

    None

    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.

  5. Overview of the activities carried out at the FEBEX site

    Missana, T.; Buil, B.; Garralon, A.; Gomez, P. [CIEMAT, Dept. de Medioambien te, 28040 Madrid (Spain); Perez-Estaun, A.; Carbonell, R. [Inst. Jaume Almera, CSIC (Spain); Suso, J.; Carretero, G.; Bueno, J.; Martinez, L. [AITEMIN (Spain) ; Hernan, P. [ENRESA (Spain)

    2007-06-15

    One of the main aim of WP 4.1 and 4.2 is to study solute migration mechanisms in crystalline host-rock in realistic conditions. Many organisations are participating in a joint study that is being performed in the FEBEX gallery (NAGRA's Grimsel Test Site, GTS, Switzerland). The FEBEX experiment reproduces at a real scale a high-level waste repository in granite and was installed more than 9 years ago. At moment, it represents the most realistic environment where the processes affecting radionuclide migration from the bentonite to granite can be studied. This paper summarises the main activities carried out at the FEBEX site during the second year of the project.

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

    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

  7. The nature of inherent bactericidal activity: insights from the nanotopology of three species of dragonfly

    Mainwaring, David E.; Nguyen, Song Ha; Webb, Hayden; Jakubov, Timur; Tobin, Mark; Lamb, Robert N.; Wu, Alex H.-F.; Marchant, Richard; Crawford, Russell J.; Ivanova, Elena P.

    2016-03-01

    While insect wings are widely recognised as multi-functional, recent work showed that this extends to extensive bactericidal activity brought about by cell deformation and lysis on the wing nanotopology. We now quantitatively show that subtle changes to this topography result in substantial changes in bactericidal activity that are able to span an order of magnitude. Notably, the chemical composition of the lipid nanopillars was seen by XPS and synchrotron FTIR microspectroscopy to be similar across these activity differences. Modelling the interaction between bacterial cells and the wing surface lipids of 3 species of dragonflies, that inhabit similar environments, but with distinctly different behavioural repertoires, provided the relationship between surface structure and antibacterial functionality. In doing so, these principal behavioural patterns correlated with the demands for antimicrobial efficiency dictated by differences in their foraging strategies. This work now reveals a new feature in the design elegance of natural multi-functional surfaces as well providing insights into the bactericidal mechanism underlying inherently antimicrobial materials, while suggesting that nanotopology is related to the evolutionary development of a species through the demands of its behavioural repertoire. The underlying relationship between the processes of wetting, adhesion and capillarity of the lipid nanopillars and bactericidal efficiency suggests new prospects for purely mechano-responsive antibacterial surfaces.While insect wings are widely recognised as multi-functional, recent work showed that this extends to extensive bactericidal activity brought about by cell deformation and lysis on the wing nanotopology. We now quantitatively show that subtle changes to this topography result in substantial changes in bactericidal activity that are able to span an order of magnitude. Notably, the chemical composition of the lipid nanopillars was seen by XPS and synchrotron

  8. Short-term meditation modulates brain activity of insight evoked with solution cue

    Tang, Yi-Yuan; Cao, Chen; Deng, Yuqin; Wang, Yan; Xin, Xiu; Posner, Michael I.

    2015-01-01

    Meditation has been shown to improve creativity in some situation. However, little is known about the brain systems underling insight into a problem when the person fails to solve the problem. Here, we examined the neural correlation using Chinese Remote Association Test, as a measure of creativity. We provide a solution following the failure of the participant to provide one. We examine how meditation in comparison with relaxation influences the reaction of the participant to a correct solution. The event-related functional magnetic resonance imaging showed greater activity, mainly distributed in the right cingulate gyrus (CG), insula, putamen, inferior frontal gyrus (IFG), and the bilateral middle frontal gyrus (MFG), the inferior parietal lobule (IPL) and the superior temporal gyrus (STG). This pattern of activation was greater following 5 h of meditation training than the same amount of relaxation. Based on prior research, we speculate on the function of this pattern of brain activity: (i) CG may be involved in detecting conflict and breaking mental set, (ii) MFG/IFG may play an important role in restructuring of the problem representation, (iii) insula, IPL and STG may be associated with error detection, problem understanding or general attentive control and (iv) putamen may be activated by ‘Aha’ feeling. PMID:24532700

  9. Insights into properties of activated carbons prepared from different raw precursors by pyrophosphoric acid activation.

    Gao, Yuan; Yue, Qinyan; Gao, Baoyu

    2016-03-01

    Low-cost activated carbons (ACs) were prepared from four kinds of solid wastes: petroleum coke, Enteromorpha prolifera, lignin from papermaking black liquid and hair, by pyrophosphoric acid (H4P2O7) activation. Thermo-gravimetric analysis of the pyrolysis of H4P2O7-precursor mixtures implied that H4P2O7 had different influences on the pyrolysis behavior of the four raw materials. N2 adsorption/desorption isotherms, scanning electron microscopy, Fourier transform infrared spectroscopy and adsorption capacities for dyes were used to characterize the prepared activated carbons. AC derived from E. prolifera exhibited the highest surface area (1094m(2)/g) and maximum monolayer adsorption capacity for malachite green (1250mg/g). Kinetic studies showed that the experimental data were in agreement with the pseudo-second-order model. The adsorption isotherms were well described by the Langmuir isotherm model, indicating the adsorption of dye onto the ACs proceeded by monolayers. PMID:26969070

  10. Caught in the act: the crystal structure of cleaved cathepsin L bound to the active site of Cathepsin L.

    Sosnowski, Piotr; Turk, Dušan

    2016-04-01

    Cathepsin L is a ubiquitously expressed papain-like cysteine protease involved in the endosomal degradation of proteins and has numerous roles in physiological and pathological processes, such as arthritis, osteoporosis, and cancer. Insight into the specificity of cathepsin L is important for elucidating its physiological roles and drug discovery. To study interactions with synthetic ligands, we prepared a presumably inactive mutant and crystallized it. Unexpectedly, the crystal structure determined at 1.4 Å revealed that the cathepsin L molecule is cleaved, with the cleaved region trapped in the active site cleft of the neighboring molecule. Hence, the catalytic mutant demonstrated low levels of catalytic activity. PMID:26992470

  11. Insights and models from medical anthropology for understanding the healing activity of the Historical Jesus

    John J. Pilch

    1995-01-01

    Full Text Available This essay sketches a basic introdution to medical anthropology as a key to understanding and interpreting  the healing activity of the historical Jesus described in the gospels. It presents select literature, leading experts, fundamental concepts, and insights and models of special value to biblical specialists. Only a cross-cultural discipline like medical anthropology allows the investigator to  interpret texts and events from other cultures with respect for their distinctive cultural contexts in order to draw more appropriate conclusions and applications in other cultures. Applications to biblical texts are not included in this essay but may be found in other articles published by the author and listed in the bibliography.

  12. 10 CFR 63.16 - Review of site characterization activities. 2

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Review of site characterization activities. 2 63.16... site characterization activities. 2 2 In addition to the review of site characterization activities... investigation and site characterization, to allow early identification of potential licensing issues for...

  13. Identification of Essential Cannabinoid-binding Domains: STRUCTURAL INSIGHTS INTO EARLY DYNAMIC EVENTS IN RECEPTOR ACTIVATION*

    Shim, Joong-Youn; Bertalovitz, Alexander C.; Kendall, Debra A.

    2011-01-01

    The classical cannabinoid agonist HU210, a structural analog of (−)-Δ9-tetrahydrocannabinol, binds to brain cannabinoid (CB1) receptors and activates signal transduction pathways. To date, an exact molecular description of the CB1 receptor is not yet available. Utilizing the minor binding pocket of the CB1 receptor as the primary ligand interaction site, we explored HU210 binding using lipid bilayer molecular dynamics (MD) simulations. Among the potential ligand contact residues, we identifie...

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

    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

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

    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

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

    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.

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

    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

  18. Study the active site of flavonoid applying radiation chemistry

    Flavonoid are a large and important class of naturally occurring, low molecular weight benzo-γ-pyrone derivatives which are reported to have a myriad of biological activities, but the study on the active sites of flavonoids is still ambiguous. In this paper, rutin, quercetin and baicalin have been selected as model compounds. It is well known that rutin is used in inhibiting arteriosclerosis and baicalin is antibacterial and antiviral. They have similar basic structure, but their medicinal properties are so different, why? As most flavonoids contain carbonyl group, which can capture electron effectively, we predict that flavonoids can capture electron to form radical anion. The formation of anion radical may have influence on the mitochondrial electron transport chain. The difference in the ability of forming anion radical may cause the difference in their medicinal effects. (author)

  19. SITE-DIRECTED MUTAGENESIS OF PROPOSED ACTIVE-SITE RESIDUES OF PENICILLIN-BINDING PROTEIN-5 FROM ESCHERICHIA-COLI

    VANDERLINDEN, MPG; DEHAAN, L; DIDEBERG, O; KECK, W

    1994-01-01

    Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser(44)), Lys(47),

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

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

    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.

  1. The role of active site aromatic residues in substrate degradation by the human chitotriosidase.

    Eide, Kristine Bistrup; Stockinger, Linn Wilhelmsen; Lewin, Anna Sofia; Tøndervik, Anne; Eijsink, Vincent G H; Sørlie, Morten

    2016-02-01

    Human chitotriosidase (HCHT) is a glycoside hydrolase family 18 chitinase synthesized and secreted in human macrophages thought be an innate part of the human immune system. It consists of a catalytic domain with the (β/α)8 TIM barrel fold having a large area of solvent-exposed aromatic amino acids in the active site and an additional family 14 carbohydrate-binding module. To gain further insight into enzyme functionality, especially the effect of the active site aromatic residues, we expressed two variants with mutations in subsites on either side of the catalytic acid, subsite -3 (W31A) and +2 (W218A), and compared their catalytic properties on chitin and high molecular weight chitosans. Exchange of Trp to Ala in subsite -3 resulted in a 12-fold reduction in extent of degradation and a 20-fold reduction in kcat(app) on chitin, while the values are 5-fold and 10-fold for subsite +2. Moreover, aromatic residue mutation resulted in a decrease of the rate of chitosan degradation contrasting previous observations for bacterial family 18 chitinases. Interestingly, the presence of product polymers of 40 sugar moieties and higher starts to disappear already at 8% degradation for HCHT50-W31A. Such behavior contrast that of the wild type and HCHT-W218A and resembles the action of endo-nonprocessive chitinases. PMID:26621384

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

    Koh-Stenta X

    2015-12-01

    Full Text Available Xiaoying Koh-Stenta,1 Joma Joy,1 Si Fang Wang,1 Perlyn Zekui Kwek,1 John Liang Kuan Wee,1 Kah Fei Wan,2 Shovanlal Gayen,1 Angela Shuyi Chen,1 CongBao Kang,1 May Ann Lee,1 Anders Poulsen,1 Subhash G Vasudevan,3 Jeffrey Hill,1 Kassoum Nacro11Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR, Singapore; 2Novartis Institute for Tropical Diseases, Singapore; 3Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, SingaporeAbstract: 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.Keywords: flavivirus protease, small molecule optimization, covalent inhibitor, active site binding, pyrazole ester derivatives

  3. Maxey Flats low-level waste disposal site closure activities

    The Maxey Flats Radioactive Waste Disposal Facility in Fleming County, Kentucky is in the process of being closed. The facility opened for commercial business in the spring of 1963 and received approximately 4.75 million cubic feet of radioactive waste by the time it was closed in December of 1977. During fourteen years of operation approximately 2.5 million curies of by-product material, 240,000 kilograms of source material, and 430 kilograms of special nuclear material were disposed. The Commonwealth purchased the lease hold estate and rights in May 1978 from the operating company. This action was taken to stabilize the facility and prepare it for closure consisting of passive care and monitoring. To prepare the site for closure, a number of remedial activities had to be performed. The remediation activities implemented have included erosion control, surface drainage modifications, installation of a temporary plastic surface cover, leachate removal, analysis, treatment and evaporation, US DOE funded evaporator concentrates solidification project and their on-site disposal in an improved disposal trench with enhanced cover for use in a humid environment situated in a fractured geology, performance evaluation of a grout injection demonstration, USGS subsurface geologic investigation, development of conceptual closure designs, and finally being added to the US EPA National Priority List for remediation and closure under Superfund. 13 references, 3 figures

  4. Prediction of P53 mutants (multiple sites transcriptional activity based on structural (2D&3D properties.

    R Geetha Ramani

    Full Text Available Prediction of secondary site mutations that reinstate mutated p53 to normalcy has been the focus of intense research in the recent past owing to the fact that p53 mutants have been implicated in more than half of all human cancers and restoration of p53 causes tumor regression. However laboratory investigations are more often laborious and resource intensive but computational techniques could well surmount these drawbacks. In view of this, we formulated a novel approach utilizing computational techniques to predict the transcriptional activity of multiple site (one-site to five-site p53 mutants. The optimal MCC obtained by the proposed approach on prediction of one-site, two-site, three-site, four-site and five-site mutants were 0.775,0.341,0.784,0.916 and 0.655 respectively, the highest reported thus far in literature. We have also demonstrated that 2D and 3D features generate higher prediction accuracy of p53 activity and our findings revealed the optimal results for prediction of p53 status, reported till date. We believe detection of the secondary site mutations that suppress tumor growth may facilitate better understanding of the relationship between p53 structure and function and further knowledge on the molecular mechanisms and biological activity of p53, a targeted source for cancer therapy. We expect that our prediction methods and reported results may provide useful insights on p53 functional mechanisms and generate more avenues for utilizing computational techniques in biological data analysis.

  5. Insights into structure-activity relationship of GABAA receptor modulating coumarins and furanocoumarins.

    Singhuber, Judith; Baburin, Igor; Ecker, Gerhard F; Kopp, Brigitte; Hering, Steffen

    2011-10-01

    The coumarins imperatorin and osthole are known to exert anticonvulsant activity. We have therefore analyzed the modulation of GABA-induced chloride currents (I(GABA)) by a selection of 18 coumarin derivatives on recombinant α(1)β(2)γ(2S) GABA(A) receptors expressed in Xenopus laevis oocytes by means of the two-microelectrode voltage clamp technique. Osthole (EC(50)=14 ± 1 μM) and oxypeucedanin (EC(50)=25 ± 8 μM) displayed the highest efficiency with I(GABA) potentiation of 116 ± 4 % and 547 ± 56 %, respectively. I(GABA) enhancement by osthole and oxypeucedanin was not inhibited by flumazenil (1 μM) indicating an interaction with a binding site distinct from the benzodiazepine binding site. In general, prenyl residues are essential for the positive modulatory activity, while longer side chains or bulkier residues (e.g. geranyl residues) diminish I(GABA) modulation. Generation of a binary classification tree revealed the importance of polarisability, which is sufficient to distinguish actives from inactives. A 4-point pharmacophore model based on oxypeucedanin - comprising three hydrophobic and one aromatic feature - identified 6 out of 7 actives as hits. In summary, (oxy-)prenylated coumarin derivatives from natural origin represent new GABA(A) receptor modulators. PMID:21749864

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

    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

  7. Active site loop conformation regulates promiscuous activity in a lactonase from Geobacillus kaustophilus HTA426.

    Yu Zhang

    Full Text Available 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.

  8. Active serine involved in the stabilization of the active site loop in the Humicola lanuginosa lipase

    Peters, Günther H.j.; Svendsen, A.; Langberg, H.; Vind, J.; Patkar, S.A.; Toxvaerd, S.; Kinnunen, P.K.J.

    1998-01-01

    reveal that the hinges of the active site lid are more flexible in the wild-type Hll than in S146A. In contrast, larger fluctuations are observed in the middle region of the active site loop in S 146A than in Hll. These findings reveal that the single mutation (S146A) of the active site serine leads to...

  9. DNA topoisomerase II structures and anthracycline activity: insights into ternary complex formation.

    Dal Ben, D; Palumbo, M; Zagotto, G; Capranico, G; Moro, S

    2007-01-01

    DNA Topoisomerase II (Top2) is an essential nuclear enzyme that regulates the topological state of the DNA, and a target of very effective anticancer drugs including anthracycline antibiotics. Even though several aspects of drug activity against Top2 are understood, the drug receptor site is not yet known. Several Top2 mutants have altered drug sensitivity and have provided information of structural features determining drug action. Here, we have revised the published crystal structures of eukaryotic and prokaryotic Top2s and relevant biochemical investigations of enzyme activity and anthracycline action. In particular, we have considered Top2 mutations conferring resistance to anthracyclines and related agents. Following a previous study (Moro et al, Biochemistry, 2004; 43: 7503-13), we have then re-built a molecular model of the entire enzyme in complex with DNA after the cleavage reaction, and used it to define the receptor site of anthracyclines. The results suggest a model wherein the drug specifically contacts the cleaved DNA as well as amino acid residues of the enzyme CAP-like domain. The findings can explain several established structure-activity relationships of antitumour anthracyclines, and provide a framework for further developments of effective Top2 poison. PMID:17897022

  10. Structural insight into activity enhancement and inhibition of H64A carbonic anhydrase II by imidazoles

    Mayank Aggarwal

    2014-03-01

    Full Text Available Human carbonic anhydrases (CAs are zinc metalloenzymes that catalyze the hydration and dehydration of CO2 and HCO3−, respectively. The reaction follows a ping-pong mechanism, in which the rate-limiting step is the transfer of a proton from the zinc-bound solvent (OH−/H2O in/out of the active site via His64, which is widely believed to be the proton-shuttling residue. The decreased catalytic activity (∼20-fold lower with respect to the wild type of a variant of CA II in which His64 is replaced with Ala (H64A CA II can be enhanced by exogenous proton donors/acceptors, usually derivatives of imidazoles and pyridines, to almost the wild-type level. X-ray crystal structures of H64A CA II in complex with four imidazole derivatives (imidazole, 1-methylimidazole, 2-methylimidazole and 4-methylimidazole have been determined and reveal multiple binding sites. Two of these imidazole binding sites have been identified that mimic the positions of the `in' and `out' rotamers of His64 in wild-type CA II, while another directly inhibits catalysis by displacing the zinc-bound solvent. The data presented here not only corroborate the importance of the imidazole side chain of His64 in proton transfer during CA catalysis, but also provide a complete structural understanding of the mechanism by which imidazoles enhance (and inhibit when used at higher concentrations the activity of H64A CA II.

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

    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

  12. A New Activity of Anti-HIV and Anti-tumor Protein GAP31: DNA Adenosine Glycosidase – Structural and Modeling Insight into its Functions

    Li, H.; Huang, P; Zhang, D; Sun, Y; Chen, H; Zhang, J; Huang, P; Kong, X; Lee-Huang, S

    2010-01-01

    We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.

  13. A new activity of anti-HIV and anti-tumor protein GAP31: DNA adenosine glycosidase - Structural and modeling insight into its functions

    Li, Hui-Guang [Department of Biochemistry, New York University School of Medicine, New York, NY 10016 (United States); Huang, Philip L. [American Biosciences, Boston, MA 02114 (United States); Zhang, Dawei; Sun, Yongtao [Department of Biochemistry, New York University School of Medicine, New York, NY 10016 (United States); Chen, Hao-Chia [Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892 (United States); Zhang, John [Department of Chemistry, New York University, New York, NY 10003 (United States); Huang, Paul L. [Department of Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114 (United States); Kong, Xiang-Peng, E-mail: xiangpeng.kong@med.nyu.edu [Department of Biochemistry, New York University School of Medicine, New York, NY 10016 (United States); Lee-Huang, Sylvia, E-mail: sylvia.lee-huang@med.nyu.edu [Department of Biochemistry, New York University School of Medicine, New York, NY 10016 (United States)

    2010-01-01

    We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.

  14. A new activity of anti-HIV and anti-tumor protein GAP31: DNA adenosine glycosidase - Structural and modeling insight into its functions

    We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.

  15. Synthesis, Purification and Characterization of Ferredoxins with Re-Designed Active Sites

    Kristensen, Jytte

    Iron-sulfur proteins with cuboidal [Fe4S4] clusters exhibit a remarkable functional diversity. Insights on the factors determining the function of the protein can be obtained by modifications of the metal site by incorporation of metals other than iron in the active site of the protein. This......, purification and characteri¬za¬tion of the two new proteins were carried out. The P. furiosus ferredoxin was studied as a reference for the two artificial proteins. The P. furiosus [Fe3S4] and [Fe4S4] ferre¬doxins were studied with cyclic voltam¬¬me¬try as re¬fe¬rence for the work on the artificial proteins....... The effects of different buffer systems and additives were tested to find the optimal conditions for electro¬chemi¬cal charac¬te¬ri¬za¬tion. Different buffer systems did not have a significant effect, but the voltammo¬grams were strongly dependent on the NaCl content as NaCl had an attenua¬ting effect...

  16. Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator

    Deng, Zengqin; Wang, Qing; Liu, Zhao; Zhang, Manfeng; Machado, Ana Carolina Dantas; Chiu, Tsu-Pei; Feng, Chong; Zhang, Qi; Yu, Lin; Qi, Lei; Zheng, Jiangge; Wang, Xu; Huo, Xinmei; Qi, Xiaoxuan; Li, Xiaorong; Wu, Wei; Rohs, Remo; Li, Ying; Chen, Zhongzhou

    2015-07-01

    Ferric uptake regulator (Fur) plays a key role in the iron homeostasis of prokaryotes, such as bacterial pathogens, but the molecular mechanisms and structural basis of Fur-DNA binding remain incompletely understood. Here, we report high-resolution structures of Magnetospirillum gryphiswaldense MSR-1 Fur in four different states: apo-Fur, holo-Fur, the Fur-feoAB1 operator complex and the Fur-Pseudomonas aeruginosa Fur box complex. Apo-Fur is a transition metal ion-independent dimer whose binding induces profound conformational changes and confers DNA-binding ability. Structural characterization, mutagenesis, biochemistry and in vivo data reveal that Fur recognizes DNA by using a combination of base readout through direct contacts in the major groove and shape readout through recognition of the minor-groove electrostatic potential by lysine. The resulting conformational plasticity enables Fur binding to diverse substrates. Our results provide insights into metal ion activation and substrate recognition by Fur that suggest pathways to engineer magnetotactic bacteria and antipathogenic drugs.

  17. 10 CFR 60.18 - Review of site characterization activities. 2

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Review of site characterization activities. 2 60.18... IN GEOLOGIC REPOSITORIES Licenses Preapplication Review § 60.18 Review of site characterization activities. 2 2 In addition to the review of site characterization activities specified in this section,...

  18. Hazardous Material Storage Facilities and Sites - WASTE_SOLID_ACTIVE_PERMITTED_IDEM_IN: Active Permitted Solid Waste Sites in Indiana (Indiana Department of Environmental Management, Point Shapefile)

    NSGIC GIS Inventory (aka Ramona) — WASTE_SOLID_ACTIVE_PERMITTED_IDEM_IN is a point shapefile that contains active permitted solid waste site locations in Indiana, provided by personnel of Indiana...

  19. Carboxylation and Decarboxylation of Active Site Lys 84 Controls the Activity of OXA-24 β-Lactamase of Acinetobacter baumannii: Raman Crystallographic and Solution Evidence

    Che, Tao; Robert A Bonomo; Shanmugam, Sivaprakash; Bethel, Christopher R.; Pusztai-Carey, Marianne; Buynak, John D.; Carey, Paul R.

    2012-01-01

    The class D β-lactamases are characterized by the presence of a carboxylated lysine in the active site that participates in catalysis. Found in Acinetobacter baumannii, OXA-24 is a class D carbapenem hydrolyzing enzyme that exhibits resistance to most available β-lactamase inhibitors. In this study, the reaction between a 6-alkylidiene penam sulfone inhibitor, SA-1-204, in single crystals of OXA-24 is followed by Raman microscopy. Details of its reaction with SA-1-204 provide insight into the...

  20. XPD Helicase Structures And Activities: Insights Into the Cancer And Aging Phenotypes From XPD Mutations

    Fan, L.; Fuss, J.O.; Cheng, Q.J.; Arvai, A.S.; Hammel, M.; Roberts, V.A.; Cooper, P.K.; Tainer, J.A.

    2009-05-18

    Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

  1. XPD Helicase Structures and Activities: Insights into the Cancer and Aging Phenotypes from XPD Mutations

    Tainer, John; Fan, Li; Fuss, Jill O.; Cheng, Quen J.; Arvai, Andrew S.; Hammel, Michal; Roberts, Victoria A.; Cooper, Priscilla K.; Tainer, John A.

    2008-06-02

    Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.

  2. Snapshots of DNA polymerase processing aberrant substrates : Structural insights into abasic site bypass and polymerization of 5-alkynylated nucleotide analogs

    Obeid, Samra

    2011-01-01

    DNA polymerases are involved in all DNA synthesis events occurring in nature. Therefore, these enzymes are essential for the maintenance of the genetic information and its stability. Structural and functional studies have added significantly to our understanding of the basic mechanisms of DNA synthesis by DNA polymerases. Thereby, X-ray crystallography has become a powerful tool to gain further insights into structure-function relationships of DNA polymerases. Based on crystal structure analy...

  3. Origin of magnetic highs at ultramafic hosted hydrothermal systems: Insights from the Yokoniwa site of Central Indian Ridge

    Fujii, Masakazu; Okino, Kyoko; Sato, Taichi; Sato, Hiroshi; Nakamura, Kentaro

    2016-05-01

    High-resolution vector magnetic measurements were performed on an inactive ultramafic-hosted hydrothermal vent field, called Yokoniwa Hydrothermal Field (YHF), using a deep-sea manned submersible Shinkai6500 and an autonomous underwater vehicle r2D4. The YHF has developed at a non-transform offset massif of the Central Indian Ridge. Dead chimneys were widely observed around the YHF along with a very weak venting of low-temperature fluids so that hydrothermal activity of the YHF was almost finished. The distribution of crustal magnetization from the magnetic anomaly revealed that the YHF is associated with enhanced magnetization, as seen at the ultramafic-hosted Rainbow and Ashadze-1 hydrothermal sites of the Mid-Atlantic Ridge. The results of rock magnetic analysis on seafloor rock samples (including basalt, dolerite, gabbro, serpentinized peridotite, and hydrothermal sulfide) showed that only highly serpentinized peridotite carries high magnetic susceptibility and that the natural remanent magnetization intensity can explain the high magnetization of Yokoniwa. These observations reflect abundant and strongly magnetized magnetite grains within the highly serpentinized peridotite. Comparisons with the Rainbow and Ashadze-1 suggest that in ultramafic-hosted hydrothermal systems, strongly magnetized magnetite and pyrrhotite form during the progression of hydrothermal alteration of peridotite. After the completion of serpentinization and production of hydrogen, pyrrhotites convert into pyrite or nonmagnetic iron sulfides, which considerably reduces their levels of magnetization. Our results revealed origins of the magnetic high and the development of subsurface chemical processes in ultramafic-hosted hydrothermal systems. Furthermore, the results highlight the use of near-seafloor magnetic field measurements as a powerful tool for detecting and characterizing seafloor hydrothermal systems.

  4. Identification of essential cannabinoid-binding domains: structural insights into early dynamic events in receptor activation.

    Shim, Joong-Youn; Bertalovitz, Alexander C; Kendall, Debra A

    2011-09-23

    The classical cannabinoid agonist HU210, a structural analog of (-)-Δ(9)-tetrahydrocannabinol, binds to brain cannabinoid (CB1) receptors and activates signal transduction pathways. To date, an exact molecular description of the CB1 receptor is not yet available. Utilizing the minor binding pocket of the CB1 receptor as the primary ligand interaction site, we explored HU210 binding using lipid bilayer molecular dynamics (MD) simulations. Among the potential ligand contact residues, we identified residues Phe-174(2.61), Phe-177(2.64), Leu-193(3.29), and Met-363(6.55) as being critical for HU210 binding by mutational analysis. Using these residues to guide the simulations, we determined essential cannabinoid-binding domains in the CB1 receptor, including the highly sought after hydrophobic pocket important for the binding of the C3 alkyl chain of classical and nonclassical cannabinoids. Analyzing the simulations of the HU210-CB1 receptor complex, the CP55940-CB1 receptor complex, and the (-)-Δ(9)-tetrahydrocannabinol-CB1 receptor complex, we found that the positioning of the C3 alkyl chain and the aromatic stacking between Trp-356(6.48) and Trp-279(5.43) is crucial for the Trp-356(6.48) rotamer change toward receptor activation through the rigid-body movement of H6. The functional data for the mutant receptors demonstrated reductions in potency for G protein activation similar to the reductions seen in ligand binding affinity for HU210. PMID:21795705

  5. Detection limit for activation measurements in ultralow background sites

    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.

  6. Insight into temperature dependence of GTPase activity in human guanylate binding protein-1.

    Anjana Rani

    Full Text Available Interferon-γ induced human guanylate binding protein-1(hGBP1 belongs to a family of dynamin related large GTPases. Unlike all other GTPases, hGBP1 hydrolyzes GTP to a mixture of GDP and GMP with GMP being the major product at 37°C but GDP became significant when the hydrolysis reaction was carried out at 15°C. The hydrolysis reaction in hGBP1 is believed to involve with a number of catalytic steps. To investigate the effect of temperature in the product formation and on the different catalytic complexes of hGBP1, we carried out temperature dependent GTPase assays, mutational analysis, chemical and thermal denaturation studies. The Arrhenius plot for both GDP and GMP interestingly showed nonlinear behaviour, suggesting that the product formation from the GTP-bound enzyme complex is associated with at least more than one step. The negative activation energy for GDP formation and GTPase assay with external GDP together indicate that GDP formation occurs through the reversible dissociation of GDP-bound enzyme dimer to monomer, which further reversibly dissociates to give the product. Denaturation studies of different catalytic complexes show that unlike other complexes the free energy of GDP-bound hGBP1 decreases significantly at lower temperature. GDP formation is found to be dependent on the free energy of the GDP-bound enzyme complex. The decrease in the free energy of this complex at low temperature compared to at high is the reason for higher GDP formation at low temperature. Thermal denaturation studies also suggest that the difference in the free energy of the GTP-bound enzyme dimer compared to its monomer plays a crucial role in the product formation; higher stability favours GMP but lower favours GDP. Thus, this study provides the first thermodynamic insight into the effect of temperature in the product formation of hGBP1.

  7. Finnsjoen study site. Scope of activities and main results

    The Finnsjoen study site was selected in 1977 to provide input to the KBS-1 and KBS-2 performance assessments. The site was later used as a test site for testing new instruments and new site characterization methods, as well as a research site for studying mainly groundwater flow and groundwater transport. All together, the Finnsjoen studies have involved 11 cored boreholes, down to max 700 m depth, and extensive borehole geophysical, geochemical and geohydraulic measurements, as well as rock stress measurements and tracer tests. This report presents the scope of the Finnsjoen studies together with main results. Conceptual uncertainties in assumptions and models are discussed with emphasis on the models used for the performance assessment SKB91. Of special interest for the Finnsjoen study site is the strong influence caused by a subhorizontal fracture zone on groundwater flow, transport and chemistry

  8. Recommended survey designs for occupancy modelling using motion-activated cameras: insights from empirical wildlife data

    Graeme Shannon

    2014-08-01

    Full Text Available Motion-activated cameras are a versatile tool that wildlife biologists can use for sampling wild animal populations to estimate species occurrence. Occupancy modelling provides a flexible framework for the analysis of these data; explicitly recognizing that given a species occupies an area the probability of detecting it is often less than one. Despite the number of studies using camera data in an occupancy framework, there is only limited guidance from the scientific literature about survey design trade-offs when using motion-activated cameras. A fuller understanding of these trade-offs will allow researchers to maximise available resources and determine whether the objectives of a monitoring program or research study are achievable. We use an empirical dataset collected from 40 cameras deployed across 160 km2 of the Western Slope of Colorado, USA to explore how survey effort (number of cameras deployed and the length of sampling period affects the accuracy and precision (i.e., error of the occupancy estimate for ten mammal and three virtual species. We do this using a simulation approach where species occupancy and detection parameters were informed by empirical data from motion-activated cameras. A total of 54 survey designs were considered by varying combinations of sites (10–120 cameras and occasions (20–120 survey days. Our findings demonstrate that increasing total sampling effort generally decreases error associated with the occupancy estimate, but changing the number of sites or sampling duration can have very different results, depending on whether a species is spatially common or rare (occupancy = ψ and easy or hard to detect when available (detection probability = p. For rare species with a low probability of detection (i.e., raccoon and spotted skunk the required survey effort includes maximizing the number of sites and the number of survey days, often to a level that may be logistically unrealistic for many studies. For common

  9. Accelerator production of tritium activities at the Savannah River Site

    The Savannah River site (SRS) has been chosen as the site to host the accelerator production of tritium (APT). This facility, which will produce tritium for national defense purposes, is a natural extension of the site's original mission. All of the tritium for U.S. weapons needs has been produced at SRS in the heavy water reactors (now shut down) that operated until 1988. Much of the tritium-handling infrastructure still exists at SRS, and the tritium recycling and purification facilities are new and fully operational. This paper summarizes the reasons for the choice of the site and describes some of the early SRS efforts to learn the new technology, to weave into it a strong operations/production ethic, and to prepare the site to host the APT

  10. Health physics and public health activities at hazardous wastes sites

    The Agency for Toxic Substances and Disease Registry (ATSDR) has worked with the U.S. Environmental Protection Agency (EPA) at several sites contaminated with radioactive materials. The Navajo Brown Vandever (B-V) uranium mine site near Bluewater, New Mexico, and the Austin Avenue Radiation Site (AAR) in Lansdowne, Pennsylvania were the subject of ATSDR health advisories. The sites were contamined with uranium or uranium byproducts but the identification of potential health effects and actions taken to prevent or reduce exposures were approached from different perspectives. At B-V contaminants included uranium and mine tailings, radium, and radon. Contaminants at the site and physical hazards were removed. At AAR, radium and radon were located in residential settings. Residents who might have had annual exposures greater than accepted standards or recommendations were relocated and contaminated building demolished

  11. Pathway activation profiling reveals new insights into Age-related Macular Degeneration and provides avenues for therapeutic interventions

    Makarev, Evgeny; Cantor, Charles; Zhavoronkov, Alex; Buzdin, Anton; Aliper, Alexander; Csoka, Antonei Benjamin

    2014-01-01

    Age-related macular degeneration (AMD) is a major cause of blindness in older people and is caused by loss of the central region of the retinal pigment epithelium (RPE). Conventional methods of gene expression analysis have yielded important insights into AMD pathogenesis, but the precise molecular pathway alterations are still poorly understood. Therefore we developed a new software program, “AMD Medicine”, and discovered differential pathway activation profiles in samples of human RPE/choro...

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

    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

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

    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

  14. SET7/9 catalytic mutants reveal the role of active site water molecules in lysine multiple methylation.

    Del Rizzo, Paul A; Couture, Jean-François; Dirk, Lynnette M A; Strunk, Bethany S; Roiko, Marijo S; Brunzelle, Joseph S; Houtz, Robert L; Trievel, Raymond C

    2010-10-01

    SET domain lysine methyltransferases (KMTs) methylate specific lysine residues in histone and non-histone substrates. These enzymes also display product specificity by catalyzing distinct degrees of methylation of the lysine ε-amino group. To elucidate the molecular mechanism underlying this specificity, we have characterized the Y245A and Y305F mutants of the human KMT SET7/9 (also known as KMT7) that alter its product specificity from a monomethyltransferase to a di- and a trimethyltransferase, respectively. Crystal structures of these mutants in complex with peptides bearing unmodified, mono-, di-, and trimethylated lysines illustrate the roles of active site water molecules in aligning the lysine ε-amino group for methyl transfer with S-adenosylmethionine. Displacement or dissociation of these solvent molecules enlarges the diameter of the active site, accommodating the increasing size of the methylated ε-amino group during successive methyl transfer reactions. Together, these results furnish new insights into the roles of active site water molecules in modulating lysine multiple methylation by SET domain KMTs and provide the first molecular snapshots of the mono-, di-, and trimethyl transfer reactions catalyzed by these enzymes. PMID:20675860

  15. SET7/9 Catalytic Mutants Reveal the Role of Active Site Water Molecules in Lysine Multiple Methylation*

    Del Rizzo, Paul A.; Couture, Jean-François; Dirk, Lynnette M. A.; Strunk, Bethany S.; Roiko, Marijo S.; Brunzelle, Joseph S.; Houtz, Robert L.; Trievel, Raymond C.

    2010-01-01

    SET domain lysine methyltransferases (KMTs) methylate specific lysine residues in histone and non-histone substrates. These enzymes also display product specificity by catalyzing distinct degrees of methylation of the lysine ϵ-amino group. To elucidate the molecular mechanism underlying this specificity, we have characterized the Y245A and Y305F mutants of the human KMT SET7/9 (also known as KMT7) that alter its product specificity from a monomethyltransferase to a di- and a trimethyltransferase, respectively. Crystal structures of these mutants in complex with peptides bearing unmodified, mono-, di-, and trimethylated lysines illustrate the roles of active site water molecules in aligning the lysine ϵ-amino group for methyl transfer with S-adenosylmethionine. Displacement or dissociation of these solvent molecules enlarges the diameter of the active site, accommodating the increasing size of the methylated ϵ-amino group during successive methyl transfer reactions. Together, these results furnish new insights into the roles of active site water molecules in modulating lysine multiple methylation by SET domain KMTs and provide the first molecular snapshots of the mono-, di-, and trimethyl transfer reactions catalyzed by these enzymes. PMID:20675860

  16. SET7/9 Catalytic Mutants Reveal the Role of Active Site Water Molecules in Lysine Multiple Methylation

    Del Rizzo, Paul A.; Couture, Jean-François; Dirk, Lynnette M.A.; Strunk, Bethany S.; Roiko, Marijo S.; Brunzelle, Joseph S.; Houtz, Robert L.; Trievel, Raymond C. (Michigan); (NWU); (Kentucky)

    2010-11-15

    SET domain lysine methyltransferases (KMTs) methylate specific lysine residues in histone and non-histone substrates. These enzymes also display product specificity by catalyzing distinct degrees of methylation of the lysine {epsilon}-amino group. To elucidate the molecular mechanism underlying this specificity, we have characterized the Y245A and Y305F mutants of the human KMT SET7/9 (also known as KMT7) that alter its product specificity from a monomethyltransferase to a di- and a trimethyltransferase, respectively. Crystal structures of these mutants in complex with peptides bearing unmodified, mono-, di-, and trimethylated lysines illustrate the roles of active site water molecules in aligning the lysine {epsilon}-amino group for methyl transfer with S-adenosylmethionine. Displacement or dissociation of these solvent molecules enlarges the diameter of the active site, accommodating the increasing size of the methylated {epsilon}-amino group during successive methyl transfer reactions. Together, these results furnish new insights into the roles of active site water molecules in modulating lysine multiple methylation by SET domain KMTs and provide the first molecular snapshots of the mono-, di-, and trimethyl transfer reactions catalyzed by these enzymes.

  17. On Terminal Alkynes That Can React with Active-Site Cysteine Nucleophiles in Proteases

    Reggy Ekkebus; van Kasteren, Sander I.; Yogesh Kulathu; Arjen Scholten; Ilana Berlin; Geurink, Paul P; Annemieke de Jong; Soenita Goerdayal; Jacques Neefjes; Heck, Albert J.R.; David Komander; Huib Ovaa

    2013-01-01

    Active-site directed probes are powerful in studies of enzymatic function. We report an active-site directed probe based on a warhead so far considered unreactive. By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was observed. The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography. Proteomic analysis of proteins boun...

  18. Human population and activities in Forsmark. Site description

    The Swedish Nuclear Fuel and Waste Management Co (SKB) is in the process of selecting a safe and environmentally acceptable location for a deep repository of radioactive waste. Two alternative locations are under investigation. These are Forsmark, Oesthammars kommun (kommun = municipality) and Simpevarp/Laxemar, Oskarshamns kommun. SKB has expressed the importance of describing the humans and their activities in these areas and therefore has this synthesis concerning the human population in Forsmark been produced.The description is a statistical synthesis, mainly based upon statistical data from SCB (Statistics Sweden) that has been collected, processed and analysed. The statistical data has not been verified through site inspections and interviews. When using statistical data, it is advisable to note that the data becomes more unreliable if the areas are small, with small populations.The data in this description is essential for future evaluations of the impact on the environment and its human population (Environmental Impact Assessments). The data is also important when modelling the potential flows of radio nuclides and calculating the risk of exposure in future safety assessments.The actual area for the study is in this report called 'the Forsmark area', an area of 19.5 km2 near Forsmark nuclear power plant. The land use in the Forsmark area differs notably from the land use in Uppsala laen (laen = county). Only 0.04% of the total area is developed (built-up) compared to 4.9% in Uppsala laen and only 4% is agricultural land compared to 25% in the county. Furthermore, there are far more forest, wetlands and water areas in the Forsmark area. The forest area represents as much as 72.5% of the total area.The Forsmark area is uninhabited, and its surroundings are very sparsely populated. In 2002, the population density in Forsmark was 1.8 inhabitants per square kilometre, which was 24 times lower than in Uppsala laen. The population density in the parish has been

  19. Human population and activities in Forsmark. Site description

    Miliander, Sofia; Punakivi, Mari; Kylaekorpi, Lasse; Rydgren, Bernt [SwedPower AB, Stockholm (Sweden)

    2004-12-01

    The Swedish Nuclear Fuel and Waste Management Co (SKB) is in the process of selecting a safe and environmentally acceptable location for a deep repository of radioactive waste. Two alternative locations are under investigation. These are Forsmark, Oesthammars kommun (kommun = municipality) and Simpevarp/Laxemar, Oskarshamns kommun. SKB has expressed the importance of describing the humans and their activities in these areas and therefore has this synthesis concerning the human population in Forsmark been produced.The description is a statistical synthesis, mainly based upon statistical data from SCB (Statistics Sweden) that has been collected, processed and analysed. The statistical data has not been verified through site inspections and interviews. When using statistical data, it is advisable to note that the data becomes more unreliable if the areas are small, with small populations.The data in this description is essential for future evaluations of the impact on the environment and its human population (Environmental Impact Assessments). The data is also important when modelling the potential flows of radio nuclides and calculating the risk of exposure in future safety assessments.The actual area for the study is in this report called 'the Forsmark area', an area of 19.5 km{sup 2} near Forsmark nuclear power plant. The land use in the Forsmark area differs notably from the land use in Uppsala laen (laen = county). Only 0.04% of the total area is developed (built-up) compared to 4.9% in Uppsala laen and only 4% is agricultural land compared to 25% in the county. Furthermore, there are far more forest, wetlands and water areas in the Forsmark area. The forest area represents as much as 72.5% of the total area.The Forsmark area is uninhabited, and its surroundings are very sparsely populated. In 2002, the population density in Forsmark was 1.8 inhabitants per square kilometre, which was 24 times lower than in Uppsala laen. The population density in the

  20. Insights into Ocean Acidification During the Middle Eocene Climatic Optimum from Boron Isotopes at Southern Ocean Site 738

    Moebius, I.; Hoenisch, B.; Friedrich, O.

    2015-12-01

    The Middle Eocene Climatic Optimum (MECO) is a ~650-kyr interval of global warming, with a brief ~50 ky long peak warming interval, and an abrupt termination. Deep sea and surface ocean temperature evolution across this interval are fairly well constrained, but thus far we have little understanding of the mechanisms responsible for the gradual warming and rapid recovery. Carbonate mass accumulation rates suggest a shoaling of the carbonate compensation depth, and studies on alkenones indicate increasing atmospheric CO2 levels during the MECO. This suggests an increase in surface ocean CO2, and consequently ocean acidification. However, the severity and timing of the proposed ocean acidification with respect to the onset, peak warming and the termination are currently not well resolved. The boron isotopic composition (δ11B) recorded in planktic foraminifer shells offers an opportunity to infer oceanic pH across this interval. We are working on a boron isotope reconstruction from Southern Ocean IODP site 738 and South Atlantic IODP site 1263, covering 42.0 to 38.5 Ma. These sites are characterized by good carbonate preservation and well-defined age models have been established. Additionally, ecology, nutrient content and bottom-water oxygenation have been shown to change significantly across the event towards a more eutrophic, periodically oxygen-depleted environment supporting different biological communities. We selected the planktic foraminifera species Acarinina spinuloinflata for this study because it is symbiont-bearing, suggesting a near-surface habitat and little vertical migration in the water column, and because of its abundance in the samples. δ11B data will be translated to surface ocean pH and atmospheric pCO2 will be approximated to refine knowledge about the carbon cycle during this time. Parallel analysis of two core sites will help to evaluate the tenacity of the data.

  1. New insights into the QuikChange™ process guide the use of Phusion DNA polymerase for site-directed mutagenesis.

    Xia, Yongzhen; Chu, Wenqiao; Qi, Qingsheng; Xun, Luying

    2015-01-01

    The QuikChange™ site-directed mutagenesis method is popular but imperfect. An improvement by using partially overlapping primers has been reported several times; however, it is incompatible with the proposed mechanism. The QuikChange™ method using complementary primers is proposed to linearly amplify a target plasmid with the products annealing to produce double-stranded DNA molecules with 5'-overhangs. The overhang annealing is supposed to form circular plasmids with staggered breaks, which can be repaired in Escherichia coli after transformation. Here, we demonstrated that the PCR enzyme fills the 5'-overhangs in the early cycles, and the product is then used as the template for exponential amplification. The linear DNA molecules with homologous ends are joined to generate the plasmid with the desired mutations through homologous recombination in E. coli. The correct understanding is important to method improvements, guiding us to use partially overlapping primers and Phusion DNA polymerase for site-directed mutagenesis. Phusion did not amplify a plasmid with complementary primers but used partially overlapping primers to amplify the plasmid, producing linear DNA molecules with homologous ends for site-directed mutagenesis. PMID:25399421

  2. Enzyme-ligand interactions that drive active site rearrangements in the Helicobacter pylori 5´-methylthioadenosine/S-adenosylhomocysteine nucleosidase

    Ronning, Donald R; Iacopelli, Natalie M; Mishra, Vidhi [Toledo

    2012-03-15

    The bacterial enzyme 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) plays a central role in three essential metabolic pathways in bacteria: methionine salvage, purine salvage, and polyamine biosynthesis. Recently, its role in the pathway that leads to the production of autoinducer II, an important component in quorum-sensing, has garnered much interest. Because of this variety of roles, MTAN is an attractive target for developing new classes of inhibitors that influence bacterial virulence and biofilm formation. To gain insight toward the development of new classes of MTAN inhibitors, the interactions between the Helicobacter pylori-encoded MTAN and its substrates and substrate analogs were probed using X-ray crystallography. The structures of MTAN, an MTAN-Formycin A complex, and an adenine bound form were solved by molecular replacement and refined to 1.7, 1.8, and 1.6 Å, respectively. The ribose-binding site in the MTAN and MTAN-adenine cocrystal structures contain a tris[hydroxymethyl]aminomethane molecule that stabilizes the closed form of the enzyme and displaces a nucleophilic water molecule necessary for catalysis. This research gives insight to the interactions between MTAN and bound ligands that promote closing of the enzyme active site and highlights the potential for designing new classes of MTAN inhibitors using a link/grow or ligand assembly development strategy based on the described H. pylori MTAN crystal structures.

  3. In silico Modeling of 1A-Adrenoceptor: Interaction of its Normal and Mutated Active Sites with Noradrenaline as well as its Agonist and Antagonist

    Ramchandran Vijayan

    2007-01-01

    Full Text Available Noradrenaline, like most other neurotransmitters, acts through various adrenoceptor subtypes. The structure and active site of adrenoceptors for the binding of noradrenaline were unknown, however, such information are crucial for understanding the molecular mechanism of action of neurotransmitters, including noradrenaline, in health and disease as well as for drug designing. In this in silico study, we modeled the α1A-adrenoceptor; a G protein coupled receptor and defined its active site. Further, molecular docking and interaction of noradrenaline and its agonist as well as antagonist with the so defined active site of the receptor was studied before and after in silico site directed mutation of several amino acid residues forming the active site. Our results indicate that the ARG166 is the most crucial residue for binding of noradrenaline and methoxamine to α1A-adrenoceptor and ILE178 is the most important residue for binding of prazosin to it. Thus, the observations provide new insights into the structure function relationship of α1A-adrenoceptor. A significant finding of this study is that the same residue of the active site may not be necessary for binding of a receptor with its natural ligand and its pharmacologically active known agonist and antagonist.

  4. A comparative study of drug resistance mechanism associated with active site and non-active site mutations: I388N and D425G mutants of acetyl-coenzyme-A carboxylase.

    Zhu, Xiao-Lei; Yang, Guang-Fu

    2012-03-01

    A major concern in the development of acetyl-CoA carboxylase-inhibiting (ACCase; EC 6.4.1.2) herbicides is the emergence of resistance as a result of the selection of distinct mutations within the CT domain. Mutations associated with resistance have been demonstrated to include both active sites and non-active sites, including Ile-1781-Leu, Trp- 2027-Cys, Ile-2041-Asn, Asp-2078-Gly, and Gly-2096-Ala (numbered according to the Alopecurus myosuroides plastid ACCase). In the present study, extensive computational simulations, including molecular dynamics (MD) simulations and molecular mechanics-Poisson-Boltzmann surface area (MM/PBSA) calculations, were carried out to compare the molecular mechanisms of active site mutation (I388N) and non-active site mutation (D425G) in Alopecurus myosuroides resistance to some commercial herbicides targeting ACCase, including haloxyfop (HF), diclofop (DF) and fenoxaprop (FR). All of the computational model and energetic results indicated that both I388N and D425G mutations have effects on the conformational change of the binding pocket. The π-π interaction between ligand and Phe377 and Tyr161' residues, which make an important contribution to the binding affinity, was decreased after mutation. As a result, the mutant-type ACCase has a lower affinity for the inhibitor than the wild-type enzyme, which accounts for the molecular basis of herbicidal resistance. The structural and mechanistic insights obtained from the present study will deepen our understanding of the interactions between ACCase and herbicides, which provides a molecular basis for the future design of a promising inhibitor with low resistance risk. PMID:22242795

  5. 76 FR 7226 - Commercial Wind Lease Issuance and Site Characterization Activities; Atlantic Outer Continental...

    2011-02-09

    ... site characterization and assessment data the lessee may submit a construction and operations plan (COP... 30 CFR 285.620-.629. Although BOEMRE does not authorize site characterization activities (i.e... on the environmental effects of reasonably foreseeable site characterization surveys that may...

  6. The status of Yucca Mountain site characterization activities

    The U.S. Department of Energy (DOE) Office of Civilian Radioactive Waste Management is continuing its studies to determine if Yucca Mountain in Nevada can safely isolate high-level nuclear waste for the next ten thousand years. As mandated by Congress in 1987, DOE is studying the rocks, the climate, and the water table at Yucca Mountain to ensure that the site is suitable before building a repository about 305 meters (1,000 feet) below the surface. Yucca Mountain, located 161 kilometers (100 miles) northwest of Las Vegas, lies on the western edge of the Nevada Test Site. Nevada and DOE have been in litigation for almost two years over three environmental permits needed to conduct studies, but recent court decisions have allowed limited work to take place. This paper will examine progress made on the Yucca Mountain Site Characterization Project (YMP) during the past year and continuing into 1992, discuss the complex legal issues that are delaying progress, and describe new site drilling work. Title I and II design work on the underground exploratory studies facility (ESF) also will be discussed

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

    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.

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

    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

  9. Are nest sites actively chosen? Testing a common assumption for three non-resource limited birds

    Goodenough, A. E.; Elliot, S. L.; Hart, A. G.

    2009-09-01

    Many widely-accepted ecological concepts are simplified assumptions about complex situations that remain largely untested. One example is the assumption that nest-building species choose nest sites actively when they are not resource limited. This assumption has seen little direct empirical testing: most studies on nest-site selection simply assume that sites are chosen actively (and seek explanations for such behaviour) without considering that sites may be selected randomly. We used 15 years of data from a nestbox scheme in the UK to test the assumption of active nest-site choice in three cavity-nesting bird species that differ in breeding and migratory strategy: blue tit ( Cyanistes caeruleus), great tit ( Parus major) and pied flycatcher ( Ficedula hypoleuca). Nest-site selection was non-random (implying active nest-site choice) for blue and great tits, but not for pied flycatchers. We also considered the relative importance of year-specific and site-specific factors in determining occupation of nest sites. Site-specific factors were more important than year-specific factors for the tit species, while the reverse was true for pied flycatchers. Our results show that nest-site selection, in birds at least, is not always the result of active choice, such that choice should not be assumed automatically in studies of nesting behaviour. We use this example to highlight the need to test key ecological assumptions empirically, and the importance of doing so across taxa rather than for single "model" species.

  10. Structural Insights into the Activation and Inhibition of Histo-Aspartic Protease from Plasmodium falciparum

    Bhaumik, Prasenjit; Xiao, Huogen; Hidaka, Koushi; Gustchina, Alla; Kiso, Yoshiaki; Yada, Rickey Y.; Wlodawer, Alexander (Guelph); (Kyoto); (NCI)

    2012-09-17

    Histo-aspartic protease (HAP) from Plasmodium falciparum is a promising target for the development of novel antimalarial drugs. The sequence of HAP is highly similar to those of pepsin-like aspartic proteases, but one of the two catalytic aspartates, Asp32, is replaced with histidine. Crystal structures of the truncated zymogen of HAP and of the complex of the mature enzyme with inhibitor KNI-10395 have been determined at 2.1 and 2.5 {angstrom} resolution, respectively. As in other proplasmepsins, the propeptide of the zymogen interacts with the C-terminal domain of the enzyme, forcing the N- and C-terminal domains apart, thereby separating His32 and Asp215 and preventing formation of the mature active site. In the inhibitor complex, the enzyme forms a tight domain-swapped dimer, not previously seen in any aspartic proteases. The inhibitor is found in an unprecedented conformation resembling the letter U, stabilized by two intramolecular hydrogen bonds. Surprisingly, the location and conformation of the inhibitor are similar to those of the fragment of helix 2 comprising residues 34p-38p in the prosegments of the zymogens of gastric aspartic proteases; a corresponding helix assumes a vastly different orientation in proplasmepsins. Each inhibitor molecule is in contact with two molecules of HAP, interacting with the carboxylate group of the catalytic Asp215 of one HAP protomer through a water molecule, while also making a direct hydrogen bond to Glu278A' of the other protomer. A comparison of the shifts in the positions of the catalytic residues in the inhibitor complex presented here with those published previously gives further hints regarding the enzymatic mechanism of HAP.

  11. Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process

    Miao, Yinglong [ORNL; Baudry, Jerome Y [ORNL

    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.

  12. Human population and activities at Simpevarp. Site description

    The Swedish Nuclear Fuel and Waste Management Co (SKB) is in the process of selecting a safe and environmentally acceptable location for a deep repository of radioactive waste. Two alternative locations are under investigation. These are Forsmark, Oesthammars kommun (kommun = municipality) and Simpevarp/Laxemar, Oskarshamns kommun. SKB has expressed the importance of describing the humans and their activities in these areas and therefore has this synthesis concerning the human population in Forsmark been produced. The description is a statistical synthesis, mainly based upon statistical data from SCB (Statistics Sweden) that has been collected, processed and analysed. The statistical data has not been verified through site inspections and interviews. When using statistical data, it is advisable to note that the data becomes more unreliable if the areas are small, with small populations. The data in this description is essential for future evaluations of the impact on the environment and its human population (environmental impacts assessments). The data is also important when modelling the potential flows of radio nuclides and calculating the risk of exposure in future safety assessments. The actual area for the study is in this report called 'the Simpevarp area', an area of 127.0 km2 near Oskarshamn nuclear power plant. The land use in Simpevarp area differs notably from the land use in Kalmar laen. The forest area is far more dominating in Simpevarp area than in Kalmar laen and it represents as much as 89% compared to 63% of the total area. Only 4.4% of the area is arable land compared to 11.6% in Kalmar laen and only 0.3% is of other type (wetlands, bare rock, quarries, pites etc) compared to 15.6% in the county. The main observation is that Simpevarp area is a sparsely populated area located in a relatively lightly populated county. In 2002, the population density was 7.4 inhabitants/km2, three times lower than in Kalmar laen. The demography statistics show no

  13. Human population and activities at Simpevarp. Site description

    Miliander, Sofia; Punakivi, Mari; Kylaekorpi, Lasse; Rydgren, Bernt [SwedPower AB, Stockholm (Sweden)

    2004-12-01

    The Swedish Nuclear Fuel and Waste Management Co (SKB) is in the process of selecting a safe and environmentally acceptable location for a deep repository of radioactive waste. Two alternative locations are under investigation. These are Forsmark, Oesthammars kommun (kommun = municipality) and Simpevarp/Laxemar, Oskarshamns kommun. SKB has expressed the importance of describing the humans and their activities in these areas and therefore has this synthesis concerning the human population in Forsmark been produced. The description is a statistical synthesis, mainly based upon statistical data from SCB (Statistics Sweden) that has been collected, processed and analysed. The statistical data has not been verified through site inspections and interviews. When using statistical data, it is advisable to note that the data becomes more unreliable if the areas are small, with small populations. The data in this description is essential for future evaluations of the impact on the environment and its human population (environmental impacts assessments). The data is also important when modelling the potential flows of radio nuclides and calculating the risk of exposure in future safety assessments. The actual area for the study is in this report called 'the Simpevarp area', an area of 127.0 km{sup 2} near Oskarshamn nuclear power plant. The land use in Simpevarp area differs notably from the land use in Kalmar laen. The forest area is far more dominating in Simpevarp area than in Kalmar laen and it represents as much as 89% compared to 63% of the total area. Only 4.4% of the area is arable land compared to 11.6% in Kalmar laen and only 0.3% is of other type (wetlands, bare rock, quarries, pites etc) compared to 15.6% in the county. The main observation is that Simpevarp area is a sparsely populated area located in a relatively lightly populated county. In 2002, the population density was 7.4 inhabitants/km{sup 2}, three times lower than in Kalmar laen. The

  14. Human population and activities at Simpevarp. Site description

    Miliander, Sofia; Punakivi, Mari; Kylaekorpi, Lasse; Rydgren, Bernt [SwedPower AB, Stockholm (Sweden)

    2004-12-01

    The Swedish Nuclear Fuel and Waste Management Co (SKB) is in the process of selecting a safe and environmentally acceptable location for a deep repository of radioactive waste. Two alternative locations are under investigation. These are Forsmark, Oesthammars kommun (kommun = municipality) and Simpevarp/Laxemar, Oskarshamns kommun. SKB has expressed the importance of describing the humans and their activities in these areas and therefore has this synthesis concerning the human population in Forsmark been produced. The description is a statistical synthesis, mainly based upon statistical data from SCB (Statistics Sweden) that has been collected, processed and analysed. The statistical data has not been verified through site inspections and interviews. When using statistical data, it is advisable to note that the data becomes more unreliable if the areas are small, with small populations. The data in this description is essential for future evaluations of the impact on the environment and its human population (environmental impacts assessments). The data is also important when modelling the potential flows of radio nuclides and calculating the risk of exposure in future safety assessments. The actual area for the study is in this report called 'the Simpevarp area', an area of 127.0 km{sup 2} near Oskarshamn nuclear power plant. The land use in Simpevarp area differs notably from the land use in Kalmar laen. The forest area is far more dominating in Simpevarp area than in Kalmar laen and it represents as much as 89% compared to 63% of the total area. Only 4.4% of the area is arable land compared to 11.6% in Kalmar laen and only 0.3% is of other type (wetlands, bare rock, quarries, pites etc) compared to 15.6% in the county. The main observation is that Simpevarp area is a sparsely populated area located in a relatively lightly populated county. In 2002, the population density was 7.4 inhabitants/km{sup 2}, three times lower than in Kalmar laen. The

  15. Human Activities in Natura 2000 Sites: A Highly Diversified Conservation Network

    Tsiafouli, Maria A.; Apostolopoulou, Evangelia; Mazaris, Antonios D.; Kallimanis, Athanasios S; Drakou, Evangelia G.; Pantis, John D.

    2013-01-01

    The Natura 2000 network was established across the European Union’s (EU) Member States with the aim to conserve biodiversity, while ensuring the sustainability of human activities. However, to what kind and to what extent Natura 2000 sites are subject to human activities and how this varies across Member States remains unspecified. Here, we analyzed 111,269 human activity records from 14,727 protected sites in 20 Member States. The frequency of occurrence of activities differs among countries...

  16. Reconstructing southern Greenland Ice Sheet history during the intensification of Northern Hemisphere glaciation: Insights from IODP Site U1307

    Blake-Mizen, Keziah; Bailey, Ian; Carlson, Anders; Stoner, Joe; Hatfield, Rob; Xuan, Chuang; Lawrence, Kira

    2016-04-01

    Should it ever melt entirely, the Greenland Ice Sheet (GIS) would contribute to ~7 metres of global sea-level rise. Understanding how the GIS might respond to anthropogenic-induced global warming over the coming century is therefore important. Central to this goal is constraining how this ice sheet has responded to radiative forcing during both warmer- and colder-than-present climate states in the geological past. Little is known in detail, however, about the GIS prior to the Late Pleistocene and large uncertainty exists in our understanding of its history across the last great climate transition during the Cenozoic, the intensification of Northern Hemisphere glaciation (iNHG; ~3.6-2.4 Ma). This time encompasses two intervals of interest: (1) the mid-Piacenzian warm period (mPWP, ~3.3-3 Ma), widely considered an analogue for a future equilibrium climate state when atmospheric CO2 levels were comparable to modern (~400 ppmv) and sea-level and global temperatures were elevated relative to today (by ~25 metres and ~2-3°C) and, (2) a subsequent gradual deterioration in global climate and decline in atmospheric CO2 that led to the development of Quaternary-magnitude glaciations from ~2.5 Ma. Important unresolved questions include: to what extent did the southern GIS retreat during the mPWP, and when did a modern-day sized GIS first develop during iNHG? To tackle these issues our project focuses on the southern GIS history that can be extracted from Eirik Drift IODP Site U1307 between ~3.3 and 2.2 Ma. To achieve this we are developing an independent orbital-resolution age model, one of the first for high-latitude marine sediments deposited during iNHG, by producing a relative paleointensity (RPI) record for Site U1307; and generating multi-proxy geochemical and sedimentological datasets that track the provenance of the sand and bulk terrigenous sediment fraction glacially eroded by the southern GIS and delivered to the study site by both ice-rafting and the Western

  17. 76 FR 24871 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    2011-05-03

    ... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department of... from eligible active uranium and thorium processing site licensees for reimbursement under Title X of the Energy Policy Act of 1992. DATES: In our Federal Register Notice of November 24, 2010, (75...

  18. Early Site Permit Demonstration Program: Recommendations for communication activities and public participation in the Early Site Permit Demonstration Program

    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.

  19. Early Site Permit Demonstration Program: Recommendations for communication activities and public participation in the Early Site Permit Demonstration Program

    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

  20. Vegetation-derived insights on the mobilization and potential transport of radionuclides from the Nopal I natural analog site, Mexico

    Leslie, B.W.; Pickett, D.A.; Pearcy, E.C.

    1999-07-01

    The Nopal I uranium (U) deposit, Pena Blanca, Mexico is a source term and contaminant transport natural analog to the proposed high-level nuclear waste repository at Yucca Mountain, Nevada. In an attempt to characterize the mobilization and potential transport of radionuclides in the unsaturated zone at the Nopal I deposit, vegetation growing on ore piles was analyzed for {sup 238}U, {sup 235}U, and {sup 232}Th decay-series isotopes. Specimens of Phacelia robusta growing on high-grade piles of U ore were collected and analyzed by alpha autoradiography, and by alpha and gamma spectrometry. Activities for U, thorium (Th), and radium (Ra) isotopes (Bq/kg dried plant) were 300, 1,000, and 7,000 for {sup 238}U, {sup 230}Th, and {sup 226}Ra, respectively. The {sup 226}Ra activities in these specimens are among the highest ever measured for plants; furthermore, the plant-to-soil {sup 226}Ra concentration ratio is higher than expected. These results demonstrate the large mobility and bio-availability of Ra in the Nopal I environment, and support previous indications of recent loss of {sup 226}Ra from the ore body. Comparison between the activities of {sup 238}U and {sup 232}Th decay-chain Th isotopes in the plants and in the ore substrate indicate that relative mobilization into pore solutions of {sup 228}Th > {sup 230}Th > {sup 232}Th, in a ratio of about 50--25:4:1, respectively. The similarity of the plant's {sup 234}U/{sup 238}U activity ratio ({approximately}1.2) to that of a caliche deposit that formed adjacent to the Nopal ore body around 54 ka suggests the {sup 234}U/{sup 238}U activity ratio of U released from the ore is approximately 1.2. The U and {sup 226}Ra isotope activities of the plants and ore substrate, and solubility considerations, are used to assess a source term model of the potential Yucca Mountain repository. These results suggest the use of a natural analog source term model in performance assessments may be non-conservative.

  1. Vegetation-derived insights on the mobilization and potential transport of radionuclides from the Nopal I natural analog site, Mexico

    The Nopal I uranium (U) deposit, Pena Blanca, Mexico is a source term and contaminant transport natural analog to the proposed high-level nuclear waste repository at Yucca Mountain, Nevada. In an attempt to characterize the mobilization and potential transport of radionuclides in the unsaturated zone at the Nopal I deposit, vegetation growing on ore piles was analyzed for 238U, 235U, and 232Th decay-series isotopes. Specimens of Phacelia robusta growing on high-grade piles of U ore were collected and analyzed by alpha autoradiography, and by alpha and gamma spectrometry. Activities for U, thorium (Th), and radium (Ra) isotopes (Bq/kg dried plant) were 300, 1,000, and 7,000 for 238U, 230Th, and 226Ra, respectively. The 226Ra activities in these specimens are among the highest ever measured for plants; furthermore, the plant-to-soil 226Ra concentration ratio is higher than expected. These results demonstrate the large mobility and bio-availability of Ra in the Nopal I environment, and support previous indications of recent loss of 226Ra from the ore body. Comparison between the activities of 238U and 232Th decay-chain Th isotopes in the plants and in the ore substrate indicate that relative mobilization into pore solutions of 228Th > 230Th > 232Th, in a ratio of about 50--25:4:1, respectively. The similarity of the plant's 234U/238U activity ratio (∼1.2) to that of a caliche deposit that formed adjacent to the Nopal ore body around 54 ka suggests the 234U/238U activity ratio of U released from the ore is approximately 1.2. The U and 226Ra isotope activities of the plants and ore substrate, and solubility considerations, are used to assess a source term model of the potential Yucca Mountain repository. These results suggest the use of a natural analog source term model in performance assessments may be non-conservative

  2. Insights into the structure activity relationship of mPGES-1 inhibitors: Hints for better inhibitor design.

    Gupta, Ashish; Aparoy, Polamarasetty

    2016-07-01

    Microsomal prostaglandin E synthase-1 (mPGES-1) is a membrane protein which plays crucial role in arachidonic acid metabolism, in the catalysis of PGH2 to PGE2. It is a potential drug target involved in variety of human cancers and inflammatory disorders. In the present study we made an attempt to identify crucial amino acid residues involved in the effective binding of its inhibitors at the active site. Molecular docking and Structure Activity Relationship (SAR) studies were performed. In the present study 127 inhibitors having significant variability in parent scaffold were considered. The results clearly indicated that in the GSH and PGH2 binding site Arg70, Arg73, Asn74, Glu77, His113, Tyr117, Arg126, Ser127, Tyr130, Thr131 and Ala138 consistently form crucial interactions with inhibitors of different classes/scaffolds. These findings are consistent with that of existing reports on the active site residues pivotal at mPGES-1 active site. Further analysis suggested that out of all important amino acid residues identified; Arg73, Asn74, His113, Tyr117, Arg126, Ser127, Tyr130, Thr131 and Ala138 play a crucial role in hydrogen and π-π interactions. The identified amino acid residues can act as target sites for the design and development of drug candidates against mPGES-1. PMID:27012893

  3. Insight into the mechanism of biological methanol activation based on the crystal structure of the methanol-cobalamin methyltransferase complex

    Hagemeier, Christoph H.; Kr̈er, Markus; Thauer, Rudolf K.; Warkentin, Eberhard; Ermler, Ulrich

    2006-01-01

    Some methanogenic and acetogenic microorganisms have the catalytic capability to cleave heterolytically the CO bond of methanol. To obtain insight into the elusive enzymatic mechanism of this challenging chemical reaction we have investigated the methanol-activating MtaBC complex from Methanosarcina barkeri composed of the zinc-containing MtaB and the 5-hydroxybenzimidazolylcobamide-carrying MtaC subunits. Here we report the 2.5-Å crystal structure of this complex organized as a (MtaBC)2 hete...

  4. Isolation of active site and antibody-binding fragments of human erythrocyte transglutaminase

    Catalytically active human erythrocyte transglutaminase (TGase) was purified using an immunoaffinity column prepared from a monoclonal antibody to guinea pig liver TGase. The enzyme activity was completely inhibited by incorporation of iodo[14C]acetamide to the level of 1 mole per 1 mole of TGase. The 14C-labeled TGase was digested with cyanogen bromide, subjected to HPLC, and four pure peptides were isolated with molecular weights ranging from 3-22 KDa. Only one of the peptides was radiolabeled and characterized as an active site peptide of 10 KDa. Another peptide of 18 KDa was identified as a monoclonal antibody-binding domain of TGase. Although the active site and the antibody-binding domain were present on different cyanogen bromide fragments, the mouse anti-TGase inhibited 100% of TGase activity. The results suggest that the antibody-binding site is not located on the enzyme active site sequence, but that the three dimensional space configuration of the antigen-antibody complex hinders substrate binding to the active site. The radiolabeled active site cysteine residue was not found in the N-terminal 21 amino acids of the 10 KDa peptide. Additional fragments of the active site peptide are currently being analyzed

  5. A roundup of SMOS validation activities at the HOBE site in the Skjern River Catchment, Denmark

    Bircher, Simone; Richaume, Philippe; Mialon, Arnaud; Berthon, Lucie; Kerr, Yann H.; Jensen, Karsten H.

    2013-04-01

    patches of the most representative land covers (agriculture, heath and forest). This communication will give an overall insight into the validation carried out at the Danish test site by means of the collected in situ data, and present data comparisons at SMOS Levels 1C, 2 and 3. The shown results include data from different soil moisture processor versions as well as the investigation of main auxiliary parameters in the retrieval algorithm. Finally, an outlook on future planned SMOS activities in the higher northern latitudes will be given.

  6. Assessment of former uranium sites and their ongoing remediation activities

    Carried out analysis on tailing's buildings operation shows that period for engineer barrier service, taking into account any catastrophic natural impacts, is too little in comparison with life-time of long-live radionuclides. Priorities should be defined by danger degree and isolation costs (protection optimization), therefore uncommon, non-traditional methods, developed taking into account natural factors for long-live waste (radionuclides) isolation are necessary. That's why, it is necessary to carry out specialized research and development, design and exploratory and other works on monitoring of social-ecological condition of these sites, as well as on demographic public diseases, living in these regions.

  7. Binding site asymmetry in human transthyretin: insights from a joint neutron and X-ray crystallographic analysis using perdeuterated protein

    Melina Haupt

    2014-11-01

    Full Text Available Human transthyretin has an intrinsic tendency to form amyloid fibrils and is heavily implicated in senile systemic amyloidosis. Here, detailed neutron structural studies of perdeuterated transthyretin are described. The analyses, which fully exploit the enhanced visibility of isotopically replaced hydrogen atoms, yield new information on the stability of the protein and the possible mechanisms of amyloid formation. Residue Ser117 may play a pivotal role in that a single water molecule is closely associated with the γ-hydrogen atoms in one of the binding pockets, and could be important in determining which of the two sites is available to the substrate. The hydrogen-bond network at the monomer–monomer interface is more extensive than that at the dimer–dimer interface. Additionally, the edge strands of the primary dimer are seen to be favourable for continuation of the β-sheet and the formation of an extended cross-β structure through sequential dimer couplings. It is argued that the precursor to fibril formation is the dimeric form of the protein.

  8. Synthesis and characterization of 18F-labeled active site inhibited factor VII (ASIS)

    Erlandsson, Maria; Nielsen, Carsten Haagen; Jeppesen, Troels Elmer;

    2015-01-01

    Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example......, thrombosis, metastasis, tumor growth, and tumor angiogenesis. The aim of this study was to develop an 18F-labeled ASIS derivative to assess TF expression in tumors. Active site inhibited factor VII was labeled using N-succinimidyl-4-[18F]fluorobenzoate, and the [18F]ASIS was purified on a PD-10 desalting...

  9. Maintenance and decline of physical activity during adolescence: insights from a qualitative study

    Filion Annie

    2011-10-01

    Full Text Available Abstract Purpose Better knowledge on why some individuals succeed in maintaining participation in physical activity throughout adolescence is needed to guide the development of effective interventions to increase and then maintain physical activity levels. Despite allowing an in-depth understanding, qualitative designs have infrequently been used to study physical activity maintenance. We explored factors contributing to the maintenance and the decline of physical activity during adolescence. Methods Questionnaires were administered to 515 grade 10-12 students. The Physical Activity Questionnaire for Adolescents was used to determine physical activity level at the end of adolescence. An adapted version of this questionnaire was used to estimate physical activity in early adolescence. Among both genders, we identified participants who maintained a high level of physical activity since grade 7 and some whose activity level declined. For each category, groups of 10 students were randomly selected to take part in focus group discussions. Results Seven focus groups with 5 to 8 participants in each were held. Both maintainers and decliners associated physical activity with positive health outcomes. Maintenance of physical activity was associated with supportive social environments and heightened feelings of competence and attractiveness. A decline in physical activity was associated with negative social validation, poor social support and barriers related to access. Conclusions Although maintainers and decliners associate physical activity with similar themes, the experiences of both groups differ substantially with regards to those themes. Taking both perspectives in consideration could help improve interventions to increase and maintain physical activity levels of adolescents.

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

    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.

  11. Assessment of activation products in the Savannah River Site environment

    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: 32P, 51Cr, 60C, and 65Zn. 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

  12. Assessment of activation products in the Savannah River Site environment

    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.

  13. Activities for gaining insight into IASCC and continuous evaluation of in-service inspection data

    The report is a documentation of the important results of various international studies conducted to gain insight into the occurrence, mechanisms, and characteristic features of irradiation-assisted stress corrosion cracking, IASCC, as well as measures preventing IASCC in light water reactors. The major information can be summarised as follows: the number of cases of damage clearly induced by IASCC is low, as compared to the damage induced by intergranular stress corrosion cracking, IGSCC. In fact, recent information from a review of documented stress corrosion cracking damage of BWR type reactor internals reveals that an increasing number of cracks formerly thought to have been caused by IASCC now can be attributed to ICSCC as the most probable cause. Generally speaking, current knowledge of the impact of ionizing radiation on the corrosion resistance of LWR materials is rather insufficient. (orig./CB)

  14. The landscape degradation in the mining sites with suspended activity

    Anca IONCE

    2009-01-01

    The extracting industry, through its extraction activities, of shipping the ores, of breaking the ores, of preparing the practical substances, of stowing the useless rock, of transporting the practical substances, etc. might modify the area’s relief and the quality of ground, of thesurface waters and of the air. Suceava County has an old tradition of mining, where the results of this activity are visible, especially the visual point of view, and where not taking certain measures of ecological...

  15. A true autoactivating enzyme. Structural insight into mannose-binding lectin-associated serine protease-2 activations.

    Gál, Péter; Harmat, Veronika; Kocsis, Andrea; Bián, Tünde; Barna, László; Ambrus, Géza; Végh, Barbara; Balczer, Júlia; Sim, Robert B; Náray-Szabó, Gábor; Závodszky, Péter

    2005-09-30

    Few reports have described in detail a true autoactivation process, where no extrinsic cleavage factors are required to initiate the autoactivation of a zymogen. Herein, we provide structural and mechanistic insight into the autoactivation of a multidomain serine protease: mannose-binding lectin-associated serine protease-2 (MASP-2), the first enzymatic component in the lectin pathway of complement activation. We characterized the proenzyme form of a MASP-2 catalytic fragment encompassing its C-terminal three domains and solved its crystal structure at 2.4 A resolution. Surprisingly, zymogen MASP-2 is capable of cleaving its natural substrate C4, with an efficiency about 10% that of active MASP-2. Comparison of the zymogen and active structures of MASP-2 reveals that, in addition to the activation domain, other loops of the serine protease domain undergo significant conformational changes. This additional flexibility could play a key role in the transition of zymogen MASP-2 into a proteolytically active form. Based on the three-dimensional structures of proenzyme and active MASP-2 catalytic fragments, we present model for the active zymogen MASP-2 complex and propose a mechanism for the autoactivation process. PMID:16040602

  16. Probing the putative active site of YjdL

    Jensen, Johanne Mørch; Ismat, Fouzia; Szakonyi, Gerda;

    2012-01-01

    with Glu388, a preliminary orientation model of a dipeptide in the YjdL cavity is presented. Single site mutations of particularly Ala281 and Trp278 support the presented orientation. A dipeptide bound in the cavity of YjdL appears to be oriented such that the N-terminal side chain protrudes into a sub...... pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278. In the...... presented orientation model, Tyr25 and Tyr58 both appear to be in proximity of the dipeptide backbone while Lys117 appears to be in proximity of the peptide C-terminus. Mutational studies of these conserved residues highlight their functional importance....

  17. Insights on organic aerosol aging and the influence of coal combustion at a regional receptor site of central eastern China

    W. W. Hu

    2013-10-01

    Full Text Available In order to understand the aging and processing of organic aerosols (OA, an intensive field campaign (Campaign of Air Pollution at Typical Coastal Areas IN Eastern China, CAPTAIN was conducted March–April at a receptor site (a Changdao island in central eastern China. Multiple fast aerosol and gas measurement instruments were used during the campaign, including a high resolution time-of-flight aerosol mass spectrometer (HR-ToF-AMS that was applied to measure mass concentrations and non-refractory chemical components of submicron particles (PM1nr. The average mass concentration of PM1(PM1nr+black carbon was 47 ± 36 μg m−3 during the campaign and showed distinct variation, depending on back trajectories and their overlap with source regions. Organic aerosol (OA is the largest component of PM1 (30%, followed by nitrate (28%, sulfate (19%, ammonium (15%, black carbon (6%, and chloride (3%. Four OA components were resolved by positive matrix factorization (PMF of the high-resolution spectra, including low-volatility oxygenated organic aerosol (LV-OOA, semi-volatile oxygenated OA (SV-OOA, hydrocarbon-like OA (HOA and a coal combustion OA (CCOA. The mass spectrum of CCOA had high abundance of fragments from polycyclic aromatic hydrocarbons (PAHs (m/z 128, 152, 178, etc.. The average atomic ratio of oxygen to carbon in OA (O / C at Changdao was 0.59, which is comparable to other field studies reported at locations downwind of large pollution sources, indicating the oxidized nature of most OA during the campaign. The evolution of OA elemental composition in the van Krevelen diagram (H / C vs. O / C showed a slope of −0.63; however, the OA influenced by coal combustion exhibits a completely different evolution that appears dominated by physical mixing. The aging of organic aerosols vs. photochemical age was investigated. It was shown that OA / ΔCO, as well as LV-OOA / ΔCO and SV-OOA / ΔCO, positively correlated with photochemical age. LV

  18. The Palaeolithic site Bistricioara-Lut\\varie III in the Romanian Carpathians - Insights from various luminescence methods

    Schmidt, Christoph; Antohi-Trandafir, Oana; Timar-Gabor, Alida; Anghelinu, Mircea; Veres, Daniel; Hambach, Ulrich

    2016-04-01

    The loess derivates on top of the terrace gravels in the Bistrita valley (Carpathians, northeastern Romania) host a large number of Palaeolithic settlements, some of which reveal several distinct cultural layers characterised by charcoal, other combustion features and/or scattered lithics. While the youngest productive layers at the site Bistricioara-Lut\\varie III (BL III) are associated with Gravettian and Epigravettian technocomplexes, the knowledge about older occupations remains diffuse. Definitely, the high density of last glacial settlements in such a harsh environment represents a puzzle. Furthermore, new excavations in 2015 exposed large (>1 m) combustion features without a related lithic inventory and of unknown origin (natural fires or fires places). The present contribution aims at fathoming the versatile applications of luminescence methods to tackle the unsolved questions at BL III. Despite methodological deficiencies concerning grain size dependent age discrepancies, optically stimulated luminescence (OSL) of quartz demonstrated the archive's chronological depth (>76 ka above terrace gravels) and placed the youngest cultural layer (CL1) in the Last Glacial Maximum, in agreement with radiocarbon (14C) dates (Trandafir et al. 2015). This cultural layer yielded a set of heated lithics (flint) during the recent excavation, providing the opportunity to directly date human presence by thermoluminescence (TL) and to reconcile these ages with (independent) methods dating different events (OSL, 14C). Such a comparison of techniques also serves at testing the accuracy of explorative TL measurement protocols under 'natural conditions'. Finally, detached from any chronological issues, the temperature-dependent sensitisation of the 110 °C quartz TL peak - in analogue to the flint TL signal - potentially allows determining the maximum heating temperature of samples from the combustion features (Göksu et al. 1989), which in turn helps elucidating whether the

  19. Active site proton delivery and the lyase activity of human CYP17A1

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.; Denisov, Ilia G.; Sligar, Stephen G., E-mail: s-sligar@illinois.edu

    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.

  20. Active site proton delivery and the lyase activity of human CYP17A1

    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

  1. Off-site medical activities, Nevada Test Site and the medical liaison officer network: a historical review

    The ''off-site'' was originally defined as ''that area surrounding the Nevada Test Site (NTS) for a radius of about 300 miles.'' Prior to 1954, the off-site radiological safety activities were conducted by the Atomic Energy Commission. In 1954, the Public Health Service was given the responsibility for off-site monitoring, and, in addition, a physician was also on temporary assignment. This physician, in addition to functioning as a monitor, also functioned part time as a physical liaison in regard to possible or alleged radiation injury. Medical concern was based upon two crude guidelines: possible radiation ''overexposure'' based upon extrapolation from surface and air radiological monitoring; and determination of actual radiation injury based upon signs and symptoms among people alleging radiation injury. The area of concern expanded to 13 areas surrounding the Nevada Test Site, and in 1956, the first Medical Liaison Officer Network (MLON) was initiated. Over the years, MLON increased to a point where there was a representative from every state in the Union; the area of concern expanded to include the entire United States, parts of the South Pacific, Hawaii, and Alaska; and sophisticated methods of evaluation were added--urine sampling, thyroid scanning, blood counts, and whole-body counting. Epidemiological studies were initiated on body burdens of radionuclides and certain disease clusters

  2. The landscape degradation in the mining sites with suspended activity

    Anca IONCE

    2009-08-01

    Full Text Available The extracting industry, through its extraction activities, of shipping the ores, of breaking the ores, of preparing the practical substances, of stowing the useless rock, of transporting the practical substances, etc. might modify the area’s relief and the quality of ground, of thesurface waters and of the air. Suceava County has an old tradition of mining, where the results of this activity are visible, especially the visual point of view, and where not taking certain measures of ecological remediation will emphasize the disappointing image of the landscape within the areas of mining activity performing.The predominant mountainous landscape, in which mining activities have been held, is being affected also by the abandoned industrial and administrative buildings, in an advanced degradation state.The hydrographic system, very rich in mining areas, has its water quality affected by the acid rock drainage- phenomenon which appeared in many mining waste deposits.

  3. Reduction of urease activity by interaction with the flap covering the active site.

    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

  4. New insights into the activation mechanism of store-operated calcium channels:roles of STIM and Orai

    Rui-wei GUO; Lan HUANG

    2008-01-01

    The activation of Ca2+ entry through store-operated channels by agonists that deplete Ca2+ from the endoplasmic reticulum (ER)is a ubiquitous signaling mechanism,the molecular basis of which has remained elusive for the past two decades.Store-operated Ca2+-release-activated Ca2+(CRAC)channels constitute the sole pathway for Ca2+ entry following antigen-receptor engagement.In a set of breakthrough studies over the past two years,stromal interaction molecule l(STIM1,tbe ER Ca2+ sensor) and Orail(a pore-forming subunit of the CRAC channel)have been identified.Here we review these recent studies and the insights they provide into the mechanism of store-operated Ca2+ channels(SOCCs).

  5. Activation of brown adipose tissue mitochondrial GDP binding sites

    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 [3H]-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

  6. Activation of brown adipose tissue mitochondrial GDP binding sites

    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.

  7. Activation of Mitogen-Activated Protein Kinase Is Required for Migration and Invasion of Placental Site Trophoblastic Tumor

    Köbel, Martin; Pohl, Gudrun; Schmitt, Wolfgang D.; Hauptmann, Steffen; Wang, Tian-Li; Shih, Ie-Ming

    2005-01-01

    Placental site trophoblastic tumor (PSTT) is a gestational neoplasm derived from the extravillous (intermediate) trophoblast of the implantation site. PSTT is characterized by a highly invasive phenotype, but the molecular mechanisms are poorly understood. In this report, we demonstrate that PSTTs expressed the activated (phosphorylated) form of mitogen-activated protein kinase (MAPK) in 84% of cases, whereas the normal extravillous trophoblastic cells did not. To characterize the role of MAP...

  8. Experience in decommissioning activities at the BR3 site

    Klein, Michel E-mail: mklein@sckcen.be; Dadoumont, Jerome; Demeulemeester, Yves; Massaut, Vincent

    2001-04-01

    We give an overview of the experience, SCK-CEN acquired during 11 years of decommissioning activities of a PWR fission reactor. Experience has been gained in decontamination and dismantling technologies and in waste management. Dismantling an old PWR is quite different from the dismantling of the future fusion reactor. However, we can try to find out some common generic features and draw some lessons. Our experience shows that it is important to take the decommissioning aspects into account as soon as possible.

  9. Lipolytic activity from bacteria prospected in polluted portuary sites

    Kaori Levy Fonseca

    2014-06-01

    This study demonstrates that these TBT resistant isolates have, at the same time, the capacity to produce enzymes with a large biotechnological potential but, nevertheless, their relationship is not well understood, representing a novel approach. It is expected for these organisms to produce highly biotechnological relevant biocatalysts, due to their severe adaptations (Suehiro et al., 2007. The fully characterization of these lipases, mostly for F3 with elevated lipolytic activity exhibited, presents also a future challenge.

  10. Current disposal planning for dry active wastes at Rokkasho Site

    Takeuchi, Mitsuo [Japan Nuclear Fuel Ltd., Aomori (Japan)

    1997-02-01

    In nuclear power stations, two kinds of low level radioactive wastes are generated: `uniform solidified waste` in which waste liquid, spent resin and so on are uniformly solidified and `solid waste` in which metals, lagging materials, plastics and others are solidified. In Rokkasho Low Level Radioactive Waste Burying Center, the burying facility for the first period for the uniform solidified waste started the operation in December, 1992, and this time as the second period plan, it has been planned to increase No. 2 waste burying facility for the solid waste. The kinds of the radioactive waste solidified in containers to be buried are the solid state radioactive waste generated by the operation of nuclear power stations and that generated accompanying the operation of this facility. The wastes are classified, cut, pressed and melted as occasion demands so that cement filling material is easily filled in containers, and solidified in the containers. As for the waste to be buried, at the time of its acceptance, 6 months or longer have elapsed since its generation in nuclear power stations, and the surface dose equivalent rate does not exceed 10 mSv/h. The acceptance plan and the expected number of burying, the total radioactivity of buried waste, and the location, geological and hydraulic features, the structure and facilities of waste burying facilities, the method of burying, the management of waste burying site and the evaluation of dose equivalent are reported. (K.I.)

  11. Current disposal planning for dry active wastes at Rokkasho Site

    In nuclear power stations, two kinds of low level radioactive wastes are generated: 'uniform solidified waste' in which waste liquid, spent resin and so on are uniformly solidified and 'solid waste' in which metals, lagging materials, plastics and others are solidified. In Rokkasho Low Level Radioactive Waste Burying Center, the burying facility for the first period for the uniform solidified waste started the operation in December, 1992, and this time as the second period plan, it has been planned to increase No. 2 waste burying facility for the solid waste. The kinds of the radioactive waste solidified in containers to be buried are the solid state radioactive waste generated by the operation of nuclear power stations and that generated accompanying the operation of this facility. The wastes are classified, cut, pressed and melted as occasion demands so that cement filling material is easily filled in containers, and solidified in the containers. As for the waste to be buried, at the time of its acceptance, 6 months or longer have elapsed since its generation in nuclear power stations, and the surface dose equivalent rate does not exceed 10 mSv/h. The acceptance plan and the expected number of burying, the total radioactivity of buried waste, and the location, geological and hydraulic features, the structure and facilities of waste burying facilities, the method of burying, the management of waste burying site and the evaluation of dose equivalent are reported. (K.I.)

  12. Insights for Exercise Adherence from a Minimal Planning Intervention to Increase Physical Activity

    Chapman, Janine; Campbell, Marianne; Wilson, Carlene

    2015-01-01

    Objective: To test the impact of a minimal, online planning intervention on physical activity in Australian office workers. Method: Employees were randomized to an implementation intention intervention (n = 124) or health information control group (n = 130). Measures of physical activity, past behavior, and motivation were taken at baseline and 6…

  13. Identifying high dose activities in industrial site radiography

    Although the radiation doses received by industrial radiographers in the UK have progressively fallen over the last few years, with most now receiving less than 1 mSv/y, a few still receive, relative to the rest, much higher doses. As a percentage of all radiographers the number stays surprisingly constant from year to year. This paper describes a survey to identify the work causing these doses and suggest possible solutions. The UK Central Index of Dose Information was interrogated to identify the industrial radiography companies having staff (not necessarily the same person) with doses of greater than 5mSv/y in the last three years for which information was available. This was 15 in total. The people on the staff receiving these doses were identified and a questionnaire sent to the companies concerned requesting information about their work. A general questionnaire about the operation of the company was also included. With the agreement of the company these questionnaires were followed up by a visit to the company to interviews a number of the management and the radiographers if available. Both groups were generally very open about their problems and every discussion had a positive outcome. Several areas of work/reasons for the doses have been identified. These are: pipeline radiography, ultra sound radiographers working on nuclear reactors, complex plant work often with several teams in the area, inability to retreat from the wind out equipment due to height or access problems, site pressure to not follow the best practices and a lack of appreciation when a dose was being received or, alternatively, carelessness. Some o these problem areas are very difficult to resolve. However ways in which the Health and Safety can help influence the doses have been identified together with practical suggestions radiographers could adopt. These will be reported. (author)

  14. Structural insight into the recognition of complement C3 activation products by integrin receptors

    Bajic, Goran

    2015-01-01

    associated with microbes and apoptotic or necrotic cells. Complement not only protects against pathogens but also maintains body homeostasis. Activation of complement leads to cleavage of the complement proteins C4, C3 and C5, and their fragments have effector functions through binding to pathogen surfaces...... small fragment C3a called anaphylatoxin. Complement leads to opsonization as the proteolytic fragment C3b becomes covalently linked to the activator surface through a reactive thioester. Self-surfaces are protected by complement regulators, whereas complement activation vividly amplifies on pathogens....... An important outcome of the regulators is the degradation of C3b to iC3b. Phagocytic receptor αMβ2 integrin (also called CR3, CD11b/CD18, or Mac-1) on leukocytes engages the opsonized activator subsequently to C3b cleavage into iC3b. Apoptotic cells activate complement leading to iC3b deposition and...

  15. School Pharmacist/School Environmental Hygienic Activities at School Site.

    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

  16. Black and Brown Bear Activity at Selected Coastal Sites in Glacier Bay National Park and Preserve, Alaska: A Preliminary Assessment Using Noninvasive Procedures

    Partridge, Steve; Smith, Tom; Lewis, Tania

    2009-01-01

    A number of efforts in recent years have sought to predict bear activity in various habitats to minimize human disturbance and bear/human conflicts. Alaskan coastal areas provide important foraging areas for bears (Ursus americanus and U. arctos), particularly following den emergence when there may be no snow-free foraging alternatives. Additionally, coastal areas provide important food items for bears throughout the year. Glacier Bay National Park and Preserve (GLBA) in southeastern Alaska has extensive coastal habitats, and the National Park Service (NPS) has been long interested in learning more about the use of these coastal habitats by bears because these same habitats receive extensive human use by park visitors, especially kayaking recreationists. This study provides insight regarding the nature and intensity of bear activity at selected coastal sites within GLBA. We achieved a clearer understanding of bear/habitat relationships within GLBA by analyzing bear activity data collected with remote cameras, bear sign mapping, scat collections, and genetic analysis of bear hair. Although we could not quantify actual levels of bear activity at study sites, agreement among measures of activity (for example, sign counts, DNA analysis, and video record) lends support to our qualitative site assessments. This work suggests that habitat evaluation, bear sign mapping, and periodic scat counts can provide a useful index of bear activity for sites of interest.

  17. Isolated metal active site concentration and stability control catalytic CO2 reduction selectivity.

    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

  18. The balance of flexibility and rigidity in the active site residues of hen egg white lysozyme

    Qi Jian-Xun; Jiang Fan

    2011-01-01

    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.

  19. 77 FR 74218 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    2012-12-13

    ... published a Notice of Availability (NOA) in the Federal Register (72 FR 62,672) of the Programmatic EIS for... Federal Register (77 FR 5560) of the Final EA for Commercial Wind Lease Issuance and Site Assessment... Bureau of Ocean Energy Management Commercial Wind Leasing and Site Assessment Activities on the...

  20. 75 FR 71677 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    2010-11-24

    ... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department of... uranium and thorium processing site licensees for reimbursement under Title X of the Energy Policy Act of... published a final rule under 10 CFR Part 765 in the Federal Register on May 23, 1994, (59 FR 26714) to...

  1. Antimicrobial activity of human α-defensin 6 analogs: insights into the physico-chemical reasons behind weak bactericidal activity of HD6 in vitro.

    Mathew, Basil; Nagaraj, Ramakrishnan

    2015-11-01

    Human α-defensin 6 (HD6), unlike other mammalian defensins, does not exhibit bactericidal activity, particularly against aerobic bacteria. Monomeric HD6 has a tertiary structure similar to other α-defensins in the crystalline state. However, the physico-chemical reasons behind the lack of antibacterial activity of HD6 are yet to be established unequivocally. In this study, we have investigated the antimicrobial activity of HD6 analogs. A linear analog of HD6, in which the distribution of arginine residues was similar to active α-defensins, shows broad-spectrum antimicrobial activity, indicating that atypical distribution of arginine residues contributes to the inactivity of HD6. Peptides spanning the N-terminal cationic segment were active against a wide range of organisms. Antimicrobial potency of these shorter analogs was further enhanced when myristic acid was conjugated at the N-terminus. Cytoplasmic localization of the analogs without fatty acylation was observed to be necessary for bacterial killing, while they exhibited fungicidal activity by permeabilizing Candida albicans membranes. Myristoylated analogs and the linear full-length arginine analog exhibited activity by permeabilizing bacterial and fungal membranes. Our study provides insights into the lack of bactericidal activity of HD6 against aerobic bacteria. PMID:26400692

  2. Poisoning Experiments Aimed at Discriminating Active and Less-Active Sites of Silica-Supported Tantalum Hydride for Alkane Metathesis

    Saggio, Guillaume

    2010-10-04

    Only 50% of the silica-supported tantalum hydride sites are active in the metathesis of propane. Indeed, more than 45% of the tantalum hydride can be eliminated by a selective oxygen poisoning of inactive sites with no significant decrease in the global turnover. Conversely, cyclopentane induces no such selective poisoning. Hence, the active tantalum hydride sites that show greater resistance to oxygen poisoning correspond to the νTa-H bands of higher wavenumbers, particularly that at 1860cm-1. These active tantalum hydride sites should correspond to tris- or monohydride species relatively far from silica surface oxygen atoms. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Structural insights into chaperone-activity enhancement by a K354E mutation in tomato acidic leucine aminopeptidase.

    DuPrez, Kevin T; Scranton, Melissa A; Walling, Linda L; Fan, Li

    2016-05-01

    Tomato plants express acidic leucine aminopeptidase (LAP-A) in response to various environmental stressors. LAP-A not only functions as a peptidase for diverse peptide substrates, but also displays chaperone activity. A K354E mutation has been shown to abolish the peptidase activity but to enhance the chaperone activity of LAP-A. To better understand this moonlighting function of LAP-A, the crystal structure of the K354E mutant was determined at 2.15 Å resolution. The structure reveals that the K354E mutation destabilizes an active-site loop and causes significant rearrangement of active-site residues, leading to loss of the catalytic metal-ion coordination required for the peptidase activity. Although the mutant was crystallized in the same hexameric form as wild-type LAP-A, gel-filtration chromatography revealed an apparent shift from the hexamer to lower-order oligomers for the K354E mutant, showing a mixture of monomers to trimers in solution. In addition, surface-probing assays indicated that the K354E mutant has more accessible hydrophobic areas than wild-type LAP-A. Consistently, computational thermodynamic estimations of the interfaces between LAP-A monomers suggest that increased exposure of hydrophobic surfaces occurs upon hexamer breakdown. These results suggest that the K354E mutation disrupts the active-site loop, which also contributes to the hexameric assembly, and destabilizes the hexamers, resulting in much greater hydrophobic areas accessible for efficient chaperone activity than in the wild-type LAP-A. PMID:27139632

  4. Recent progress in volcanism studies: Site characterization activities for the Yucca Mountain site characterization project

    Significant progress has been made on volcanism studies over the past calendar year. There are a number of major highlights from this work. Geochronology data have been obtained for the Lathrop Wells center using a range of isotopic, radiogenic, and age-calibrated methods. Initial work is encouraging but still insufficient to resolve the age of the center with confidence. Geologic mapping of the Sleeping Butte volcanic centers was completed and a report issued on the geology and chronology data. Twenty shallow trenches have been constructed in volcanic units of the Lathrop Wells volcanic center. Results of detailed studies of the trenches support a polycyclic eruptive history. New soil data from the trenches continue to support a late Pleistocene or Holocene age for many of the volcanic units at the center. Geochemical data (trace element and isotopic analysis) show that the volcanic units of the Lathrop Wells center cannot be related to one another by fractional crystallization of a single magma batch, supporting a polycyclic model of volcanism. Structural models using existing data are used to evaluate the probability of magmatic disruption of a potential repository. Several permissive models have been developed but none lead to significant differences in calculating the disruption ratio. Work was initiated on the eruptive and subsurface effects of magmatic activity on a repository. (author)

  5. [NiFe] dithiolene diphosphine complex for hydrogen gas activation: a Theoretic Insight

    GuYan, Jing

    2015-01-01

    A diphosphino-nickel-iron dithiolene complex, [Ni(bdt)(dppf)] (bdt = 1,2-benzenedithiolate, dppf = 1,1-bis(diphenylphosphino)ferrocene), has been recently found to be reasonably active on proton reduction to dihydrogen (J. Am. Chem. Soc. 2015, 137, 1109). Interestingly, this exceptional complex was found to be also reactive towards dihydrogen activation as indicated by the electrochemical investigation. However, a pure nickel dithiolene diphosphine theoretical mode, excluding the contributions from iron moiety, was applied to attribute the experimental catalytic observation. We have re-visited the theoretical approach in details for this [NiFe] catalyst and compared it with the non-active nickel dithiolene diphosphine complexes. We found that both nickel and iron moieties in this newly developed complex were imperative for the observed catalytic per-formance, particularly towards the activation of dihydrogen.

  6. Site specific rationale for technical impracticability of active groundwater restoration at a former manufactured gas plant site

    The National Contingency Plan (40 CFR Part 300 ) requires that remedial strategies must, at minimum, protect human health and the environment and meet applicable and relevant or appropriate requirements (ARARs). Where groundwater is impacted, maximum contaminant levels (MCLs) and maximum contaminant level goals (MCLGs) set under the Safe Drinking Water Act are often used as ARARs, whether or not the aquifer is a reasonably anticipated future source of drinking water. The US Environmental Protection Agency now recognizes the difficulty of groundwater restoration at sites where dense nonaqueous phase liquids are present, particularly in certain complex hydrogeological settings (EPA 1993). However, demonstration of impracticability generally does not occur until active remediation (e.g., pump and treat) has been shown to be ineffective. A case study of a former manufactured gas plant (MGP) is used to demonstrate how physical and chemical properties of the aquifer and coal tar, the major waste product from MGP sites, influence the feasibility of active restoration. Field characterization investigations, laboratory studies, and groundwater modeling are integrated into a demonstration following EPA guidelines. Laboratory studies included microbiological characterization and natural biodegradation and suggest that intrinsic bioremediation is occurring at this site. This work will be useful as EPA continues to develop presumptive remedies for cleanup under Superfund

  7. 1993 annual report of hazardous waste activities for the Oak Ridge K-25 site

    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

  8. Insights into Gulf of Mexico Gas Hydrate Study Sites GC955 and WR313 from New Multicomponent and High-Resolution 2D Seismic Data

    Haines, S. S.; Hart, P. E.; Collett, T. S.; Shedd, W. W.; Frye, M.

    2014-12-01

    In 2013, the U.S. Geological Survey led a seismic acquisition expedition in the Gulf of Mexico, acquiring multicomponent data and high-resolution 2D multichannel seismic (MCS) data at Green Canyon 955 (GC955) and Walker Ridge 313 (WR313). Based on previously collected logging-while-drilling (LWD) borehole data, these gas hydrate study sites are known to include high concentrations of gas hydrate within sand layers. At GC955 our new 2D data reveal at least three features that appear to be fluid-flow pathways (chimneys) responsible for gas migration and thus account for some aspects of the gas hydrate distribution observed in the LWD data. Our new data also show that the main gas hydrate target, a Pleistocene channel/levee complex, has an areal extent of approximately 5.5 square kilometers and that a volume of approximately 3 x 107 cubic meters of this body lies within the gas hydrate stability zone. Based on LWD-inferred values and reasonable assumptions for net sand, sand porosity, and gas hydrate saturation, we estimate a total equivalent gas-in-place volume of approximately 8 x 108 cubic meters for the inferred gas hydrate within the channel/levee deposits. At WR313 we are able to map the thin hydrate-bearing sand layers in considerably greater detail than that provided by previous data. We also can map the evolving and migrating channel feature that persists in this area. Together these data and the emerging results provide valuable new insights into the gas hydrate systems at these two sites.

  9. Coordination environment of the active-site metal ion of liver alcohol dehydrogenase.

    Makinen, M W; Yim, M B

    1981-01-01

    The coordination environment of the catalytically active metal ion of horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) has been investigated by electron paramagnetic resonance (EPR) methods with use of the active-site-specific Co2+-reconstituted enzyme. The EPR absorption spectrum of the metal-substituted enzyme is characteristic of a rhombically distorted environment. The spectrum of the enzyme--NAD+ complex shows approximate axial symmetry of the metal ion site, i...

  10. Denaturation studies of active-site labeled papain using electron paramagnetic resonance and fluorescence spectroscopy.

    Ping, Z A; Butterfiel, D A

    1991-01-01

    A spin-labeled p-chloromercuribenzoate (SL-PMB) and a fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan), both of which bind to the single SH group located in the active site of papain, were used to investigate the interaction of papain (EC 3.4.22.2) with two protein denaturants. It was found that the active site of papain was highly stable in urea solution, but underwent a large conformational change in guanidine hydrochloride solution. Electron paramagnetic resonance and ...

  11. Molecular dynamics explorations of active site structure in designed and evolved enzymes

    Osuna Oliveras, Sílvia; Jiménez-Osés, Gonzalo; Noey, Elizabeth L.; Houk, Kendall N.

    2015-01-01

    This Account describes the use of molecular dynamics (MD) simulations to reveal how mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient when functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. We describe our explora...

  12. New Insights into the Antibacterial Activity of Hydroxycoumarins against Ralstonia solanacearum.

    Yang, Liang; Ding, Wei; Xu, Yuquan; Wu, Dousheng; Li, Shili; Chen, Juanni; Guo, Bing

    2016-01-01

    Coumarins are important plant-derived natural products with wide-ranging bioactivities and extensive applications. In this study, we evaluated for the first time the antibacterial activity and mechanisms of action of coumarins against the phytopathogen Ralstonia solanacearum, and investigated the effect of functional group substitution. We first tested the antibacterial activity of 18 plant-derived coumarins with different substitution patterns, and found that daphnetin, esculetin, xanthotol, and umbelliferone significantly inhibited the growth of R. solanacearum. Daphnetin showed the strongest antibacterial activity, followed by esculetin and umbelliferone, with MICs of 64, 192, and 256 mg/L, respectively, better than the archetypal coumarin with 384 mg/L. We further demonstrated that the hydroxylation of coumarins at the C-6, C-7 or C-8 position significantly enhanced the antibacterial activity against R. solanacearum. Transmission electron microscope (TEM) and fluorescence microscopy images showed that hydroxycoumarins may interact with the pathogen by mechanically destroying the cell membrane and inhibiting biofilm formation. The antibiofilm effect of hydroxycoumarins may relate to the repression of flagellar genes fliA and flhC. These physiological changes in R. solanacearum caused by hydroxycoumarins can provide information for integral pathogen control. The present findings demonstrated that hydroxycoumarins have superior antibacterial activity against the phytopathogen R. solanacearum, and thus have the potential to be applied for controlling plant bacterial wilt. PMID:27070570

  13. New Insights into the Antibacterial Activity of Hydroxycoumarins against Ralstonia solanacearum

    Liang Yang

    2016-04-01

    Full Text Available Coumarins are important plant-derived natural products with wide-ranging bioactivities and extensive applications. In this study, we evaluated for the first time the antibacterial activity and mechanisms of action of coumarins against the phytopathogen Ralstonia solanacearum, and investigated the effect of functional group substitution. We first tested the antibacterial activity of 18 plant-derived coumarins with different substitution patterns, and found that daphnetin, esculetin, xanthotol, and umbelliferone significantly inhibited the growth of R. solanacearum. Daphnetin showed the strongest antibacterial activity, followed by esculetin and umbelliferone, with MICs of 64, 192, and 256 mg/L, respectively, better than the archetypal coumarin with 384 mg/L. We further demonstrated that the hydroxylation of coumarins at the C-6, C-7 or C-8 position significantly enhanced the antibacterial activity against R. solanacearum. Transmission electron microscope (TEM and fluorescence microscopy images showed that hydroxycoumarins may interact with the pathogen by mechanically destroying the cell membrane and inhibiting biofilm formation. The antibiofilm effect of hydroxycoumarins may relate to the repression of flagellar genes fliA and flhC. These physiological changes in R. solanacearum caused by hydroxycoumarins can provide information for integral pathogen control. The present findings demonstrated that hydroxycoumarins have superior antibacterial activity against the phytopathogen R. solanacearum, and thus have the potential to be applied for controlling plant bacterial wilt.

  14. Low-frequency earthquakes and tomography in western Japan: Insight into fluid and magmatic activity

    Zhao, Dapeng; Wei, Wei; Nishizono, Yukihisa; Inakura, Hirohito

    2011-11-01

    Low-frequency (LF) microearthquakes are detected in the lower crust and uppermost mantle beneath western Japan, which may reflect fluid and magmatic activity in the subduction zone. In this work we combine seismic tomography and the LF earthquakes to study the crustal and upper-mantle structure and fluid and magmatic activity in western Japan. High-resolution tomographic images under this region are determined using high-quality arrival-time data. In Kyushu the subducting Philippine Sea slab is detected clearly as a high-velocity zone, and the arc-magma related low-velocity (low- V) anomalies under the active arc and back-arc volcanoes are imaged clearly. Prominent low- V zones are visible in the crust and uppermost mantle beneath the Quaternary volcanoes along the Japan Sea coast, and LF events occur actively under some of those volcanoes. LF events are also detected in the source areas of the 1995 Kobe earthquake (M 7.2) and the 2000 Tottori earthquake (M 7.3), supporting the hypothesis that the large crustal earthquakes were triggered by the crustal fluids. These results indicate that fluid and magmatic activities exist widely in western Japan, which are caused by dehydration of the subducting Philippine Sea slab and corner flow in the mantle wedge.

  15. Active site dynamics of toluene hydroxylation by cytochrome P-450

    Rat liver cytochrome P-450 hydroxylates toluene to benzyl alcohol plus o-, m-, and p-cresol. Deuterated toluenes were incubated under saturating conditions with liver microsomes from phenobarbital-pretreated rats, and product yields and ratios were measured. Stepwise deuteration of the methyl leads to stepwise decreases in the alcohol/cresol ratio without changing the cresol isomer ratios. Extensive deuterium retention in the benzyl alcohols from PhCH2D and PhCHD2 suggests there is a large intrinsic isotope effect for benzylic hydroxylation. After replacement of the third benzylic H by D, the drop in the alcohol/cresol ratio was particularly acute, suggsting that metabolic switching from D to H within the methyl group was easier than switching from the methyl to the ring. Comparison of the alcohol/cresol ratio for PhCH3 vs PhCD3 indicated a net isotope effect of 6.9 for benzylic hydroxylation. From product yield data for PhCH3 and PhCD3, DV for benzyl alcohol formation is only 1.92, whereas DV for total product formation is 0.67 (i.e., inverse). From competitive incubations of PhCH3/PhCD3 mixtures D(V/K) isotope effects on benzyl alcohol formation and total product formation (3.6 and 1.23, respectively) are greatly reduced, implying strong commitment to catalysis. In contrast, D(V/K) for the alcohol/cresol ratio is 6.3, indicating that the majority of the intrinsic isotope effect is expressed through metabolic switching. Overall, these data are consistent with reversible formation of a complex between toluene and the active oxygen form of cytochrome P-450, which rearranges internally and reacts to form products faster than it dissociates back to release substrate

  16. 'Unconventional' coordination chemistry by metal chelating fragments in a metalloprotein active site.

    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

  17. Recent insights into the biological activities and drug delivery systems of tanshinones.

    Cai, Yuee; Zhang, Wenji; Chen, Zirong; Shi, Zhi; He, Chengwei; Chen, Meiwan

    2016-01-01

    Tanshinones, the major lipid-soluble pharmacological constituents of the Chinese medicinal herb Tanshen (Salvia miltiorrhiza), have attracted growing scientific attention because of the prospective biomedical applications of these compounds. Numerous pharmacological activities, including anti-inflammatory, anticancer, and cardio-cerebrovascular protection activities, are exhibited by the three primary bioactive constituents among the tanshinones, ie, tanshinone I (TNI), tanshinone IIA (TNIIA), and cryptotanshinone (CPT). However, due to their poor solubility and low dissolution rate, the clinical applications of TNI, TNIIA, and CPT are limited. To solve these problems, many studies have focused on loading tanshinones into liposomes, nanoparticles, microemulsions, cyclodextrin inclusions, solid dispersions, and so on. In this review, we aim to offer an updated summary of the biological activities and drug delivery systems of tanshinones to provide a reference for these constituents in clinical applications. PMID:26792989

  18. Insights into the interactions between enzyme and co-solvents: stability and activity of stem bromelain.

    Rani, Anjeeta; Venkatesu, Pannuru

    2015-02-01

    In present study, an attempt is made to elucidate the effects of various naturally occurring osmolytes and denaturants on BM at pH 7.0. The effects of the varying concentrations of glycerol, sorbitol, sucrose, trehalose, urea and guanidinium chloride (GdnHCl) on structure, stability and activity of BM are explored by fluorescence spectroscopy, circular dichroism (CD), UV-vis spectroscopy and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Our experimental observations reveal that glycerol and sorbitol are acting as stabilizers at all concentrations while sucrose and trehalose are found to be destabilizers at lower concentrations, however, acted as stabilizers at higher concentrations. On the other hand, urea and GdnHCl are denaturants except at lower concentrations. There is a direct relationship between activity and conformational stability as the activity data are found to be in accordance with conformational stability parameters (ΔGu, Tm, ΔCp) and BM profile on SDS-PAGE. PMID:25434803

  19. Geomorphic signal of active faulting at the northern edge of Lut Block: Insights on the kinematic scenario of Central Iran

    Calzolari, Gabriele; Della Seta, Marta; Rossetti, Federico; Nozaem, Reza; Vignaroli, Gianluca; Cosentino, Domenico; Faccenna, Claudio

    2016-01-01

    Recent works documented Neogene to Quaternary dextral strike-slip tectonics along the Kuh-e-Sarhangi and Kuh-e-Faghan intraplate strike-slip faults at the northern edge of the Lut Block of Central Iran, previously thought to be dominated by sinistral strike-slip deformation. This work focuses on the evidence of Quaternary activity of one of these fault systems, in order to provide new spatiotemporal constraints on their role in the active regional kinematic scenario. Through geomorphological and structural investigation, integrated with optically stimulated luminescence dating of three generations of alluvial fans and fluvial terraces (at ~53, ~25, and ~6 ka), this study documents (i) the topographic inheritance of the long-term (Myr) punctuated history of fault nucleation, propagation, and exhumation along the northern edge of Lut Block; (ii) the tectonic control on drainage network evolution, pediment formation, fluvial terraces, and alluvial fan architecture; (iii) the minimum Holocene age of Quaternary dextral strike-slip faulting; and (iv) the evidence of Late Quaternary fault-related uplift localized along the different fault strands. The documented spatial and temporal constraints on the active dextral strike-slip tectonics at the northern edge of Lut Block provide new insights on the kinematic model for active faulting in Central Iran, which has been reinterpreted in an escape tectonic scenario.

  20. New insights into membrane-active action in plasma membrane of fungal hyphae by the lipopeptide antibiotic bacillomycin L.

    Zhang, Bao; Dong, Chunjuan; Shang, Qingmao; Han, Yuzhu; Li, Pinglan

    2013-09-01

    Bacillomycin L, a natural iturinic lipopeptide produced by Bacillus amyloliquefaciens, is characterized by strong antifungal activities against a variety of agronomically important filamentous fungi including Rhizoctonia solani Kühn. Prior to this study, the role of membrane permeabilization in the antimicrobial activity of bacillomycin L against plant pathogenic fungi had not been investigated. To shed light on the mechanism of this antifungal activity, the permeabilization of R. solani hyphae by bacillomycin L was investigated and compared with that by amphotericin B, a polyene antibiotic which is thought to act primarily through membrane disruption. Our results derived from electron microscopy, various fluorescent techniques and gel retardation experiments revealed that the antifungal activity of bacillomycin L may be not solely a consequence of fungal membrane permeabilization, but related to the interaction of it with intracellular targets. Our findings provide more insights into the mode of action of bacillomycin L and other iturins, which could in turn help to develop new or improved antifungal formulations or result in novel strategies to prevent fungal spoilage. PMID:23756779

  1. XAFS Study of the Photo-Active Site of Mo/MCM-41

    An Mo/MCM-41 catalyst was prepared and used for study of propene and 1-butene photo-metathesis reactions. XAFS analysis revealed that hydrogen reduction leads to a decreased role for the Mo=O site. The Mo-O site plays an important role for the olefin photo-metathesis reaction on the H2 reduced Mo/MCM-41. From EXAFS analysis, the active site of photo-metathesis reaction is the Mo=O part for oxidized Mo/MCM-41, whereas it is the Mo-O site for reduced Mo/MCM-41

  2. Active sites for NO reduction over Fe-ZSM-5 catalysts.

    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

  3. Benzene Hydroxylation over FeZSM-5 Catalysts: Which Fe-sites Are Active?

    Yuranov, I.; Bulushev, D. A.; Renken, A.; Kiwi-Minsker, L.

    2004-01-01

    FeZSM-5 with a wide range of Fe content (0.015–2.1 wt%) were studied in the benzene hydroxylation to phenol with nitrous oxide (C6H6:N2O = 1:5) at low temperatures (98%) was obtained within 3 h without any deactivation of the catalyst. Three types of Fe(II) sites were formed in the zeolites extraframework due to activation and are attributed to: (1) Fe(II) sites in mononuclear species, (2) oligonuclear species with at least two oxygen-bridged Fe(II) sites, and (3) Fe(II) sites within Fe2O3 na...

  4. Concept for calculating dose rates from activated groundwater at accelerator sites

    Prolingheuer, N; Vanderborght, J; Schlögl, B; Nabbi, R; Moormann, R

    Licensing of particle accelerators requires the proof that the groundwater outside of the site will not be significantly contaminated by activation products formed below accelerator and target. In order to reduce the effort for this proof, a site independent simplified but conservative method is under development. The conventional approach for calculation of activation of soil and groundwater is shortly described on example of a site close to Forschungszentrum Juelich, Germany. Additionally an updated overview of a data library for partition coefficients for relevant nuclides transported in the aquifer at the site is presented. The approximate model for transport of nuclides with ground water including exemplary results on nuclide concentrations outside of the site boundary and of resulting effective doses is described. Further applications and developments are finally outlined.

  5. New insight into the solution structures of wheat gluten proteins from Raman optical activity

    Blanch, E.W.; Kasarda, D.D.; Hecht, L.;

    2003-01-01

    Vibrational Raman optical activity (ROA) spectra of the wheat proteins a-gliadin (A-gliadin), omega-liadin, and a 30 kDa peptide called T-A-1 from the high molecular weight glutenin subunit (HMW-GS) Dx5 were measured to obtain new information about their solution structures. The spectral data sho...

  6. Marketing Activities to Support ‘Moderately Novel’ Product Innovation: Insights from the Chemical Industry

    Smits, A.A.J.; Vissers, G.A.N.; Dankbaar, B.

    2015-01-01

    Scholars often follow a contingency approach to study which marketing activities are suitable for a particular type of product innovation project, thereby making a distinction between incremental and radical innovation only. ‘Moderately novel’ projects, which have intermediate levels of newness, hav

  7. Functional insights into modulation of BKCa channel activity to alter myometrial contractility

    RamónALorca

    2014-07-01

    Full Text Available The large-conductance voltage- and Ca2+-activated K+ channel (BKCa is an important regulator of membrane excitability in a wide variety of cells and tissues. In myometrial smooth muscle, activation of BKCa plays essential roles in buffering contractility to maintain uterine quiescence during pregnancy and in the transition to a more contractile state at the onset of labor. Multiple mechanisms of modulation have been described to alter BKCa channel activity, expression, and cellular localization. In the myometrium, BKCa is regulated by alternative splicing, protein targeting to the plasma membrane, compartmentation in membrane microdomains, and posttranslational modifications. In addition, interaction with auxiliary proteins (i.e., β1- and β2-subunits, association with G-protein coupled receptor signaling pathways, such as those activated by adrenergic and oxytocin receptors, and hormonal regulation provide further mechanisms of variable modulation of BKCa channel function in myometrial smooth muscle. Here, we provide an overview of these mechanisms of BKCa channel modulation and provide a context for them in relation to myometrial function.

  8. Functional characterization of autophosphorylation sites of the activated insulin receptor-tyrosine kinase

    Insulin receptor, solubilized from 3T3-L1 cellular membranes and then purified, was autophosphorylated with [γ-32P]ATP in the absence or presence of insulin. Specific phosphopeptides generated by trypsin digestion of the 32P-labeled β-subunit were identified and separated by reverse phase HPLC. In the absence of insulin, radioactivity of the phosphopeptides is evenly distributed among four major peaks designated as sites I, II, III and IV, according to their order of elution. This pattern is maintained for at least the first 30 min of autophosphorylation. When the reaction is carried out in the presence of insulin, > 50% of the total 32P radioactivity is found in site I and the rate of 32P incorporation into this site is markedly higher than into sites II, III and IV. Maximal activation of tyrosine kinase activity, as estimated by substrate phosphorylation, is coincident with the nearly complete phosphorylation of site I. Delayed activation of previously autophosphorylated receptor by insulin, but not by EGF or IGF-I, produced a similar pattern where phosphorylated site I predominates. These observations indicate that one major insulin-regulated autophosphorylation site in the β-subunit is responsible for activation of the insulin receptor tyrosine kinase. The isolation of this phosphopeptide on a preparative scale and its characterization are now in progress

  9. Functional characterization of autophosphorylation sites of the activated insulin receptor-tyrosine kinase

    Flores-Riveros, J.R.; Lane, M.D.

    1987-05-01

    Insulin receptor, solubilized from 3T3-L1 cellular membranes and then purified, was autophosphorylated with (..gamma..-/sup 32/P)ATP in the absence or presence of insulin. Specific phosphopeptides generated by trypsin digestion of the /sup 32/P-labeled ..beta..-subunit were identified and separated by reverse phase HPLC. In the absence of insulin, radioactivity of the phosphopeptides is evenly distributed among four major peaks designated as sites I, II, III and IV, according to their order of elution. This pattern is maintained for at least the first 30 min of autophosphorylation. When the reaction is carried out in the presence of insulin, > 50% of the total /sup 32/P radioactivity is found in site I and the rate of /sup 32/P incorporation into this site is markedly higher than into sites II, III and IV. Maximal activation of tyrosine kinase activity, as estimated by substrate phosphorylation, is coincident with the nearly complete phosphorylation of site I. Delayed activation of previously autophosphorylated receptor by insulin, but not by EGF or IGF-I, produced a similar pattern where phosphorylated site I predominates. These observations indicate that one major insulin-regulated autophosphorylation site in the ..beta..-subunit is responsible for activation of the insulin receptor tyrosine kinase. The isolation of this phosphopeptide on a preparative scale and its characterization are now in progress.

  10. Site-specific targeting of antibody activity in vivo mediated by disease-associated proteases

    Erster, Oran; Thomas, Jerry M; Hamzah, Juliana; Jabaiah, Abeer M.; Getz, Jennifer A.; Schoep, Tobias; Hall, Sejal S.; Ruoslahti, Erkki; Daugherty, Patrick S.

    2012-01-01

    As a general strategy to selectively target antibody activity in vivo, a molecular architecture was designed to render binding activity dependent upon proteases in disease tissues. A protease-activated antibody (pro-antibody) targeting vascular cell adhesion molecule 1 (VCAM-1), a marker of atherosclerotic plaques, was constructed by tethering a binding site-masking peptide to the antibody via a matrix metalloprotease (MMP) susceptible linker. Pro-antibody activation in vitro by MMP-1 yielded...

  11. Global Indicators Analysis and Consultancy Experience Insights into Correlation between Entrepreneurial Activities and Business Environment

    Jovan Krivokapić

    2015-02-01

    Full Text Available Many researches and practical experiences clearly indicate the existence of a strong relationship between entrepreneurial activities and the business environment in which these activities are initiated. Although this topic has been quite ignored until the late twentieth century, a lot of studies and consulting practice have contributed to the fact that there are now a number of theories concerning mentioned correlation. These theories aim to offer a model that would provide better utilization of the possibilities from the business environment which could be very important for the development from both macroeconomic and microeconomic aspects. An increasing number of articles on this topic says enough about its importance, and numerous researches by many reputable globally recognized institutions go in favor of this claim. There are many indicators that observe the economic situation in a country or a region from different aspects, so the analyses of these indicators make it possible to determine the specific relationships between entrepreneurial activities and the local and the global business environment. Given the complexity of these relations, the impact cannot be observed partially, without taking into consideration other important factors, but more detailed analyses, however, result in some useful conclusions, which in the proper context can have a positive impact on many economic factors. It is very important to emphasize the fact that the correlation between the business environment and entrepreneurial activities is bidirectional, since this influence is mutual, so that changes in one of these factors can and usually cause some modifications in the other. Frequent series of such iterations actually lead to changes in the business environment, while entrepreneurial activity changes its shape and affects the economy of a country or a region, which is of particular importance for its competitiveness in the era of globalization.

  12. Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions

    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.

  13. Streaming potential modeling in fractured rock: Insights into the identification of hydraulically active fractures

    Roubinet, D; Jougnot, D; Irving, J

    2016-01-01

    Numerous field experiments suggest that the self-potential (SP) geophysical method may allow for the detection of hydraulically active fractures and provide information about fracture properties. However, a lack of suitable numerical tools for modeling streaming potentials in fractured media prevents quantitative interpretation and limits our understanding of how the SP method can be used in this regard. To address this issue, we present a highly efficient two-dimensional discrete-dual-porosity approach for solving the fluid flow and associated self-potential problems in fractured rock. Our approach is specifically designed for complex fracture networks that cannot be investigated using standard numerical methods. We then simulate SP signals associated with pumping conditions for a number of examples to show that (i) accounting for matrix fluid flow is essential for accurate SP modeling and (ii) the sensitivity of SP to hydraulically active fractures is intimately linked with fracture-matrix fluid interaction...

  14. Metabolic Activation of Rhein: Insights into the Potential Toxicity Induced by Rhein-Containing Herbs.

    Yuan, Yuan; Zheng, Jiyue; Wang, Meiyu; Li, Yuan; Ruan, Jianqing; Zhang, Hongjian

    2016-07-20

    Rhein is a major component of the many medicinal herbs such as rhubarb. Despite wide use, intoxication cases associated with rhein-containing herbs are often reported. The present work aimed to investigate if rhein was subject to metabolic activation leading to toxicity. Upon incubations with different species of liver microsomes, three monoglucuronides were identified, corresponding to two hydroxyl glucuronides and one acyl glucuronide via the carboxyl group, respectively. Further study revealed that rhein acyl glucuronide was chemically reactive, and showed cytotoxicity toward hepatocarcinoma cells. In addition, significant species differences in glucuronidation of rhein were observed between laboratory animals and humans. Reaction phenotyping experiments demonstrated that rhein acyl glucuronide was catalyzed predominantly by uridine 5'-diphospho-glucuronosyltransferase 1A1, 1A9, and 2B7. Taken together, the present study confirmed that rhein could be metabolically activated via the formation of acyl glucuronide, especially in human. PMID:27362917

  15. Insights into the amplification of bacterial resistance to erythromycin in activated sludge.

    Guo, Mei-Ting; Yuan, Qing-Bin; Yang, Jian

    2015-10-01

    Wastewater treatment plants are significant reservoirs for antimicrobial resistance. However, little is known about wastewater treatment effects on the variation of antibiotic resistance. The shifts of bacterial resistance to erythromycin, a macrolide widely used in human medicine, on a lab-scale activated sludge system fed with real wastewater was investigated from levels of bacteria, community and genes, in this study. The resistance variation of total heterotrophic bacteria was studied during the biological treatment process, based on culture dependent method. The alterations of bacterial community resistant to erythromycin and nine typical erythromycin resistance genes were explored with molecular approaches, including high-throughput sequencing and quantitative polymerase chain reaction. The results revealed that the total heterotrophs tolerance level to erythromycin concentrations (higher than 32 mg/L) was significantly amplified during the activated sludge treatment, with the prevalence increased from 9.6% to 21.8%. High-throughput sequencing results demonstrated an obvious increase of the total heterotrophic bacterial diversity resistant to erythromycin. Proteobacteria and Bacteroidetes were the two dominant phyla in the influent and effluent of the bioreactor. However, the prevalence of Proteobacteria decreased from 76% to 59% while the total phyla number increased greatly from 18 to 29 through activated sludge treatment. The gene proportions of erm(A), mef(E) and erm(D) were greatly amplified after biological treatment. It is proposed that the transfer of antibiotic resistance genes through the variable mixtures of bacteria in the activated sludge might be the reason for the antibiotic resistance amplification. The amplified risk of antibiotic resistance in wastewater treatment needs to be paid more attention. PMID:25957255

  16. Recent insights into the biological activities and drug delivery systems of tanshinones

    Cai Y

    2016-01-01

    Full Text Available Yuee Cai,1,* Wenji Zhang,2,* Zirong Chen,3 Zhi Shi,1,2 Chengwei He,1 Meiwan Chen1 1State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, People’s Republic of China; 2Department of Cell Biology & Institute of Biomedicine, National Engineering Research Center of Genetic Medicine, Guangdong Provincial Key Laboratory of Bioengineering Medicine, College of Life Science and Technology, Jinan University, Guangzhou, Guangdong, People’s Republic of China; 3Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL, USA *These authors contributed equally to this work Abstract: Tanshinones, the major lipid-soluble pharmacological constituents of the Chinese medicinal herb Tanshen (Salvia miltiorrhiza, have attracted growing scientific attention because of the prospective biomedical applications of these compounds. Numerous pharmacological activities, including anti-inflammatory, anticancer, and cardio-cerebrovascular protection activities, are exhibited by the three primary bioactive constituents among the tanshinones, ie, tanshinone I (TNI, tanshinone IIA (TNIIA, and cryptotanshinone (CPT. However, due to their poor solubility and low dissolution rate, the clinical applications of TNI, TNIIA, and CPT are limited. To solve these problems, many studies have focused on loading tanshinones into liposomes, nanoparticles, microemulsions, cyclodextrin inclusions, solid dispersions, and so on. In this review, we aim to offer an updated summary of the biological activities and drug delivery systems of tanshinones to provide a reference for these constituents in clinical applications. Keywords: tanshinones, biological activities, drug delivery systems, bioavailability, solubility

  17. Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases.

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette

    2016-03-29

    Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze theo-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme's interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate-enzyme complexes were performed, and a key residue was identified that influences the plant PPO's acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their--so far unknown--natural substrates in vivo. PMID:26976571

  18. Baroreflex Activation Therapy in Congestive Heart Failure: Novel Findings and Future Insights.

    Grassi, Guido; Brambilla, GianMaria; Pizzalla, Daniela Prata; Seravalle, Gino

    2016-08-01

    Congestive heart failure is characterized by hemodynamic and non-hemodynamic abnormalities, the latter including an activation of the sympathetic influences to the heart and peripheral circulation coupled with an impairment of baroreceptor control of autonomic function. Evidence has been provided that both these alterations are hallmark features of the disease with a specific relevance for the disease progression as well as for the development of life-threatening cardiac arrhythmias. In addition, a number of studies have documented in heart failure the adverse prognostic role of the sympathetic and baroreflex alterations, which both are regarded as major independent determinants of cardiovascular morbidity and mortality. This represents the pathophysiological and clinical background for the use of carotid baroreceptor activation therapy in the treatment of congestive heart failure. Promising data collected in experimental animal models of heart failure have supported the recent performance of pilot small-scale clinical studies, aimed at providing initial information in this area. The results of these studies demonstrated the clinical safety and efficacy of the intervention which has been tested in large-scale clinical studies. The present paper will critically review the background and main results of the published studies designed at defining the clinical impact of baroreflex activation therapy in congestive heart failure patients. Emphasis will be given to the strengths and limitations of such studies, which represent the background for the ongoing clinical trials testing the long-term effects of the device in heart failure patients. PMID:27334011

  19. Insights into the mechanism of human papillomavirus E2-induced procaspase-8 activation and cell death.

    Singh, Nitu; Senapati, Sanjib; Bose, Kakoli

    2016-01-01

    High-risk human papillomavirus (HR-HPV) E2 protein, the master regulator of viral life cycle, induces apoptosis of host cell that is independent of its virus-associated regulatory functions. E2 protein of HR-HPV18 has been found to be involved in novel FADD-independent activation of caspase-8, however, the molecular basis of this unique non-death-fold E2-mediated apoptosis is poorly understood. Here, with an interdisciplinary approach that involves in silico, mutational, biochemical and biophysical probes, we dissected and characterized the E2-procasapse-8 binding interface. Our data demonstrate direct non-homotypic interaction of HPV18 E2 transactivation domain (TAD) with α2/α5 helices of procaspase-8 death effector domain-B (DED-B). The observed interaction mimics the homotypic DED-DED complexes, wherein the conserved hydrophobic motif of procaspase-8 DED-B (F122/L123) occupies a groove between α2/α3 helices of E2 TAD. This interaction possibly drives DED oligomerization leading to caspase-8 activation and subsequent cell death. Furthermore, our data establish a model for E2-induced apoptosis in HR-HPV types and provide important clues for designing E2 analogs that might modulate procaspase-8 activation and hence apoptosis. PMID:26906543

  20. Structural insights into Ca(2+)-activated long-range allosteric channel gating of RyR1.

    Wei, Risheng; Wang, Xue; Zhang, Yan; Mukherjee, Saptarshi; Zhang, Lei; Chen, Qiang; Huang, Xinrui; Jing, Shan; Liu, Congcong; Li, Shuang; Wang, Guangyu; Xu, Yaofang; Zhu, Sujie; Williams, Alan J; Sun, Fei; Yin, Chang-Cheng

    2016-09-01

    Ryanodine receptors (RyRs) are a class of giant ion channels with molecular mass over 2.2 mega-Daltons. These channels mediate calcium signaling in a variety of cells. Since more than 80% of the RyR protein is folded into the cytoplasmic assembly and the remaining residues form the transmembrane domain, it has been hypothesized that the activation and regulation of RyR channels occur through an as yet uncharacterized long-range allosteric mechanism. Here we report the characterization of a Ca(2+)-activated open-state RyR1 structure by cryo-electron microscopy. The structure has an overall resolution of 4.9 Å and a resolution of 4.2 Å for the core region. In comparison with the previously determined apo/closed-state structure, we observed long-range allosteric gating of the channel upon Ca(2+) activation. In-depth structural analyses elucidated a novel channel-gating mechanism and a novel ion selectivity mechanism of RyR1. Our work not only provides structural insights into the molecular mechanisms of channel gating and regulation of RyRs, but also sheds light on structural basis for channel-gating and ion selectivity mechanisms for the six-transmembrane-helix cation channel family. PMID:27573175

  1. New insights into the aquatic photochemistry of fluoroquinolone antibiotics: Direct photodegradation, hydroxyl-radical oxidation, and antibacterial activity changes.

    Ge, Linke; Na, Guangshui; Zhang, Siyu; Li, Kai; Zhang, Peng; Ren, Honglei; Yao, Ziwei

    2015-09-15

    The ubiquity and photoreactivity of fluoroquinolone antibiotics (FQs) in surface waters urge new insights into their aqueous photochemical behavior. This study concerns the photochemistry of 6 FQs: ciprofloxacin, danofloxacin, levofloxacin, sarafloxacin, difloxacin and enrofloxacin. Methods were developed to calculate their solar direct photodegradation half-lives (td,E) and hydroxyl-radical oxidation half-lives (tOH,E) in sunlit surface waters. The td,E values range from 0.56 min to 28.8 min at 45° N latitude, whereas tOH,E ranges from 3.24h to 33.6h, suggesting that most FQs tend to undergo fast direct photolysis rather than hydroxyl-radical oxidation in surface waters. However, a case study for levofloxacin and sarafloxacin indicated that the hydroxyl-radical oxidation induced risky photochlorination and resulted in multi-degradation pathways, such as piperazinyl hydroxylation and clearage. Changes in the antibacterial activity of FQs caused by photodegradation in various waters were further examined using Escherichia coli, and it was found that the activity evolution depended on primary photodegradation pathways and products. Primary intermediates with intact FQ nuclei retained significant antibacterial activity. These results are important for assessing the fate and risk of FQs in surface waters. PMID:25956144

  2. Crystal structure of type I 3-dehydroquinate dehydratase of Aquifex aeolicus suggests closing of active site flap is not essential for enzyme action.

    Devi, Aribam Swarmistha; Ebihara, Akio; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Kumarevel, Thirumananseri; Ponnuraj, Karthe

    2013-03-01

    Structural analyses of enzymes involved in biosynthetic pathways that are present in micro-organisms, but absent from mammals (for example Shikimate pathway) are important in developing anti-microbial drugs. Crystal structure of the Shikimate pathway enzyme, type I 3-dehydroquinate dehydratase (3-DHQase) from the hyperthermophilic bacterium Aquifex aeolicus was solved both as an apo form and in complex with a ligand. The complex structure revealed an interesting structural difference when compared to other ligand-bound type I 3-DHQases suggesting that closure of the active site loop is not essential for catalysis. This provides new insights into the catalytic mechanism of type I 3-DHQases. PMID:23396056

  3. Mechanistic and Structural Insights into the Prion-Disaggregase Activity of Hsp104.

    Sweeny, Elizabeth A; Shorter, James

    2016-05-01

    Hsp104 is a dynamic ring translocase and hexameric AAA+ protein found in yeast, which couples ATP hydrolysis to disassembly and reactivation of proteins trapped in soluble preamyloid oligomers, disordered protein aggregates, and stable amyloid or prion conformers. Here, we highlight advances in our structural understanding of Hsp104 and how Hsp104 deconstructs Sup35 prions. Although the atomic structure of Hsp104 hexamers remains uncertain, volumetric reconstruction of Hsp104 hexamers in ATPγS, ADP-AlFx (ATP hydrolysis transition-state mimic), and ADP via small-angle x-ray scattering has revealed a peristaltic pumping motion upon ATP hydrolysis. This pumping motion likely drives directional substrate translocation across the central Hsp104 channel. Hsp104 initially engages Sup35 prions immediately C-terminal to their cross-β structure. Directional pulling by Hsp104 then resolves N-terminal cross-β structure in a stepwise manner. First, Hsp104 fragments the prion. Second, Hsp104 unfolds cross-β structure. Third, Hsp104 releases soluble Sup35. Deletion of the Hsp104 N-terminal domain yields a hypomorphic disaggregase, Hsp104(∆N), with an altered pumping mechanism. Hsp104(∆N) fragments Sup35 prions without unfolding cross-β structure or releasing soluble Sup35. Moreover, Hsp104(∆N) activity cannot be enhanced by mutations in the middle domain that potentiate disaggregase activity. Thus, the N-terminal domain is critical for the full repertoire of Hsp104 activities. PMID:26608812

  4. The nature of the volcanic activity at Loki: Insights from Galileo NIMS and PPR data

    Howell, Robert R.; Lopes, Rosaly M. C.

    2007-02-01

    Loki is the largest patera and the most energetic hotspot on Jupiter's moon Io, in turn the most volcanically active body in the Solar System, but the nature of the activity remains enigmatic. We present detailed analysis of Galileo Near-Infrared Mapping Spectrometer (NIMS) and PhotoPolarimeter/Radiometer (PPR) observations covering the 1.5-100 μm wavelength range during the I24, I27, and I32 flybys. The general pattern of activity during these flybys is consistent with previously proposed models of a resurfacing wave periodically crossing a silicate lava lake. In particular our analysis of the I32 NIMS observations shows, over much of the observed patera, surface temperatures and implied ages closely matching those expected for a wave advancing counterclockwise at 0.94-1.38 km/day. The age pattern is different than other published analyses which do not show as clearly this azimuthal pattern. Our analysis also shows two additional distinctly different patera surfaces. The first is located along the inner and outer margins where components with a 3.00-4.70-μm color temperature of 425 K exist. The second is located at the southwestern margin where components with a 550-K color temperature exist. Although the high temperatures could be caused by disruption of a lava lake crust, some additional mechanism is required to explain why the southwest margin is different from the inner or outer ones. Finally, analysis of the temperature profiles across the patera reveal a smoothness that is difficult to explain by simple lava cooling models. Paradoxically, at a subpixel level, wide temperature distributions exist which may be difficult to explain by just the presence of hot cracks in the lava crust. The resurfacing wave and lava cooling models explain well the overall characteristics of the observations. However, additional physical processes, perhaps involving heat transport by volatiles, are needed to explain the more subtle features.

  5. Active reward processing during human sleep: insights from sleep-related eating disorder

    Lampros ePerogamvros

    2012-11-01

    Full Text Available In this paper, we present two carefully documented cases of patients with sleep-related eating disorder (SRED, a parasomnia which is characterized by involuntary compulsive eating during the night and whose pathophysiology is not known. Using video-polysomnography and psychometric examination, we found that both patients present elevated novelty seeking and increased reward sensitivity on reward-related questionnaires. In light of new evidence on the mesolimbic dopaminergic implication in compulsive eating disorders, our findings suggest a role of an active reward system during sleep in the manifestation of SRED.

  6. Theoretical insights on the antioxidant activity of edaravone free radical scavengers derivatives

    Cerón-Carrasco, José P.; Roy, Hélène M.; Cerezo, Javier; Jacquemin, Denis; Laurent, Adèle D.

    2014-04-01

    The prediction of antioxidant properties is not straightforward due to the complexity of the in vivo systems. Here, we use theoretical descriptors, including the potential of ionization, the electrodonating power and the spin density distribution, to characterize the antioxidant capacity of edaravone (EDV) derivatives. Our computations reveal the relationship between these parameters and their potential bioactivity as free radical scavengers. We conclude that more efficient antioxidants could be synthesized by tuning the R1 and R2 positions of the EDV structure, rather than modifying the R3 group. Such modifications might improve the antioxidant activity in neutral and deprotonated forms.

  7. Insights into the mechanism of human papillomavirus E2-induced procaspase-8 activation and cell death

    Nitu Singh; Sanjib Senapati; Kakoli Bose

    2016-01-01

    High-risk human papillomavirus (HR-HPV) E2 protein, the master regulator of viral life cycle, induces apoptosis of host cell that is independent of its virus-associated regulatory functions. E2 protein of HR-HPV18 has been found to be involved in novel FADD-independent activation of caspase-8, however, the molecular basis of this unique non-death-fold E2-mediated apoptosis is poorly understood. Here, with an interdisciplinary approach that involves in silico, mutational, biochemical and bioph...

  8. Modeling Steroid 5alpha-reductase and Characterizing Its Potential Active Sites

    OU Min-Rui; LI Jun-Qian

    2012-01-01

    Steroid 5alpha-reductase of human is an enzyme in the biosynthetic pathway from testosterone (T) to dihydrotestosterone (DHT). Up to now, no crystal structure of this enzyme has been reported. However, knowledge of the tertiary structure and possible active sites is essential for understanding the catalysis mechanism and for the design of inhibitors. A model with putative active sites has been created and evaluated by using homology modeling and molecular docking techniques based on the bioinformatics knowledge. The homology model is optimized in Swiss PDB Viewer with MM method and substrate structures before docking are also optimized on HF/6-31G. The active site for the docking of NADP, T, DHT and Finasteride is located near the N-terminus of enzyme. Four active amino acids in the active site are identified as Ala26, Arg53, Arg176 and Lys177. Reaction procedure, binding pattern of active sites, the types of weak interaction and so on are also discussed.

  9. Effects of resource activities upon repository siting and waste containment with reference to bedded salt

    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

  10. A caspase active site probe reveals high fractional inhibition needed to block DNA fragmentation.

    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

  11. Metal ion site engineering indicates a global toggle switch model for seven-transmembrane receptor activation

    Elling, Christian E; Frimurer, Thomas M; Gerlach, Lars-Ole;

    2006-01-01

    to form high affinity metal ion sites in both the bidentate and potential tridentate settings. This indicates that the residues involved in the main ligand-binding pocket will have to move closer to each other during receptor activation. On the basis of the distance constraints from these activating metal...

  12. 77 FR 39508 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    2012-07-03

    ... specific project proposals on those leases) in an identified Wind Energy Area (WEA) on the OCS offshore... Activities on the Atlantic OCS Offshore RI and MA'' to: Program Manager, Office of Renewable Energy Programs... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on...

  13. An Insight into the Anticancer Activities of Ru(II-Based Metallocompounds Using Docking Methods

    Peter A. Ajibade

    2013-09-01

    Full Text Available Unlike organic molecules, reports on docking of metal complexes are very few; mainly due to the inadequacy of force fields in docking packages to appropriately characterize the metal atoms that consequentially hinder the rational design of metal-based drug complexes. In this study we have made used Molegro and Autodock to predict the anticancer activities of selected Ru(II complexes against twelve anticancer targets. We observed that introducing the quantum calculated atomic charges of the optimized geometries significantly improved the docking predictions of these anticancer metallocompounds. Despite several limitations in the docking of metal-based complexes, we obtained results that are highly correlated with the available experimental results. Most of our newly proposed metallocompounds are found theoretically to be better anticancer metallocompounds than all the experimentally proposed RAPTA complexes. An interesting features of a strong interactions of new modeled of metallocompounds against the two base edges of DNA strands suggest similar mechanisms of anticancer activities similar to that of cisplatin. There is possibility of covalent bonding between the metal center of the metallocompounds and the residues of the receptors DNA-1, DNA-2, HDAC7, HIS and RNR. However, the general results suggest the possibility of metals positioning the coordinated ligands in the right position for optimal receptor interactions and synergistic effects, rather than forming covalent bonds.

  14. Insights into the antioxidant activity of some flavones on silver nanoparticles using the chemiluminescence method

    Voicescu, Mariana, E-mail: voicescu@icf.ro [Romanian Academy, Institute of Physical Chemistry “Ilie Murgulescu”, Splaiul Independentei 202, 060021 Bucharest (Romania); Nistor, Cristina L. [Polymer Department, National R and D Institute for Chemistry and Petrochemistry ICECHIM, Splaiul Independentei 202, 060021 Bucharest (Romania); Meghea, Aurelia [University POLITEHNICA of Bucharest, Faculty of Applied Chemistry and Materials Sciences, Polizu 1, 78126 Bucharest (Romania)

    2015-01-15

    The work aims to simulate in vitro the effects caused by oxidation of five hydroxyflavones (HF) (some typical models of flavonols), (3-HF, 6-HF, 7-HF, 3,6-diHF and 3,7-diHF) on silver nanoparticles (SNPs) using the chemiluminescent system luminol–hydrogen peroxide, in phosphate buffer, pH 7.4. The contribution of bovine and human serum albumins to the antioxidant activity of the mentioned flavones, and the effect on the SNPs support, in the chemiluminescent system luminol–hydrogen peroxide, has been also investigated. The results are discussed with relevance to the oxidative stress process. - Highlights: • The effects caused by oxidation of five hydroxiflavones (HF) (3-HF, 6-HF, 7-HF, 3,6-diHF and 3,7-diHF) on silver nanoparticles (SNPs) using the chemiluminescent (CL) system luminol–hydrogen peroxide, in phosphate buffer, pH 7.4. • The contribution of bovine and human serum albumins to the antioxidant activity of the mentioned flavones, and the effect on the SNPs support, in the CL system luminol–hydrogen peroxide, are discussed. • The results have relevance to the oxidative stress process.

  15. Insights into the antioxidant activity of some flavones on silver nanoparticles using the chemiluminescence method

    The work aims to simulate in vitro the effects caused by oxidation of five hydroxyflavones (HF) (some typical models of flavonols), (3-HF, 6-HF, 7-HF, 3,6-diHF and 3,7-diHF) on silver nanoparticles (SNPs) using the chemiluminescent system luminol–hydrogen peroxide, in phosphate buffer, pH 7.4. The contribution of bovine and human serum albumins to the antioxidant activity of the mentioned flavones, and the effect on the SNPs support, in the chemiluminescent system luminol–hydrogen peroxide, has been also investigated. The results are discussed with relevance to the oxidative stress process. - Highlights: • The effects caused by oxidation of five hydroxiflavones (HF) (3-HF, 6-HF, 7-HF, 3,6-diHF and 3,7-diHF) on silver nanoparticles (SNPs) using the chemiluminescent (CL) system luminol–hydrogen peroxide, in phosphate buffer, pH 7.4. • The contribution of bovine and human serum albumins to the antioxidant activity of the mentioned flavones, and the effect on the SNPs support, in the CL system luminol–hydrogen peroxide, are discussed. • The results have relevance to the oxidative stress process

  16. Insights on the Phytochemical Profile (Cyclopeptides and Biological Activities of Calotropis procera Latex Organic Fractions

    Thiago Lustosa Jucá

    2013-01-01

    Full Text Available Calotropis procera is a medicinal plant whose pharmacological properties are associated with its latex. Here, the Calotropis procera latex fractions were investigated in an attempt to trace its phytochemical profile and measure its anti-inflammatory and toxicity activity. The crude latex was partitioned, yielding five fractions (49.4% hexane, 5.2% dichloromethane, 2.0% ethyl acetate, 2.1% n-butanol, and 41.1% aqueous. Phytochemical screening and spectroscopy analysis revealed that dichloromethane is the most chemically diverse fraction. Triterpenes were detected in both the hexane and dichloromethane fractions, while flavonoids were detected in the dichloromethane and ethyl acetate fractions. These fractions were cytotoxic to cancer cell lines (LD50 0.05 to 3.9 μg/mL and lethal to brine shrimp (LD50 10.9 to 65.7 μg/mL. Reduced neutrophil migration in rats was observed in carrageenan-induced peritonitis for the dichloromethane (67%, ethyl acetate (56%, and aqueous (72% fractions. A positive reaction with tolidine and ninhydrin suggested that cyclopeptides are in the ethyl acetate fraction. It is therefore concluded that Calotropis procera latex dichloromethane and ethyl acetate fractions exhibit both in vitro and in vivo activities as well as anti-inflammatory properties. Cyclopeptide detection is especially interesting because previous attempts to investigate these low-molecular cyclic amino acid sequences in C. procera have failed.

  17. The structure of a novel thermophilic esterase from the Planctomycetes species, Thermogutta terrifontis reveals an open active site due to a minimal ‘cap’ domain.

    Jennifer Ann Littlechild

    2015-11-01

    Full Text Available A carboxyl esterase (TtEst2 has been identified in a novel thermophilic bacterium, Thermogutta terrifontis from the phylum Planctomycetes and has been cloned and over-expressed in Escherichia coli. The enzyme has been characterised biochemically and shown to have activity towards small p-nitrophenyl (pNP carboxylic esters with optimal activity for pNP-acetate. The enzyme shows moderate thermostability retaining 75% activity after incubation for 30 minutes at 70°C. The crystal structures have been determined for the native TtEst2 and its complexes with the carboxylic acid products propionate, butyrate and valerate. TtEst2 differs from most enzymes of the α/β-hydrolase family 3 as it lacks the majority of the ‘cap’ domain and its active site cavity is exposed to the solvent. The bound ligands have allowed the identification of the carboxyl pocket in the enzyme active site. Comparison of TtEst2 with structurally related enzymes has given insight into how differences in their substrate preference can be rationalised based upon the properties of their active site pockets.

  18. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    Parashar, Abhinav [Center for Biomedical Research, VIT University, Vellore, Tamil Nadu, 632014 India (India); Venkatachalam, Avanthika [REDOx Lab, PSG Institute of Advanced Studies, Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, 641004 (India); Gideon, Daniel Andrew [Center for Biomedical Research, VIT University, Vellore, Tamil Nadu, 632014 India (India); Manoj, Kelath Murali, E-mail: satyamjayatu@yahoo.com [REDOx Lab, PSG Institute of Advanced Studies, Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, 641004 (India)

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: 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.

  19. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: 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

  20. Some insights about the activity of the Ceboruco Volcano (Nayarit, Mexico) from recent seismic low-frequency activity

    Rodríguez Uribe, María Carolina; Núñez-Cornú, Francisco Javier; Nava Pichardo, Fidencio Alejandro; Suárez-Plascencia, Carlos

    2013-10-01

    The Ceboruco stratovolcano (2,280 m.a.s.l.) is located in Nayarit, Mexico, at the western end of the Mexican volcanic belt, near several population centers and by the side of a strategic highway. During the last 1,000 years it has had, on the average, one eruption every 125 years. It last eruptive activity began in 1870, and during the following 5 years it presented superficial activity including vapor emissions, ash falls, and rhyodacitic lava flows along the southeast side. A data set consisting of 139 low-frequency volcanic-type earthquakes, recorded from March 2003 to July 2008 at the CEBN triaxial short period digital station on the southwestern side of the volcano, was classified according to waveform and spectral characteristics into four families: short duration, extended coda, bobbin, and modulated amplitude. Approximate hypocentral locations indicate that there is no particular location for events of any family, but rather that all events occur at different points within the volcano. The presence of ongoing volcanic-earthquake activity together with the ongoing vapor emissions indicate that the Ceboruco volcano continues to be active, and the higher occurrence rates of short-duration events, as compared with those for the other families, could indicate an increase in the stress in the volcanic edifice. This apparent stress increase, together with the fact that the last eruption occurred 143 years ago, tell us that the Ceboruco may be approaching a critical state, and may represent a hazard to the surrounding communities and economic activities.

  1. Silicate species of water glass and insights for alkali-activated green cement

    Jansson, Helén; Bernin, Diana; Ramser, Kerstin

    2015-06-01

    Despite that sodium silicate solutions of high pH are commonly used in industrial applications, most investigations are focused on low to medium values of pH. Therefore we have investigated such solutions in a broad modulus range and up to high pH values (˜14) by use of infrared (IR) spectroscopy and silicon nuclear magnetic resonance (29Si-NMR). The results show that the modulus dependent pH value leads to more or less charged species, which affects the configurations of the silicate units. This in turn, influences the alkali-activation process of low CO2 footprint cements, i.e. materials based on industrial waste or by-products.

  2. Silicate species of water glass and insights for alkali-activated green cement

    Helén Jansson

    2015-06-01

    Full Text Available Despite that sodium silicate solutions of high pH are commonly used in industrial applications, most investigations are focused on low to medium values of pH. Therefore we have investigated such solutions in a broad modulus range and up to high pH values (∼14 by use of infrared (IR spectroscopy and silicon nuclear magnetic resonance (29Si-NMR. The results show that the modulus dependent pH value leads to more or less charged species, which affects the configurations of the silicate units. This in turn, influences the alkali-activation process of low CO2 footprint cements, i.e. materials based on industrial waste or by-products.

  3. Silicate species of water glass and insights for alkali-activated green cement

    Despite that sodium silicate solutions of high pH are commonly used in industrial applications, most investigations are focused on low to medium values of pH. Therefore we have investigated such solutions in a broad modulus range and up to high pH values (∼14) by use of infrared (IR) spectroscopy and silicon nuclear magnetic resonance (29Si-NMR). The results show that the modulus dependent pH value leads to more or less charged species, which affects the configurations of the silicate units. This in turn, influences the alkali-activation process of low CO2 footprint cements, i.e. materials based on industrial waste or by-products

  4. Physical Activity, Exercise, and Mammalian Testis Function: Emerging Preclinical Protein Biomarker and Integrative Biology Insights.

    Gomes, Mariana; Freitas, Maria João; Fardilha, Margarida

    2015-09-01

    Exercise and physical activity have long been recognized for health promotion and to delay the onset of many pathological situations such as diabetes and cancers. Still, there appears to be an upper limit on the beneficial health effects regarding intensity and frequency of exercise training. In humans, the effect of exercise training in the male reproductive system has been studied mainly through the analysis of semen quality parameters, with inconsistent results. Less is known on molecular biomarkers of exercise-related changes in testis at the protein/proteome level. This review offers an in-depth analysis on the small scale protein studies available primarily from the preclinical studies and interprets their functional impact on the reproductive health with a view to humans. In all, exercise training in preclinical models seems to negatively modulate, in the course of health, critical functions that directly affect spermatogenesis, such as testosterone biosynthesis, energy supply, and antioxidant system components. Exercise training induces apoptosis, leading to the impairment of spermatogenesis and, consequently, to male infertility. In pathological conditions, an improvement in the testicular functions is observed by increases in steroidogenic enzymes and antioxidant defenses, and reductions in activity of inflammatory pathways. Importantly, the mechanisms by which exercise training modulates the reproductive function are far from being fully understood. The analyses of the testis proteome in varying exercise conditions would inform the molecular mechanisms involved and identify putative theranostics opportunities. Such future research is a cornerstone for health promotion in the pursuit of reproductive health informed by omics systems sciences. PMID:26284990

  5. Molecular insights into cold active polygalacturonase enzyme for its potential application in food processing.

    Ramya, L N; Pulicherla, K K

    2015-09-01

    Pectin is a complex structural heteropolysaccharide that require numerous pectinolytic enzymes for its complete degradation. Polygalacturonase from mesophilic or thermophilic origin are being widely used in fruit and vegetable processing in the recent decades to degrade pectic substances. Recently cold active pectinases are finding added advantages over meso and thermophilic counterparts, to use in industrial scale particularly in food processing industry. They facilitate in conservation of several properties of foods so that the end product retains its naturality and also generates economic benefits. In the present study, Pseudoalteromonas haloplanktis, a well reported marine psychrophile is taken as a model organism for cold active polygalacturonase and is evaluated in comparision to the routinely used mesophilic and thermophilic enzymes by insicio approach. Polygalacturonase sequences from industrially important microbial sources were subjected to MEME and Pfam wherein motifs and domains involved in the conservation were analyzed. Dendrogram revealed sequence level similarity and motifs showed uniform distribution of conserved regions that are involved in important functions. It was also observed through clustalW analysis that the amount of arginine content of psychrophiles is less when compared with thermophiles. Finally, all the modeled enzyme structures were subjected to docking studies using Autodock 4.2 with the substrate polygalacturonic acid and binding energies were found to be -5.73, -6.22 and -7.27 KCals/mole for meso, thermo and psychrophiles respectively which indicates the efficiency of psychrophilic enzymes when compared with its counterparts giving scope for further experimentation to find their better usage in various food industry applications. PMID:26344963

  6. Climate impacts on human settlement and agricultural activities in northern Norway: new insights from biogeochemistry

    D'anjou, R. M.; Bradley, R. S.; Balascio, N. L.; Finkelstein, D. B.

    2012-12-01

    Disentangling the effects of climate change and anthropogenic activities on the environment is a major challenge in paleoenvironmental research. Here, we used fecal sterols and other biogeochemical compounds in lake sediments from northern Norway to identify both natural and anthropogenic signals of environmental change during the late Holocene. The area was first occupied by humans and their grazing animals at ~2,250±75 cal yr BP. The arrival of humans is indicated by an abrupt increase in coprostanol (and its epimer epicoprostanol) in the sediments, and an associated increase in 5β-stigmastanol (and 5β-epistigmastanol), which resulted from human and animal feces washing into the lake. Human settlement was accompanied by an abrupt increase in landscape fires (indicated by the rise in pyrolytic polycyclic aromatic hydrocarbons, PAHs) and a decline in woodland (registered by a change in n-alkane chain lengths from leaf waxes), accelerating a process that began earlier in the Holocene. Human activity and associated landscape changes in the region over the last two millennia were mainly driven by summer temperatures, as indicated by independent tree-ring reconstructions, though there were periods when socio-economic factors played an equally important role. This is the first time that fecal sterols in lake sediments have been used to provide a record of human occupancy through time. This approach may be useful in many archeological studies, both to confirm the presence of humans and grazing animals, and to distinguish between anthropogenic and natural factors that have influenced the environment in the past.

  7. Dynamic Contacts of U2, RES, Cwc25, Prp8 and Prp45 Proteins with the Pre-mRNA Branch-Site and 3' Splice Site during Catalytic Activation and Step 1 Catalysis in Yeast Spliceosomes.

    Cornelius Schneider

    Full Text Available Little is known about contacts in the spliceosome between proteins and intron nucleotides surrounding the pre-mRNA branch-site and their dynamics during splicing. We investigated protein-pre-mRNA interactions by UV-induced crosslinking of purified yeast B(act spliceosomes formed on site-specifically labeled pre-mRNA, and analyzed their changes after conversion to catalytically-activated B* and step 1 C complexes, using a purified splicing system. Contacts between nucleotides upstream and downstream of the branch-site and the U2 SF3a/b proteins Prp9, Prp11, Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site. A comparison of the B(act crosslinking pattern versus that of B* and C complexes revealed that U2 and RES protein interactions with the intron are dynamic. Upon step 1 catalysis, Cwc25 contacts with the branch-site region, and enhanced crosslinks of Prp8 and Prp45 with nucleotides surrounding the branch-site were observed. Cwc25's step 1 promoting activity was not dependent on its interaction with pre-mRNA, indicating it acts via protein-protein interactions. These studies provide important insights into the spliceosome's protein-pre-mRNA network and reveal novel RNP remodeling events during the catalytic activation of the spliceosome and step 1 of splicing.

  8. Insights into the pathogenic character of a common NUBPL branch-site mutation associated with mitochondrial disease and complex I deficiency using a yeast model

    Mateusz M. Wydro

    2013-09-01

    Complex I deficiencies are the most common causes of mitochondrial disorders. They can result from mutations not only in the structural subunits but also in a growing number of known assembly factors. A branch-site mutation in the human gene encoding assembly factor NUBPL has recently been associated with mitochondrial encephalopathy and complex I deficiency in seven independent cases. Moreover, the mutation is present in 1.2% of European haplotypes. To investigate its pathogenicity, we have reconstructed the altered C-terminus that results from the branch-site mutation and frameshift in the homologous Ind1 protein in the respiratory yeast Yarrowia lipolytica. We demonstrate that the altered sequence did not affect IND1 mRNA stability, yet it led to a decrease in Ind1 protein level. The instability of mutant Ind1 resulted in a strong decrease in complex I activity and caused slow growth, resembling the phenotype of the deletion strain of IND1. The presented data confirms the deleterious impact of the altered C-terminus resulting from the branch-site mutation. Furthermore, our approach demonstrates the great potential of Y. lipolytica as a model to investigate complex I deficiencies, especially in cases with genetic complexity.

  9. Elucidation of tonic and activated B-cell receptor signaling in Burkitt's lymphoma provides insights into regulation of cell survival.

    Corso, Jasmin; Pan, Kuan-Ting; Walter, Roland; Doebele, Carmen; Mohr, Sebastian; Bohnenberger, Hanibal; Ströbel, Philipp; Lenz, Christof; Slabicki, Mikolaj; Hüllein, Jennifer; Comoglio, Federico; Rieger, Michael A; Zenz, Thorsten; Wienands, Jürgen; Engelke, Michael; Serve, Hubert; Urlaub, Henning; Oellerich, Thomas

    2016-05-17

    Burkitt's lymphoma (BL) is a highly proliferative B-cell neoplasm and is treated with intensive chemotherapy that, because of its toxicity, is often not suitable for the elderly or for patients with endemic BL in developing countries. BL cell survival relies on signals transduced by B-cell antigen receptors (BCRs). However, tonic as well as activated BCR signaling networks and their relevance for targeted therapies in BL remain elusive. We have systematically characterized and compared tonic and activated BCR signaling in BL by quantitative phosphoproteomics to identify novel BCR effectors and potential drug targets. We identified and quantified ∼16,000 phospho-sites in BL cells. Among these sites, 909 were related to tonic BCR signaling, whereas 984 phospho-sites were regulated upon BCR engagement. The majority of the identified BCR signaling effectors have not been described in the context of B cells or lymphomas yet. Most of these newly identified BCR effectors are predicted to be involved in the regulation of kinases, transcription, and cytoskeleton dynamics. Although tonic and activated BCR signaling shared a considerable number of effector proteins, we identified distinct phosphorylation events in tonic BCR signaling. We investigated the functional relevance of some newly identified BCR effectors and show that ACTN4 and ARFGEF2, which have been described as regulators of membrane-trafficking and cytoskeleton-related processes, respectively, are crucial for BL cell survival. Thus, this study provides a comprehensive dataset for tonic and activated BCR signaling and identifies effector proteins that may be relevant for BL cell survival and thus may help to develop new BL treatments. PMID:27155012

  10. Crystal structure of an avian influenza polymerase PA[subscript N] reveals an endonuclease active site

    Yuan, Puwei; Bartlam, Mark; Lou, Zhiyong; Chen, Shoudeng; Zhou, Jie; He, Xiaojing; Lv, Zongyang; Ge, Ruowen; Li, Xuemei; Deng, Tao; Fodor, Ervin; Rao, Zihe; Liu, Yingfang; (NU Sinapore); (Nankai); (Oxford); (Chinese Aca. Sci.); (Tsinghua)

    2009-11-10

    The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site and reportedly harbours endonuclease activity, whereas PB2 is responsible for cap binding. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease and protease activities as well as viral RNA/complementary RNA promoter binding. Here we report the 2.2 angstrom (A) crystal structure of the N-terminal 197 residues of PA, termed PA(N), from an avian influenza H5N1 virus. The PA(N) structure has an alpha/beta architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DX(N)(D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PA(N) provide further evidence that PA(N) holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PA(N) holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PA(N) is an important target for the design of new anti-influenza therapeutics.

  11. Substrate Shuttling Between Active Sites of Uroporphyrinogen Decarboxylase in Not Required to Generate Coproporphyrinogen

    Phillips, J.; Warby, C; Whitby, F; Kushner, J; Hill, C

    2009-01-01

    Uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.

  12. In silico analysis of Pycnoporus cinnabarinus laccase active site with toxic industrial dyes.

    Prasad, Nirmal K; Vindal, Vaibhav; Narayana, Siva Lakshmi; Ramakrishna, V; Kunal, Swaraj Priyaranjan; Srinivas, M

    2012-05-01

    Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in various industrial oxidative processes like production of fine chemicals to bioremediation of contaminated soil and water. In order to understand the mechanisms of substrate binding and interaction between substrates and Pycnoporus cinnabarinus laccase, a homology model was generated. The resulted model was further validated and used for docking studies with toxic industrial dyes- acid blue 74, reactive black 5 and reactive blue 19. Interactions of chemical mediators with the laccase was also examined. The docking analysis showed that the active site always cannot accommodate the dye molecules, due to constricted nature of the active site pocket and steric hindrance of the residues whereas mediators are relatively small and can easily be accommodated into the active site pocket, which, thereafter leads to the productive binding. The binding properties of these compounds along with identification of critical active site residues can be used for further site-directed mutagenesis experiments in order to identify their role in activity and substrate specificity, ultimately leading to improved mutants for degradation of these toxic compounds. PMID:21877154

  13. Export of earthquake-triggered landslides in active mountain ranges: insights from 2D morphodynamic modelling.

    Croissant, Thomas; Lague, Dimitri; Davy, Philippe; Steer, Philippe

    2016-04-01

    In active mountain ranges, large earthquakes (Mw > 5-6) trigger numerous landslides that impact river dynamics. These landslides bring local and sudden sediment piles that will be eroded and transported along the river network causing downstream changes in river geometry, transport capacity and erosion efficiency. The progressive removal of landslide materials has implications for downstream hazards management and also for understanding landscape dynamics at the timescale of the seismic cycle. The export time of landslide-derived sediments after large-magnitude earthquakes has been studied from suspended load measurements but a full understanding of the total process, including the coupling between sediment transfer and channel geometry change, still remains an issue. Note that the transport of small sediment pulses has been studied in the context of river restoration, but the magnitude of sediment pulses generated by landslides may make the problem different. Here, we study the export of large volumes (>106 m3) of sediments with the 2D hydro-morphodynamic model, Eros. This model uses a new hydrodynamic module that resolves a reduced form of the Saint-Venant equations with a particle method. It is coupled with a sediment transport and lateral and vertical erosion model. Eros accounts for the complex retroactions between sediment transport and fluvial geometry, with a stochastic description of the floods experienced by the river. Moreover, it is able to reproduce several features deemed necessary to study the evacuation of large sediment pulses, such as river regime modification (single-thread to multi-thread), river avulsion and aggradation, floods and bank erosion. Using a synthetic and simple topography we first present how granulometry, landslide volume and geometry, channel slope and flood frequency influence 1) the dominance of pulse advection vs. diffusion during its evacuation, 2) the pulse export time and 3) the remaining volume of sediment in the catchment

  14. Locomotor activity influences muscle architecture and bone growth but not muscle attachment site morphology

    Rabey, Karyne N.; Green, David J; Taylor, Andrea B.; Begun, David R.; Richmond, Brian G.; McFarlin, Shannon C.

    2014-01-01

    The ability to make behavioural inferences from skeletal remains is critical to understanding the lifestyles and activities of past human populations and extinct animals. Muscle attachment site (enthesis) morphology has long been assumed to reflect muscle strength and activity during life, but little experimental evidence exists to directly link activity patterns with muscle development and the morphology of their attachments to the skeleton. We used a mouse model to experimentally test how t...

  15. New insights into dietary supplements used in sport: active substances, pharmacological and side effects.

    Koncic, Marijana Zovko; Tomczyk, Michal

    2013-08-01

    As a society we are increasingly concerned about our physical appearance. For example, as much as 24% of people in developed countries admittedly exercise to improve their performance. Professional sportsmen and amateurs alike are in a constant search for new means that will enable them better sport results in shorter time. Among those means, a prominent place belongs to dietary supplements. However, the producers often advertise products whose use in sports is neither scientifically founded nor safe. This brings on an irrational use of herbal supplements which sometimes leads to unwanted side effects, but is more often of little use. Thus, the aim of this review will be to systematically evaluate some of the herbal supplements that are used as adaptogenic and ergogenic aids in sport. The review will include available data on Rhodiola rosea, Withania somnifera, Schisandra chinensis, Tribulus terrestris, Vitis vinifera, Citrus aurantium, and others. Their effects, active ingredients as well as possible adverse effects will be discussed with special focus on clinical studies. PMID:23574283

  16. Commentary on the Liquid Metallic Hydrogen Model of the Sun: Insight Relative to Coronal Holes, Sunspots, and Solar Activity

    Robitaille P.-M.

    2013-04-01

    Full Text Available While mankind will always remain unable to sample the interior of the Sun, the presence of sunspots and coronal holes can provide clues as to its subsurface structure. Insight relative to the solar body can also be gained by recognizing that the Sun must exist in the condensed state and support a discrete lattice structure, as required for the production of its continuous spectrum. In this regard, the layered liquid metallic hydrogen lattice advanced as a condensed model of the Sun (Robitaille P.M. Liquid Metallic Hydrogen: A Building Block for the Liquid Sun. Progr. Phys ., 2011, v. 3, 60–74; Robitaille P.M. Liquid Metallic Hydrogen II: A Critical Assessment of Current and Primordial Helium Levels in Sun. Progr. Phys ., 2013, v. 2, 35–47; Robitaille J.C. and Robitaille P.M. Liquid Metallic Hydrogen III. Intercalation and Lattice Exclusion Versus Gravitational Settling and Their Consequences Relative to Internal Structure, Surface Activity, and Solar Winds in the Sun. Progr. Phys ., 2013, v. 2, in press provides the ability to add structure to the solar interior. This constitutes a significant advantage over the gaseous solar models. In fact, a layered liquid metallic hydrogen lattice and the associated intercalation of non-hydrogen elements can help to account for the position of sunspots and coronal holes. At the same time, this model provides a greater understanding of the mechanisms which drive solar winds and activity.

  17. Nuclear waste: Issues concerning DOE's postponement of second repository siting activities

    In May 1986 the Department of Energy announced it was postponing site-specific work for a second nuclear waste repository because of progress made in siting the first repository and the uncertainty about when a second repository might be needed. A DOE draft report, which tentatively identified potential sites in seven different states, received considerable comment and objections by concerned states, Indian tribes, and individuals. Following the postponement decision, DOE initiated actions to redirect and restructure the second repository program, concentrating on technical issues rather than site-specific work. This reduction includes a phase-down of siting activities and the formulation of a Technology Development Program with an emphasis on cooperative efforts in international research

  18. Large zinc cation occupancy of octahedral sites in mechanically activated zinc ferrite powders

    The cation site occupancy of a mechanically activated nanocrystalline zinc ferrite powder was determined as (Zn0.552+Fe0.183+)tet[Zr0.452+Fe1.823+]octO4 through analysis of extended x-ray absorption fine structure measurements, showing a large redistribution of cations between sites compared to normal zinc ferrite samples. The overpopulation of cations in the octahedral sites was attributed to the ascendance in importance of the ionic radii over the crystal energy and bonding coordination in determining which interstitial sites are occupied in this structurally disordered powder. Slight changes are observed in the local atomic environment about the zinc cations, but not the iron cations, with respect to the spinel structure. The presence of Fe3+ on both sites is consistent with the measured room temperature magnetic properties. (c) 2000 American Institute of Physics

  19. New insights into the aquatic photochemistry of fluoroquinolone antibiotics: Direct photodegradation, hydroxyl-radical oxidation, and antibacterial activity changes

    The ubiquity and photoreactivity of fluoroquinolone antibiotics (FQs) in surface waters urge new insights into their aqueous photochemical behavior. This study concerns the photochemistry of 6 FQs: ciprofloxacin, danofloxacin, levofloxacin, sarafloxacin, difloxacin and enrofloxacin. Methods were developed to calculate their solar direct photodegradation half-lives (td,E) and hydroxyl-radical oxidation half-lives (t·OH,E) in sunlit surface waters. The td,E values range from 0.56 min to 28.8 min at 45° N latitude, whereas t·OH,E ranges from 3.24 h to 33.6 h, suggesting that most FQs tend to undergo fast direct photolysis rather than hydroxyl-radical oxidation in surface waters. However, a case study for levofloxacin and sarafloxacin indicated that the hydroxyl-radical oxidation induced risky photochlorination and resulted in multi-degradation pathways, such as piperazinyl hydroxylation and clearage. Changes in the antibacterial activity of FQs caused by photodegradation in various waters were further examined using Escherichia coli, and it was found that the activity evolution depended on primary photodegradation pathways and products. Primary intermediates with intact FQ nuclei retained significant antibacterial activity. These results are important for assessing the fate and risk of FQs in surface waters. - Highlights: • It is first reported on hydroxyl-radical oxidation of 6 fluoroquinolone antibiotics. • Methods were developed to assess photolysis and oxidation fate in surface waters. • The neutral form reacted faster with hydroxyl radical than protonated forms. • The main oxidation intermediates and transformation pathways were clarified. • The antibacterial activity changes depend on dominant photolysis pathways

  20. New insights into the aquatic photochemistry of fluoroquinolone antibiotics: Direct photodegradation, hydroxyl-radical oxidation, and antibacterial activity changes

    Ge, Linke; Na, Guangshui [Key Laboratory for Ecological Environment in Coastal Areas (SOA), National Marine Environmental Monitoring Center, Dalian 116023 (China); Zhang, Siyu [Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016 (China); Li, Kai [Key Laboratory for Ecological Environment in Coastal Areas (SOA), National Marine Environmental Monitoring Center, Dalian 116023 (China); Zhang, Peng, E-mail: pzhang@nmemc.org.cn [Key Laboratory for Ecological Environment in Coastal Areas (SOA), National Marine Environmental Monitoring Center, Dalian 116023 (China); Ren, Honglei; Yao, Ziwei [Key Laboratory for Ecological Environment in Coastal Areas (SOA), National Marine Environmental Monitoring Center, Dalian 116023 (China)

    2015-09-15

    The ubiquity and photoreactivity of fluoroquinolone antibiotics (FQs) in surface waters urge new insights into their aqueous photochemical behavior. This study concerns the photochemistry of 6 FQs: ciprofloxacin, danofloxacin, levofloxacin, sarafloxacin, difloxacin and enrofloxacin. Methods were developed to calculate their solar direct photodegradation half-lives (t{sub d,E}) and hydroxyl-radical oxidation half-lives (t{sub ·OH,E}) in sunlit surface waters. The t{sub d,E} values range from 0.56 min to 28.8 min at 45° N latitude, whereas t{sub ·OH,E} ranges from 3.24 h to 33.6 h, suggesting that most FQs tend to undergo fast direct photolysis rather than hydroxyl-radical oxidation in surface waters. However, a case study for levofloxacin and sarafloxacin indicated that the hydroxyl-radical oxidation induced risky photochlorination and resulted in multi-degradation pathways, such as piperazinyl hydroxylation and clearage. Changes in the antibacterial activity of FQs caused by photodegradation in various waters were further examined using Escherichia coli, and it was found that the activity evolution depended on primary photodegradation pathways and products. Primary intermediates with intact FQ nuclei retained significant antibacterial activity. These results are important for assessing the fate and risk of FQs in surface waters. - Highlights: • It is first reported on hydroxyl-radical oxidation of 6 fluoroquinolone antibiotics. • Methods were developed to assess photolysis and oxidation fate in surface waters. • The neutral form reacted faster with hydroxyl radical than protonated forms. • The main oxidation intermediates and transformation pathways were clarified. • The antibacterial activity changes depend on dominant photolysis pathways.

  1. Biogeochemical Activity of Siderophilic Cyanobacteria and Insights from their Genomes Implications for the Development of New Biosignatures

    Brown, I. I.; Bryant, D. A.; Thomas,-Keprta, K. L.; Tringe, S. G.; Sarkisova, S. A.; Galindo, C., Jr.; Malley, K.; Sosa, O.; Garrison, D. H.; McKay, David S.

    2010-01-01

    Verifying the links between genomie features in living organisms and their mineralization/demineralization activity will help to reveal traces of life on Earth and beyond. Among contemporary environments, iron-depositing hot springs (IDHS) may represent one of the most appropriate natural models for insights into ancient life since organisms may have originated on Earth and possibly Mars in association with hydrothennal activity and high [Fe(2+)]. Siderophilic or "iron-loving" cyanobacteria (CB) inhabiting IDHS may have genomic features and properties similar to those of ancient organisms because abundant Fe(2+) in IDHS has a strong potential to increase the magnitude of oxidative stress. That is why specific and/or additional proteins involved in Fe mineralization by siderophilic CB are expected. Inorganic polyphosphates (PPi) are known to increase the viability of prokaryotes Linder heavy metal concentrations and UV stress conditions. PPi have also been proposed as biosignatures. Ancient CB could have also been stressed by occasional migrations from the Fe(2+) rich Ocean to the basaltic land which was almost devoid of dissolved Fe(2+). Thus, the study of the adaptation reactions of siderophilic CB to fluctuation of dissolved Fe level may shed light on the paleophysiology of ancient oxygenic prokaryotes. Moreover, bioweathered Fe, Al, P, Cu, Ti and rare earth elements can be thought of as candidate organomarkers that document the effects of or ganic molecules in weathered rocks. However, the molecular mechanisms of the maintenance of Fe homeostasis in siderophilic CB, the role of PPi for this process and bioweathering activities are poorly understood. Here we present preliminary results describing a new mechanism of Fe mineralization in siderophilic CB, the effect of Fe on the generation of PPi bodies in siderophilic CB, their bioweathering activity and preliminary analysis of the diversity of proteins involved in the prevention of oxidative stress in phototrophs

  2. The toponymy of communal activity: Anglo-Saxon assembly sites and their functions

    Baker, John

    2014-01-01

    The paper builds on earlier discussion of the multiple functions of medieval judicial assembly sites, providing a comprehensive evaluation of relevant English hundred-names, and making reference to associated microtoponymy. While religious, military, commercial, and recreational activities may all have occurred at assembly-sites, it can be hard to delineate the evidence so clearly along these lines, and attempts to do so may be anachronistic in some instances; nevertheless, the analysis of di...

  3. Anticancer activity of galactoxyloglucan polysaccharide-conjugated doxorubicin nanoparticles: Mechanistic insights and interactome analysis.

    Joseph, Manu M; Aravind, S R; George, Suraj K; Raveendran Pillai, K; Mini, S; Sreelekha, T T

    2015-06-01

    Toxicity associated with chemotherapeutic drugs such as doxorubicin (Dox), is one of the major obstacles that is currently affecting patients. PST-Dox (Galactoxyloglucan, PST001-conjugated Dox) nanoparticles were synthesized by encapsulating Dox with polysaccharide PST001, isolated from Tamarindus indica (Ti) by ionic gelation with tripolyphosphate (TPP). Herein, we demonstrate a detailed mechanistic and interactome network analysis that is specific to PST-Dox action in cancer cells and normal lymphocytes. Our results show that PST-Dox is superior to its parental counterparts, exhibiting a greater cytotoxicity by the induction of apoptosis against a wide variety of cancers by enhanced cellular uptake of Dox from the nanoparticle conjugates. Also, PST-Dox nanoparticles were non-toxic to normal lymphocytes with limited immunostimulatory effects up to certain doses. Elucidation of molecular mechanism by whole genome microarray in cancer cells and lymphocytes revealed that a large number of genes were dysregulated specifically in cancer cells. Specifically, a unique target gene EGR1, contextually determined translational activation of P53 in the cancerous and non-cancerous cells. Most of the key downregulated genes were tyrosine kinases, indicating the potential inhibitory action of PST-Dox on tyrosine kinase oncogenic pathways. Western blotting of proteins corresponding to the genes that were altered at the genomic level was very well correlated in the majority of them, except in a few that demonstrated post-transcriptional modifications. The important findings and highly disciplined approaches highlighted in the present study will speed up the therapeutic potential of this augmented nanoparticle formulation for more robust clinical studies and testing in several cancers. PMID:25864443

  4. Alkaline phosphatase activity in the subtropical ocean: insights from nutrient, dust and trace metal addition experiments

    Claire eMahaffey

    2014-12-01

    Full Text Available Phosphorus is an essential nutrient for all life on earth. In the ocean, the most bioavailable form of phosphorus is inorganic phosphate, but in the extensive subtropical gyres, phosphate concentrations can be chronically low and limit primary productivity and nitrogen fixation. In these regions, organisms produce hydrolytic enzymes, such as alkaline phosphatase (AP, that enable them to utilize the more replete dissolved organic phosphorus (DOP pool to meet their cellular phosphorus demands. In this study, we synthesized data from 14 published studies and present our own findings from two research cruises (D326 and D361 in the eastern subtropical Atlantic to explore the relationship between AP activity (APA and nutrients, Saharan dust and trace metals. We found that below a threshold phosphate concentration of ~ 30 nM, APA increased with an inverse hyperbolic relationship with phosphate concentration. Meanwhile, DOP concentrations decreased with enhanced APA, indicating utilization of the DOP pool. We found APA rates were significantly higher in the subtropical Atlantic compared to the subtropical Pacific Ocean, even over the same low phosphate concentration range (0 to 50 nM. While the phosphate concentration may have a first order control on the APA rates, we speculate that other factors influence this basin scale contrast. Using bioassay experiments, we show that the addition of Saharan dust and zinc significantly increased the rate of APA. To our knowledge, our results are the first direct field-based evidence that APA is limited by zinc in the subtropical ocean. Further work is required to explore the relationship between trace metals such as iron and zinc, which are co-factors of phosphohydrolytic enzymes, specifically PhoX and PhoA, respectively, and APA in the ocean.

  5. Hydroxylation of p-substituted phenols by tyrosinase: further insight into the mechanism of tyrosinase activity.

    Muñoz-Muñoz, Jose Luis; Berna, Jose; García-Molina, María del Mar; Garcia-Molina, Francisco; Garcia-Ruiz, Pedro Antonio; Varon, Ramon; Rodriguez-Lopez, Jose N; Garcia-Canovas, Francisco

    2012-07-27

    A study of the monophenolase activity of tyrosinase by measuring the steady state rate with a group of p-substituted monophenols provides the following kinetic information: k(cat)(m) and the Michaelis constant, K(M)(m). Analysis of these data taking into account chemical shifts of the carbon atom supporting the hydroxyl group (δ) and σ(p)(+), enables a mechanism to be proposed for the transformation of monophenols into o-diphenols, in which the first step is a nucleophilic attack on the copper atom on the form E(ox) (attack of the oxygen of the hydroxyl group of C-1 on the copper atom) followed by an electrophilic attack (attack of the hydroperoxide group on the ortho position with respect to the hydroxyl group of the benzene ring, electrophilic aromatic substitution with a reaction constant ρ of -1.75). These steps show the same dependency on the electronic effect of the substituent groups in C-4. Furthermore, a study of a solvent deuterium isotope effect on the oxidation of monophenols by tyrosinase points to an appreciable isotopic effect. In a proton inventory study with a series of p-substituted phenols, the representation of [Formula: see text] / [Formula: see text] against n (atom fractions of deuterium), where [Formula: see text] is the catalytic constant for a molar fraction of deuterium (n) and [Formula: see text] is the corresponding kinetic parameter in a water solution, was linear for all substrates. These results indicate that only one of the proton transfer processes from the hydroxyl groups involved the catalytic cycle is responsible for the isotope effects. We suggest that this step is the proton transfer from the hydroxyl group of C-1 to the peroxide of the oxytyrosinase form (E(ox)). After the nucleophilic attack, the incorporation of the oxygen in the benzene ring occurs by means of an electrophilic aromatic substitution mechanism in which there is no isotopic effect. PMID:22732412

  6. Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4-Oxalocrotonate Tautomerase More Effectively than Distant Mutations.

    Rahimi, Mehran; van der Meer, Jan-Ytzen; Geertsema, Edzard M; Poddar, Harshwardhan; Baas, Bert-Jan; Poelarends, Gerrit J

    2016-07-01

    The enzyme 4-oxalocrotonate tautomerase (4-OT), which catalyzes enol-keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon-carbon bond-forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4-OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000-fold improvement in catalytic efficiency (kcat /Km ) and a >10(7) -fold change in reaction specificity, by exploring small libraries in which only "hotspots" are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4-OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site. PMID:27238293

  7. Hydroxylation of p-substituted phenols by tyrosinase: Further insight into the mechanism of tyrosinase activity

    Highlights: ► The action the copper complexes and tyrosinase on phenols is equivalent. ► Isotope effect showed that nucleophilic attack to copper atom may be the slower step. ► The value of ρ (Hammett constant) supports an electrophilic aromatic substitution. ► Data obtained in steady state pH 7 conditions support the mechanism of Scheme 1SM. -- Abstract: A study of the monophenolase activity of tyrosinase by measuring the steady state rate with a group of p-substituted monophenols provides the following kinetic information: kcatm and the Michaelis constant, KMm. Analysis of these data taking into account chemical shifts of the carbon atom supporting the hydroxyl group (δ) and σp+, enables a mechanism to be proposed for the transformation of monophenols into o-diphenols, in which the first step is a nucleophilic attack on the copper atom on the form Eox (attack of the oxygen of the hydroxyl group of C-1 on the copper atom) followed by an electrophilic attack (attack of the hydroperoxide group on the ortho position with respect to the hydroxyl group of the benzene ring, electrophilic aromatic substitution with a reaction constant ρ of −1.75). These steps show the same dependency on the electronic effect of the substituent groups in C-4. Furthermore, a study of a solvent deuterium isotope effect on the oxidation of monophenols by tyrosinase points to an appreciable isotopic effect. In a proton inventory study with a series of p-substituted phenols, the representation of kcatfn/kcatf0 against n (atom fractions of deuterium), where kcatfn is the catalytic constant for a molar fraction of deuterium (n) and kcatf0 is the corresponding kinetic parameter in a water solution, was linear for all substrates. These results indicate that only one of the proton transfer processes from the hydroxyl groups involved the catalytic cycle is responsible for the isotope effects. We suggest that this step is the proton transfer from the hydroxyl group of C-1 to the

  8. Hydroxylation of p-substituted phenols by tyrosinase: Further insight into the mechanism of tyrosinase activity

    Munoz-Munoz, Jose Luis [GENZ - Grupo de Investigacion Enzimologia, Departamento de Bioquimica y Biologia Molecular-A, Facultad de Biologia, Campus Internacional de Excelencia Campus Mare Nostrum, Universidad de Murcia, E-30100 Espinardo, Murcia (Spain); Berna, Jose [Grupo de Quimica Organica Sintetica, Departamento de Quimica Organica, Facultad de Quimica Campus Internacional de Excelencia Campus Mare Nostrum, Universidad de Murcia (Spain); Garcia-Molina, Maria del Mar; Garcia-Molina, Francisco [GENZ - Grupo de Investigacion Enzimologia, Departamento de Bioquimica y Biologia Molecular-A, Facultad de Biologia, Campus Internacional de Excelencia Campus Mare Nostrum, Universidad de Murcia, E-30100 Espinardo, Murcia (Spain); Garcia-Ruiz, Pedro Antonio [QCPAI - Grupo de Quimica de Carbohidratos, Polimeros y Aditivos Industriales, Departamento de Quimica Organica, Facultad de Quimica Campus Internacional de Excelencia Campus Mare Nostrum, Universidad de Murcia (Spain); Varon, Ramon [Departamento de Quimica-Fisica, Escuela de Ingenieros Industriales de Albacete, Universidad de Castilla la Mancha, Avda. Espana s/n. Campus Universitario, E-02071 Albacete (Spain); and others

    2012-07-27

    Highlights: Black-Right-Pointing-Pointer The action the copper complexes and tyrosinase on phenols is equivalent. Black-Right-Pointing-Pointer Isotope effect showed that nucleophilic attack to copper atom may be the slower step. Black-Right-Pointing-Pointer The value of {rho} (Hammett constant) supports an electrophilic aromatic substitution. Black-Right-Pointing-Pointer Data obtained in steady state pH 7 conditions support the mechanism of Scheme 1SM. -- Abstract: A study of the monophenolase activity of tyrosinase by measuring the steady state rate with a group of p-substituted monophenols provides the following kinetic information: k{sub cat}{sup m} and the Michaelis constant, K{sub M}{sup m}. Analysis of these data taking into account chemical shifts of the carbon atom supporting the hydroxyl group ({delta}) and {sigma}{sub p}{sup +}, enables a mechanism to be proposed for the transformation of monophenols into o-diphenols, in which the first step is a nucleophilic attack on the copper atom on the form E{sub ox} (attack of the oxygen of the hydroxyl group of C-1 on the copper atom) followed by an electrophilic attack (attack of the hydroperoxide group on the ortho position with respect to the hydroxyl group of the benzene ring, electrophilic aromatic substitution with a reaction constant {rho} of -1.75). These steps show the same dependency on the electronic effect of the substituent groups in C-4. Furthermore, a study of a solvent deuterium isotope effect on the oxidation of monophenols by tyrosinase points to an appreciable isotopic effect. In a proton inventory study with a series of p-substituted phenols, the representation of k{sub cat}{sup f{sub n}}/k{sub cat}{sup f{sub 0}} against n (atom fractions of deuterium), where k{sub cat}{sup f{sub n}} is the catalytic constant for a molar fraction of deuterium (n) and k{sub cat}{sup f{sub 0}} is the corresponding kinetic parameter in a water solution, was linear for all substrates. These results indicate that

  9. Improving the neutral phytase activity from Bacillus amyloliquefaciens DSM 1061 by site-directed mutagenesis.

    Xu, Wei; Shao, Rong; Wang, Zupeng; Yan, Xiuhua

    2015-03-01

    Neutral phytase is used as a feed additive for degradation of anti-nutritional phytate in aquatic feed industry. Site-directed mutagenesis of Bacillus amyloliquefaciens DSM 1061 phytase was performed with an aim to increase its activity. Mutation residues were chosen based on multiple sequence alignments and structure analysis of neutral phytsaes from different microorganisms. The mutation sites on surface (D148E, S197E and N156E) and around the active site (D52E) of phytase were selected. Analysis of the phytase variants showed that the specific activities of mutants D148E and S197E remarkably increased by about 35 and 13% over a temperature range of 40-75 °C at pH 7.0, respectively. The k cat of mutants D148E and S197E were 1.50 and 1.25 times than that of the wild-type phytase, respectively. Both D148E and S197E showed much higher thermostability than that of the wild-type phytase. However, mutants N156E and D52E led to significant loss of specific activity of the enzyme. Structural analysis revealed that these mutations may affect conformation of the active site of phytase. The present mutant phytases D148E and S197E with increased activities and thermostabilities have application potential as additives in aquaculture feed. PMID:25613522

  10. Stringency of the 2-His-1-Asp active-site motif in prolyl 4-hydroxylase.

    Kelly L Gorres

    Full Text Available The non-heme iron(II dioxygenase family of enzymes contain a common 2-His-1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C-H bonds. Prolyl 4-hydroxylase (P4H is an alpha-ketoglutarate-dependent iron(II dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His-1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change.

  11. Quantum delocalization of protons in the hydrogen bond network of an enzyme active site

    Wang, Lu; Boxer, Steven G; Markland, Thomas E

    2015-01-01

    Enzymes utilize protein architectures to create highly specialized structural motifs that can greatly enhance the rates of complex chemical transformations. Here we use experiments, combined with ab initio simulations that exactly include nuclear quantum effects, to show that a triad of strongly hydrogen bonded tyrosine residues within the active site of the enzyme ketosteroid isomerase (KSI) facilitates quantum proton delocalization. This delocalization dramatically stabilizes the deprotonation of an active site tyrosine residue, resulting in a very large isotope effect on its acidity. When an intermediate analog is docked, it is incorporated into the hydrogen bond network, giving rise to extended quantum proton delocalization in the active site. These results shed light on the role of nuclear quantum effects in the hydrogen bond network that stabilizes the reactive intermediate of KSI, and the behavior of protons in biological systems containing strong hydrogen bonds.

  12. Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

    Nakamichi, Yusuke; Oiki, Sayoko; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

    2016-08-01

    Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst. PMID:27402448

  13. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity.

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-12-15

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  14. Testing the applicability of rapid on-site enzymatic activity detection for surface water monitoring

    Stadler, Philipp; Vogl, Wolfgang; Juri, Koschelnik; Markus, Epp; Maximilian, Lackner; Markus, Oismüller; Monika, Kumpan; Peter, Strauss; Regina, Sommer; Gabriela, Ryzinska-Paier; Farnleitner Andreas, H.; Matthias, Zessner

    2015-04-01

    On-site detection of enzymatic activities has been suggested as a rapid surrogate for microbiological pollution monitoring of water resources (e.g. using glucuronidases, galactosidases, esterases). Due to the possible short measuring intervals enzymatic methods have high potential as near-real time water quality monitoring tools. This presentation describes results from a long termed field test. For twelve months, two ColiMinder devices (Vienna Water Monitoring, Austria) for on-site determination of enzymatic activity were tested for stream water monitoring at the experimental catchment HOAL (Hydrological Open Air Laboratory, Center for Water Resource Systems, Vienna University of Technology). The devices were overall able to follow and reflect the diverse hydrological and microbiological conditions of the monitored stream during the test period. Continuous data in high temporal resolution captured the course of enzymatic activity in stream water during diverse rainfall events. The method also proofed sensitive enough to determine diurnal fluctuations of enzymatic activity in stream water during dry periods. The method was able to capture a seasonal trend of enzymatic activity in stream water that matches the results gained from Colilert18 analysis for E. coli and coliform bacteria of monthly grab samples. Furthermore the comparison of ColiMinder data with measurements gained at the same test site with devices using the same method but having different construction design (BACTcontrol, microLAN) showed consistent measuring results. Comparative analysis showed significant differences between measured enzymatic activity (modified fishman units and pmol/min/100ml) and cultivation based analyses (most probable number, colony forming unit). Methods of enzymatic activity measures are capable to detect ideally the enzymatic activity caused by all active target bacteria members, including VBNC (viable but nonculturable) while cultivation based methods cannot detect VBNC

  15. Preliminary examination of the impacts of repository site characterization activities and facility construction and operation activities on Hanford air quality

    Glantz, C.S.; Ramsdell, J.V.

    1986-04-01

    Air quality impacts that would result from site characterization activities and from the construction and operation of a high-level nuclear wste repository at Hanford are estimated using two simple atmospheric dispersion models, HANCHI and CHISHORT. Model results indicate that pollutant concentrations would not exceed ambient air quality standards at any point outside the Hanford fenceline or at any publicly accessible location within the Hanford Site. The increase in pollutant concentrations in nearby communities due to site activities would be minimal. HANCHI and CHISHORT are documented in the appendices of this document. Further study of the repository's impact on air quality will be conducted when more detailed project plans and work schedules are available.

  16. Preliminary examination of the impacts of repository site characterization activities and facility construction and operation activities on Hanford air quality

    Air quality impacts that would result from site characterization activities and from the construction and operation of a high-level nuclear wste repository at Hanford are estimated using two simple atmospheric dispersion models, HANCHI and CHISHORT. Model results indicate that pollutant concentrations would not exceed ambient air quality standards at any point outside the Hanford fenceline or at any publicly accessible location within the Hanford Site. The increase in pollutant concentrations in nearby communities due to site activities would be minimal. HANCHI and CHISHORT are documented in the appendices of this document. Further study of the repository's impact on air quality will be conducted when more detailed project plans and work schedules are available

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

    Soares Alexei S

    2007-11-01

    Full Text Available Abstract Background Ricin is a potent toxin and known bioterrorism threat with no available antidote. The ricin A-chain (RTA acts enzymatically to cleave a specific adenine base from ribosomal RNA, thereby blocking translation. To understand better the relationship between ligand binding and RTA active site conformational change, we used a fragment-based approach to find a minimal set of bonding interactions able to induce rearrangements in critical side-chain positions. Results We found that the smallest ligand stabilizing an open conformer of the RTA active site pocket was an amide group, bound weakly by only a few hydrogen bonds to the protein. Complexes with small amide-containing molecules also revealed a switch in geometry from a parallel towards a splayed arrangement of an arginine-tryptophan cation-pi interaction that was associated with an increase and red-shift in tryptophan fluorescence upon ligand binding. Using the observed fluorescence signal, we determined the thermodynamic changes of adenine binding to the RTA active site, as well as the site-specific binding of urea. Urea binding had a favorable enthalpy change and unfavorable entropy change, with a ΔH of -13 ± 2 kJ/mol and a ΔS of -0.04 ± 0.01 kJ/(K*mol. The side-chain position of residue Tyr80 in a complex with adenine was found not to involve as large an overlap of rings with the purine as previously considered, suggesting a smaller role for aromatic stacking at the RTA active site. Conclusion 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

  18. Threatened and endangered wildlife species of the Hanford Site related to CERCLA characterization activities

    Fitzner, R.E. [Pacific Northwest Lab., Richland, WA (United States); Weiss, S.G.; Stegen, J.A. [Westinghouse Hanford Co., Richland, WA (United States)

    1994-06-01

    The US Department of Energy`s (DOE) Hanford Site has been placed on the National Priorities List, which requires that it be remediated under the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) or Superfund. Potentially contaminated areas of the Hanford Site were grouped into operable units, and detailed characterization and investigation plans were formulated. The DOE Richland Operations Office requested Westinghouse Hanford Company (WHC) to conduct a biological assessment of the potential impact of these characterization activities on the threatened, endangered, and sensitive wildlife species of the Hanford Site. Additional direction for WHC compliances with wildlife protection can be found in the Environmental Compliance Manual. This document is intended to meet these requirements, in part, for the CERCLA characterization activities, as well as for other work comparable in scope. This report documents the biological assessment and describes the pertinent components of the Hanford Site as well as the planned characterization activities. Also provided are accounts of endangered, threatened, and federal candidate wildlife species on the Hanford Site and information as to how human disturbances can affect these species. Potential effects of the characterization activities are described with recommendations for mitigation measures.

  19. The active site of low-temperature methane hydroxylation in iron-containing zeolites.

    Snyder, Benjamin E R; Vanelderen, Pieter; Bols, Max L; Hallaert, Simon D; Böttger, Lars H; Ungur, Liviu; Pierloot, Kristine; Schoonheydt, Robert A; Sels, Bert F; Solomon, Edward I

    2016-08-18

    An efficient catalytic process for converting methane into methanol could have far-reaching economic implications. Iron-containing zeolites (microporous aluminosilicate minerals) are noteworthy in this regard, having an outstanding ability to hydroxylate methane rapidly at room temperature to form methanol. Reactivity occurs at an extra-lattice active site called α-Fe(ii), which is activated by nitrous oxide to form the reactive intermediate α-O; however, despite nearly three decades of research, the nature of the active site and the factors determining its exceptional reactivity are unclear. The main difficulty is that the reactive species-α-Fe(ii) and α-O-are challenging to probe spectroscopically: data from bulk techniques such as X-ray absorption spectroscopy and magnetic susceptibility are complicated by contributions from inactive 'spectator' iron. Here we show that a site-selective spectroscopic method regularly used in bioinorganic chemistry can overcome this problem. Magnetic circular dichroism reveals α-Fe(ii) to be a mononuclear, high-spin, square planar Fe(ii) site, while the reactive intermediate, α-O, is a mononuclear, high-spin Fe(iv)=O species, whose exceptional reactivity derives from a constrained coordination geometry enforced by the zeolite lattice. These findings illustrate the value of our approach to exploring active sites in heterogeneous systems. The results also suggest that using matrix constraints to activate metal sites for function-producing what is known in the context of metalloenzymes as an 'entatic' state-might be a useful way to tune the activity of heterogeneous catalysts. PMID:27535535

  20. Designing anti-influenza aptamers: novel quantitative structure activity relationship approach gives insights into aptamer-virus interaction.

    Boaz Musafia

    Full Text Available This study describes the development of aptamers as a therapy against influenza virus infection. Aptamers are oligonucleotides (like ssDNA or RNA that are capable of binding to a variety of molecular targets with high affinity and specificity. We have studied the ssDNA aptamer BV02, which was designed to inhibit influenza infection by targeting the hemagglutinin viral protein, a protein that facilitates the first stage of the virus' infection. While testing other aptamers and during lead optimization, we realized that the dominant characteristics that determine the aptamer's binding to the influenza virus may not necessarily be sequence-specific, as with other known aptamers, but rather depend on general 2D structural motifs. We adopted QSAR (quantitative structure activity relationship tool and developed computational algorithm that correlate six calculated structural and physicochemical properties to the aptamers' binding affinity to the virus. The QSAR study provided us with a predictive tool of the binding potential of an aptamer to the influenza virus. The correlation between the calculated and actual binding was R2 = 0.702 for the training set, and R2 = 0.66 for the independent test set. Moreover, in the test set the model's sensitivity was 89%, and the specificity was 87%, in selecting aptamers with enhanced viral binding. The most important properties that positively correlated with the aptamer's binding were the aptamer length, 2D-loops and repeating sequences of C nucleotides. Based on the structure-activity study, we have managed to produce aptamers having viral affinity that was more than 20 times higher than that of the original BV02 aptamer. Further testing of influenza infection in cell culture and animal models yielded aptamers with 10 to 15 times greater anti-viral activity than the BV02 aptamer. Our insights concerning the mechanism of action and the structural and physicochemical properties that govern the interaction

  1. Structural Insights into an Oxalate-producing Serine Hydrolase with an Unusual Oxyanion Hole and Additional Lyase Activity.

    Oh, Juntaek; Hwang, Ingyu; Rhee, Sangkee

    2016-07-15

    In Burkholderia species, the production of oxalate, an acidic molecule, is a key event for bacterial growth in the stationary phase. Oxalate plays a central role in maintaining environmental pH, which counteracts inevitable population-collapsing alkaline toxicity in amino acid-based culture medium. In the phytopathogen Burkholderia glumae, two enzymes are responsible for oxalate production. First, the enzyme oxalate biosynthetic component A (ObcA) catalyzes the formation of a tetrahedral C6-CoA adduct from the substrates acetyl-CoA and oxaloacetate. Then the ObcB enzyme liberates three products from the C6-CoA adduct: oxalate, acetoacetate, and CoA. Interestingly, these two stepwise reactions are catalyzed by a single bifunctional enzyme, Obc1, from Burkholderia thailandensis and Burkholderia pseudomallei Obc1 has an ObcA-like N-terminal domain and shows ObcB activity in its C-terminal domain despite no sequence homology with ObcB. We report the crystal structure of Obc1 in its apo and glycerol-bound form at 2.5 Å and 2.8 Å resolution, respectively. The Obc1 N-terminal domain is essentially identical both in structure and function to that of ObcA. Its C-terminal domain has an α/β hydrolase fold that has a catalytic triad for oxalate production and a novel oxyanion hole distinct from the canonical HGGG motif in other α/β hydrolases. Functional analyses through mutagenesis studies suggested that His-934 is an additional catalytic acid/base for its lyase activity and liberates two additional products, acetoacetate and CoA. These results provide structural and functional insights into bacterial oxalogenesis and an example of divergent evolution of the α/β hydrolase fold, which has both hydrolase and lyase activity. PMID:27226606

  2. Structural investigation of heteroyohimbine alkaloid synthesis reveals active site elements that control stereoselectivity.

    Stavrinides, Anna; Tatsis, Evangelos C; Caputi, Lorenzo; Foureau, Emilien; Stevenson, Clare E M; Lawson, David M; Courdavault, Vincent; O'Connor, Sarah E

    2016-01-01

    Plants produce an enormous array of biologically active metabolites, often with stereochemical variations on the same molecular scaffold. These changes in stereochemistry dramatically impact biological activity. Notably, the stereoisomers of the heteroyohimbine alkaloids show diverse pharmacological activities. We reported a medium chain dehydrogenase/reductase (MDR) from Catharanthus roseus that catalyses formation of a heteroyohimbine isomer. Here we report the discovery of additional heteroyohimbine synthases (HYSs), one of which produces a mixture of diastereomers. The crystal structures for three HYSs have been solved, providing insight into the mechanism of reactivity and stereoselectivity, with mutation of one loop transforming product specificity. Localization and gene silencing experiments provide a basis for understanding the function of these enzymes in vivo. This work sets the stage to explore how MDRs evolved to generate structural and biological diversity in specialized plant metabolism and opens the possibility for metabolic engineering of new compounds based on this scaffold. PMID:27418042

  3. Utility experiences in redevelopment of formerly used sites -- Wisconsin Electric's risk management and economic development activities

    Wisconsin Electric Power Company, which recently celebrated its 100th anniversary, has actively promoted the redevelopment of its former sites as well as those of its customers. Serving Milwaukee and southeast Wisconsin, Wisconsin Electric's (WE) sites include former power plants, landfills, right-of-ways, and manufactured gas plant sites. In setting an example for others, as well as seeking to maximize the economic value of these sites, WE has either redeveloped or promoted the redevelopment of these sites by others. Examples include the East Wells Power Plant (now home of the Milwaukee Repertory Theater), the Lakeside Power Plant Site (now the home of Harnischfeger Corporation's headquarters), and the Commerce Street Power Plant located on the Milwaukee River near downtown Milwaukee. In each case the company evaluated the potential environmental liabilities against the unrealized asset value derived from facility location, site size, architectural uniqueness, or other characteristics. At the Commerce Street Power Plant, walking distance to the downtown Milwaukee business district combined with river frontage, were significant site values leveraged against a $5 million asbestos and lead-based paint removal project done to prepare the plant for marketing. More recently, WE has used its experience in promoting the redevelopment of the Menomonee River Valley, the original core of Milwaukee's industrial community, and in advancing a more practical regulatory approach to redeveloping older sites. Finally, the company is working with a non-profit community health clinic, community groups and local foundations in linking these redevelopment activities with the economic and physical health of inner city residents

  4. In Silico Structure Prediction of Human Fatty Acid Synthase-Dehydratase: A Plausible Model for Understanding Active Site Interactions.

    John, Arun; Umashankar, Vetrivel; Samdani, A; Sangeetha, Manoharan; Krishnakumar, Subramanian; Deepa, Perinkulam Ravi

    2016-01-01

    Fatty acid synthase (FASN, UniProt ID: P49327) is a multienzyme dimer complex that plays a critical role in lipogenesis. Consequently, this lipogenic enzyme has gained tremendous biomedical importance. The role of FASN and its inhibition is being extensively researched in several clinical conditions, such as cancers, obesity, and diabetes. X-ray crystallographic structures of some of its domains, such as β-ketoacyl synthase, acetyl transacylase, malonyl transacylase, enoyl reductase, β-ketoacyl reductase, and thioesterase, (TE) are already reported. Here, we have attempted an in silico elucidation of the uncrystallized dehydratase (DH) catalytic domain of human FASN. This theoretical model for DH domain was predicted using comparative modeling methods. Different stand-alone tools and servers were used to validate and check the reliability of the predicted models, which suggested it to be a highly plausible model. The stereochemical analysis showed 92.0% residues in favorable region of Ramachandran plot. The initial physiological substrate β-hydroxybutyryl group was docked into active site of DH domain using Glide. The molecular dynamics simulations carried out for 20 ns in apo and holo states indicated the stability and accuracy of the predicted structure in solvated condition. The predicted model provided useful biochemical insights into the substrate-active site binding mechanisms. This model was then used for identifying potential FASN inhibitors using high-throughput virtual screening of the National Cancer Institute database of chemical ligands. The inhibitory efficacy of the top hit ligands was validated by performing molecular dynamics simulation for 20 ns, where in the ligand NSC71039 exhibited good enzyme inhibition characteristics and exhibited dose-dependent anticancer cytotoxicity in retinoblastoma cancer cells in vitro. PMID:27559295

  5. Human Activities in Natura 2000 Sites: A Highly Diversified Conservation Network

    Tsiafouli, Maria A.; Apostolopoulou, Evangelia; Mazaris, Antonios D.; Kallimanis, Athanasios S.; Drakou, Evangelia G.; Pantis, John D.

    2013-05-01

    The Natura 2000 network was established across the European Union's (EU) Member States with the aim to conserve biodiversity, while ensuring the sustainability of human activities. However, to what kind and to what extent Natura 2000 sites are subject to human activities and how this varies across Member States remains unspecified. Here, we analyzed 111,269 human activity records from 14,727 protected sites in 20 Member States. The frequency of occurrence of activities differs among countries, with more than 86 % of all sites being subjected to agriculture or forestry. Activities like hunting, fishing, urbanization, transportation, and tourism are more frequently recorded in south European sites than in northern or eastern ones. The observed variations indicate that Natura 2000 networks are highly heterogeneous among EU Member States. Our analysis highlights the importance of agriculture in European landscapes and indicates possible targets for policy interventions at national, European, or "sub-European" level. The strong human presence in the Natura 2000 network throughout Member States, shows that conservation initiatives could succeed only by combining social and ecological sustainability and by ensuring the integration of policies affecting biodiversity.

  6. Human activities in Natura 2000 sites: a highly diversified conservation network.

    Tsiafouli, Maria A; Apostolopoulou, Evangelia; Mazaris, Antonios D; Kallimanis, Athanasios S; Drakou, Evangelia G; Pantis, John D

    2013-05-01

    The Natura 2000 network was established across the European Union's (EU) Member States with the aim to conserve biodiversity, while ensuring the sustainability of human activities. However, to what kind and to what extent Natura 2000 sites are subject to human activities and how this varies across Member States remains unspecified. Here, we analyzed 111,269 human activity records from 14,727 protected sites in 20 Member States. The frequency of occurrence of activities differs among countries, with more than 86 % of all sites being subjected to agriculture or forestry. Activities like hunting, fishing, urbanization, transportation, and tourism are more frequently recorded in south European sites than in northern or eastern ones. The observed variations indicate that Natura 2000 networks are highly heterogeneous among EU Member States. Our analysis highlights the importance of agriculture in European landscapes and indicates possible targets for policy interventions at national, European, or "sub-European" level. The strong human presence in the Natura 2000 network throughout Member States, shows that conservation initiatives could succeed only by combining social and ecological sustainability and by ensuring the integration of policies affecting biodiversity. PMID:23571828

  7. A facile reflux procedure to increase active surface sites form highly active and durable supported palladium@platinum bimetallic nanodendrites

    Wang, Qin; Li, Yingjun; Liu, Baocang; Xu, Guangran; Zhang, Geng; Zhao, Qi; Zhang, Jun

    2015-11-01

    A series of well-dispersed bimetallic Pd@Pt nanodendrites uniformly supported on XC-72 carbon black are fabricated by using different capping agents. These capping agents are essential for the branched morphology control. However, the surfactant adsorbed on the nanodendrites surface blocks the access of reactant molecules to the active surface sites, and the catalytic activities of these bimetallic nanodendrites are significantly restricted. Herein, a facile reflux procedure to effectively remove the capping agent molecules without significantly affecting their sizes is reported for activating supported nanocatalysts. More significantly, the structure and morphology of the nanodendrites can also be retained, enhancing the numbers of active surface sites, catalytic activity and stability toward methanol and ethanol electro-oxidation reactions. The as-obtained hot water reflux-treated Pd@Pt/C catalyst manifests superior catalytic activity and stability both in terms of surface and mass specific activities, as compared to the untreated catalysts and the commercial Pt/C and Pd/C catalysts. We anticipate that this effective and facile removal method has more general applicability to highly active nanocatalysts prepared with various surfactants, and should lead to improvements in environmental protection and energy production.

  8. Immobilized low-activity waste site borehole 299-E17-21

    The Tank Waste Remediation System (TWRS) is the group at the Hanford Site responsible for the safe underground storage of liquid waste from previous Hanford Site operations, the storage and disposal of immobilized tank waste, and closure of underground tanks. The current plan is to dispose of immobilized low-activity tank waste (ILAW) in new facilities in the southcentral part of 200-East Area and in four existing vaults along the east side of 200-East Area. Boreholes 299-E17-21, B8501, and B8502 were drilled at the southwest corner of the ILAW site in support of the Performance Assessment activities for the disposal options. This report summarizes the initial geologic findings, field tests conducted on those boreholes, and ongoing studies. One deep (480 feet) borehole and two shallow (50 feet) boreholes were drilled at the southwest corner of the ILAW site. The primary factor dictating the location of the boreholes was their characterization function with respect to developing the geohydrologic model for the site and satisfying associated Data Quality Objectives. The deep borehole was drilled to characterize subsurface conditions beneath the ILAW site, and two shallow boreholes were drilled to support an ongoing environmental tracer study. The tracer study will supply information to the Performance Assessment. All the boreholes provide data on the vadose zone and saturated zone in a previously uncharacterized area

  9. Surface binding sites in carbohydrate active enzymes: An emerging picture of structural and functional diversity

    Svensson, Birte; Cockburn, Darrell

    2013-01-01

    universal and is in fact rare among some families of enzymes. In some cases an alternative to possessing a CBM is for the enzyme to bind to the substrate at a site on the catalytic domain, but away from the active site. Such a site is termed a surface (or secondary) binding site (SBS). SBSs have been...... identified in enzymes from a wide variety of families, though almost half are found in the α-amylase family GH13. The roles attributed to SBSs are not limited to targeting the enzyme to the substrate, but also include a variety of others such as guiding the substrate into the active site, altering enzyme...... specificity and acting as an allosteric site. Although SBSs share many roles with CBMs they may not simply be an alternative to CBMs, but rather complementary as SBSs and CBMs frequently co-occur in enzymes. Despite a relatively long history, it is only in recent years that SBSs have been studied in great...

  10. Immobilized low-activity waste site borehole 299-E17-21

    Reidel, S.P.; Reynolds, K.D.; Horton, D.G.

    1998-08-01

    The Tank Waste Remediation System (TWRS) is the group at the Hanford Site responsible for the safe underground storage of liquid waste from previous Hanford Site operations, the storage and disposal of immobilized tank waste, and closure of underground tanks. The current plan is to dispose of immobilized low-activity tank waste (ILAW) in new facilities in the southcentral part of 200-East Area and in four existing vaults along the east side of 200-East Area. Boreholes 299-E17-21, B8501, and B8502 were drilled at the southwest corner of the ILAW site in support of the Performance Assessment activities for the disposal options. This report summarizes the initial geologic findings, field tests conducted on those boreholes, and ongoing studies. One deep (480 feet) borehole and two shallow (50 feet) boreholes were drilled at the southwest corner of the ILAW site. The primary factor dictating the location of the boreholes was their characterization function with respect to developing the geohydrologic model for the site and satisfying associated Data Quality Objectives. The deep borehole was drilled to characterize subsurface conditions beneath the ILAW site, and two shallow boreholes were drilled to support an ongoing environmental tracer study. The tracer study will supply information to the Performance Assessment. All the boreholes provide data on the vadose zone and saturated zone in a previously uncharacterized area.

  11. POISONING OF ACTIVE SITES ON ZIEGLER-NATTA CATALYST FOR PROPYLENE POLYMERIZATION

    Kitti Tangjituabun; Sang Yull Kim; Yuichi Hiraoka; Toshiaki Taniike; Minoru Terano; Bunjerd Jongsomjit; Piyasan Praserthdam

    2008-01-01

    The effects of poisoning materials on catalytic activity and isospecificity of the supported Ziegler-Natta catalyst were investigated.A minor amount of simple structure of Lewis base,i.e.,methanol,acetone,ethyl acetate,was introduced into the catalyst slurry for partial poisoning catalytic active centers.It was found that the variations in deactivation power were in the order of methanol>acetone>ethyl acetate.The kinetic investigation via stopped-flow polymerization showed that poisoning compounds caused a decrease in activity through the reduction of the number of active sites whereas no effect on the degree of isotacticity was observed.

  12. Sphingosine-1-phosphate receptor 1 reporter mice reveal receptor activation sites in vivo

    Kono, Mari; Tucker, Ana E.; Tran, Jennifer; Bergner, Jennifer B.; Turner, Ewa M; Proia, Richard L.

    2014-01-01

    Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a ...

  13. Molecular Basis of Enhanced Activity in Factor VIIa-Trypsin Variants Conveys Insights into Tissue Factor-mediated Allosteric Regulation of Factor VIIa Activity

    Sorensen, Anders B.; Madsen, Jesper Jonasson; Svensson, L. Anders; Pedersen, Anette A.; Østergaard, Henrik; Overgaard, Michael T.; Olsen, Ole H.; Gandhi, Prafull S

    2016-01-01

    The complex of coagulation factor VIIa (FVIIa), a trypsin-like serine protease, and membrane-bound tissue factor (TF) initiates blood coagulation upon vascular injury. Binding of TF to FVIIa promotes allosteric conformational changes in the FVIIa protease domain and improves its catalytic...... properties. Extensive studies have revealed two putative pathways for this allosteric communication. Here we provide further details of this allosteric communication by investigating FVIIa loop swap variants containing the 170 loop of trypsin that display TF-independent enhanced activity. Using x......-ray crystallography, we show that the introduced 170 loop from trypsin directly interacts with the FVIIa active site, stabilizing segment 215-217 and activation loop 3, leading to enhanced activity. Molecular dynamics simulations and novel fluorescence quenching studies support that segment 215-217 conformation is...

  14. Theoretical insights on the catalytic activity and mechanism for oxygen reduction reaction at Fe and P codoped graphene.

    He, Feng; Li, Kai; Xie, Guangyou; Wang, Ying; Jiao, Menggai; Tang, Hao; Wu, Zhijian

    2016-05-14

    The non-precious metal graphene catalyst doped with Fe-Px are recently proposed as a promising candidate in substituting Pt for catalyzing oxygen reduction reaction (ORR) in fuel cells. Systematic DFT calculations are performed to investigate the catalytic activity and the ORR mechanism on the Fe-Px (x = 1-4) system in acid medium in this work. Our results indicated that the configuration with one Fe and two P atoms codoped at zigzag edge site (Fe-P2-zig-G) is the most stable, in excellent agreement with the experimental observation that the ratio of Fe and P is nearly 1 : 2. The four-electron reduction mechanism for ORR on the Fe-P2-zig-G is via the competing OOH hydrogenation pathways (to form either OH + OH or O + H2O). The rate determining step is the O2 hydrogenation with an energy barrier of 0.43 eV, much smaller that of calculated 0.80 eV for pure Pt. In addition, the highest energy barrier of the studied ORR mechanism is the O2 dissociation with an energy barrier of 0.70 eV, a value also smaller than that of pure Pt. This demonstrated that the zigzag edge site of the Fe-P2 codoped graphene should be active for the ORR. PMID:27094325

  15. Insights into the Molecular Activation Mechanism of the RhoA-specific Guanine Nucleotide Exchange Factor, PDZRhoGEF

    Bielnicki, Jakub A.; Shkumatov, Alexander V.; Derewenda, Urszula; Somlyo, Avril V.; Svergun, Dmitri I.; Derewenda, Zygmunt S. (EMBL); (UV)

    2012-10-09

    PDZRhoGEF (PRG) belongs to a small family of RhoA-specific nucleotide exchange factors that mediates signaling through select G-protein-coupled receptors via G{alpha}{sub 12/13} and activates RhoA by catalyzing the exchange of GDP to GTP. PRG is a multidomain protein composed of PDZ, regulators of G-protein signaling-like (RGSL), Dbl-homology (DH), and pleckstrin-homology (PH) domains. It is autoinhibited in cytosol and is believed to undergo a conformational rearrangement and translocation to the membrane for full activation, although the molecular details of the regulation mechanism are not clear. It has been shown recently that the main autoregulatory elements of PDZRhoGEF, the autoinhibitory 'activation box' and the 'GEF switch,' which is required for full activation, are located directly upstream of the catalytic DH domain and its RhoA binding surface, emphasizing the functional role of the RGSL-DH linker. Here, using a combination of biophysical and biochemical methods, we show that the mechanism of PRG regulation is yet more complex and may involve an additional autoinhibitory element in the form of a molten globule region within the linker between RGSL and DH domains. We propose a novel, two-tier model of autoinhibition where the activation box and the molten globule region act synergistically to impair the ability of RhoA to bind to the catalytic DH-PH tandem. The molten globule region and the activation box become less ordered in the PRG-RhoA complex and dissociate from the RhoA-binding site, which may constitute a critical step leading to PRG activation.

  16. Quantification of active mitochondrial permeability transition pores using GNX-4975 inhibitor titrations provides insights into molecular identity.

    Richardson, Andrew P; Halestrap, Andrew P

    2016-05-01

    Inhibition of the mitochondrial permeability transition pore (MPTP) by the novel inhibitor GNX-4975 was characterized. Titration of MPTP activity in de-energized rat liver mitochondria allowed determination of the number of GNX-4975-binding sites and their dissociation constant (Ki). Binding sites increased in number when MPTP opening was activated by increasing [Ca(2+)], phenylarsine oxide (PAO) or KSCN, and decreased when MPTP opening was inhibited with bongkrekic acid (BKA) or ADP. Values ranged between 9 and 50 pmol/mg of mitochondrial protein, but the Ki remained unchanged at ∼1.8 nM when the inhibitor was added before Ca(2+) However, when GNX-4975 was added after Ca(2+) it was much less potent with a Ki of ∼140 nM. These data imply that a protein conformational change is required to form the MPTP complex and generate the GNX-4975-binding site. Occupation of the latter with GNX-4975 prevents the Ca(2+) binding that triggers pore opening. We also demonstrated that GNX-4975 stabilizes an interaction between the adenine nucleotide translocase (ANT), held in its 'c' conformation with carboxyatractyloside (CAT), and the phosphate carrier (PiC) bound to immobilized PAO. No components of the F1Fo-ATP synthase bound significantly to immobilized PAO. Our data are consistent with our previous proposal that the MPTP may form at an interface between the PiC and ANT (or other similar mitochondrial carrier proteins) when they adopt novel conformations induced by factors that sensitize the MPTP to [Ca(2+)]. We propose that GNX-4975 binds to this interface preventing a calcium-triggered event that opens the interface into a pore. PMID:26920024

  17. Molecular dioxygen enters the active site of 12/15-lipoxygenase via dynamic oxygen access channels.

    Saam, Jan; Ivanov, Igor; Walther, Matthias; Holzhütter, Hermann-Georg; Kuhn, Hartmut

    2007-08-14

    Cells contain numerous enzymes that use molecular oxygen for their reactions. Often, their active sites are buried deeply inside the protein, which raises the question whether there are specific access channels guiding oxygen to the site of catalysis. Choosing 12/15-lipoxygenase as a typical example for such oxygen-dependent enzymes, we determined the oxygen distribution within the protein and defined potential routes for oxygen access. For this purpose, we have applied an integrated strategy of structural modeling, molecular dynamics simulations, site-directed mutagenesis, and kinetic measurements. First, we computed the 3D free-energy distribution for oxygen, which led to identification of four oxygen channels in the protein. All channels connect the protein surface with a region of high oxygen affinity at the active site. This region is localized opposite to the nonheme iron providing a structural explanation for the reaction specificity of this lipoxygenase isoform. The catalytically most relevant path can be obstructed by L367F exchange, which leads to a strongly increased Michaelis constant for oxygen. The blocking mechanism is explained in detail by reordering the hydrogen-bonding network of water molecules. Our results provide strong evidence that the main route for oxygen access to the active site of the enzyme follows a channel formed by transiently interconnected cavities whereby the opening and closure are governed by side chain dynamics. PMID:17675410

  18. Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage

    Yuan,Y.; Pei, Y.; Ma, J.; Kuryavyi, V.; Zhadina, M.; Meister, G.; Chen, H.; Dauter, Z.; Tuschi, T.; Patel, D.

    2005-01-01

    Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies, which establish this eubacterial Ago as a bona fide guide DNA strand-mediated site-specific RNA endonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the 5' phosphate of the guide strand can be anchored in a basic pocket, and the mRNA can be positioned for site-specific cleavage by RNase H-type divalent cation-coordinated catalytic Asp residues of the PIWI domain. Domain swap experiments involving chimeras of human Ago (hAgo1) and cleavage-competent hAgo2 reinforce the role of the PIWI domain in 'slicer' activity. We propose a four-step Ago-mediated catalytic cleavage cycle model, which provides distinct perspectives into the mechanism of guide strand-mediated mRNA cleavage within the RISC.

  19. Site Energies of Active and Inactive Pheophytins in the Reaction Center of Photosystem II from Chlamydomonas Reinhardtii

    It is widely accepted that the primary electron acceptor in various Photosystem II (PSII) reaction center (RC) preparations is pheophytin α (Pheo α) within the D1 protein (PheoD1), while PheoD2 (within the D2 protein) is photochemically inactive. The Pheo site energies, however, have remained elusive, due to inherent spectral congestion. While most researchers over the past two decades placed the Qy-states of PheoD1 and PheoD2 bands near 678-684 and 668-672 nm, respectively, recent modeling (Raszewski et al. Biophys. J. 2005, 88, 986-998; Cox et al. J. Phys. Chem. B 2009, 113, 12364-12374) of the electronic structure of the PSII RC reversed the assignment of the active and inactive Pheos, suggesting that the mean site energy of PheoD1 is near 672 nm, whereas PheoD2 (∼677.5 nm) and ChlD1 (∼680 nm) have the lowest energies (i.e., the PheoD2-dominated exciton is the lowest excited state). In contrast, chemical pigment exchange experiments on isolated RCs suggested that both pheophytins have their Qy absorption maxima at 676-680 nm (Germano et al. Biochemistry 2001, 40, 11472-11482; Germano et al. Biophys. J. 2004, 86, 1664-1672). To provide more insight into the site energies of both PheoD1 and PheoD2 (including the corresponding Qx transitions, which are often claimed to be degenerate at 543 nm) and to attest that the above two assignments are most likely incorrect, we studied a large number of isolated RC preparations from spinach and wild-type Chlamydomonas reinhardtii (at different levels of intactness) as well as the Chlamydomonas reinhardtii mutant (D2-L209H), in which the active branch PheoD1 is genetically replaced with chlorophyll α (Chl α). We show that the Qx-/Qy-region site energies of PheoD1 and PheoD2 are ∼545/680 nm and ∼541.5/670 nm, respectively, in good agreement with our previous assignment (Jankowiak et al. J. Phys. Chem. B 2002, 106, 8803?8814). The latter values should be used to model excitonic structure and excitation energy

  20. Site Energies of Active and Inactive Pheophytins in the Reaction Center of Photosystem II from Chlamydomonas Reinhardtii

    Acharya, K.; Neupane, B.; Zazubovich, V.; Sayre, R. T.; Picorel, R.; Seibert, M.; Jankowiak, R.

    2012-03-29

    It is widely accepted that the primary electron acceptor in various Photosystem II (PSII) reaction center (RC) preparations is pheophytin {alpha} (Pheo {alpha}) within the D1 protein (Pheo{sub D1}), while Pheo{sub D2} (within the D2 protein) is photochemically inactive. The Pheo site energies, however, have remained elusive, due to inherent spectral congestion. While most researchers over the past two decades placed the Q{sub y}-states of Pheo{sub D1} and Pheo{sub D2} bands near 678-684 and 668-672 nm, respectively, recent modeling [Raszewski et al. Biophys. J. 2005, 88, 986-998; Cox et al. J. Phys. Chem. B 2009, 113, 12364-12374] of the electronic structure of the PSII RC reversed the assignment of the active and inactive Pheos, suggesting that the mean site energy of Pheo{sub D1} is near 672 nm, whereas Pheo{sub D2} ({approx}677.5 nm) and Chl{sub D1} ({approx}680 nm) have the lowest energies (i.e., the Pheo{sub D2}-dominated exciton is the lowest excited state). In contrast, chemical pigment exchange experiments on isolated RCs suggested that both pheophytins have their Q{sub y} absorption maxima at 676-680 nm [Germano et al. Biochemistry 2001, 40, 11472-11482; Germano et al. Biophys. J. 2004, 86, 1664-1672]. To provide more insight into the site energies of both Pheo{sub D1} and Pheo{sub D2} (including the corresponding Q{sub x} transitions, which are often claimed to be degenerate at 543 nm) and to attest that the above two assignments are most likely incorrect, we studied a large number of isolated RC preparations from spinach and wild-type Chlamydomonas reinhardtii (at different levels of intactness) as well as the Chlamydomonas reinhardtii mutant (D2-L209H), in which the active branch Pheo{sub D1} is genetically replaced with chlorophyll {alpha} (Chl {alpha}). We show that the Q{sub x}-/Q{sub y}-region site energies of Pheo{sub D1} and Pheo{sub D2} are {approx}545/680 nm and {approx}541.5/670 nm, respectively, in good agreement with our previous assignment

  1. Active catalytic sites in the ammoxidation of propane and propene over V-Sb-O catalysts

    Buchholz, S.A.; Zanthoff, H.W. [Bochum Univ. (Germany). Lehrstuhl fuer Technische Chemie

    1998-12-31

    The ammoxidation of propane over VSb{sub y}O{sub x} catalysts (y=1, 2, 5) was investigated with respect to the role of different oxygen species in the selective and non selective reaction steps using transient experiments in the Temporal Analysis of Products (TAP) reactor. Only lattice oxygen is involved in the oxidation reactions. Using isotopic labelled oxygen it is shown that two different active sites exist on the surface. On site A, which can be reoxidized faster by gas phase oxygen compared to site B, mainly CO is formed. On site B CO{sub 2} and acrolein as well as NO and N{sub 2}O in the presence of ammonia in the feed gas are formed and reoxidation mainly occurs with bulk lattice oxygen. (orig.)

  2. A three-dimensional model of mammalian tyrosinase active site accounting for loss of function mutations.

    Schweikardt, Thorsten; Olivares, Concepción; Solano, Francisco; Jaenicke, Elmar; García-Borrón, José Carlos; Decker, Heinz

    2007-10-01

    Tyrosinases are the first and rate-limiting enzymes in the synthesis of melanin pigments responsible for colouring hair, skin and eyes. Mutation of tyrosinases often decreases melanin production resulting in albinism, but the effects are not always understood at the molecular level. Homology modelling of mouse tyrosinase based on recently published crystal structures of non-mammalian tyrosinases provides an active site model accounting for loss-of-function mutations. According to the model, the copper-binding histidines are located in a helix bundle comprising four densely packed helices. A loop containing residues M374, S375 and V377 connects the CuA and CuB centres, with the peptide oxygens of M374 and V377 serving as hydrogen acceptors for the NH-groups of the imidazole rings of the copper-binding His367 and His180. Therefore, this loop is essential for the stability of the active site architecture. A double substitution (374)MS(375) --> (374)GG(375) or a single M374G mutation lead to a local perturbation of the protein matrix at the active site affecting the orientation of the H367 side chain, that may be unable to bind CuB reliably, resulting in loss of activity. The model also accounts for loss of function in two naturally occurring albino mutations, S380P and V393F. The hydroxyl group in S380 contributes to the correct orientation of M374, and the substitution of V393 for a bulkier phenylalanine sterically impedes correct side chain packing at the active site. Therefore, our model explains the mechanistic necessity for conservation of not only active site histidines but also adjacent amino acids in tyrosinase. PMID:17850513

  3. Recent Experience Using Active Love Wave Techniques to Characterize Seismographic Station Sites

    Martin, A. J.; Yong, A.; Salomone, L.

    2014-12-01

    Active-source Love waves recorded by the multi-channel analysis of surface wave (MASLW) technique were recently analyzed in two site characterization projects. Between 2010 and 2011, the 2009 American Recovery and Reinvestment Act (ARRA) funded GEOVision to conduct geophysical investigations at 189 seismographic stations—185 in California and 4 in the Central Eastern U.S. (CEUS). The original project plan was to utilize active and passive Rayleigh wave-based techniques to obtain shear-wave velocity (VS) profiles to a minimum depth of 30 m and the time-averaged VS of the upper 30 meters (VS30). Early in the investigation it became evident that Rayleigh wave techniques, such as multi-channel analysis of surface waves (MASRW), were not effective at characterizing all sites. Shear-wave seismic refraction and MASLW techniques were therefore applied. The MASLW technique was deployed at a total of 38 sites, in addition to other methods, and used as the primary technique to characterize 22 sites, 5 of which were also characterized using Rayleigh wave techniques. In 2012, the Electric Power Research Institute funded characterization of 33 CEUS station sites. Based on experience from the ARRA investigation, both MASRW and MASLW data were acquired by GEOVision at 24 CEUS sites—the remaining 9 sites and 2 overlapping sites were characterized by University of Texas, Austin. Of the 24 sites characterized by GEOVision, 16 were characterized using MASLW data, 4 using both MASLW and MASRW data and 4 using MASRW data. Love wave techniques were often found to perform better, or at least yield phase velocity data that could be more readily modeled using the fundamental mode assumption, at shallow rock sites, sites with steep velocity gradients, and, sites with a thin, low velocity, surficial soil layer overlying stiffer sediments. These types of velocity structure often excite dominant higher modes in Rayleigh wave data, but not in Love wave data. At such sites, it may be possible

  4. Variants of the cell recognition site of fibronectin that retain attachment-promoting activity

    1985-01-01

    A tetrapeptide sequence, Arg-Gly-Asp-Ser, is the minimal structure recognized by cells in the large, adhesive glycoprotein fibronectin. We now have defined the structural requirements for this cell recognition site by testing several synthetic variants of the active tetrapeptide sequence. The conservative substitutions of lysine for arginine, alanine for glycine, or glutamic acid for aspartic acid each resulted in abrogation of the cell attachment-promoting activity characteristic of the natu...

  5. Screening Approach to the Activation of Soil and Contamination of Groundwater at Linear Proton Accelerator Sites

    Otto, Thomas

    The activation of soil and the contamination of groundwater at proton accelerator sites with the radionuclides 3H and 22Na are estimated with a Monte-Carlo calculation and a conservative soil- and ground water model. The obtained radionuclide concentrations show that the underground environment of future accelerators must be adequately protected against a migration of activation products. This study is of particular importance for the proton driver accelerator in the planned EURISOL facility.

  6. Study on the active sites of Cu-ZSM-5 in trichloroethylene catalytic combustion with air

    Cheng Hua Xu; Chuan Qi Liu; Yan Zhong; Xiu Zhou Yang; Jian Ying Liu; Ying Chun Yang; Zhi Xiang Ye

    2008-01-01

    The catalytic activity of Cu-ZSM-5 in trichloroethylene (TCE) combustion increases with the increasing skeletal Cu amount and however decreases with the increase of surface amorphous CuO,which is detected by infrared spectroscopy (IR) and diffuse reflectance ultraviolet-visible spectroscopy (DRS-UV-vis),therefore the skeletal Cu species are concluded to be the active sites for the TCE combustion.

  7. Remediation of uranium contaminated sites: clean-up activities in Serbia

    One of the serious environmental problems in Serbia represent sites contaminated with depleted uranium (DU) during past war activities. According to UNEP reports and our findings there are two types of contamination: (i) localized points of high, concentrated contamination where DU penetrators enter the soil, and (ii) low level of widespread DU contamination, which indicates that during the conflict DU dust was dispersed into the environment. Remediation of these sites is an urgent need because they represent a permanent threat to the population living in this area. Here we give a brief description of approaches commonly used in remediation of DU contaminated sites, and an overview of current clean-up activities performed in Serbia. (author)

  8. Identification and characterization of radioactively contaminated sites in Ukraine and planning for environmental restoration activities

    In the Pridniprovsk-Krivoy Rog region uranium, titanium, iron and manganese ores were mined and milled beginning in the 1950s. These activities have caused radioactive contamination of the environment at some sites. In recent times intensive works concerning the surveying of contaminated areas and substantiating the need for remediation have been initiated. The research methodologies applied and the results from radiation surveys are presented for the site of the first uranium mine in the Ukraine, for tailings originating from the Pridniprovsk Chemical Plant (PChP), for the recultivated dump-site of the former 'O'-mine, as well as for the wastes, raw materials and production of the Nicopol Ferro-Alloy Plant. The planning procedure for the remediation activities at the town of Zhovty Vody is described. (author)

  9. Insights into the Thiamine Diphosphate Enzyme Activation Mechanism: Computational Model for Transketolase Using a Quantum Mechanical/Molecular Mechanical Method.

    Nauton, Lionel; Hélaine, Virgil; Théry, Vincent; Hecquet, Laurence

    2016-04-12

    We propose the first computational model for transketolase (TK), a thiamine diphosphate (ThDP)-dependent enzyme, using a quantum mechanical/molecular mechanical method on the basis of crystallographic TK structures from yeast and Escherichia coli, together with experimental kinetic data reported in the literature with wild-type and mutant TK. This model allowed us to define a new route for ThDP activation in the enzyme environment. We evidenced a strong interaction between ThDP and Glu418B of the TK active site, itself stabilized by Glu162A. The crucial point highlighted here is that deprotonation of ThDP C2 is not performed by ThDP N4' as reported in the literature, but by His481B, involving a HOH688A molecule bridge. Thus, ThDP N4' is converted from an amino form to an iminium form, ensuring the stabilization of the C2 carbanion or carbene. Finally, ThDP activation proceeds via an intermolecular process and not by an intramolecular one as reported in the literature. More generally, this proposed ThDP activation mechanism can be applied to some other ThDP-dependent enzymes and used to define the entire TK mechanism with donor and acceptor substrates more accurately. PMID:26998737

  10. Active sites residues of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT-II).

    Nic a'Bháird, N; Yankovskaya, V; Ramsay, R R

    1998-01-01

    The carnitine acyltransferases which catalyse the reversible transfer of fatty acyl groups between carnitine and coenzyme A have been proposed to contain a catalytic histidine. Here, the chemical reactivity of active site groups has been used to demonstrate differences between the active sites of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase-II (CPT-II). Treatment of CPT-II with the histidine-selective reagent, diethyl pyrocarbonate (DEPC), resulted in simple linear pseudo-first-order kinetics. The reversal of the inhibition by hydroxylamine and the pKa (7.1) of the modified residue indicated that the residue was a histidine. The order of the inactivation kinetics showed that 1mol of histidine was modified per mol of CPT-II.When COT was treated with DEPC the kinetics of inhibition were biphasic with an initial rapid loss of activity followed by a slower loss of activity. The residue reacting in the faster phase of inhibition was not a histidine but possibly a serine. The modification of this residue did not lead to complete loss of activity suggesting that a direct role in catalysis is unlikely. It was deduced that the residue modified by DEPC in the slower phase was a lysine and indeed fluorodinitrobenzene (FDNB) inactivated COT with linear pseudo-first-order kinetics. The COT peptide containing the FDNB-labelled lysine was isolated and sequenced. Alignment of this sequence placed it 10 amino acids downstream of the putative active-site histidine. PMID:9480926

  11. Molecular Interactions Between the Active Sites of RGD (Arg-Gly-Asp with its Receptor (Integrine

    E. Jauregui

    2000-03-01

    Full Text Available A study of the molecular interactions between the active sites of RGD (Arg-Gly-Asp with it Receptor using simultaions is reported. Our calculations indicate that the guanidine-carboxylate complex is energetically favourd with respect to the guanidine-methyl tetrazole complex.

  12. 78 FR 8190 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    2013-02-05

    ... published the Notice in the Federal Register (77 FR 74218) inviting Federal, state, local government... Bureau of Ocean Energy Management Commercial Wind Leasing and Site Assessment Activities on the Atlantic... Notice of Intent to Prepare an Environmental Assessment (EA) for Commercial Wind Leasing and...

  13. 77 FR 5830 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    2012-02-06

    ... (NOA) in the Federal Register (72 FR 62,672) of the Programmatic EIS for Alternative Energy Development... FR 30,616) of the EA for Issuance of Leases for Wind Resource Data Collection on the Outer... Bureau of Ocean Energy Management Commercial Wind Leasing and Site Assessment Activities on the...

  14. 76 FR 40925 - Commercial Wind Lease Issuance and Site Characterization Activities on the Atlantic Outer...

    2011-07-12

    ... renewable energy leases (which includes reasonably foreseeable site characterization activities--geophysical...-Atlantic WEAs (76 FR 7226), which requested public input with regard to the identification of the important... Bureau of Ocean Energy Management, Regulation and Enforcement Commercial Wind Lease Issuance and...

  15. Dynamics of water molecules in the active-site cavity of human cytochromes P450

    Rydberg, Patrik; Rod, Thomas Holm; Olsen, Lars;

    2007-01-01

    We have studied the dynamics of water molecules in six crystal structures of four human cytochromes P450, 2A6, 2C8, 2C9, and 3A4, with molecular dynamics simulations. In the crystal structures, only a few water molecules are seen and the reported sizes of the active-site cavity vary a lot. In the...

  16. Organized Agents: Canadian Teacher Unions as Alternative Sites for Social Justice Activism

    Rottmann, Cindy

    2008-01-01

    Historically teachers' federations have been some of the major organizational sites for social justice leadership in K-12 public education. Despite this history of activism, social justice teacher unionism remains a relatively underdeveloped concept. This article merges four philosophical conceptions of social justice in education: liberal…

  17. Aberration-corrected imaging of active sites on industrial catalyst nanoparticles

    Gontard, Lionel Cervera; Chang, L-Y; Hetherington, CJD;

    2007-01-01

    Picture perfect: Information about the local topologies of active sites on commercial nanoparticles can be gained with atomic resolution through spherical-aberration-corrected transmission electron microscopy (TEM). A powder of Pt nanoparticles on carbon black was examined with two advanced TEM...

  18. Biosynthesis of selenosubtilisin: A novel way to target selenium into the active site of subtilisin

    LI Jing; LIU XiaoMan; JI YueTong; QI ZhenHui; GE Yan; XU JiaYun; LIU JunQiu; LUO GuiMin; SHEN JiaCong

    2008-01-01

    Glutathione peroxidase (GPx,EC1.11.1.9),an important anti-oxidative selenoenzyme,can catalyze the reduction of harmful hydroperoxides with concomitant glutathione,thereby protecting cells and other biological issues against oxidative damage.It captures considerable interest in redesign of its function for either the mechanism study or the pharmacological development as an antioxidant.In order to de-velop a general strategy for specifically targeting and operating selenium in active sites of enzymes,the catalytically essential residue selenocysteine (Sec) was first successfully bioincorporated into the catalytic center of subtilisin by using an auxotrophic expression system.The studies of the catalytic activity and the steady-state kinetics demonstrated that selenosubtilisin is an excellent GPx-like bio-catalyst.In comparison with the chemically modified method,biosynthesis exhibits obvious advan-tages:Sec could be site-directly incorporated into active sites of enzymes to overcome the non-speci-ficity generated by chemical modification.This study provides an important strategy for specifically targeting and operating selenium in the active site of an enzyme.

  19. 77 FR 3460 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    2012-01-24

    ...This Notice announces the Department of Energy's (DOE) acceptance of claims in FY 2012 from eligible active uranium and thorium processing site licensees for reimbursement under Title X of the Energy Policy Act of 1992. In FY 2009, Congress appropriated $70 million for Title X in the American Recovery and Reinvestment Act of 2009 (Recovery Act). Also in FY 2009, Congress provided $10 million......

  20. 78 FR 33908 - Commercial Wind Lease Issuance and Site Assessment Activities on the Atlantic Outer Continental...

    2013-06-05

    ... identified Wind Energy Area (WEA) on the OCS offshore Rhode Island (RI) and Massachusetts (MA). The revised... Bureau of Ocean Energy Management Commercial Wind Lease Issuance and Site Assessment Activities on the.... BOEM may issue one or more commercial wind energy leases in the WEA. The competitive lease process...

  1. 40 CFR 35.6260 - Combining Cooperative Agreement sites and activities.

    2010-07-01

    ... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Combining Cooperative Agreement sites and activities. 35.6260 Section 35.6260 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Contracts for Superfund Response Actions Combining Cooperative Agreements § 35.6260 Combining...

  2. The role of the catalysts with highly dispersed and isolated active sites in the selective oxidation of light hydrocarbons

    WANG Hongxuan; ZHAO Zhen

    2005-01-01

    This review summarizes the role of catalysts with highly dispersed and isolated active sites (active sites: supported atoms f≤0.5 % ) in the selective oxidation of light hydrocarbons, such as methane, ethane and propane, into oxygenatesand the epoxidation of olefins. The plausible structures of the highly dispersed and isolated active species, as well as their effects on the catalytic performances are discussed. The special physico-chemical properties and the functional mechanism of the catalysts with highly dispersed and isolated active sites, as well as the preparation, characterization of the catalysts with highly dispersed and isolated active sites and their applications in other types of reactions of lower hydrocarbons are summarized.

  3. Stress hormone levels in a freshwater turtle from sites differing in human activity

    Polich, Rebecca L.

    2016-01-01

    Glucocorticoids, such as corticosterone (CORT), commonly serve as a measure of stress levels in vertebrate populations. These hormones have been implicated in regulation of feeding behaviour, locomotor activity, body mass, lipid metabolism and other crucial behaviours and physiological processes. Thus, understanding how glucocorticoids fluctuate seasonally and in response to specific stressors can yield insight into organismal health and the overall health of populations. I compared circulating CORT concentrations between two similar populations of painted turtle, Chrysemys picta, which differed primarily in the level of exposure to human recreational activities. I measured basal CORT concentrations as well as the CORT stress response and did not find any substantive difference between the two populations. This similarity may indicate that painted turtles are not stressed by the presence of humans during the nesting season. The results of this study contribute to our understanding of CORT concentrations in freshwater reptiles, a group that is historically under-represented in studies of circulating hormone concentrations; specifically, studies that seek to use circulating concentrations of stress hormones, such as CORT, as a measure of the effect of human activities on wild populations. They also give insight into how these species as a whole may respond to human recreational activities during crucial life-history stages, such as the nesting season. Although there was no discernable difference between circulating CORT concentrations between the urban and rural populations studied, I did find a significant difference in circulating CORT concentrations between male and female C. picta. This important finding provides better understanding of the sex differences between male and female painted turtles and adds to our understanding of this species and other species of freshwater turtle. PMID:27293763

  4. Characterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase

    The pyridoxal phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid (ACC) synthase the key enzyme in ethylene biosynthesis, is inactivated by its substrate S-adenosylmethionine (AdoMet). Apple ACC synthase was purified with an immunoaffinity gel, and its active site was probed with NaB3H4 or Ado[14C]Met. Peptide sequencing of both 3H- and 14C-labeled peptides revealed a common dodecapeptide of Ser-Leu-Ser-Xaa-Asp-Leu-Gly-Leu-Pro-Gly-Phe-Arg, where Xaa was the modified, radioactive residue in each case. Acid hydrolysis of the 3H-labeled enzyme released radioactive N-pyridoxyllysine, indicating that the active-site peptide contained lysine at position 4. Mass spectrometry of the 14C-labeled peptide indicated a protonated molecular ion at m/z 1390.6, from which the mass of Xaa was calculated to be 229, a number that is equivalent to the mass of a lysine residue alkylated by the 2-aminobutyrate portion of AdoMet, as we previously proposed. These results indicate that the same active-site lysine binds the PLP and convalently links to the 2-aminobutyrate portion of AdoMet during inactivation. The active site of tomato ACC synthase was probed in the same manner with Ado [14C]Met. Sequencing of the tomato active-site peptide revealed two highly conserved dodecapeptides; the minor peptide possessed a sequence identical to that of the apple enzyme, whereas the major peptide differed from the minor peptide in that methionine replaced leucine at position 6

  5. Active sites for methane activation in MgO and Li doped MgO

    Kwapień, Karolina

    2012-01-01

    Die vorliegende Dissertation präsentiert eine detaillierte quantenchemische Untersuchung der H-Abstraktion von Methan durch MgO und Li dotiertes MgO. Motiviert wurde die durch das UniCat-Excellenz-Cluster, welches sich zum Ziel gesetzt hat, die oxidative Kupplung von Methan (OCM) im Detail zu verstehen. Basierend auf der Hypothese, dass Li+O•– Spezies für den H-Abstraktion Schritt verantwortlich sind, wurden kleine kationische MgO- und Li dotierte MgO-Cluster (die O•– Sites modellieren) unt...

  6. A split active site couples cap recognition by Dcp2 to activation

    Floor, Stephen N.; Jones, Brittnee N.; Hernandez, Gail A.; Gross, John D.

    2010-01-01

    Decapping by Dcp2 is an essential step in 5′-3′ mRNA decay. In yeast, decapping requires an open-to-closed transition in Dcp2, though the link between closure and catalysis remains elusive. Here we show using NMR that cap binds conserved residues on both the catalytic and regulatory domains of Dcp2. Lesions in the cap-binding site on the regulatory domain reduce the catalytic step two orders of magnitude and block formation of the closed state whereas Dcp1 enhances the catalytic step by a fac...

  7. Integral Public Activities as a Support to the Site Selection Process for LILW Repository

    The first site selection process for low and intermediate level radioactive waste (LILW) repository took place between 1990 and 1993 in Slovenia was stopped unsuccessfully with very strong public opposition at local level, followed by political withdrawal on national level. As one of the consequences ARAO started to develop new approach to the site selection based also on the findings from sociology, psychology and other human sciences. The recommendations on public involvement and transparency were so strong that ARAO started with first limited public relation (PR) activities which later grew to the PR process which supports all technical activities in ARAO. Presently the PR process covers communication, information and research activities and assures careful planning, prompt responds and involvement of the highest responsible persons at ARAO. Integral public relation activities are divided in several parts. Majority of activities support the on-going site selection process where activities are presently focused on functioning of local partnerships developed as a basic communication tool to involve as much citizens and public as possible on local level. Presently two local partnerships are working in Krsko and Brezice community with clear role to enhance public involvement according to Aarchus convention. Each of the partnerships is organized in a specific way adjusted to the local needs. Communication activities are organized also for different other projects and are preparing the necessary basis for the work with different groups of stake holders and in different situations. As a foundation very broad information material, such as books, leaflets, reports, magazines, video cassettes, CD and DVD on the radioactive waste management is prepared and used for different purposes. We also try to be proactive with web pages and have a well organized visitors' center. Improvement of public relation process is achieved through constant survey and feed-back information

  8. Number of active transcription factor binding sites is essential for the Hes7 oscillator

    de Angelis Martin

    2006-02-01

    Full Text Available Abstract Background It is commonly accepted that embryonic segmentation of vertebrates is regulated by a segmentation clock, which is induced by the cycling genes Hes1 and Hes7. Their products form dimers that bind to the regulatory regions and thereby repress the transcription of their own encoding genes. An increase of the half-life of Hes7 protein causes irregular somite formation. This was shown in recent experiments by Hirata et al. In the same work, numerical simulations from a delay differential equations model, originally invented by Lewis, gave additional support. For a longer half-life of the Hes7 protein, these simulations exhibited strongly damped oscillations with, after few periods, severely attenuated the amplitudes. In these simulations, the Hill coefficient, a crucial model parameter, was set to 2 indicating that Hes7 has only one binding site in its promoter. On the other hand, Bessho et al. established three regulatory elements in the promoter region. Results We show that – with the same half life – the delay system is highly sensitive to changes in the Hill coefficient. A small increase changes the qualitative behaviour of the solutions drastically. There is sustained oscillation and hence the model can no longer explain the disruption of the segmentation clock. On the other hand, the Hill coefficient is correlated with the number of active binding sites, and with the way in which dimers bind to them. In this paper, we adopt response functions in order to estimate Hill coefficients for a variable number of active binding sites. It turns out that three active transcription factor binding sites increase the Hill coefficient by at least 20% as compared to one single active site. Conclusion Our findings lead to the following crucial dichotomy: either Hirata's model is correct for the Hes7 oscillator, in which case at most two binding sites are active in its promoter region; or at least three binding sites are active, in which

  9. Transcriptional activities of mammalian genomes at sites of recombination with foreign DNA

    The nucleotide sequences of several sites of recombination between adenovirus DNA and hamster, mouse, or human cell DNAs were determined. These sites of recombination had been cloned from adenovirus type 2 (Ad2)- or type 12 (Ad12)-transformed cells, from Ad12-induced tumor cells, or from a symmetric recombinant between Ad12 DNA and human cell DNA. One important precondition for the generation of recombinants between host and foreign DNAs might be the establishment of a chromatin configuration that permits access of foreign DNA and of the recombination machinery to cellular DNA. Such favorable chromatin structures might arise during cellular DNA replication or transcription or both. As a first approach toward investigating these more complex problems of foreign DNA insertion. The authors determined transcriptional activities of cellular DNA sequences at viral junction sites. The results presented demonstrate that the cellular DNA sequences involved in linkage to viral DNA at five completely different sites in DNA from three different species are transcribed into RNAs even in cells which have not been transformed or infected by adenovirus. These results are consistent with the notion that at least some of the cellular DNA sequences at sites of insertion of adenovirus (foreign) DNA are transcriptionally active and thus provide an opportunity for recombination

  10. Role of active sites in the reaction of methanol to olefin over modified ZSM-5 zeolite

    ZSM-5 zeolites were modified with metals Mg, K, Ca, Ba and La by an incipient impregnation and characterized by X-ray diffraction, N/sub 2/ adsorption and temperature - programmed desorption of NH/sub 3/ and CO/sub 2/. The obtained samples were investigated for their selectivity and catalytic stability in the methanol-to-olefin (MTO) reaction. The catalytic performance was influenced by the properties of active sites on the catalyst, which was indicated by the changes of the light olefin selectivity and catalytic stability in the methanol conversion. Correlating the reaction evaluation with the catalyst characterization, it can be seen that the catalyst with too strong basic sites shows an inferior catalytic performance. In addition, steam treatment further increased the catalytic performance, especially for Ca modified ZSM-5. It may be concluded that basic sites must suitably match with acidic sites for a special ZSM-5 in the methanol conversion and the formed active sites were relative with the modification metal based on the obtained results. (author)

  11. Management of Ground and Groundwater Contamination on a Compact Site Constrained by Ongoing Activities

    Sellafield Site is a compact and complex site which since the 1940's has been home to a range of facilities associated with the production and reprocessing of fissile material. The site contains the UK equivalent of the Chicago Pile-1 reactor, Hanford B Reactor, Rocky Flats Buildings 771 and 774, West Valley Main Process Plant Building, Savannah River Vitrification Plant, Savannah River MOX Plant, Savannah River F Canyon, Hanford 222 Analytical Laboratory, Savannah River K-, L-, and P-Basins, and the Fort St. Vrain Reactor all in an area of approximately 1000 acres. Spent fuel reprocessing is still undertaken on site; however waste management and decommissioning activities are of increasing importance. These include the emptying and removal of fragile ponds and silos containing significant radioactive inventories, the decommissioning of reactors (including the world's first commercial reactor for power generation and the Windscale Piles, the site of a reactor fire in the late 1950's) and the construction of a new generation of vitrification and encapsulation plants. Leaks, spills and on-site disposals during the site's industrial lifetime have resulted in a legacy of fission products and other radionuclides in the ground and groundwater. Volumes of contaminated ground have been estimated as being as much as 18 million m3 and an estimated below ground inventory of approximately 1.8 E16 Bq. These have all occurred within close proximity to a range of receptors including farm land and the sea. The cramped nature of the facilities on site, overlapping source terms and ongoing decommissioning, waste management and operating activities all raise significant challenges in the management and remediation of contaminated land and groundwater. The strategy to address these challenges includes: 1. Data collection, management and interpretation. The congested nature of the site and the age of some of the monitoring facilities has resulted in particular difficulties. For example

  12. Activities of Secreted Aryl Alcohol Quinone Oxidoreductases from Pycnoporus cinnabarinus Provide Insights into Fungal Degradation of Plant Biomass.

    Mathieu, Yann; Piumi, Francois; Valli, Richard; Aramburu, Juan Carro; Ferreira, Patricia; Faulds, Craig B; Record, Eric

    2016-04-15

    Auxiliary activities family 3 subfamily 2 (AA3_2) from the CAZy database comprises various functions related to ligninolytic enzymes, such as fungal aryl alcohol oxidases (AAO) and glucose oxidases, both of which are flavoenzymes. The recent study of thePycnoporus cinnabarinusCIRM BRFM 137 genome combined with its secretome revealed that four AA3_2 enzymes are secreted during biomass degradation. One of these AA3_2 enzymes, scf184803.g17, has recently been produced heterologously inAspergillus niger Based on the enzyme's activity and specificity, it was assigned to the glucose dehydrogenases (PcinnabarinusGDH [PcGDH]). Here, we analyze the distribution of the other three AA3_2 enzymes (scf185002.g8, scf184611.g7, and scf184746.g13) to assess their putative functions. These proteins showed the highest homology with aryl alcohol oxidase fromPleurotus eryngii Biochemical characterization demonstrated that they were also flavoenzymes harboring flavin adenine dinucleotide (FAD) as a cofactor and able to oxidize a wide variety of phenolic and nonphenolic aryl alcohols and one aliphatic polyunsaturated primary alcohol. Though presenting homology with fungal AAOs, these enzymes exhibited greater efficiency in reducing electron acceptors (quinones and one artificial acceptor) than molecular oxygen and so were defined as aryl-alcohol:quinone oxidoreductases (AAQOs) with two enzymes possessing residual oxidase activity (PcAAQO2 andPcAAQO3). Structural comparison ofPcAAQO homology models withP. eryngiiAAO demonstrated a wider substrate access channel connecting the active-site cavity to the solvent, explaining the absence of activity with molecular oxygen. Finally, the ability ofPcAAQOs to reduce radical intermediates generated by laccase fromP. cinnabarinuswas demonstrated, shedding light on the ligninolytic system of this fungus. PMID:26873317

  13. Ionizing Groups at the Active Site of the Alkaline Phosphatase from Ostrea cucullate

    CHEN Qiao; WANG Qin; LIAO Jinhua; CHEN Qingxi

    2006-01-01

    The ionizing groups at the active site of alkaline phosphatase (ALP, EC 3.1.3.1) from Ostrea cucullate were studied using kinetic methods. The ionization constant, pKe, of the ionizing groups at the active site of the enzyme was found to be 10.11 at 37.0℃ and the organic solvent, dioxin, had no effect on the pKe. The standard dissociation enthalpy (△Ho) was determined to be 10.57 kcal/mol (1 cal=4.18 J). The results show that the dissociation group of the enzyme active center is the з-NH3+ of lysine. Chemical modification of the enzyme by acetic anhydride and succinic anhydride demonstrates that the amino group is one of the enzyme's functional groups.

  14. Active space decomposition with multiple sites: Density matrix renormalization group algorithm

    Parker, Shane M

    2014-01-01

    We extend the active space decomposition method, recently developed by us, to more than two active sites using the density matrix renormalization group algorithm. The fragment wave functions are described by complete or restricted active-space wave functions. Numerical results are shown on a benzene pentamer and a perylene diimide trimer. It is found that the truncation errors in our method decrease almost exponentially with respect to the number of renormalization states M, allowing for numerically exact calculations (to a few {\\mu}Eh or less) with M = 128 in both cases, which is in contrast to conventional ab initio density matrix renormalization group.

  15. Communication: Active space decomposition with multiple sites: Density matrix renormalization group algorithm

    Parker, Shane M.; Shiozaki, Toru [Department of Chemistry, Northwestern University, 2145 Sheridan Rd., Evanston, Illinois 60208 (United States)

    2014-12-07

    We extend the active space decomposition method, recently developed by us, to more than two active sites using the density matrix renormalization group algorithm. The fragment wave functions are described by complete or restricted active-space wave functions. Numerical results are shown on a benzene pentamer and a perylene diimide trimer. It is found that the truncation errors in our method decrease almost exponentially with respect to the number of renormalization states M, allowing for numerically exact calculations (to a few μE{sub h} or less) with M = 128 in both cases. This rapid convergence is because the renormalization steps are used only for the interfragment electron correlation.

  16. Communication: Active space decomposition with multiple sites: Density matrix renormalization group algorithm

    We extend the active space decomposition method, recently developed by us, to more than two active sites using the density matrix renormalization group algorithm. The fragment wave functions are described by complete or restricted active-space wave functions. Numerical results are shown on a benzene pentamer and a perylene diimide trimer. It is found that the truncation errors in our method decrease almost exponentially with respect to the number of renormalization states M, allowing for numerically exact calculations (to a few μEh or less) with M = 128 in both cases. This rapid convergence is because the renormalization steps are used only for the interfragment electron correlation

  17. Mapping Topoisomerase IV Binding and Activity Sites on the E. coli Genome.

    El Sayyed, Hafez; Le Chat, Ludovic; Lebailly, Elise; Vickridge, Elise; Pages, Carine; Cornet, Francois; Cosentino Lagomarsino, Marco; Espéli, Olivier

    2016-05-01

    Catenation links between sister chromatids are formed progressively during DNA replication and are involved in the establishment of sister chromatid cohesion. Topo IV is a bacterial type II topoisomerase involved in the removal of catenation links both behind replication forks and after replication during the final separation of sister chromosomes. We have investigated the global DNA-binding and catalytic activity of Topo IV in E. coli using genomic and molecular biology approaches. ChIP-seq revealed that Topo IV interaction with the E. coli chromosome is controlled by DNA replication. During replication, Topo IV has access to most of the genome but only selects a few hundred specific sites for its activity. Local chromatin and gene expression context influence site selection. Moreover strong DNA-binding and catalytic activities are found at the chromosome dimer resolution site, dif, located opposite the origin of replication. We reveal a physical and functional interaction between Topo IV and the XerCD recombinases acting at the dif site. This interaction is modulated by MatP, a protein involved in the organization of the Ter macrodomain. These results show that Topo IV, XerCD/dif and MatP are part of a network dedicated to the final step of chromosome management during the cell cycle. PMID:27171414

  18. The Molybdenum Active Site of Formate Dehydrogenase Is Capable of Catalyzing C-H Bond Cleavage and Oxygen Atom Transfer Reactions.

    Hartmann, Tobias; Schrapers, Peer; Utesch, Tillmann; Nimtz, Manfred; Rippers, Yvonne; Dau, Holger; Mroginski, Maria Andrea; Haumann, Michael; Leimkühler, Silke

    2016-04-26

    Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal-containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pKa of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase. PMID:27054466

  19. Regulatory inspection activities on nuclear power plant sites during construction in the United Kingdom

    The work of regulatory inspection of the construction of the plant on the site is performed not only by the inspector who has been allocated to inspection duties for that site but also by the specialist staff who are involved with the safety assessment of the plant. The coordination of this work is described in the paper and examples are given of inspection activities associated with the enforcement requirements of license conditions as well as those related to the inspection of the plant itself

  20. Technical basis for classification of low-activity waste fraction from Hanford site tanks

    Petersen, C.A., Westinghouse Hanford

    1996-07-17

    The overall objective of this report is to provide a technical basis to support a U.S. Nuclear Regulatory Commission determination to classify the low-activity waste from the Hanford Site single-shell and double-shell tanks as `incidental` wastes after removal of additional radionuclides and immobilization.The proposed processing method, in addition to the previous radionuclide removal efforts, will remove the largest practical amount of total site radioactivity, attributable to high-level wastes, for disposal in a deep geologic repository. The remainder of the waste would be considered `incidental` waste and could be disposed onsite.

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

    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

  2. Assessment of national systems for obtaining local acceptance of nuclear waste management siting activities

    On behalf of the United States Department of Energy (DOE), International Energy Associates Limited (IEAL) of Washington, D.C. has conducted surveys and analyses of fourteen countries' plans and approaches for dealing with the problems of obtaining local siting acceptance for nuclear waste management facilities. It was determined that the following elements of the formal systems generally facilitate and/or expedite waste management siting decisions: (1) a clear-cut pro-nuclear power position on the part of the government; (2) a willingness on the part of the central government to exert (with prudence and restraint) its pre-emptive rights in nuclear matters; (3) political structures in which the heads of regional or provincial governments are appointed by the central government; (4) national laws that link reactor licensing with a detailed plan for waste management; (5) an established and stable policy with regard to reprocessing. In contrast, it was determined that the following elements of the formal system generally hinder waste management siting activities: (1) historically strong local land used veto laws; (2) the use of national referenda for making nuclear decisions; (3) requirements for public hearings. The informal approaches fall into the following five categories: (1) political: e.g. assertion of will by political leaders, activities to enlist support of local politicians, activities to broaden involvement in decision-making; (2) economic: e.g. emphasis on normal benefits, provision for additional economic benefits; (3) siting: e.g. at or near existing nuclear facilities, on government or utility property, at multiple locations to spread the political burden; (4) timing: e.g. decoupling drilling activities from ultimate repository site decision, deliberate deferral to (long-range) future; (5) education: e.g. creation of special government programmes, enlisting of media support

  3. The role of an active site Mg2+ in HDV ribozyme self-cleavage: insights from QM/MM calculations

    Mlýnský, V.; Walter, Nils G.; Šponer, Jiří; Otyepka, Michal; Banáš, Pavel

    2015-01-01

    Roč. 17, č. 1 (2015), s. 670-679. ISSN 1463-9076 R&D Projects: GA ČR(CZ) GAP208/12/1878 Institutional support: RVO:68081707 Keywords : HEPATITIS-DELTA-VIRUS * MOLECULAR-DYNAMICS SIMULATIONS * 2'-HYDROXYL GROUP Subject RIV: BO - Biophysics Impact factor: 4.493, year: 2014

  4. Models for the active site of vanadium-dependent haloperoxidases: insight into the solution structure of peroxo vanadium compounds.

    Conte, V; Bortolini, O; Carraro, M; Moro, S

    2000-05-30

    A combined use of electrospray ionization-mass spectrometry (ESI-MS), 51V NMR spectroscopy and ab initio calculations has been proved to be a powerful tool for obtaining direct information of the structure and the chemistry of peroxo vanadates in solutions. The analysis of acid solutions containing monoperoxo vanadates showed the occurrence of exchange reactions between solvent molecules in the coordination sphere of the metal. On the other hand, bisperoxo vanadates appear to be less prone to coordinate more than one water or alcohol molecule. The bisperoxo complex [VO5]- in the presence of histidine and histidine-like ligands, at near neutral conditions, has been studied. Coordination of one and two molecules of ligand is observed affording [VO5L]- and [VO5L2]-, respectively. Characterization of these species has been obtained by MSn experiments, which allowed us to distinguish specific fragmentations of the peroxidic moiety. PMID:10885462

  5. Linde FUSRAP Site Remediation: Engineering Challenges and Solutions of Remedial Activities on an Active Industrial Facility - 13506

    The Linde FUSRAP Site (Linde) is located in Tonawanda, New York at a major research and development facility for Praxair, Inc. (Praxair). Successful remediation activities at Linde combines meeting cleanup objectives of radiological contamination while minimizing impacts to Praxair business operations. The unique use of Praxair's property coupled with an array of active and abandoned utilities poses many engineering and operational challenges; each of which has been overcome during the remedial action at Linde. The U.S. Army Corps of Engineers - Buffalo District (USACE) and CABRERA SERVICES, INC. (CABRERA) have successfully faced engineering challenges such as relocation of an aboveground structure, structural protection of an active water line, and installation of active mechanical, electrical, and communication utilities to perform remediation. As remediation nears completion, continued success of engineering challenges is critical as remaining activities exist in the vicinity of infrastructure essential to business operations; an electrical substation and duct bank providing power throughout the Praxair facility. Emphasis on engineering and operations through final remediation and into site restoration will allow for the safe and successful completion of the project. (authors)

  6. Active-site titration analysis of surface influence on immobilized Candida antarctica Lipase B activity

    Matrix morphology and surface polarity effects were investigated for Candida antarctica lipase B immobilization. Measurements of the amount of lipase immobilized (bicinchoninic acid method) and the catalyst’s tributyrin hydrolysis activity, coupled with a determination of the lipase’s functional fr...

  7. RCRA and CERCLA requirements affecting cleanup activities at a federal facility superfund site

    The Fernald Environmental Management Project (FEMP) achieved success on an integrated groundwater monitoring program which addressed both RCRA and CERCLA requirements. The integrated plan resulted in a cost savings of approximately $2.6 million. At present, the FEMP is also working on an integrated closure process to address Hazardous Waste Management Units (HWMUs) at the site. To date, Ohio EPA seems willing to discuss an integrated program with some stipulations. If an integrated program is implemented, a cost savings of several million dollars will be realized since the CERCLA documents can be used in place of a RCRA closure plan. The success of an integrated program at the FEMP is impossible without the support of DOE and the regulators. Since DOE is an owner/operator of the facility and Ohio EPA regulates hazardous waste management activities at the FEMP, both parties must be satisfied with the proposed integration activities. Similarly, US EPA retains CERCLA authority over the site along with a signed consent agreement with DOE, which dictates the schedule of the CERCLA activities. Another federal facility used RCRA closure plans to satisfy CERCLA activities. This federal facility was in a different US EPA Region than the FEMP. While this approach was successful for this site, an integrated approach was required at the FEMP because of the signed Consent Agreement and Consent Decree. For federal facilities which have a large number of HWMUs along with OUs, an integrated approach may result in a timely and cost-effective cleanup

  8. Structural and kinetic contributions of the oxyanion binding site to the catalytic activity of acylaminoacyl peptidase.

    Kiss, András L; Palló, Anna; Náray-Szabó, Gábor; Harmat, Veronika; Polgár, László

    2008-05-01

    It is widely accepted that the catalytic activity of serine proteases depends primarily on the Asp-His-Ser catalytic triad and other residues within the vicinity of this motif. Some of these residues form the oxyanion binding site that stabilizes the tetrahedral intermediate by hydrogen bonding to the negatively charged oxyanion. In acylaminoacyl peptidase from the thermophile Aeropyrum pernix, the main chain NH group of Gly369 is one of the hydrogen bond donors forming the oxyanion binding site. The side chain of His367, a conserved residue in acylaminoacyl peptidases across all species, fastens the loop holding Gly369. Determination of the crystal structure of the H367A mutant revealed that this loop, including Gly369, moves away considerably, accounting for the observed three orders of magnitude decrease in the specificity rate constant. For the wild-type enzyme ln(k(cat)/K(m)) vs. 1/T deviates from linearity indicating greater rate enhancement with increasing temperature for the dissociation of the enzyme-substrate complex compared with its decomposition to product. In contrast, the H367A variant provided a linear Arrhenius plot, and its reaction was associated with unfavourable entropy of activation. These results show that a residue relatively distant from the active site can significantly affect the catalytic activity of acylaminoacyl peptidase without changing the overall structure of the enzyme. PMID:18325786

  9. Progress report on decommissioning activities at the Fernald Environmental Management Project (FEMP) site

    NONE

    1998-07-01

    The Fernald Environmental Management Project (FEMP), is located about 18 miles northwest of Cincinnati, Ohio. Between 1953 and 1989, the facility, then called the Feed Material Production Center or FMPC, produced uranium metal products used in the eventual production of weapons grade material for use by other US Department of Energy (DOE) sites. In 1989, FMPC`s production was suspended by the federal government in order to focus resources on environmental restoration versus defense production. In 1992, Fluor Daniel Fernald assumed responsibility for managing all cleanup activities at the FEMP under contract to the DOE. In 1990, as part of the remediation effort, the site was divided into five operable units based on physical proximity of contaminated areas, similar amounts of types of contamination, or the potential for a similar technology to be used in cleanup activities. This report continues the outline of the decontamination and decommissioning (D and D) activities at the FEMP site Operable Unit 3 (OU3) and provides an update on the status of the decommissioning activities. OU3, the Facilities Closure and Demolition Project, involves the remediation of more than 200 uranium processing facilities. The mission of the project is to remove nuclear materials stored in these buildings, then perform the clean out of the buildings and equipment, and decontaminate and dismantle the facilities.

  10. Progress report on decommissioning activities at the Fernald Environmental Management Project (FEMP) site

    The Fernald Environmental Management Project (FEMP), is located about 18 miles northwest of Cincinnati, Ohio. Between 1953 and 1989, the facility, then called the Feed Material Production Center or FMPC, produced uranium metal products used in the eventual production of weapons grade material for use by other US Department of Energy (DOE) sites. In 1989, FMPC's production was suspended by the federal government in order to focus resources on environmental restoration versus defense production. In 1992, Fluor Daniel Fernald assumed responsibility for managing all cleanup activities at the FEMP under contract to the DOE. In 1990, as part of the remediation effort, the site was divided into five operable units based on physical proximity of contaminated areas, similar amounts of types of contamination, or the potential for a similar technology to be used in cleanup activities. This report continues the outline of the decontamination and decommissioning (D and D) activities at the FEMP site Operable Unit 3 (OU3) and provides an update on the status of the decommissioning activities. OU3, the Facilities Closure and Demolition Project, involves the remediation of more than 200 uranium processing facilities. The mission of the project is to remove nuclear materials stored in these buildings, then perform the clean out of the buildings and equipment, and decontaminate and dismantle the facilities

  11. Human glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site

    Misquitta Stephanie A

    2004-02-01

    Full Text Available Abstract Background Glutaminyl cyclase (QC forms the pyroglutamyl residue at the amino terminus of numerous secretory peptides and proteins. We previously proposed the mammalian QC has some features in common with zinc aminopeptidases. We now have generated a structural model for human QC based on the aminopeptidase fold (pdb code 1AMP and mutated the apparent active site residues to assess their role in QC catalysis. Results The structural model proposed here for human QC, deposited in the protein databank as 1MOI, is supported by a variety of fold prediction programs, by the circular dichroism spectrum, and by the presence of the disulfide. Mutagenesis of the six active site residues present in both 1AMP and QC reveal essential roles for the two histidines (140 and 330, QC numbering and the two glutamates (201 and 202, while the two aspartates (159 and 248 appear to play no catalytic role. ICP-MS analysis shows less than stoichiometric zinc (0.3:1 in the purified enzyme. Conclusions We conclude that human pituitary glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site residues. In contrast to the aminopeptidase, however, QC does not appear to require zinc for enzymatic activity.

  12. Multiple nucleophilic elbows leading to multiple active sites in a single module esterase from Sorangium cellulosum

    Udatha, D.B.R.K. Gupta; Madsen, Karina Marie; Panagiotou, Gianni; Olsson, Lisbeth

    2015-01-01

    The catalytic residues in carbohydrate esterase enzyme families constitute a highly conserved triad: serine, histidine and aspartic acid. This catalytic triad is generally located in a very sharp turn of the protein backbone structure, called the nucleophilic elbow and identified by the consensus...... sequence GXSXG. An esterase from Sorangium cellulosum Soce56 that contains five nucleophilic elbows was cloned and expressed in Escherichia coli and the function of each nucleophilic elbowed site was characterized. In order to elucidate the function of each nucleophilic elbow, site directed mutagenesis was...... used to generate variants with deactivated nucleophilic elbows and the functional promiscuity was analyzed. In silico analysis together with enzymological characterization interestingly showed that each nucleophilic elbow formed a local active site with varied substrate specificities and affinities. To...

  13. Role of a cysteine residue in the active site of ERK and the MAPKK family

    Kinases of mitogen-activated protein kinase (MAPK) cascades, including extracellular signal-regulated protein kinase (ERK), represent likely targets for pharmacological intervention in proliferative diseases. Here, we report that FR148083 inhibits ERK2 enzyme activity and TGFβ-induced AP-1-dependent luciferase expression with respective IC50 values of 0.08 and 0.05 μM. FR265083 (1'-2' dihydro form) and FR263574 (1'-2' and 7'-8' tetrahydro form) exhibited 5.5-fold less and no activity, respectively, indicating that both the α,β-unsaturated ketone and the conformation of the lactone ring contribute to this inhibitory activity. The X-ray crystal structure of the ERK2/FR148083 complex revealed that the compound binds to the ATP binding site of ERK2, involving a covalent bond to Sγ of ERK2 Cys166, hydrogen bonds with the backbone NH of Met108, Nζ of Lys114, backbone C=O of Ser153, Nδ2 of Asn154, and hydrophobic interactions with the side chains of Ile31, Val39, Ala52, and Leu156. The covalent bond motif in the ERK2/FR148083 complex assures that the inhibitor has high activity for ERK2 and no activity for other MAPKs such as JNK1 and p38MAPKα/β/γ/δ which have leucine residues at the site corresponding to Cys166 in ERK2. On the other hand, MEK1 and MKK7, kinases of the MAPKK family which also can be inhibited by FR148083, contain a cysteine residue corresponding to Cys166 of ERK2. The covalent binding to the common cysteine residue in the ATP-binding site is therefore likely to play a crucial role in the inhibitory activity for these MAP kinases. These findings on the molecular recognition mechanisms of FR148083 for kinases with Cys166 should provide a novel strategy for the pharmacological intervention of MAPK cascades

  14. Soil pollution with trace elements at selected sites in Romania studied by instrumental neutron activation analysis

    Instrumental neutron activation analysis (INAA) was used to determine concentrations of 42 elements in samples of surface soil collected at seven sites polluted from various anthropogenic activities and a control site in a relatively clean area. Elements studied were Ag, Al, As, Au, Ba, Br, Ca, Cd, Ce, Co, Cr, Cs, Eu, Fe, Gd, Hf, Hg, K, La, Lu, Mg, Mn, Mo, Na, Nd, Ni, Rb, Sb, Sc, Se, Sm, Sr, Ta, Tb, Th, Ti, U, V, W, Yb, Zn, and Zr. The results are compared with data for trace elements atmospheric deposition in lichen transplants from the same sites. The most severe soil contamination was observed at Copsa Mica from non-ferrous metallurgy. Appreciable soil contamination was also indicated at Baia Mare (non-ferrous mining and metallurgy), Deva (coal-fired power plant, cement and building materials industry), Galati (ferrous metallurgy), Magurele and Afumati (general urban pollution), and Oradea (chemical and light industries). In most cases excessive levels of toxic metals in soils matched correspondingly high values in lichen transplants. Compared to Romanian norms, legal upper limits were exceeded for Zn and Cd at Copsa Mica. Also, As and Sb occurred in excessive levels at given sites. (orig.)

  15. Assessment of Tritium Activity in Groundwater at the Nuclear Objects Sites in Lithuania

    Vigilija Cidzikienė

    2014-01-01

    Full Text Available The assessment of nuclear objects sites in Lithuania, including groundwater characterization, took place in the last few years. Tritium activity in groundwater is a very useful tool for determining how groundwater systems function. Natural and artificial tritium was measured in 8 wells in different layers (from 1.5 to 15 meters depth. The results were compared with other regions of Lithuania also. The evaluated tritium activities varied from 1.8 to 6.4 Bq/L at nuclear objects sites in Lithuania and they are coming to background level (1.83 Bq/L and other places in Lithuania. The data show, that groundwater at the nuclear power objects sites is not contaminated with artificial tritium. In this work, the vertical tritium transfer from soil water to the groundwater well at nuclear objects site was estimated. The data show that the main factor for vertical tritium transfer to the well depends on the depth of wells.

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

    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

  17. Soil pollution with trace elements at selected sites in Romania studied by instrumental neutron activation analysis

    Pantelica, A. [Horia Hulubei National Institute for Physics and Nuclear Engineering (IFIN-HH), Magurele, Ilfov County (Romania); Carmo Freitas, M. do [Technological and Nuclear Institute (ITN), Sacavem (Portugal); Ene, A. [Dunarea de Jos Univ. of Galati (Romania). Dept. of Chemistry, Physics and Environment; Steinnes, E. [Norwegian Univ. of Science and Technology, Trondheim (Norway). Dept. of Chemistry

    2013-03-01

    Instrumental neutron activation analysis (INAA) was used to determine concentrations of 42 elements in samples of surface soil collected at seven sites polluted from various anthropogenic activities and a control site in a relatively clean area. Elements studied were Ag, Al, As, Au, Ba, Br, Ca, Cd, Ce, Co, Cr, Cs, Eu, Fe, Gd, Hf, Hg, K, La, Lu, Mg, Mn, Mo, Na, Nd, Ni, Rb, Sb, Sc, Se, Sm, Sr, Ta, Tb, Th, Ti, U, V, W, Yb, Zn, and Zr. The results are compared with data for trace elements atmospheric deposition in lichen transplants from the same sites. The most severe soil contamination was observed at Copsa Mica from non-ferrous metallurgy. Appreciable soil contamination was also indicated at Baia Mare (non-ferrous mining and metallurgy), Deva (coal-fired power plant, cement and building materials industry), Galati (ferrous metallurgy), Magurele and Afumati (general urban pollution), and Oradea (chemical and light industries). In most cases excessive levels of toxic metals in soils matched correspondingly high values in lichen transplants. Compared to Romanian norms, legal upper limits were exceeded for Zn and Cd at Copsa Mica. Also, As and Sb occurred in excessive levels at given sites. (orig.)

  18. The active sites in the heterogeneous Baeyer-Villiger oxidation of cyclopentanone by hydrotalcite catalysts

    Ueno, Shinji; Ebitani, Kohki; Ookubo, Akira; Kaneda, Kiyotomi

    1997-11-01

    The active sites of hydrotalcites, [ Mg1-x2+Alx3+( OH) 2] x+[ Ax/nn-·m H 2O ] x-, A″ n-; CO 32-, Cl -, etc., were studied in the heterogeneous Baeyer-Villiger oxidation of cyclopentanone to δ-valerolactone with a combination oxidant system of molecular oxygen and benzaldehyde (O 2/benzaldehyde) in a 1,2-dichloroethane solvent. The hydrotalcites with different basicity were prepared by changing element ratios of Al 3+ to Mg 2+ in the Brucite-like layer and by changing anionic compounds (CO 32-, Cl -, and SO 42-) in the interlayer. The basicities of hydrotalcites were evaluated by measuring the calorimetric heats of benzoic acid adsorption and the zeta-potential of potassium chloride and by the indicator titration method. Yields of δ-valerolactone were almost proportional to the basicities of hydrotalcites, i.e., the heats of benzoic acid adsorption on hydrotalcites, which suggests that basic sites of hydrotalcites are active sites for the oxidation. Yields of δ-valerolactone were also dependent on the basicities of hydrotalcites using m-chloroperbenzoic acid ( m-CPBA) as an oxidant instead of O 2/benzaldehyde. Basic sites of hydrotalcites play an important role in the oxygen transfer from perbenzoic acid to ketone.

  19. 78 FR 21352 - Update on Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    2013-04-10

    ... on Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY... reimbursement for cleanup work performed by licensees at eligible uranium and thorium processing sites in... licensees of eligible uranium and thorium processing sites. If licensees submit claims in FY 2013,...

  20. Characterization of the interactions between the active site of a protein tyrosine kinase and a divalent metal activator

    Ayrapetov Marina K

    2005-11-01

    Full Text Available Abstract Background Protein tyrosine kinases are important enzymes for cell signalling and key targets for anticancer drug discovery. The catalytic mechanisms of protein tyrosine kinase-catalysed phosphorylation are not fully understood. Protein tyrosine kinase Csk requires two Mg2+ cations for activity: one (M1 binds to ATP, and the other (M2 acts as an essential activator. Results Experiments in this communication characterize the interaction between M2 and Csk. Csk activity is sensitive to pH in the range of 6 to 7. Kinetic characterization indicates that the sensitivity is not due to altered substrate binding, but caused by the sensitivity of M2 binding to pH. Several residues in the active site with potential of binding M2 are mutated and the effect on metal activation studied. An active mutant of Asn319 is generated, and this mutation does not alter the metal binding characteristics. Mutations of Glu236 or Asp332 abolish the kinase activity, precluding a positive or negative conclusion on their role in M2 coordination. Finally, the ability of divalent metal cations to activate Csk correlates to a combination of ionic radius and the coordination number. Conclusion These studies demonstrate that M2 binding to Csk is sensitive to pH, which is mainly responsible for Csk activity change in the acidic arm of the pH response curve. They also demonstrate critical differences in the metal activator coordination sphere in protein tyrosine kinase Csk and a protein Ser/Thr kinase, the cAMP-dependent protein kinase. They shed light on the physical interactions between a protein tyrosine kinase and a divalent metal activator.

  1. Synthesis of supported bimetallic nanoparticles with controlled size and composition distributions for active site elucidation

    Hakim, Sikander H.; Sener, Canan; Alba Rubio, Ana C.; Gostanian, Thomas M.; O' neill, Brandon J; Ribeiro, Fabio H.; Miller, Jeffrey T.; Dumesic, James A

    2015-08-01

    Elucidation of active sites in supported bimetallic catalysts is complicated by the high level of dispersity in the nanoparticle size and composition that is inherent in conventional methods of catalyst preparation. We present a synthesis strategy that leads to highly dispersed, bimetallic nanoparticles with uniform particle size and composition by means of controlled surface reactions. We demonstrate the synthesis of three systems, RhMo, PtMo, and RhRe, consisting of a highly reducible metal with an oxophilic promoter. These catalysts are characterized by FTIR, CO chemisorption, STEM/EDS, TPR, and XAS analysis. The catalytic properties of these bimetallic nanoparticles were probed for the selective CO hydrogenolysis of (hydroxymethyl)tetrahydropyran to produce 1,6 hexanediol. Based on the characterization results and reactivity trends, the active sites in the hydrogenolysis reaction are identified to be small ensembles of the more noble metal (Rh, Pt) adjacent to highly reduced moieties of the more oxophilic metal (Mo, Re).

  2. Use of the Multi-Agency Radiological Laboratory Analytical Protocols Manual (MARLAP) for site cleanup activities

    MARLAP is being developed as a multi-agency guidance manual for project managers and radioanalytical laboratories. The document uses a performance based approach and will provide guidance and a framework to assure that laboratory radioanalytical data meets the specific project or program needs and requirements. MARLAP supports a wide range of data collection activities including site characterization and compliance demonstration activities. Current participants include: US Environmental Protection Agency (EPA), US Department of Energy (DOE), US Nuclear Regulatory Commission (NRC), US Department of Defense (DoD), US National Institutes of Standards and Technology (NIST), US Geologic Survey (USGS), US Food and Drug Administration (FDA), Commonwealth of Kentucky, and the State of California. MARLAP is the radioanalytical laboratory counterpart to the Multi-Agency Radiological Survey and Site Investigation Manual (MARSSIM). MARLAP is currently in a preliminary draft stage. (author)

  3. Computation of Rate Constants for Diffusion of Small Ligands to and from Buried Protein Active Sites.

    Wang, P-H; De Sancho, D; Best, R B; Blumberger, J

    2016-01-01

    The diffusion of ligands to actives sites of proteins is essential to enzyme catalysis and many cellular signaling processes. In this contribution we review our recently developed methodology for calculation of rate constants for diffusion and binding of small molecules to buried protein active sites. The diffusive dynamics of the ligand obtained from molecular dynamics simulation is coarse grained and described by a Markov state model. Diffusion and binding rate constants are then obtained either from the reactive flux formalism or by fitting the time-dependent population of the Markov state model to a phenomenological rate law. The method is illustrated by applications to diffusion of substrate and inhibitors in [NiFe] hydrogenase, CO-dehydrogenase, and myoglobin. We also discuss a recently developed sensitivity analysis that allows one to identify hot spots in proteins, where mutations are expected to have the strongest effects on ligand diffusion rates. PMID:27497172

  4. Chemistry related to the actives sites of the [Fe]- and [FeFe]-hydrogenases

    Hill, Amanda D.

    2016-01-01

    Hydrogenases are an important group of enzymes found in a range of microorganisms. There are three phylogenetically distinct classes of hydrogenase all of which feature iron-containing complexes. The work contained in this thesis has two main focuses: the synthesis and characterization of novel mimics of the [Fe]-hydrogenase active site, and spectroscopic studies on the interaction of iron-sulfur clusters with CO and CN− relevant to the biosynthesis of the H-cluster of [FeFe]-hydrogenase. ...

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

    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.

  6. 76 FR 30696 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    2011-05-26

    ...This Notice announces revisions to the Department of Energy (DOE) acceptance of claims in FY 2011 from eligible active uranium and thorium processing site licensees for reimbursement under Title X of the Energy Policy Act of 1992. In our Federal Register Notice of November 24, 2010 (75 FR 71677), the Department announced the closing date for the submission of claims in FY 2011 as April 29,......

  7. Identification of the provenience of Majolica from sites in the Caribbean using neutron activation analysis

    Olin, J.S.; Sayre, E.V.

    1975-01-01

    Tin-enamelled earthenware pottery from five early Spanish Colonial sites in the Dominican Republic and Venezuela were sampled and analyzed by neutron activation analysis in an attempt to determine whether these sherds had a common source. The tentative conclusion was that although several sources were indicated for the specimens analyzed the overall similarity in composition indicated that these sources were probably closely related. (JSR)

  8. Measurement of human tissue-type plasminogen activator by a two-site immunoradiometric assay

    A two-site immunoradiometric assay for human extrinsic (tissue-type) plasminogen activator was developed by using rabbit antibodies raised against plasminogen activator purified from human melanoma cell culture fluid. Samples of 100 μl containing 1 to 100 ng/ml plasminogen activator were incubated in the wells of polyvinyl chloride microtiter plates coated with antibody. The amount of bound extrinsic plasminogen activator was quantitated by the subsequent binding of 125I-labeled affinospecific antibody. The mean level of plasma samples taken at rest was 6.6 +/- 2.9 ng/ml (n = 54). This level increased approximately threefold by exhaustive physical exercise, venous occlusion, or infusion of DDAVP. Extrinsic plasminogen activator in plasma is composed of a fibrin-adsorbable and active component (1.9 +/- 1.1 ng/ml, n = 54, in resting conditions) and an inactive component that does not bind to a fibrin clot (probably extrinsic plasminogen activator-proteinase inhibitor complexes). The fibrin-adsorbable fraction increased approximately fivefold to eightfold after physical exercise, venous occlusion, or DDAVP injections. Potential applications of the immunoradiometric assay are illustrated by the measurement of extrinsic plasminogen activator in different tissue extracts, body fluids, and cell culture fluids and in oocyte translation products after injection with mRNA for plasminogen activator

  9. Days of dismantling activities of installations and rehabilitation of contaminated sites in France

    The objective of these days, organized by the section environment of the French society of radiation protection, is to present a panorama of the activities of nuclear installations dismantling and contaminated sites rehabilitation in France, by leaning in the same time on practical cases and by stating the French rule and the national and international recommendations on the subject. These days have also for object to approach the stakes associated with the sectors of waste management and the materials generated by these activities and in a more general way, the stakes to come for the different actors of the dismantling and the rehabilitation. (N.C.)

  10. Comparative active-site mutation study of human and Caenorhabditis elegans thymidine kinase 1

    Skovgaard, Tine; Uhlin, Ulla; Munch-Petersen, Birgitte

    2012-01-01

    ligands. To improve our understanding of TK1 substrate specificity, we performed a detailed, mutation-based comparative structure-function study of the active sites of two thymidine kinases: HuTK1 and Caenorhabditis elegans TK1 (CeTK1). Specifically, mutations were introduced into the hydrophobic pocket...... surrounding the substrate base. In CeTK1, some of these mutations led to increased activity with deoxycytidine and deoxyguanosine, two unusual substrates for TK1-like kinases. In HuTK1, mutation of T163 to S resulted in a kinase with a 140-fold lower K(m) for the antiviral nucleoside analogue 3'-azido-3...

  11. Reaction of argininosuccinase with bromomesaconic acid: role of an essential lysine in the active site

    We have undertaken studies on bovine liver argininosuccinase (L-argininosuccinate arginine-lyase with the active site-directed reagent bromo[U-14C]mesaconic acid, an analogue of fumaric acid. Reactivity, measured by enzyme inactivation, followed pseudo-first-order kinetics, and the rate increased with reagent concentration. Argininosuccinate completely protected the enzyme against inactivation, but neither arginine nor fumarate was protective. A plot of the degree of inactivation as a function of alkyl groups incorporated was extrapolated to 4 mol per mol of enzyme, or 1 mol per active site. After large-scale alkylation of the enzyme (and digestion with trypsin), two 14C-labeled tryptic peptides were isolated. These were chemically sequenced by the Edman method. The amino acid sequences proved to be identical with regions of the deduced amino acid sequences or argininosuccinases from human and yeast sources The 14C-labeled tryptic peptide in the active site region had the sequence Gly-Leu-Glu-Xaa-Ala-Gly-Leu-Leu-Thr-Lys; Xaa represents an unknown phenylthiohydantoin derivative detected in cycle 4. The corresponding amino acid was identified as lysine-51 on the basis of sequence similarity with human and yeast amino acid sequences in this region. The reaction of the enzyme with the alkylating agent and the specific protection against inactivation by argininosuccinate suggest that this lysine residue has an essential role in the binding of argininosuccinate to the enzyme and, consequently, is essential for catalysis

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

    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

  13. Methods for Determining the Activity Concentration Calibration Factor for Ventilation Duct in Cyclotron Sites

    Cyclotrons are commonly used for production of radioactive isotopes for nuclear medicine purposes. The nuclear process requires installation of a ventilation stack. According to the regulations in many countries, the released activity should be monitored and controlled in order to limit air pollution. This requires a stuck radiation detector and determining a converting factor that will translate the radiation detector reading into activity concentration units. Calibrating the conversion factor is done mainly by releasing a known amount of activity. Having a preliminary estimation of the conversion factor, during the site construction stage, is an important option for the duct configuration design, in order to achieve the required detection sensitivity. An algorithm for estimating the stack concentration calibration factor in positron emitting isotopes producing sites was developed. The algorithm which is described in this article is based on three independent methods that consist of MCNP simulations analytical calculations and experimental setup with controlled static calibration releases of gaseous 18F which was produced especially for this purpose by the cyclotron site in Soreq Nuclear Research Center Israel. The specified methods applied on few stack sections with different shapes and sizes

  14. Radiological audit of remedial action activities at the processing site, transfer site, and Cheney disposal site Grand Junction, Colorado: Audit date, August 9--11, 1993. Final report

    1993-08-01

    The Uranium Mill Tailing Remedial Action (UMTRA) Project`s Technical Assistance Contractor (TAC) performed a radiological audit of the Remedial Action Contractor (RAC), MK-Ferguson and CWM Federal Environmental Services, Inc., at the processing site, transfer site, and Cheney disposal site in Grand Junction, Colorado. Jim Hylko and Bill James of the TAC conducted this audit August 9 through 11, 1993. Bob Cornish and Frank Bosiljevec represented the US Department of Energy (DOE). This report presents one programmatic finding, eleven site-specific observations, one good practice, and four programmatic observations.

  15. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J. (Abbott)

    2012-03-02

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

  16. Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation.

    Yamano, Koji; Queliconi, Bruno B; Koyano, Fumika; Saeki, Yasushi; Hirokawa, Takatsugu; Tanaka, Keiji; Matsuda, Noriyuki

    2015-10-16

    Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser(65) by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity. PMID:26260794

  17. A kinetic description for sodium and potassium effects on (Na+ plus K+)-adenosine triphosphatase: a model for a two-nonequivalent site potassium activation and an analysis of multiequivalent site models for sodium activation.

    Lindenmayer, G E; Schwartz, A; Thompson, H K

    1974-01-01

    1. Dissociation constants for sodium and potassium of a site that modulates the rate of ouabain-(Na(+)+K(+))-ATPase interaction were applied to models for potassium activation of (Na(+)+K(+))-ATPase. The constants for potassium (0.213 mM) and for sodium (13.7 mM) were defined, respectively, as activation constant, K(a) and inhibitory constant, K(i).2. Tests of the one- and the two-equivalent site models, that describe sodium and potassium competition, revealed that neither model adequately predicts the activation effects of potassium in the presence of 100 or 200 mM sodium.3. The potassium-activation data, obtained at low potassium and high sodium, were explained by a two-nonequivalent site model where the dissociation constants of the first site are 0.213 mM for potassium and 13.7 mM for sodium. The second site was characterized by dissociation constants of 0.091 mM for potassium and 74.1 mM for sodium.4. The two-nonequivalent site model adequately predicted the responses to concentrations of potassium between 0.25 and 5 mM in the presence of 100-500 mM sodium. At lower sodium concentrations the predicted responses formed an upper limit for the function of observed activities. This limit was reached at lower concentrations of potassium and higher concentrations of sodium, which inferred saturation of the sodium-activation sites with sodium.5. Sodium-activation data were corrected for sodium interaction with potassium-activation sites by use of the two-nonequivalent site model for potassium activation. Tests of equivalent site models suggested that the corrected data for sodium activation may be most consistent with a model that has three-equivalent sites. Other multiequivalent site models (n = 2, 4, 5 or 6), however, cannot be statistically eliminated as possibilities. The three-equivalent site activation model was characterized by dissociation constants of 1.39 mM for sodium and 11.7 mM for potassium. The system theoretically would be half-maximally activated by

  18. Switching activation barriers: new insights in E-field driven processes at the interface: perspectives in physical chemistry and catalysis

    Susarrey Arce, A.

    2014-01-01

    The research described in this thesis aimed to explore new concepts in catalysis with the use of a homemade ATR-IR silicon-based microreactor. During this journey we have performed multidisciplinary research at the interface between physics and chemistry. New insights in the fabrication, integration

  19. A GTPase chimera illustrates an uncoupled nucleotide affinity and release rate, Providing insight into the activation mechanism

    Guilfoyle, Amy P.; Deshpande, Chandrika N.; Font Sadurni, Josep;

    2014-01-01

    GDP release, or, alternatively, the movement is a consequence of release. To gain additional insight into the sequence of events leading to GDP release, we have created a chimeric protein comprised of Escherichia coli NFeoB and the G5 loop from the human Giα1 protein. The protein chimera retains...

  20. Three-dimensional representations of salt-dome margins at four active strategic petroleum reserve sites.

    Rautman, Christopher Arthur; Stein, Joshua S.

    2003-01-01

    Existing paper-based site characterization models of salt domes at the four active U.S. Strategic Petroleum Reserve sites have been converted to digital format and visualized using modern computer software. The four sites are the Bayou Choctaw dome in Iberville Parish, Louisiana; the Big Hill dome in Jefferson County, Texas; the Bryan Mound dome in Brazoria County, Texas; and the West Hackberry dome in Cameron Parish, Louisiana. A new modeling algorithm has been developed to overcome limitations of many standard geological modeling software packages in order to deal with structurally overhanging salt margins that are typical of many salt domes. This algorithm, and the implementing computer program, make use of the existing interpretive modeling conducted manually using professional geological judgement and presented in two dimensions in the original site characterization reports as structure contour maps on the top of salt. The algorithm makes use of concepts of finite-element meshes of general engineering usage. Although the specific implementation of the algorithm described in this report and the resulting output files are tailored to the modeling and visualization software used to construct the figures contained herein, the algorithm itself is generic and other implementations and output formats are possible. The graphical visualizations of the salt domes at the four Strategic Petroleum Reserve sites are believed to be major improvements over the previously available two-dimensional representations of the domes via conventional geologic drawings (cross sections and contour maps). Additionally, the numerical mesh files produced by this modeling activity are available for import into and display by other software routines. The mesh data are not explicitly tabulated in this report; however an electronic version in simple ASCII format is included on a PC-based compact disk.

  1. Functional properties of the two redox-active sites in yeast protein disulphide isomerase in vitro and in vivo

    Westphal, V; Darby, N J; Winther, Jakob R.

    1999-01-01

    Protein folding catalysed by protein disulphide isomerase (PDI) has been studied both in vivo and in vitro using different assays. PDI contains a CGHC active site in each of its two catalytic domains (a and a'). The relative importance of each active site in PDI from Saccharomyces cerevisiae (y...

  2. Targeting mechanisms at sites of complement activation for imaging and therapy.

    Holers, V Michael

    2016-06-01

    The complement system plays a key role in many acute injury states as well as chronic autoimmune and inflammatory diseases. Localized complement activation and alternative pathway-mediated amplification on diverse target surfaces promote local recruitment of pro-inflammatory cells and elaboration of other mediators. Despite a general understanding of the architecture of the system, though, many of the mechanisms that underlie site-specific complement activation and amplification in vivo are incompletely understood. In addition, there is no capability yet to measure the level of local tissue site-specific complement activation in patients without performing biopsies to detect products using immunohistochemical techniques. Herein is reviewed emerging evidence obtained through clinical research studies of human rheumatoid arthritis along with translational studies of its disease models which demonstrate that several parallel mechanisms are involved in site-specific amplification of activation of the complement system in vivo. Among these processes are de-regulation of the alternative pathway, effector pathway-catalyzed amplification of proximal complement activation, recognition of injury-associated ligands by components of the lectin pathway, and engagement of pathogenic natural antibodies that recognize a limited set of injury-associated neoepitopes. Studies suggest that each of these inter-related processes can play key roles in amplification of complement-dependent injury on self-tissues in vivo. These findings, in addition to development of an imaging strategy described herein designed to quantitatively measure local complement C3 fixation, have relevance to therapeutic and diagnostic strategies targeting the complement system. PMID:25979851

  3. Modeling emissions and dispersion of contaminants from cleanup activities at a mixed waste site to estimate air impacts and risks

    The transport and dispersion of contaminants via the air pathway is a major concern during cleanup of contaminated sites. Impacts to air quality and human health during cleanup were evaluated for the Weldon Spring site by using site-specific information for source areas, activities, and receptor locations. In order to ensure protection of human health and the environment, results are being used to focus on those cleanup activities for which release controls should be emphasized

  4. Comparative analysis of the impact of geological activity on astronomical sites of the Canary Islands, Hawaii and Chile

    Eff-Darwich, A.; Garcia-Lorenzo, B.; Rodriguez-Losada, J. A.; de la Nuez, J.; Hernandez-Gutierrez, L. E.; Romero-Ruiz, M. C.

    2009-01-01

    An analysis of the impact of seismic and volcanic activity was carried out at selected astronomical sites, namely the observatories of El Teide (Tenerife, Canary Islands), Roque de los Muchachos (La Palma, Canary Islands), Mauna Kea (Hawaii) and Paranal (Chile) and the candidate site of Cerro Ventarrones (Chile). Hazard associated to volcanic activity is low or negligible at all sites, whereas seismic hazard is very high in Chile and Hawaii. The lowest geological hazard in both seismic and vo...

  5. Insights into an intriguing gas sorption mechanism in a polar metal–organic framework with open-metal sites and narrow channels

    Forrest, Katherine A.

    2014-01-01

    Simulations of H2 and CO2 sorption were performed in the metal-organic framework (MOF), [Cu(Me-4py-trz-ia)]. This MOF was recently shown experimentally to exhibit high uptake for H2 and CO2 sorption and this was reproduced and elucidated through the simulations performed herein. Consistent with experiment, the theoretical isosteric heat of adsorption, Qst, values were nearly constant across all loadings for both sorbates. The simulations revealed that sorption directly onto the open-metal sites was not observed in this MOF, ostensibly a consequence of the low partial positive charges of the Cu2+ ions as determined through electronic structure calculations. Sorption was primarily observed between adjacent carboxylate oxygen atoms (site 1) and between nearby methyl groups (site 2) of the organic linkers. In addition, saturation of the most energetically favorable sites (site 1) is possible only after filling a nearby site (site 2) first due to the MOF topology. This suggests that the lack of dependence on loading for the Qst is due to the concurrent filling of sites 1 and 2, leading to an observed average Qst value. © 2014 the Partner Organisations.

  6. Insights into the "pair of sugar tongs" surface binding site in barley alpha-amylase isozymes and crystallization of appropriate sugar tongs mutants

    Tranier, S.; Deville, K.; Robert, X.; Bozonnet, Sophie; Haser, R.; Svensson, Birte; Aghajari, N.

    2005-01-01

    additional surface binding site called a "pair of sugar tongs" due to the sugar capturing by Tyr380 which is situated in domain C of AMYL For the first time, a biological role for the domain C was suggested as well as a hypothetical explanation of enzymatic differences between the two barley a...... mutational engineering. Also, the contribution of domain A could be suggested in addition to minor differences implying interacting residues at the "sugar tongs" binding site. Finally, the successful crystallization of five mutants in the "sugar tongs" binding site is reported.......-amylase isozymes. However, no sugar was bound at the "sugar tongs" site in the AMY2/acarbose complex. Comparative studies of this domain on the basis of sequence, secondary structure and spatial organization allow to propose factors needed for such a site. One of the most obvious is the replacement of Ser378(AMY1...

  7. Altered binding of thioflavin t to the peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos

    The peripheral anionic site of acetylcholinesterase, when occupied by a ligand, is known to modulate reaction rates at the active site of this important enzyme. The current report utilized the peripheral anionic site specific fluorogenic probe thioflavin t to determine if the organophosphates chlorpyrifos oxon and dichlorvos bind to the peripheral anionic site of human recombinant acetylcholinesterase, since certain organophosphates display concentration-dependent kinetics when inhibiting this enzyme. Incubation of 3 nM acetylcholinesterase active sites with 50 nM or 2000 nM inhibitor altered both the Bmax and Kd for thioflavin t binding to the peripheral anionic site. However, these changes resulted from phosphorylation of Ser203 since increasing either inhibitor from 50 nM to 2000 nM did not alter further thioflavin t binding kinetics. Moreover, the organophosphate-induced decrease in Bmax did not represent an actual reduction in binding sites, but instead likely resulted from conformational interactions between the acylation and peripheral anionic sites that led to a decrease in the rigidity of bound thioflavin t. A drop in fluorescence quantum yield, leading to an apparent decrease in Bmax, would accompany the decreased rigidity of bound thioflavin t molecules. The organophosphate-induced alterations in Kd represented changes in binding affinity of thioflavin t, with diethylphosphorylation of Ser203 increasing Kd, and dimethylphosphorylation of Ser203 decreasing Kd. These results indicate that chlorpyrifos oxon and dichlorvos do not bind directly to the peripheral anionic site of acetylcholinesterase, but can affect binding to that site through phosphorylation of Ser203

  8. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-01-01

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser696 and Ser698 in the JM (juxtamembrane) region and probably Ser886 and/or Ser893 in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser717 in the JM, and at Ser733, Thr752, Ser783, Ser864, Ser911, Ser958 and Thr9...

  9. Cernavoda Unit 2: - BOP 3D model proposal for a possible organization of site activities

    The scope of this activity is to define characteristics and advantages of the 3D model of Cernavoda BOP to this set up at site for engineering and construction activities. This model will provide a modern and proven tool able to strongly support the site activities with particular regard to the following: 1. engineering activities, - plant arrangement 'double check' for resolution of clashing; - easy management of future design changes; - real time plant configuration updating as soon as any design modification is approved and integrated in the model; - preparation of high quality documentation for procurement, construction and commissioning; - prompt availability of the as built configuration of the plant as soon as the last modification is frozen; 2. material procurement activities, - definition of the priorities in the construction material procurement according to the construction planning by area; - inventory list of equipment, pipes, fittings, valves, cable trays and ventilation ducts to be installed in each construction area; 3. construction activities, - definition of construction sequences, with particular reference in the congested areas, for piping cable trays (electrical and C-and-I) and ventilations ducts; - definition of piping spools by construction contractors; - follow-up of the activities in each area (i.e. construction, painting, insulation, flushing, pressure testing, etc); 4. turn-over and commissioning, - check of the progress. The success of this approach is based on the following: i) proper management of the remote workstations providing easy and reliable access to the model; ii) subdivision of the Integrated Building in construction areas, whose detail design may be allotted to Romanian organizations with multidisciplinary tasks; iii) integration in the model of the remote developed engineering in order to validate the details of the design. (authors)

  10. Tricyclic covalent inhibitors selectively target Jak3 through an active site thiol.

    Goedken, Eric R; Argiriadi, Maria A; Banach, David L; Fiamengo, Bryan A; Foley, Sage E; Frank, Kristine E; George, Jonathan S; Harris, Christopher M; Hobson, Adrian D; Ihle, David C; Marcotte, Douglas; Merta, Philip J; Michalak, Mark E; Murdock, Sara E; Tomlinson, Medha J; Voss, Jeffrey W

    2015-02-20

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases. PMID:25552479

  11. Exploring the active site structure of photoreceptor proteins by Raman optical activity

    Unno, Masashi

    2015-03-01

    Understanding protein function at the atomic level is a major challenge in a field of biophysics and requires the combined efforts of structural and functional methods. We use photoreceptor proteins as a model system to understand in atomic detail how a chromophore and a protein interact to sense light and send a biological signal. A potential technique for investigating molecular structures is Raman optical activity (ROA), which is a spectroscopic method with a high sensitivity to the structural details of chiral molecules. However, its application to photoreceptor proteins has not been reported. Thus we have constructed ROA spectrometer using near-infrared (NIR) laser excitation at 785 nm. The NIR excitation enables us to measure ROA spectra for a variety of biological samples, including photoreceptor proteins, without fluorescence from the samples. In the present study, we have applied the NIR-ROA to bacteriorhodopsin (BR) and photoactive yellow protein (PYP). BR is a light-driven proton pump and contains a protonated Schiff base of retinal as a chromophore. PYP is a blue light receptor, and this protein has the 4-hydroxycinnamyl chromophore, which is covalently linked to Cys69 through a thiolester bond. We have successfully obtained the ROA spectra of the chromophore within a protein environment. Furthermore, calculations of the ROA spectra utilizing density functional theory provide detailed structural information, such as data on out-of-plane distortions of the chromophore. The structural information obtained from the ROA spectra includes the positions of hydrogen atoms, which are usually not detected in the crystal structures of biological samples.

  12. Integral communication activities in support of the repository site selection in Slovenia

    The siting and licensing of a radioactive waste repository requires a complete public consensus that is very difficult to obtain. The main reasons for the public reluctance to accept the radioactive waste repository are the feeling of being ignored in the decision making process and inadequate understanding of radioactivity. Therefore communication and information activities, as early as possible in the siting process, are very important for reducing the potential conflicts of interests between the local community and the investor of the radioactive waste repository on the potential repository sites. ARAO communication activities are based on research on public opinion and public knowledge about radioactive waste management. Communication strategies that provide two-way communication channels, such as interactive web pages, workshops, study circles, visitors' centre, are preferred. Different educational materials (leaflets, CD-ROMs, articles in the local newspapers, yearly magazine, and posters) are also being produced. Collaboration with non-governmental environmental organizations has also proved to be helpful in confidence building, as well as in informing the public. It is also very important for establishing competent public participation in the decision making process. (author)

  13. Stress-induced enhancement of leukocyte trafficking into sites of surgery or immune activation

    Viswanathan, Kavitha; Dhabhar, Firdaus S.

    2005-04-01

    Effective immunoprotection requires rapid recruitment of leukocytes into sites of surgery, wounding, infection, or vaccination. In contrast to immunosuppressive chronic stressors, short-term acute stressors have immunoenhancing effects. Here, we quantify leukocyte infiltration within a surgical sponge to elucidate the kinetics, magnitude, subpopulation, and chemoattractant specificity of an acute stress-induced increase in leukocyte trafficking to a site of immune activation. Mice acutely stressed before sponge implantation showed 200-300% higher neutrophil, macrophage, natural killer cell, and T cell infiltration than did nonstressed animals. We also quantified the effects of acute stress on lymphotactin- (LTN; a predominantly lymphocyte-specific chemokine), and TNF-- (a proinflammatory cytokine) stimulated leukocyte infiltration. An additional stress-induced increase in infiltration was observed for neutrophils, in response to TNF-, macrophages, in response to TNF- and LTN, and natural killer cells and T cells in response to LTN. These results show that acute stress initially increases trafficking of all major leukocyte subpopulations to a site of immune activation. Tissue damage-, antigen-, or pathogen-driven chemoattractants subsequently determine which subpopulations are recruited more vigorously. Such stress-induced increases in leukocyte trafficking may enhance immunoprotection during surgery, vaccination, or infection, but may also exacerbate immunopathology during inflammatory (cardiovascular disease or gingivitis) or autoimmune (psoriasis, arthritis, or multiple sclerosis) diseases. chemokine | psychophysiological stress | surgical sponge | wound healing | lymphotactin

  14. Synthesis and characterization of (18)F-labeled active site inhibited factor VII (ASIS).

    Erlandsson, Maria; Nielsen, Carsten H; Jeppesen, Troels E; Kristensen, Jesper B; Petersen, Lars C; Madsen, Jacob; Kjaer, Andreas

    2015-05-15

    Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example, thrombosis, metastasis, tumor growth, and tumor angiogenesis. The aim of this study was to develop an (18)F-labeled ASIS derivative to assess TF expression in tumors. Active site inhibited factor VII was labeled using N-succinimidyl-4-[(18)F]fluorobenzoate, and the [(18)F]ASIS was purified on a PD-10 desalting column. The radiochemical yield was 25 ± 6%, the radiochemical purity was >97%, and the pseudospecific radioactivity was 35 ± 9 GBq/µmol. The binding efficacy was evaluated in pull-down experiments, which monitored the binding of unlabeled ASIS and [(18)F]ASIS to TF and to a specific anti-factor VII antibody (F1A2-mAb). No significant difference in binding efficacy between [(18)F]ASIS and ASIS could be detected. Furthermore, [(18)F]ASIS was relatively stable in vitro and in vivo in mice. In conclusion, [(18)F]ASIS has for the first time been successfully synthesized as a possible positron emission tomography tracer to image TF expression levels. In vivo positron emission tomography studies to evaluate the full potential of [(18)F]ASIS are in progress. PMID:25820758

  15. Benzodiazepines: rat pinealocyte binding sites and augmentation of norepinephrine-stimulated N-acetyltransferase activity

    Matthew, E.; Parfitt, A.G.; Sugden, D.; Engelhardt, D.L.; Zimmerman, E.A.; Klein, D.C.

    1984-02-01

    Studies of (/sup 3/H)diazepam binding to intact rat pineal cells were carried out in tissue culture preparations. The binding was saturable, reversible and proportional to the number of cells used. Scatchard analysis resulted in a linear plot (Kd . 23 nM, maximum binding sites (Bmax) . 1.56 pmol/mg of protein for cells in monolayer culture; Kd . 7 nM, Bmax . 1.3 pmol/mg of protein for cells in suspension culture). Inhibition constants (Ki) for clonazepam (500 nM), flunitrazepam (38 nM) and Ro-5-4864 (5 nM) indicated that the binding sites were probably of the ''peripheral'' type. In addition, the effects of diazepam on norepinephrine-stimulated N-acetyltransferase (NAT) activity were studied in organ culture and dissociated cell culture. Diazepam (10-50 microM) both prolonged and increased the magnitude of the norepinephrine-induced increase in NAT activity but did not affect the initial rate of rise of enzyme activity. The effect was dose-dependent and was also seen with clonazepam, flunitrazepam and Ro-5-4864, but not with Ro-15-1788. Diazepam, by itself, at these concentrations, had no effect on NAT, but enzyme activity was increased by higher concentrations (0.1-1 mM). Although a relationship between the (/sup 3/H)diazepam binding sites described here and the effect of benzodiazepines on NAT cannot be established from these studies, the data suggest that the benzodiazepines may alter melatonin levels through their action on NAT.

  16. Benzodiazepines: rat pinealocyte binding sites and augmentation of norepinephrine-stimulated N-acetyltransferase activity

    Studies of [3H]diazepam binding to intact rat pineal cells were carried out in tissue culture preparations. The binding was saturable, reversible and proportional to the number of cells used. Scatchard analysis resulted in a linear plot [Kd . 23 nM, maximum binding sites (Bmax) . 1.56 pmol/mg of protein for cells in monolayer culture; Kd . 7 nM, Bmax . 1.3 pmol/mg of protein for cells in suspension culture]. Inhibition constants (Ki) for clonazepam (500 nM), flunitrazepam (38 nM) and Ro-5-4864 (5 nM) indicated that the binding sites were probably of the ''peripheral'' type. In addition, the effects of diazepam on norepinephrine-stimulated N-acetyltransferase (NAT) activity were studied in organ culture and dissociated cell culture. Diazepam (10-50 microM) both prolonged and increased the magnitude of the norepinephrine-induced increase in NAT activity but did not affect the initial rate of rise of enzyme activity. The effect was dose-dependent and was also seen with clonazepam, flunitrazepam and Ro-5-4864, but not with Ro-15-1788. Diazepam, by itself, at these concentrations, had no effect on NAT, but enzyme activity was increased by higher concentrations (0.1-1 mM). Although a relationship between the [3H]diazepam binding sites described here and the effect of benzodiazepines on NAT cannot be established from these studies, the data suggest that the benzodiazepines may alter melatonin levels through their action on NAT

  17. Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase

    N-bromoacetylethanolamine phosphate was found to act as a specific affinity label for the active center of glucosephosphate isomerase. The inactivation process followed pseudo-first order kinetics, was irreversible, and exhibited rate saturation kinetics with minimal half-lives of inactivation of 4.5 and 6.3 min for the enzyme isolated from human placenta and rabbit muscle, respectively. The pH dependence of the inactivation process closely paralleled the pH dependence of the overall catalytic process with pK/sub a/ values at pH 6.4 and 9.0. The stoichiometry of labeling of either enzyme, as determined with N-bromo[14C2]acetylethanolamine phosphate, was 1 eq of the affinity label/subunit of enzyme. After acid hydrolysis and amino acid analysis of the radioactive affinity-labeled human enzyme, only radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive derivative obtained was 1-carboxymethyl histidine. Active site tryptic peptides were isolated by solvent extraction, thin layer peptide fingerprinting, and ion exchange chromatography before and after removal of the phosphate from the active site peptide. Amino acid analysis of the labeled peptides from the two species were very similar. Using high sensitivity methods for sequence analysis, the primary structure of the active site was established as Val-Leu-His-Ala-Glu-Asn-Val-Asp (Gly,Thr,Ser) Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr-Phe. Apparent sequence homology between the catalytic center of glucosephosphate isomerase and triosephosphate isomerase suggest that the two enzymes may have evolved from a common ancestral gene

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

    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

  19. Molecular Study of the Effects of Chemical Processing on Heterogeneous Ice Nucleation: Role of Active Sites and Product Formation

    Sihvonen, S.; Schill, G. P.; Murphy, K. A.; Mueller, K.; Tolbert, M. A.; Freedman, M. A.

    2014-12-01

    Mineral dust aerosol is the largest global source of ice nuclei, but the identity of the active sites for nucleation is unknown. During atmospheric transport, mineral dust aerosol can encounter and react with sulfuric acid, which affects the ice nucleation activity either due to changes to reactive surface sites or product formation. In this study, we reacted two types of clays found in mineral dust, kaolinite and montmorillonite, with sulfuric acid. Variation in the mineral due to acid treatment was separated from product formation through rinsing techniques. The samples were subsequently reacted with a probe molecule, (3,3,3-trifluoropropyl)dimethylchlorosilane, that selectively binds to edge hydroxyl groups that are bonded to a silicon atom with three bridging oxygens. Hydroxyl groups are considered potential active sites, because they can hydrogen bond with water and facilitate ice nucleation. Attachment to these sites was quantified by 19F magic angle spinning nuclear magnetic resonance (MAS NMR) of the 19F atoms on the probe molecule, which provided a direct correlation of the number of hydroxyl groups. Our results indicate that the number of edge-site hydroxyl groups increases with exposure to acid. Ice nucleation measurements indicate that the sulfuric acid-treated mineral is less ice active than the untreated mineral. Surprisingly, no difference between the nucleation activity of the untreated mineral and acid-treated, rinsed mineral is observed. As a result, we hypothesize that once a critical density of active sites is reached for ice nucleation, there is no further change in nucleation activity despite a continued increase in active sites. We additionally propose that the reduced activity of the acid-treated mineral is due to product formation that blocks active sites on the mineral, rather than changes to active sites.

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

    Farman Ullah Dawar

    2016-08-01

    Full Text Available 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.

  1. Active site studies of Escherichia coli 2-keto-4-hydroxyglutarate aldolase

    Vlahos, C.J.

    1987-01-01

    The data presented delineate the complete amino acid sequence of E. coli KHG aldolase and also identify Lys-133, Glu-45, and Arg-49 as aminoacyl residues required for catalytic activity. Incubation of E. coli KHG aldolase with (/sup 14/C)pyruvate in the presence of NaCNBH/sub 3/ results in the incorporation of one mol of /sup 14/C per mol of enzyme subunit. Digestion of this enzyme-adduct with trypsin, followed by purification of the peptides, allowed for the isolation of a unique radioactive peptide. Its amino acid sequence showed that the pyruvate-binding (i.e., Schiff-base forming) lysine residue is located at position 133 in the intact enzyme. E. coli KHG aldolase activity is lost when the enzyme is reacted with bromopyruvate; saturation kinetics are observed. The substrates, pyruvate and KHG, protect the enzyme from inactivation. Both facts suggest that the reagent is active-site specific. Incubation of the aldolase with (3-/sup 14/C)bromopyruvate is associated with a concomitant loss of enzymatic activity and esterification of Glu-45; if the enzyme is denatured in the presence of excess bromopyruvate, Cys-159 and Cys-180 are also alkylated. Blocking the active-site lysine residue with pyruvate prevents Glu-45 from being esterified but does not eliminate alkylation of these two cysteine residues. Woodward's Reagent K was also found to inactivate the aldolase under conditions that are usually specific for carboxyl group modification. This aldolase is also inactivated by 1,2-cyclohexanedione. Loss of enzymatic activity occurs concomitantly with modification of one arginine residue per enzyme subunit. Treatment of the aldolase with the arginine-specific reagent, 4-(oxyacetyl)phenoxyacetic acid, followed by digestion with trypsin allowed for the isolation of a unique peptide and the identification of Arg-49 as the specific residue involved.

  2. Three dimensional visualization in support of Yucca Mountain Site characterization activities

    An understanding of the geologic and hydrologic environment for the proposed high-level nuclear waste repository at Yucca Mountain, NV is a critical component of site characterization activities. Conventional methods allow visualization of geologic data in only two or two and a half dimensions. Recent advances in computer workstation hardware and software now make it possible to create interactive three dimensional visualizations. Visualization software has been used to create preliminary two-, two-and-a-half-, and three-dimensional visualizations of Yucca Mountain structure and stratigraphy. The three dimensional models can also display lithologically dependent or independent parametric data. Yucca Mountain site characterization studies that will be supported by this capability include structural, lithologic, and hydrologic modeling, and repository design

  3. Mechanical Control of ATP Synthase Function: Activation Energy Difference between Tight and Loose Binding Sites

    Beke-Somfai, Tamás

    2010-01-26

    Despite exhaustive chemical and crystal structure studies, the mechanistic details of how FoF1-ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations using a recent highresolution X-ray structure, we conclude that formation of the P-O bond may be achieved through a transition state (TS) with a planar PO3 - ion. Surprisingly, there is a more than 40 kJ/mol difference between barrier heights of the loose and tight binding sites of the enzyme. This indicates that even a relatively small change in active site conformation, induced by the γ-subunit rotation, may effectively block the back reaction in βTP and, thus, promote ATP. © 2009 American Chemical Society.

  4. Structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase in complex with the feedback inhibitor CoA reveals only one active-site conformation

    Wubben, T.; Mesecar, A.D. (Purdue); (UIC)

    2014-10-02

    Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine to form dephosphocoenzyme A (dPCoA). To complement recent biochemical and structural studies on Mycobacterium tuberculosis PPAT (MtPPAT) and to provide further insight into the feedback regulation of MtPPAT by CoA, the X-ray crystal structure of the MtPPAT enzyme in complex with CoA was determined to 2.11 {angstrom} resolution. Unlike previous X-ray crystal structures of PPAT-CoA complexes from other bacteria, which showed two distinct CoA conformations bound to the active site, only one conformation of CoA is observed in the MtPPAT-CoA complex.

  5. Otter (Lontra longicaudis) Spraint and Mucus Depositions: Early Ecological Insights into the Differences in Marking Site Selection and Implications for Monitoring Prey Availability

    Nathan James Roberts; Ryan Matthew Clark; Delyth Williams

    2016-01-01

    Otters are not territorial in the classical sense of marking territory boundaries. Instead they mark key resource areas within the territory with faeces, or spraint, for olfactory communication. There is a high level of site fidelity in otter marking behaviour and the function of scent marks may explain their spatial distribution. Typically marking sites are in proximity to deep water which provides key resource areas for energy-efficient foraging. Occasionally spraint is laden with mucus, wh...

  6. HydPred: a novel method for the identification of protein hydroxylation sites that reveals new insights into human inherited disease.

    Li, Shuyan; Lu, Jun; Li, Jiazhong; Chen, Ximing; Yao, Xiaojun; Xi, Lili

    2016-01-26

    The disruption of protein hydroxylation is highly associated with several serious diseases and consequently the identification of protein hydroxylation sites has attracted significant attention recently. Here, we report the development of an improved method, called HydPred, to identify protein hydroxylation sites (hydroxyproline and hydroxylysine) based on the synthetic minority over-sampling technique (SMOTE), the random forest (RF) algorithm and four blocks of newly composed features that are derived from the protein primary sequence. The HydPred method achieved the best prediction performance reported until now with Matthew's correlation coefficient values of 0.770 and 0.857 for hydroxyproline and hydroxylysine, respectively, according to jack-knife cross-validation. This represents an improvement of 8% for hydroxyproline and 19% for hydroxylysine compared to the best results of available predictors. The prediction performance of HydPred for the external validation of hydroxyproline and hydroxylysine was also improved compared with other published methods. We subsequently applied HydPred to study the association of disruption of hydroxylation sites with human inherited disease. The analyses suggested that the loss of hydroxylation sites is more likely to cause disease instead of the gain of hydroxylation sites and 52 different human inherited diseases were found to be highly associated with the loss of hydroxylation sites. Therefore, HydPred represents a new strategy to discover the molecular basis of pathogenesis associated with abnormal hydroxylation. HydPred is now available online as a user-friendly web server at . PMID:26661679

  7. The active site of oxidative phosphorylation and the origin of hyperhomocysteinemia in aging and dementia.

    McCully, Kilmer S

    2015-01-01

    The active site of oxidative phosphorylation and adenosine triphosphate (ATP) synthesis in mitochondria is proposed to consist of two molecules of thioretinamide bound to cobalamin, forming thioretinaco, complexed with ozone, oxygen, nicotinamide adenine dinucleotide. and inorganic phosphate, TR2CoO3O2NAD(+)H2PO4(-). Reduction of the pyridinium nitrogen of the nicotinamide group by an electron from electron transport complexes initiates polymerization of phosphate with adenosine diphosphate, yielding nicotinamide riboside and ATP bound to thioretinaco ozonide oxygen. A second electron reduces oxygen to hydroperoxyl radical, releasing ATP from the active site. A proton gradient is created within F1F0 ATPase complexes of mitochondria by reaction of protons with reduced nicotinamide riboside and with hydroperoxyl radical, yielding reduced nicotinamide riboside and hydroperoxide. The hyperhomocysteinemia of aging and dementia is attributed to decreased synthesis of adenosyl methionine by thioretinaco ozonide and ATP, causing decreased allosteric activation of cystathionine synthase and decreased allosteric inhibition of methylenetetrahydrofolate reductase and resulting in dysregulation of methionine metabolism. PMID:25887881

  8. PCR-based site-specific mutagenesis of peptide antibiotics FALL-39 and its biologic activities

    Yun-xia YANG; Yun FENG; Bo-yao WANG; Qi WU

    2004-01-01

    AIM: To construct PGEX-1λT-FALL-39 expression vector and its mutant vector, and study the relationship of function and structure. METHODS: A cDNA encoding mature FALL-39 was cloned from SPCA- 1 cell mRNA and the prokaryotic expression vector PGEX- 1λT-FALL-39 was constructed. Two kinds of polymerase chain reaction (PCR) for the site-direction mutagenesis were used to construct FALL-39 mutant expression vector, FALL-39-Lys-32 and FALL-39-Lys-24. Minimal effective concentration, minimal inhibitory concentration, and minimal bactericidal concentration were used to assay the antibacterial activities of these peptides. Effects of different solution on the antibacterial activity of FALL-39 and FALL-39-Lys-32 were observed by CFU determination. The hemolytic effects of these peptides were also examined on human red blood cells. RESULTS: Two site-specific mutants FALL-39-Lys-32 and FALL-39-Lys24 were obtained by PCR-induced mutagenesis. In comparison with two-step PCR which required two pairs of primers, one step PCR which required one pair of primers is a simple and efficient method for the PCR based site-specific mutagenesis. Using the prokaryotic expression system, the E coli-based products of recombinant FALL39 and its mutant peptides were also obtained. The antibacterial assay showed that FALL-39-Lys-32 and FALL-39-Lys24 were more potential in the antibacterial activity against E coli ML35p and Pseltdomonas aeruginosa ATCC27853 than that of FALL-39, and no increase in hemolysis was observed at the antibacterial concentrations. The antibacterial activity of FALL-39-Lys-32 against E coli was more potent than that of FALL-39 in NaCl-containing LB medium, while its activity was almost the same as FALL-39 in SO2-4 containing Medium E. CONCLUSION: PCR-based mutagensis is a useful model system for studying the structure and function relationship of antimicrobial peptides. Keeping α-helical conformation of FALL-39 and increasing net positive charge can increase the

  9. Water molecule network and active site flexibility of apo protein tyrosine phosphatase 1B

    Pedersen, A.K.; Peters, Günther H.J.; Møller, K.B.;

    2004-01-01

    Protein tyrosine phosphatase 1B (PTP1B) plays a key role as a negative regulator of insulin and leptin signalling and is therefore considered to be an important molecular target for the treatment of type 2 diabetes and obesity. Detailed structural information about the structure of PTP1B, including...... the conformation and flexibility of active-site residues as well as the water-molecule network, is a key issue in understanding ligand binding and enzyme kinetics and in structure-based drug design. A 1.95 Angstrom apo PTP1B structure has been obtained, showing four highly coordinated water molecules...

  10. How to awaken your nanomachines: Site-specific activation of focal adhesion kinases through ligand interactions

    Walkiewicz, Katarzyna W.

    2015-06-17

    The focal adhesion kinase (FAK) and the related protein-tyrosine kinase 2-beta (Pyk2) are highly versatile multidomain scaffolds central to cell adhesion, migration, and survival. Due to their key role in cancer metastasis, understanding and inhibiting their functions are important for the development of targeted therapy. Because FAK and Pyk2 are involved in many different cellular functions, designing drugs with partial and function-specific inhibitory effects would be desirable. Here, we summarise recent progress in understanding the structural mechanism of how the tug-of-war between intramolecular and intermolecular interactions allows these protein ‘nanomachines’ to become activated in a site-specific manner.

  11. Determination of blood Cd in subjects living near dismessed mines and active industrial sites

    Madeddu, Roberto Beniamino; Tolu, Paola; Asara, Yolande; Farace, Cristiano; Forte, Giovanni; Bocca, Beatrice

    2012-01-01

    The environmental exposure to Cd in 265 subjects living in a South-Western area of Sardinia (Sulcis-Iglesiente) with a great history of mining activities and large industrial settings was assessed. Individuals living near the industrial plants had geometric means (GM) of blood Cd (0.79 μg/l) significantly higher than controls (0.47 μg/l) and than residents of the mining sites (0.54 μg/l). Demographic and lifestyle variables were also investigated and data show...

  12. Application of instrumental neutron activation analysis to study ceramic fragments from Damascus Castle site, Syria

    Thirty-three archaeological ceramic fragment samples from Damascus Castle archaeological site, Damascus city, Syria, were analyzed using instrumental neutron activation analysis (INAA). 36 elements were determined. These elemental concentrations have been processed using two multivariate statistical methods, cluster and factor analysis in order to determine similarities and correlation between the various samples. Factor analysis confirms that 84.8% of the ceramics samples classified by cluster analysis are correctly classified by cluster analysis. The results provided persuasive evidence that Castle pottery used at least four different clay sources. Moreover, by means of systematic local analysis it will be clear whether these sources are local or not. (author)

  13. Consumer Insights

    JANKOT

    2004-01-01

    Fang Jun, the head of consumer and market insights of Unilever Shanghai, has summarized his early life as a market in two sentences: rush about to study market changes;act all day to observe consumer behavior. And now?"Tell stories, conduct interviews and piece together different data; calculate numbers,build models and write reports."

  14. Science insights.

    Tanabe, Kazuyuki

    2015-06-01

    "Below is an essay by Prof. Tanabe originally written in Japanese. It gives an insight to Prof. Tanabe's inquiring mind and his approach to science. He also seek, as always, to inspire and nudge the young to scientific discovery". PMID:25463310

  15. Non-native ligands define the active site of Pennisetum glaucum (L.) R. Br dehydroascorbate reductase.

    Krishna Das, Bhaba; Kumar, Amit; Maindola, Priyank; Mahanty, Srikrishna; Jain, S K; Reddy, Mallireddy K; Arockiasamy, Arulandu

    2016-05-13

    Dehydroascorbate reductase (DHAR), a member of the glutathione-S-transferase (GST) family, reduces dehydroascorbate (DHA) to ascorbate (AsA; Vitamin-C) in a glutathione (GSH)-dependent manner and in doing so, replenishes the critical AsA pool of the cell. To understand the enzyme mechanism in detail, we determined the crystal structure of a plant DHAR from Pennisetum glaucum (PgDHAR) using Iodide-Single Anomalous Dispersion (SAD) and Molecular replacement methods, in two different space groups. Here, we show PgDHAR in complex with two non-native ligands, viz. an acetate bound at the G-site, which resembles the γ-carboxyl moiety of GSH, and a glycerol at the H-site, which shares the backbone of AsA. We also show that, in the absence of bound native substrates, these non-native ligands help define the critical 'hook points' in the DHAR enzyme active site. Further, our data suggest that these non-native ligands can act as the logical bootstrapping points for iterative design of inhibitors/analogs for DHARs. PMID:27067046

  16. Active urea transport by the skin of Bufo viridis: Amiloride- and phloretin-sensitive transport sites

    Urea is actively transported inwardly (Ji) across the skin of the green toad Bufo viridis. Ji is markedly enhanced in toads adapted to hypertonic saline. The authors studied urea transport across the skin of Bufo viridis under a variety of experimental conditions, including treatment with amiloride and phloretin, agents that inhibit urea permeability in the bladder of Bufo marinus. Amiloride (10-4 M) significantly inhibited Ji in both adapted and unadapted animals and was unaffected by removal of sodium from the external medium. Phloretin (10-4 M) significantly inhibited Ji in adapted animals by 23-46%; there was also a reduction in Ji in unadapted toads at 10-4 and 5 x 10-4 M phloretin. A dose-response study revealed that the concentration of phloretin causing half-maximal inhibition (K1/2) was 5 x 10-4 M for adapted animals. Ji was unaffected by the substitution of sucrose for Ringer solution or by ouabain. They conclude (1) the process of adaptation appears to involve an increase in the number of amiloride- and phloretin-inhibitable urea transport sites in the skin, with a possible increase in the affinity of the sites for phloretin; (2) the adapted skin resembles the Bufo marinus urinary bladder with respect to amiloride and phloretin-inhibitable sites; (3) they confirm earlier observations that Ji is independent of sodium transport

  17. Prioritizing conservation activities using reserve site selection methods and population viability analysis.

    Newbold, Stephen C; Siikamäki, Juha

    2009-10-01

    In recent years a large literature on reserve site selection (RSS) has developed at the interface between ecology, operations research, and environmental economics. Reserve site selection models use numerical optimization techniques to select sites for a network of nature reserves for protecting biodiversity. In this paper, we develop a population viability analysis (PVA) model for salmon and incorporate it into an RSS framework for prioritizing conservation activities in upstream watersheds. We use spawner return data for three closely related salmon stocks in the upper Columbia River basin and estimates of the economic costs of watershed protection from NOAA to illustrate the framework. We compare the relative cost-effectiveness of five alternative watershed prioritization methods, based on various combinations of biological and economic information. Prioritization based on biological benefit-economic cost comparisons and accounting for spatial interdependencies among watersheds substantially outperforms other more heuristic methods. When using this best-performing prioritization method, spending 10% of the cost of protecting all upstream watersheds yields 79% of the biological benefits (increase in stock persistence) from protecting all watersheds, compared to between 20% and 64% for the alternative methods. We also find that prioritization based on either costs or benefits alone can lead to severe reductions in cost-effectiveness. PMID:19831069

  18. Relating subsurface temperature changes to microbial activity at a crude oil-contaminated site

    Warren, Ean; Bekins, Barbara A.

    2015-11-01

    Crude oil at a spill site near Bemidji, Minnesota has been undergoing aerobic and anaerobic biodegradation for over 30 years, creating a 150-200 m plume of primary and secondary contaminants. Microbial degradation generates heat that should be measurable under the right conditions. To measure this heat, thermistors were installed in wells in the saturated zone and in water-filled monitoring tubes in the unsaturated zone. In the saturated zone, a thermal groundwater plume originates near the residual oil body with temperatures ranging from 2.9 °C above background near the oil to 1.2 °C down gradient. Temperatures in the unsaturated zone above the oil body were up to 2.7 °C more than background temperatures. Previous work at this site has shown that methane produced from biodegradation of the oil migrates upward and is oxidized in a methanotrophic zone midway between the water table and the surface. Enthalpy calculations and observations demonstrate that the temperature increases primarily result from aerobic methane oxidation in the unsaturated zone above the oil. Methane oxidation rates at the site independently estimated from surface CO2 efflux data are comparable to rates estimated from the observed temperature increases. The results indicate that temperature may be useful as a low-cost measure of activity but care is required to account for the correct heat-generating reactions, other heat sources and the effects of focused recharge.

  19. The influence of a small amount of active sites on the adsorption kinetics of nitrogen on ruthenium

    Recently, Dahl et al. [S. Dahl, A. Logadottir, R.C. Egeberg, J.H. Larsen, I. Chorkendorff, E. Toernqvist, J.K. Norskov, Phys. Rev. Lett. 83 (1999) 1814; S. Dahl, E. Toernqvist, I. Chorkendorff, J. Catal. 192 (2000) 381] have proposed very interesting hypothesis that the rate of dissociation and adsorption of nitrogen on Ru(0 0 0 1) facet is totally dominated by the presence of a small amount of step sites on Ru(0 0 0 1) terraces. Following this idea, a kinetic model, based on applying the Statistical Rate Theory approach, was developed in order to explain if such mechanism is able to explain the observed features of the system N2/Ru(0 0 0 1). As a result, it was stated that the activation barrier for adsorption on the active (step) sites is equal to 36 kJ/mol; in turn, the adsorption energy of nitrogen atoms on the active sites is 43 kJ/mol. It implies that the rate of adsorption via the active sites is much faster than direct adsorption on the three-fold hollow sites; moreover, the occupation of the active sites is always close to zero at the investigated temperatures, so they are not blocked and may act as an indirect channel for adsorption. Thus, the rate of nitrogen adsorption on Ru(0 0 0 1) surface is governed by the rate of diffusion of nitrogen atoms from the active sites into the three-fold hollow sites. The analysis of thermodesorption spectra revealed an important role of repulsive interactions between the N atoms adsorbed on the hollow sites, the associated interaction parameter between nearest neighbors was estimated to be 5 kJ/mol. The presence of small amount of gold on Ru(0 0 0 1), apart of blocking the active sites, seems to remove the repulsion between nitrogen atoms

  20. Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity.

    Weir, Marion E; Mann, Jacqueline E; Corwin, Thomas; Fulton, Zachary W; Hao, Jennifer M; Maniscalco, Jeanine F; Kenney, Marie C; Roman Roque, Kristal M; Chapdelaine, Elizabeth F; Stelzl, Ulrich; Deming, Paula B; Ballif, Bryan A; Hinkle, Karen L

    2016-04-01

    Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. PMID:27001024

  1. Comparative analysis of the impact of geological activity on astronomical sites of the Canary Islands, Hawaii and Chile

    Eff-Darwich, A; Rodriguez-Losada, J A; de la Nuez, J; Hernandez-Gutierrez, L E; Romero-Ruiz, M C

    2009-01-01

    An analysis of the impact of seismic and volcanic activity was carried out at selected astronomical sites, namely the observatories of El Teide (Tenerife, Canary Islands), Roque de los Muchachos (La Palma, Canary Islands), Mauna Kea (Hawaii) and Paranal (Chile) and the candidate site of Cerro Ventarrones (Chile). Hazard associated to volcanic activity is low or negligible at all sites, whereas seismic hazard is very high in Chile and Hawaii. The lowest geological hazard in both seismic and volcanic activity was found at Roque de los Muchachos observatory, in the island of La Palma.

  2. Acetylene is an active-site-directed, slow-binding, reversible inhibitor of Azotobacter vinelandii hydrogenase

    The inhibition of purified and membrane-bound hydrogenase from Azotobacter vinelandii by dihydrogen-free acetylene was investigated. The inhibition was a time-dependent process which exhibited first-order kinetics. Both H2 and CO protected against the inhibition by acetylene. K/sub protect(app)/ values of 0.41 and 24 μM were derived for these gases, respectively. Both H2-oxidizing activity and the tritium exchange capacity of the purified enzyme were inhibited at the same rate by acetylene. Removal of acetylene reversed the inhibition for both the purified and the membrane-associated form of the enzyme. The purified hydrogenases from both Rhizobium japonicum and Alcaligenes eutrophus H16 were also inhibited by acetylene in a time-dependent fashion. These findings suggest that acetylene is an active-site-directed, slow-binding, reversible inhibitor of some membrane-bound hydrogenases from aerobic bacteria

  3. Active site coupling in PDE:PKA complexes promotes resetting of mammalian cAMP signaling.

    Krishnamurthy, Srinath; Moorthy, Balakrishnan Shenbaga; Xin Xiang, Lim; Xin Shan, Lim; Bharatham, Kavitha; Tulsian, Nikhil Kumar; Mihalek, Ivana; Anand, Ganesh S

    2014-09-16

    Cyclic 3'5' adenosine monophosphate (cAMP)-dependent-protein kinase (PKA) signaling is a fundamental regulatory pathway for mediating cellular responses to hormonal stimuli. The pathway is activated by high-affinity association of cAMP with the regulatory subunit of PKA and signal termination is achieved upon cAMP dissociation from PKA. Although steps in the activation phase are well understood, little is known on how signal termination/resetting occurs. Due to the high affinity of cAMP to PKA (KD ∼ low nM), bound cAMP does not readily dissociate from PKA, thus begging the question of how tightly bound cAMP is released from PKA to reset its signaling state to respond to subsequent stimuli. It has been recently shown that phosphodiesterases (PDEs) can catalyze dissociation of bound cAMP and thereby play an active role in cAMP signal desensitization/termination. This is achieved through direct interactions with the regulatory subunit of PKA, thereby facilitating cAMP dissociation and hydrolysis. In this study, we have mapped direct interactions between a specific cyclic nucleotide phosphodiesterase (PDE8A) and a PKA regulatory subunit (RIα isoform) in mammalian cAMP signaling, by a combination of amide hydrogen/deuterium exchange mass spectrometry, peptide array, and computational docking. The interaction interface of the PDE8A:RIα complex, probed by peptide array and hydrogen/deuterium exchange mass spectrometry, brings together regions spanning the phosphodiesterase active site and cAMP-binding sites of RIα. Computational docking combined with amide hydrogen/deuterium exchange mass spectrometry provided a model for parallel dissociation of bound cAMP from the two tandem cAMP-binding domains of RIα. Active site coupling suggests a role for substrate channeling in the PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. This is the first instance, to our knowledge, of PDEs directly interacting with a cAMP-receptor protein in a mammalian system, and

  4. Crystal Structure of Liganded Rat Peroxisomal Multifunctional Enzyme Type 1: A FLEXIBLE MOLECULE WITH TWO INTERCONNECTED ACTIVE SITES*

    Kasaragod, Prasad; Venkatesan, Rajaram; Kiema, Tiila R.; Hiltunen, J. Kalervo; Wierenga, Rik K.

    2010-01-01

    The crystal structure of the full-length rat peroxisomal multifunctional enzyme, type 1 (rpMFE1), has been determined at 2.8 Å resolution. This enzyme has three catalytic activities and two active sites. The N-terminal part has the crotonase fold, which builds the active site for the Δ3,Δ2-enoyl-CoA isomerase and the Δ2-enoyl-CoA hydratase-1 catalytic activities, and the C-terminal part has the (3S)-hydroxyacyl-CoA dehydrogenase fold and makes the (3S)-hydroxyacyl-CoA dehydrogenase active sit...

  5. Human holocarboxylase synthetase with a start site at methionine-58 is the predominant nuclear variant of this protein and has catalytic activity

    Highlights: → Unambiguous evidence is provided that methionine-58 serves as an in-frame alternative translation site for holocarboxylase synthetase (HLCS58). → Full-length HLCS and HLCS58 enter the nucleus, but HLCS58 is the predominant variant. → HLCS58 has biological activity as biotin protein ligase. -- Abstract: Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to both carboxylases in extranuclear structures and histones in cell nuclei, thereby mediating important roles in intermediary metabolism, gene regulation, and genome stability. HLCS has three putative translational start sites (methionine-1, -7, and -58), but lacks a strong nuclear localization sequence that would explain its participation in epigenetic events in the cell nucleus. Recent evidence suggests that small quantities of HLCS with a start site in methionine-58 (HLCS58) might be able to enter the nuclear compartment. We generated the following novel insights into HLCS biology. First, we generated a novel HLCS fusion protein vector to demonstrate that methionine-58 is a functional translation start site in human cells. Second, we used confocal microscopy and western blots to demonstrate that HLCS58 enters the cell nucleus in meaningful quantities, and that full-length HLCS localizes predominantly in the cytoplasm but may also enter the nucleus. Third, we produced recombinant HLCS58 to demonstrate its biological activity toward catalyzing the biotinylation of both carboxylases and histones. Collectively, these observations are consistent with roles of HLCS58 and full-length HLCS in nuclear events. We conclude this report by proposing a novel role for HLCS in epigenetic events, mediated by physical interactions between HLCS and other chromatin proteins as part of a larger multiprotein complex that mediates gene repression.

  6. Mapping the ribonucleolytic active site of bovine seminal ribonuclease. The binding of pyrimidinyl phosphonucleotide inhibitors.

    Dossi, Kyriaki; Tsirkone, Vicky G; Hayes, Joseph M; Matousek, Josef; Poucková, Pavla; Soucek, Josef; Zadinova, Marie; Zographos, Spyros E; Leonidas, Demetres D

    2009-11-01

    Bovine seminal ribonuclease (BS-RNase) is a 27kDa homodimeric enzyme and a member of the pancreatic RNase A superfamily. It is the only RNase with a quaternary structure and it is a mixture of two dimeric forms. In the most abundant form the active site is formed by the swapping of the N-terminal segments. BS-RNase is a potent antitumor agent with severe side effects such as aspermatogenicity, and immunosuppression. As a first step towards the design of potent inhibitors of this enzyme we mapped its active site through the study of the binding of uridine 2'-phosphate (U2'p), uridine 3'-phosphate (U3'p), uridine 5'-diphosphate (UDP), cytidine 3'-phosphate (C3'p), and cytidine 5-phosphate (C5'p), by kinetics, and X-ray crystallography. These phosphonucleotides are potent inhibitors with C3'p being the most potent with a K(i) value of 22 microM. Absorption, distribution, metabolism, and excretion pharmacokinetic property predictions reveal U2'p, U3'p, and C5'p as the most promising with respect to oral bioavailability. In vivo studies on the aspermatogenic effect have shown that C3'p and C5'p inhibit significantly this biological action of BS-RNase. PMID:19643512

  7. Identification of the active site histidine in Staphylococcus hyicus lipase using chemical modification and mass spectrometry.

    Boots, J W; van Dongen, W D; Verheij, H M; de Haas, G H; Haverkamp, J; Slotboom, A J

    1995-04-01

    Staphylococcus hyicus lipase is a serine hydrolase. In order to identify the active site histidine of S. hyicus lipase we have chemically modified S. hyicus lipase with 1-bromo-octan-2-one. The enzyme is rapidly inactivated by this inhibitor with a half-time of 578 s at pH 6.5 and 30 degrees C. Addition of the enzyme's cofactor calcium increases the inactivation rate approx. 2-fold. When n-hexadecylphosphocholine, a non-hydrolysable substrate analogue, is added the inactivation rate decreases about 3-fold, suggesting that a residue in the active site of S. hyicus lipase is involved in the inactivation reaction. Inactivation of S. hyicus lipase with 14C-labelled 1-bromo-octan-2-one shows that 1.4 moles of inhibitor per mole of lipase are incorporated. The results of an electrospray mass spectrometric study of the inactivated enzyme are consistent with this finding. In order to identify the modified residue, both the inactivated and the unmodified lipase were digested with cyanogen bromide followed by trypsin. The resulting peptides were analysed using HPLC and fast atom bombardment mass spectrometry. The results allow the modified residue to be assigned to the peptide Gly597-Lys612. Collision induced dissociation mass spectrometry allowed the modified residue to be identified as His-600. From these results we conclude that this residue forms part of the catalytic triad of S. hyicus lipase. PMID:7711054

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

    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

  9. Characterization of the Dielectric Constant in the Trichoderma reesei Cel7B Active Site.

    Song, Xiangfei; Wang, Yefei; Zhang, Shujun; Yan, Shihai; Li, Tong; Yao, Lishan

    2015-07-27

    An attempt is made to evaluate the dielectric constant of the Trichoderma reesei Cel7B active site. Through kinetic measurements, the pKa value of the catalytic acid E201 is determined. Mutations (away from E201) with net charge changes are introduced to perturb the E201 pKa. It is shown that the mutation with a +1 charge change (including G225R, G230R, and A335R) decreases the pKa of E201, whereas the mutation with a -1 charge change (including Q149E, A222D, G225D, and G230D) increases the pKa. This effect is consistent with the electrostatic interaction between the changed charge and the E201 side chain. The fitting of the experimental data yields an apparent dielectric constant of 25-80. Molecular dynamics simulations with explicit water molecules indicate that the high solvent accessibility of the active site contributes largely to the high dielectric constant. ONIOM calculations show that high dielectric constant benefits the catalysis through decreasing the energy of the transition state relative to that of the enzyme substrate complex. PMID:26114648

  10. New Insights Into the Transmissibility of Leishmania infantum From Dogs to Sand Flies: Experimental Vector-Transmission Reveals Persistent Parasite Depots at Bite Sites.

    Aslan, Hamide; Oliveira, Fabiano; Meneses, Claudio; Castrovinci, Philip; Gomes, Regis; Teixeira, Clarissa; Derenge, Candace A; Orandle, Marlene; Gradoni, Luigi; Oliva, Gaetano; Fischer, Laurent; Valenzuela, Jesus G; Kamhawi, Shaden

    2016-06-01

    Canine leishmaniasis (CanL) is a chronic fatal disease of dogs and a major source of human infection through propagation of parasites in vectors. Here, we infected 8 beagles through multiple experimental vector transmissions with Leishmania infantum-infected Lutzomyia longipalpis. CanL clinical signs varied, although live parasites were recovered from all dog spleens. Splenic parasite burdens correlated positively with Leishmania-specific interleukin 10 levels, negatively with Leishmania-specific interferon γ and interleukin 2 levels, and negatively with Leishmania skin test reactivity. A key finding was parasite persistence for 6 months in lesions observed at the bite sites in all dogs. These recrudesced following a second transmission performed at a distal site. Notably, sand flies efficiently acquired parasites after feeding on lesions at the primary bite site. In this study, controlled vector transmissions identify a potentially unappreciated role for skin at infectious bite sites in dogs with CanL, providing a new perspective regarding the mechanism of Leishmania transmissibility to vector sand flies. PMID:26768257

  11. On-site characterization and verification for removal of cesium contamination along an active rail spur

    A rapid, cost-effective sampling and analysis approach was used to conduct on-site analysis of soil and ballast for 137Cs contamination along an active rail spur in Oak Ridge, Tennessee. Savings in time and analytical costs were realized for both the characterization and cleanup phases of this project. For characterization, a procedure was developed to conduct gamma-logging of shallow boreholes. Portable gamma radiation survey instruments consisting of a 5- by 5-cm NaI detector coupled to a digital ratemeter/scaler with a single-channel analyzer were calibrated to a 137Cs source, then used for both surface and subsurface gamma radiation surveys. A correlation was made between the direct radiation measurements and 137Cs concentrations in surface and subsurface soil and ballast. Borehole logs of gamma-ray activity as a function of depth were used to estimate vertical profiles of the subsurface distribution of 137Cs. Information from these surveys was used to develop accurate 3-dimensional isopleths of contaminated areas, including estimates of waste volumes. After a remedial alternative was selected, on-site analytical methods were again used to verify that the cleanup level was achieved. Samples from excavated areas were analyzed for 137Cs by comparative gamma-ray counting using a multi-channel pulse height analyzer and a 3-in. diameter by 2-in. thick NaI crystal. The counting system was housed in a mobile field laboratory. To better direct ongoing excavation, a correlation factor between samples analyzed by gamma spectroscopy and measurements made by field instruments was determined. In both the characterization and the cleanup phases, sample results were verified by independent off-site laboratory analyses

  12. Accommodation of GDP-Linked Sugars in the Active Site of GDP-Perosamine Synthase

    Cook, Paul D.; Carney, Amanda E.; Holden, Hazel M. (UW)

    2009-01-12

    Perosamine (4-amino-4,6-dideoxy-d-mannose), or its N-acetylated form, is one of several dideoxy sugars found in the O-antigens of such infamous Gram-negative bacteria as Vibrio cholerae O1 and Escherichia coli O157:H7. It is added to the bacterial O-antigen via a nucleotide-linked version, namely GDP-perosamine. Three enzymes are required for the biosynthesis of GDP-perosamine starting from mannose 1-phosphate. The focus of this investigation is GDP-perosamine synthase from Caulobacter crescentus, which catalyzes the final step in GDP-perosamine synthesis, the conversion of GDP-4-keto-6-deoxymannose to GDP-perosamine. The enzyme is PLP-dependent and belongs to the aspartate aminotransferase superfamily. It contains the typically conserved active site lysine residue, which forms a Schiff base with the PLP cofactor. Two crystal structures were determined for this investigation: a site-directed mutant protein (K186A) complexed with GDP-perosamine and the wild-type enzyme complexed with an unnatural ligand, GDP-3-deoxyperosamine. These structures, determined to 1.6 and 1.7 {angstrom} resolution, respectively, revealed the manner in which products, and presumably substrates, are accommodated within the active site pocket of GDP-perosamine synthase. Additional kinetic analyses using both the natural and unnatural substrates revealed that the K{sub m} for the unnatural substrate was unperturbed relative to that of the natural substrate, but the k{sub cat} was lowered by a factor of approximately 200. Taken together, these studies shed light on why GDP-perosamine synthase functions as an aminotransferase whereas another very similar PLP-dependent enzyme, GDP-4-keto-6-deoxy-d-mannose 3-dehydratase or ColD, catalyzes a dehydration reaction using the same substrate.

  13. Research in Support of Remediation Activities at the Savannah River Site

    The USDOE Savannah River Site (SRS), an 803-km2 (310-mile2) facility located south of Aiken, SC on the upper Atlantic Coastal Plain and bounded to the west by the Savannah River, was established in the 1950s for the production and refinement of nuclear materials. To fulfill this mission during the past 50 years SRS has operated five nuclear reactors, two large chemical separation areas, waste disposal facilities (landfills, waste ponds, waste tanks, and waste stabilization), and a large number of research and logistics support facilities. Contaminants of concern (COC) resulting from site operations include chlorinated solvents, radionuclides, metals, and metalloids, often found as complex mixtures that greatly complicate remediation efforts when compared with civilian industries. The objective of this article is to provide a description of the lithology and hydrostratigraphy of the SRS, as well as a brief history of site operations and research activities as a preface to the current special section of Vadose Zone Journal (VZJ) dedicated to SRS, focusing mainly on issues that are unique to the USDOE complex. Contributions to the special section reflect a diverse range of topics, from hydrologic tracer experiments conducted both within the vadose and saturated zones to studies specifically aimed at identifying geochemical processes controlling the migration and partitioning of specific contaminants (e.g., TCE, 137Cs, U, and Pu) in SRS subsurface environments. Addressing the diverse environmental challenges of the SRS provides a unique opportunity to conduct both fundamental and applied research across a range of experimental scales. Hence, the SRS has been a pioneering force in several areas of environmental research and remediation, often through active interdisciplinary collaboration with researchers from other USDOE facilities, academic and federal institutions, and commercial entities

  14. Active Sites of Spinoxin, a Potassium Channel Scorpion Toxin, Elucidated by Systematic Alanine Scanning.

    Peigneur, Steve; Yamaguchi, Yoko; Kawano, Chihiro; Nose, Takeru; Nirthanan, Selvanayagam; Gopalakrishnakone, Ponnampalam; Tytgat, Jan; Sato, Kazuki

    2016-05-31

    Peptide toxins from scorpion venoms constitute the largest group of toxins that target the voltage-gated potassium channel (Kv). Spinoxin (SPX) isolated from the venom of scorpion Heterometrus spinifer is a 34-residue peptide neurotoxin cross-linked by four disulfide bridges. SPX is a potent inhibitor of Kv1.3 potassium channels (IC50 = 63 nM), which are considered to be valid molecular targets in the diagnostics and therapy of various autoimmune disorders and cancers. Here we synthesized 25 analogues of SPX and analyzed the role of each amino acid in SPX using alanine scanning to study its structure-function relationships. All synthetic analogues showed similar disulfide bond pairings and secondary structures as native SPX. Alanine replacements at Lys(23), Asn(26), and Lys(30) resulted in loss of activity against Kv1.3 potassium channels, whereas replacements at Arg(7), Met(14), Lys(27), and Tyr(32) also largely reduced inhibitory activity. These results suggest that the side chains of these amino acids in SPX play an important role in its interaction with Kv1.3 channels. In particular, Lys(23) appears to be a key residue that underpins Kv1.3 channel inhibition. Of these seven amino acid residues, four are basic amino acids, suggesting that the positive electrostatic potential on the surface of SPX is likely required for high affinity interaction with Kv1.3 channels. This study provides insight into the structure-function relationships of SPX with implications for the rational design of new lead compounds targeting potassium channels with high potency. PMID:27159046

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

    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

  16. The "Simeiz-Katzively" co-location site of space geodesy techniques: current state and future activity

    Yatskiv, Y.; Odynets, P.; Volvach, O.

    2014-12-01

    Two Satellite Laser Ranging (SLR) stations, two Global Navigation Satellite System (GNSS) stations and Very Long Base Interferometry (VLBI) station are placed on the "Simeiz-Katzively" co-location site. The activity of these space geodesy techniques in 2010-2012 is presented. Special attention is paid on results of new local tie surveys at this co-location site.

  17. Examining guidelines for the site selection for geological disposal. Research activities principle for future risk assessment and quality assurance

    A draft for the committee in charge of nuclear safety regulations regarding geological disposal of high-level radioactive wastes from nuclear facilities is presented. The report particularly concerns with a line of policy or principle at the stage of investigation activities for the site selection and site characterization. IAEA guidelines are consulted. (S. Ohno)

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

    Beke-Somfai, T.

    2013-01-23

    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.

  19. Analysis of surface binding sites (SBSs) in carbohydrate active enzymes with focus on glycoside hydrolase families 13 and 77

    Cockburn, Darrell; Wilkens, Casper; Ruzanski, Christian;

    2014-01-01

    Surface binding sites (SBSs) interact with carbohydrates outside of the enzyme active site. They are frequently situated on catalytic domains and are distinct from carbohydrate binding modules (CBMs). SBSs are found in a variety of enzymes and often seen in crystal structures. Notably about half ...

  20. The active site of hen egg-white lysozyme: flexibility and chemical bonding

    Chemical bonding at the active site of lysozyme is analyzed on the basis of a multipole model employing transferable multipole parameters from a database. Large B factors at low temperatures reflect frozen-in disorder, but therefore prevent a meaningful free refinement of multipole parameters. Chemical bonding at the active site of hen egg-white lysozyme (HEWL) is analyzed on the basis of Bader’s quantum theory of atoms in molecules [QTAIM; Bader (1994 ▶), Atoms in Molecules: A Quantum Theory. Oxford University Press] applied to electron-density maps derived from a multipole model. The observation is made that the atomic displacement parameters (ADPs) of HEWL at a temperature of 100 K are larger than ADPs in crystals of small biological molecules at 298 K. This feature shows that the ADPs in the cold crystals of HEWL reflect frozen-in disorder rather than thermal vibrations of the atoms. Directly generalizing the results of multipole studies on small-molecule crystals, the important consequence for electron-density analysis of protein crystals is that multipole parameters cannot be independently varied in a meaningful way in structure refinements. Instead, a multipole model for HEWL has been developed by refinement of atomic coordinates and ADPs against the X-ray diffraction data of Wang and coworkers [Wang et al. (2007), Acta Cryst. D63, 1254–1268], while multipole parameters were fixed to the values for transferable multipole parameters from the ELMAM2 database [Domagala et al. (2012), Acta Cryst. A68, 337–351] . Static and dynamic electron densities based on this multipole model are presented. Analysis of their topological properties according to the QTAIM shows that the covalent bonds possess similar properties to the covalent bonds of small molecules. Hydrogen bonds of intermediate strength are identified for the Glu35 and Asp52 residues, which are considered to be essential parts of the active site of HEWL. Furthermore, a series of weak C

  1. Microquake activity associated with underground nuclear testing at the Nevada Test Site

    In the fall of 1976, the Los Alamos Close-In Seismic Network was added to the existing strong motion net deployed around each nuclear test conducted by Los Alamos. Six to ten stations, including both accelerometers and seismometers, are installed within a two-DOB (depth of burial) circle around SGZ (surface ground zero) and operated until the major portion of the microquake activity ceases, usually within 48 hours. Epicentral locations are determined and local magnitudes are calculated from event durations. Four primary conclusions have been reached on the basis of the data analyzed to date: (1) major faults bounding the immediate site of a nuclear test confine the observed microquake activity to the structural block in which the test is conducted; (2) microquake epicenters are generally distributed around the cavity created by the nuclear test with the peak occurrence generally occurring about three cavity radii away from the working point; (3) magnitudes of locatable microquakes apparently distribute randomly over the entire region of activity; and (4) the microquake activity as a function of time appears to be controlled by the collapse phenomenology

  2. A selective, slow binding inhibitor of factor VIIa binds to a nonstandard active site conformation and attenuates thrombus formation in vivo.

    Olivero, Alan G; Eigenbrot, Charles; Goldsmith, Richard; Robarge, Kirk; Artis, Dean R; Flygare, John; Rawson, Thomas; Sutherlin, Daniel P; Kadkhodayan, Saloumeh; Beresini, Maureen; Elliott, Linda O; DeGuzman, Geralyn G; Banner, David W; Ultsch, Mark; Marzec, Ulla; Hanson, Stephen R; Refino, Canio; Bunting, Stuart; Kirchhofer, Daniel

    2005-03-11

    The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF.FVIIa is also implicated in thrombosis-related disorders and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF.FVIIa (Ki = 0.35 +/- 0.11 nM). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF.FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa.G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217-Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a "flipped" Lys192-Gly193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by approximately 70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo. PMID:15632123

  3. Short-time dynamics of pH-dependent conformation and substrate binding in the active site of beta-glucosidases: A computational study.

    Flannelly, David F; Aoki, Thalia G; Aristilde, Ludmilla

    2015-09-01

    The complete degradation of cellulose to glucose is essential to carbon turnover in terrestrial ecosystems and to engineered biofuel production. A rate-limiting step in this pathway is catalyzed by beta-glucosidase (BG) enzymes, which convert cellulobiose into two glucose molecules. The activity of these enzymes has been shown to vary with solution pH. However, it is not well understood how pH influences the enzyme conformation required for catalytic action on the substrate. A structural understanding of this pH effect is important for predicting shifts in BG activity in bioreactors and environmental matrices, in addition to informing targeted protein engineering. Here we applied molecular dynamics simulations to explore conformational and substrate binding dynamics in two well-characterized BGs of bacterial (Clostridium cellulovorans) and fungal (Trichoderma reesei) origins as a function of pH. The enzymes were simulated in an explicit solvated environment, with NaCl as electrolytes, at their prominent ionization states obtained at pH 5, 6, 7, and 7.5. Our findings indicated that pH-dependent changes in the ionization states of non-catalytic residues localized outside of the immediate active site led to pH-dependent disruption of the active site conformation. This disruption interferes with favorable H-bonding interactions with catalytic residues required to initiate catalysis on the substrate. We also identified specific non-catalytic residues that are involved in stabilizing the substrate at the optimal pH for enzyme activity. The simulations further revealed the dynamics of water-bridging interactions both outside and inside the substrate binding cleft during structural changes in the enzyme-substrate complex. These findings provide new structural insights into the pH-dependent substrate binding specificity in BGs. PMID:26160737

  4. Unusual non-fluorescent broad spectrum siderophore activity (SID EGYII) by Pseudomonas aeruginosa strain EGYII DSM 101801 and a new insight towards simple siderophore bioassay.

    Embaby, Amira M; Heshmat, Yasmin; Hussein, Ahmed

    2016-03-01

    Present study highlights an unusual non-fluorescent hydroxamate broad spectrum siderophore (SID EGYII) activity from Pseudomonas aeruginosa strain EGYII DSM 101801, a soil bacterial isolate, along with simple low cost effective siderophore bioassay. Detection of SID EGYII activity qualitatively was proved by masking this activity against Erwinia amylovora strain EGY1 DSM 101800, an indicator strain, in well-cut diffusion assay containing 100 µM FeCl3. SID EGYII activity was expressed quantitatively as arbitrary units [Siderophore arbitrary units (SAU)] 380 SAU/mL against E. amylovora strain EGY1 DSM 101800. Maximal SID EGYII activity was achieved upon growing P. aeruginosa strain EGYII DSM 101801 in PYB broth at 180 rpm for 24 h. SID EGYII displayed a broad spectrum antimicrobial activity against some human pathogens (i.e., Gram-positive bacteria, Gram-negative bacteria and yeasts) and a fireblight plant pathogen. Interestingly, transformants of Escherichia coli JM109 (DE3)pSID/EGYII harboring P. aeruginosa strain EGYII DSM 101801 plasmid demonstrated a perceivable antimicrobial activity against E. amylovora strain EGY1 DSM 101800. The broad spectrum antimicrobial activity of the unusual non-fluorescent SID EGYII would underpin its high potential in targeting bacterial pathogens posing probable threats to human health and agricultural economy. The present simple low cost effective bioassay is a new insight towards an alternative to the expensive cumbersome siderophore Chrome Azurol S assay. PMID:27015845

  5. Human uroporphyrinogen III synthase: NMR-based mapping of the active site.

    Cunha, Luis; Kuti, Miklos; Bishop, David F; Mezei, Mihaly; Zeng, Lei; Zhou, Ming-Ming; Desnick, Robert J

    2008-05-01

    Uroporphyrinogen III synthase (URO-synthase) catalyzes the cyclization and D-ring isomerization of hydroxymethylbilane (HMB) to uroporphyrinogen (URO'gen) III, the cyclic tetrapyrrole and physiologic precursor of heme, chlorophyl, and corrin. The deficient activity of human URO-synthase results in the autosomal recessive cutaneous disorder, congenital erythropoietic porphyria. Mapping of the structural determinants that specify catalysis and, potentially, protein-protein interactions is lacking. To map the active site and assess the enzyme's possible interaction in a complex with hydroxymethylbilane-synthase (HMB-synthase) and/or uroporphyrinogen-decarboxylase (URO-decarboxylase) by NMR, an efficient expression and purification procedure was developed for these cytosolic enzymes of heme biosynthesis that enabled preparation of special isotopically-labeled protein samples for NMR characterization. Using an 800 MHz instrument, assignment of the URO-synthase backbone (13)C(alpha) (100%), (1)H(alpha) (99.6%), and nonproline (1)H(N) and (15)N resonances (94%) was achieved as well as 85% of the side-chain (13)C and (1)H resonances. NMR analyses of URO-synthase titrated with competitive inhibitors N(D)-methyl-1-formylbilane (NMF-bilane) or URO'gen III, revealed resonance perturbations of specific residues lining the cleft between the two major domains of URO synthase that mapped the enzyme's active site. In silico docking of the URO-synthase crystal structure with NMF-bilane and URO'gen III was consistent with the perturbation results and provided a 3D model of the enzyme-inhibitor complex. The absence of chemical shift changes in the (15)N spectrum of URO-synthase mixed with the homogeneous HMB-synthase holoenzyme or URO-decarboxylase precluded occurrence of a stable cytosolic enzyme complex. PMID:18004775

  6. New insights into the QuikChangeTM process guide the use of Phusion DNA polymerase for site-directed mutagenesis

    Xia, Yongzhen; Chu, Wenqiao; Qi, Qingsheng; Xun, Luying

    2014-01-01

    The QuikChangeTM site-directed mutagenesis method is popular but imperfect. An improvement by using partially overlapping primers has been reported several times; however, it is incompatible with the proposed mechanism. The QuikChangeTM method using complementary primers is proposed to linearly amplify a target plasmid with the products annealing to produce double-stranded DNA molecules with 5′-overhangs. The overhang annealing is supposed to form circular plasmids with staggered breaks, whic...

  7. Crystal Structure of A. aeolicus Argonaute, a Site-Specific DNA-Guided Endoribonuclease, Provides Insights into RISC-Mediated mRNA Cleavage

    Yuan, Yu-Ren; Pei, Yi; Ma, Jin-Biao; Kuryavyi, Vitaly; Zhadina, Maria; Meister, Gunter; Chen, Hong-Ying; Dauter, Zbigniew; Tuschl, Thomas; Patel, Dinshaw J.

    2005-01-01

    Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies, which establish this eubacterial Ago as a bona fide guide DNA strand-mediated site-specific RNA endonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the ...

  8. Otter (Lontra longicaudis Spraint and Mucus Depositions: Early Ecological Insights into the Differences in Marking Site Selection and Implications for Monitoring Prey Availability

    Nathan James Roberts

    2016-04-01

    Full Text Available Otters are not territorial in the classical sense of marking territory boundaries. Instead they mark key resource areas within the territory with faeces, or spraint, for olfactory communication. There is a high level of site fidelity in otter marking behaviour and the function of scent marks may explain their spatial distribution. Typically marking sites are in proximity to deep water which provides key resource areas for energy-efficient foraging. Occasionally spraint is laden with mucus, which may also be deposited in isolation without any faecal material. Any difference in the microhabitat variables predisposing site selection by otters with mucus present and absent in depositions has not yet been quantitatively investigated. This study serves as a primary exploration of these selective processes. Here we show that habitat selection by the Neotropical otter (Lontra longicaudis is different when mucus is deposited at a site compared to when it is absent. The cause of mucus deposition has been suggested in other otter species to indicate reduced prey availability or reproductive state. Depositions here were associated with deep water as in other studies, and temporally, the relative abundance of those with mucus present was highest toward the end of the dry season when prey availability is assumed relatively low. Here we infer how the monitoring of mucus prevalence may be used as a valuable efficient indirect index of the status of otters and their prey. For species whose primary threats include reduced prey availability, such as the Neotropical otter, research attention to these aspects of behavioural ecology are particularly significant in applied conservation. Furthermore, research to validate indirect indices of prey availability and species-habitat interactions may extend to benefit recreational interests and broader human-wildlife conflict mitigation strategies.

  9. A new luminescence dating chronology for the Rhafas cave site (NE Morocco): Insights into Palaeolithic human cultural change under varying palaeoenvironmental conditions in the Maghreb

    Dörschner, Nina; Fitzsimmons, Kathryn E.; Ditchfield, Peter; McLaren, Sue J.; Steele, Teresa E.; Zielhofer, Christoph; McPherron, Shannon P.; Bouzouggar, Abdeljalil; Hublin, Jean-Jacques

    2016-04-01

    Archaeological sites in northern Africa provide a rich record that is of increasing importance for current debates relating to the origins of modern human behaviour and to Out of Africa human dispersal events. Particular interest is placed on the cultural transition between the North African Middle Stone Age (MSA) and Late Stone Age (LSA), and the need for accurately defined chronologies, however the timing and nature of Palaeolithic human behaviour and dispersal across north-western Africa (the Maghreb) and potential correlation with environmental conditions remain poorly understood. The inland cave site of Rhafas (Morocco) preserves a long stratified sequence providing valuable chronological information about cultural changes in the Maghreb spanning the North African MSA through to the Neolithic. In this study, we apply optically stimulated luminescence (OSL) dating on sand-sized quartz grains to the cave deposits of Rhafas as well as to a section on the terrace in front of the cave entrance. Single grain OSL dating reliably constrains the timing of technocomplexes beyond the limits of radiocarbon by directly dating sediment associated with archaeological traces. We combine OSL dating with multi-proxy geological investigations (XRF, grain size analyses, stable isotopes, thin sections) to investigate site formation processes and reconstruct palaeoenvironmental conditions during human occupation phases at Rhafas. Our results indicate that the occupation of the site started at least in MIS 6 during a phase of relatively arid environmental conditions. Climatic amelioration after c.140 ka is associated with a change in sediment geochemistry at the site, most likely linked to a change in sediment source due to shifting wind directions. Tanged pieces - typical for the classical Aterian technocomplex - start to occur in the archaeological sequence in MIS 5, consistent with previously published chronological data from the Maghreb. From 55 ka, climatic conditions were

  10. Relevance of Co, Ag-ferrierite catalysts acidity and cation siting to CH4-NOx-SCR activity

    The influence of acidity on Ag.Co exchanged ferrierite obtained from different parent forms was tested in CH4-DeNOx reaction. Ag and Co cation siting distribution and residual zeolite acidity were evaluated by means of a quantitative evaluation of catalyst acidity through NH3-TPD experiments and a detailed structural catalyst characterization by Rietveld refinement. A new nomenclature for the cation sites in hydrated and dehydrated cation exchange ferrierites was introduced for sake of clarity. The sites relative populations obtained by the UV-Vis spectra did not agree with the values given by the Rietveld refinement and the SCR. activity scale since the high abundance of Co cations in the retained most active position. Co2a, was shown by the less active catalyst obtained from the Na,K form. It was concluded that SCR activity does not only depend on Co and Ag siting within the zeolite framework but also by the presence of residual acidity evidenced on the most active catalysts. CH4 combustion tests showed that the presence of residual acidity appears relevant to SCR catalytic performances, likely related to its ability in methane activation. The importance of the coexistence of Co and zeolitic, acid sites for the HC-SCR suggested that SCR reaction could proceed on a dual site.

  11. Holocene local forest history at two sites in Småland, southern Sweden - insights from quantitative reconstructions using the Landscape Reconstruction Algorithm

    Cui, Qiaoyu; Gaillard, Marie-José; Lemdahl, Geoffrey; Olsson, Fredrik; Sugita, Shinya

    2010-05-01

    Quantitative reconstruction of past vegetation using fossil pollen was long very problematic. It is well known that pollen percentages and pollen accumulation rates do not represent vegetation abundance properly because pollen values are influenced by many factors of which inter-taxonomic differences in pollen productivity and vegetation structure are the most important ones. It is also recognized that pollen assemblages from large sites (lakes or bogs) record the characteristics of the regional vegetation, while pollen assemblages from small sites record local features. Based on the theoretical understanding of the factors and mechanisms that affect pollen representation of vegetation, Sugita (2007a and b) proposed the Landscape Reconstruction Algorithm (LRA) to estimate vegetation abundance in percentage cover for well defined spatial scales. The LRA includes two models, REVEALS and LOVE. REVEALS estimates regional vegetation abundance at a spatial scale of 100 km x 100 km. LOVE estimates local vegetation abundance at the spatial scale of the relevant source area of pollen (RSAP sensu Sugita 1993) of the pollen site. REVEALS estimates are needed to apply LOVE in order to calculate the RSAP and the vegetation cover within the RSAP. The two models were validated theoretically and empirically. Two small bogs in southern Sweden were studied for pollen, plant macrofossil, charcoal, and coleoptera in order to reconstruct the local Holocene forest and fire history (e.g. Greisman and Gaillard 2009; Olsson et al. 2009). We applied the LOVE model in order to 1) compare the LOVE estimates with pollen percentages for a better understanding of the local forest history; 2) obtain more precise information on the local vegetation to explain between-sites differences in fire history. We used pollen records from two large lakes in Småland to obtain REVEALS estimates for twelve continuous 500-yrs time windows. Following the strategy of the Swedish VR LANDCLIM project (see Gaillard

  12. Sport for All? Insight into Stratification and Compensation Mechanisms of Sporting Activity in the 27 European Union Member States

    Van Tuyckom, Charlotte; Scheerder, Jeroen

    2010-01-01

    Physical activity is an important public health issue and the benefits of an active lifestyle in relation to well-being and health have been strongly emphasised in recent years in Europe, as well as in most parts of the world. However, previous research has shown that physical activity within Europe and its member states is stratified. The present…

  13. Multiple-site mutations of phage Bp7 endolysin improves its activities against target bacteria

    Can; Zhang; Yuanchao; Wang; Huzhi; Sun; Huiying; Ren

    2015-01-01

    The widespread use of antibiotics has caused serious drug resistance. Bacteria that were once easily treatable are now extremely difficult to treat. Endolysin can be used as an alternative to antibiotics for the treatment of drug-resistant bacteria. To analyze the antibacterial activity of the endolysin of phage Bp7(Bp7e), a 489-bp DNA fragment of endolysin Bp7e was PCR-amplified from a phage Bp7 genome and cloned, and then a p ET28a-Bp7e prokaryotic expression vector was constructed. Two amino acids were mutated(L99A, M102E) to construct p ET28a-Bp7Δe, with p ET28a-Bp7e as a template. Phylogenetic analysis suggested that BP7e belongs to a T4-like phage endolysin group. Bp7e and its mutant Bp7Δe were expressed in Escherichia coli BL21(DE3) as soluble proteins. They were purified by affinity chromatography, and then their antibacterial activities were analyzed. The results demonstrated that the recombinant proteins Bp7e and Bp7Δe showed obvious antibacterial activity against Micrococcus lysodeikticus but no activity against Staphylococcus aureus. In the presence of malic acid, Bp7e and Bp7Δe exhibited an effect on most E. coli strains which could be lysed by phage Bp7, but no effect on Salmonella paratyphi or Pseudomonas aeruginosa. Moreover, Bp7Δe with double-site mutations showed stronger antibacterial activity and a broader lysis range than Bp7e.

  14. Biochemical characterization of mutants in the active site residues of the β-galactosidase enzyme of Bacillus circulans ATCC 31382

    Bultema, Jelle B; Bas J.H. Kuipers; Lubbert Dijkhuizen

    2014-01-01

    The Bacillus circulans ATCC 31382 β-galactosidase (BgaD) is a retaining-type glycosidase of glycoside hydrolase family 2 (GH2). Its commercial enzyme preparation, Biolacta N5, is used for commercial-scale production of galacto-oligosaccharides (GOS). The BgaD active site and catalytic amino acid residues have not been studied. Using bioinformatic routines we identified two putative catalytic glutamates and two highly conserved active site histidines. The site-directed mutants E447N, E532Q, an...

  15. Site- and horizon-specific patterns of microbial community structure and enzyme activities in permafrost-affected soils of Greenland

    Gittel, Antje; Barta, Jiri; Kohoutova, Iva;

    2014-01-01

    its impact on the carbon budget are thus still highly uncertain. However, the fate of OC is not only determined by abiotic factors, but closely tied to microbial activity. We investigated eight soil profiles in northeast Greenland comprising two sites with typical tundra vegetation and one wet fen...... site. We assessed microbial community structure and diversity (SSU rRNA gene tag sequencing, quantification of bacteria, archaea and fungi), and measured hydrolytic and oxidative enzyme activities. Sampling site and thus abiotic factors had a significant impact on microbial community structure...

  16. Assessment of national systems for obtaining local acceptance of waste management siting and routing activities

    Paige, H.W.; Lipman, D.S.; Owens, J.E.

    1980-07-01

    There is a rich mixture of formal and informal approaches being used in our sister nuclear democracies in their attempts to deal with the difficulties of obtaining local acceptance for siting of waste management facilities and activities. Some of these are meeting with a degree of success not yet achieved in the US. Although this survey documents and assesses many of these approaches, time did not permit addressing in any detail their relevance to common problems in the US. It would appear the US could benefit from a periodic review of the successes and failures of these efforts, including analysis of their applicability to the US system. Of those countries (Germany, Sweden, Switzerland, Japan, Belgium, and the US) who are working to a time table for the preparation of a high-level waste (HLW) repository, Germany is the only country to have gained local siting acceptance for theirs. With this (the most difficult of siting problems) behind them they appear to be in the best overall condition relative to waste management progress and plans. This has been achieved without a particularly favorable political structure, made up for by determination on the part of the political leadership. Of the remaining three countries studied (France, UK and Canada) France, with its AVM production facility, is clearly the world leader in the HLW immobilization aspect of waste management. France, Belgium and the UK appear to have the least favorable political structures and environments for arriving at waste management decisions. US, Switzerland and Canada appear to have the least favorable political structures and environments for arriving at waste management decisions.

  17. Natural resource management activities at the Savannah River Site. Environmental Assessment

    1993-07-01

    This environmental assessment (EA) reviews the environmental consequences of ongoing natural resource management activities on the Savannah River Site (SRS). Appendix A contains the Natural Resources Management Plant (NRMP). While several SRS organizations have primary responsibilities for different elements of the plan, the United States Department of Agriculture (USDA), Forest Service, Savannah River Forest Station (SRFS) is responsible for most elements. Of the river scenarios defined in 1985, the High-Intensity Management alternative established the upper bound of environmental consequences; it represents a more intense level of resource management than that being performed under current resource management activities. This alternative established compliance mechanisms for several natural resource-related requirements and maximum practical timber harvesting. Similarly, the Low-Intensity Management alternative established the lower bound of environmental consequences and represents a less intense level of resource management than that being performed under current resource management activities. This alternative also established compliance mechanisms, but defined a passively managed natural area. The Proposed Action of this EA describes the current level of multiple-natural resource management. This EA reviews the proposed action, and the high and low intensity alternative scenarios.

  18. Microbial community changes along the active seepage site of one cold seep in the Red Sea.

    Cao, Huiluo

    2015-07-21

    The active seepage of the marine cold seeps could be a critical process for the exchange of energy between the submerged geosphere and the sea floor environment through organic-rich fluids, potentially even affecting surrounding microbial habitats. However, few studies have investigated the associated microbial community changes. In the present study, 16S rRNA genes were pyrosequenced to decipher changes in the microbial communities from the Thuwal seepage point in the Red Sea to nearby marine sediments in the brine pool, normal marine sediments and water, and benthic microbial mats. An unexpected number of reads from unclassified groups were detected in these habitats; however, the ecological functions of these groups remain unresolved. Furthermore, ammonia-oxidizing archaeal community structures were investigated using the ammonia monooxygenase subunit A (amoA) gene. Analysis of amoA showed that planktonic marine habitats, including seeps and marine water, hosted archaeal ammonia oxidizers that differed from those in microbial mats and marine sediments, suggesting modifications of the ammonia oxidizing archaeal (AOA) communities along the environmental gradient from active seepage sites to peripheral areas. Changes in the microbial community structure of AOA in different habitats (water vs. sediment) potentially correlated with changes in salinity and oxygen concentrations. Overall, the present results revealed for the first time unanticipated novel microbial groups and changes in the ammonia-oxidizing archaea in response to environmental gradients near the active seepages of a cold seep.

  19. Analysis of the activity of virus internal ribosome entry site in silkworm Bombyx mori

    Lupeng Ye; Lanfang Zhuang; Jisheng Li; Zhengying You; Jianshe Liang; Hao Wei; Jianrong Lin

    2013-01-01

    Internal ribosome entry site (IRES) has been widely used in genetic engineering; however,the application in silkworm (Bombyx mori) has hardly been reported.In this study,the biological activity of partial sequence of Encephalomyocardltis virus (EMCV) IRES,Rhopalosiphum padi virus (RhPV)IRES,and the hybrid of IRES of EMCV and RhPV were investigated in Spodoptera frugiperda (Sf9) cell line and silkworm tissues.The hybrid IRES of EMCV and RhPV showed more effective than EMCV IRES or RhPV IRES in promoting downstream gene expression in insect and silkworm.The activities of all IRESs in middle silk gland of silkworm were higher than those in the fat body and posterior silk gland.The hybrid IRES of EMCV and RhPV was integrated into silkworm genome by transgenic technology to test biological activity of IRES.Each of the positive transgenic individuals had significant expression of report gene EGFP.These results suggested that IRES has a potential to be used in the genetic engineering research of silkworm.

  20. Microbial community changes along the active seepage site of one cold seep in the Red Sea

    Huiluo eCao

    2015-07-01

    Full Text Available The active seepage of the marine cold seeps could be a critical process for the exchange of energy between the submerged geosphere and the sea floor environment through organic-rich fluids, potentially even affecting surrounding microbial habitats. However, few studies have investigated the associated microbial community changes. In the present study, 16S rRNA genes were pyrosequenced to decipher changes in the microbial communities from the Thuwal seepage point in the Red Sea to nearby marine sediments in the brine pool, normal marine sediments and water, and benthic microbial mats. An unexpected number of reads from unclassified groups were detected in these habitats; however, the ecological functions of these groups remain unresolved. Furthermore, ammonia-oxidizing archaeal community structures were investigated using the ammonia monooxygenase subunit A (amoA gene. Analysis of amoA showed that planktonic marine habitats, including seeps and marine water, hosted archaeal ammonia oxidizers that differed from those in microbial mats and marine sediments, suggesting modifications of the ammonia oxidizing archaeal communities along the environmental gradient from active seepage sites to peripheral areas. Changes in the microbial community structure of ammonia oxidizing archaea in different habitats (water versus sediment potentially correlated with changes in salinity and oxygen concentrations. Overall, the present results revealed for the first time unanticipated novel microbial groups and changes in the ammonia-oxidizing archaea in response to environmental gradients near the active seepages of a cold seep.